Replacement of IGE-FCERI

Abstract

Disclosed herein are single domain antibody-based constructs that are useful in the treatment of IgE-related diseases or disorders by promoting the dissociation of IgE from high affinity IgE receptors. The present application further discloses a composition comprising the construct. The present application also provides nucleic acids and vectors, and methods of treating IgE related diseases.

Description

[Field of the Invention]

[0001] The present invention relates to single domain antibody based constructs for treating IgE related diseases or disorders by promoting the dissociation of IgE from high affinity IgE receptors. [Background of the invention]

[0003] Allergic diseases are inflammatory diseases in which immunoglobulin E (IgE) antibodies play a key role. IgE recognizes allergens through its Fab region, while its effector function is controlled by the interaction of the Fc region with two major cell surface receptors: the high affinity IgE receptor (FcεRI) and the low affinity IgE receptor (FcεRII / CD23). IgE binds to FcεRI with high affinity (KD of 0.01 to 0.1 nM) at a site involving the Cε3 domain of IgE-Fc, providing the basis for the long-term stability of effector cells and a half-life of ∼10 days (Chang et al., 2000). IgE has much lower binding affinity to CD23 at sites involving the Cε3 and Cε4 domains of IgE-Fc. IgE bound by allergen binding to cross-linked FcεRI leads to activation and degranulation of effector cells (e.g., mast cells and basophils), triggering the release of histamine and other inflammatory mediators and the synthesis of many cytokines and other factors that can produce inflammatory responses. IgE also binds to CD23 located on cell types such as B cells, macrophages, platelets, and epithelial cells and plays a variety of roles, for example, in regulating IgE synthesis, allergen transcytosis, transport of IgE:allergen immune complexes through the intestine and airways, and promoting antigen presentation on antigen-presenting cells.

[0004] Recent insights have revealed that IgE cannot bind to both types of receptors simultaneously due to the conformational flexibility of its Cε3 domain (Holdom M., 2011, Dhaliwal et al., 2012). FcεRI binds to IgE only when the Cε3 domain adopts the so-called "open" conformation, while CD23 binds to IgE only when the Cε3 domain adopts the so-called "closed" conformation. This conformational selectivity has been further described using an engineered IgE-Fc Cε3-4, which has a disulfide bond at the Cys-335 (IgE-Fc Cε3-4, 335) position, maintaining IgE-Fc in a "closed" conformation. FcεRI can only bind to this construct after the disulfide bond is reduced (Wurzburg et al., 2012). Importantly, FcεRI stabilizes IgE in the open conformation, thereby preventing CD23 from binding to IgE. In contrast, CD23 binding to IgE stabilizes a closed conformation that prevents FcεRI from binding to IgE. This makes the binding of the two receptors to IgE mutually exclusive and prevents the two pathways from overlapping (Drinkwater et al., 2014).

[0005] IgE adopts a compact, bent conformation, as evident from the crystal structure of IgE Fc Cε2-Cε4 (PDB: 2WQR), where the backfold of the Cε2 domain makes extensive contacts with the Cε3 and even Cε4 domains. However, molecular dynamics simulations and biophysical studies revealed a transient extended conformation in which the Cε2 domain “flips” from one side to the other. Moreover, this energetically less favorable extended conformation was recently stabilized by two anti-IgE Fab fragments, resulting in FcεRI inhibition, revealing a potential new mode of action for anti-IgE molecules (Drinkwater et al., 2014).

[0006] However, only one therapeutic anti-IgE antibody (Omalizumab sdAb sold by Novartis / Genentech, ) is approved for the treatment of allergic diseases including asthma, nasal polyposis, and chronic spontaneous urticaria (CSU). It is a humanized monoclonal IgG1 antibody that binds to the Cε3 domain of free IgE, thereby inhibiting the binding of IgE to FcεRI and CD23. As a result, the binding of circulating IgE to the FcεRI receptor and the CD23 receptor is impaired. Omalizumab sdAb has several disadvantages, including frequent administration (e.g., once every 2 to 4 weeks), the need for large injections, immunogenicity, and immune complex formation. Since Omalizumab sdAb only processes free IgE, Omalizumab sdAb treatment may not be effective in preventing all types of allergic reactions, such as anaphylaxis. It has been shown that even trace amounts of allergen-specific IgE bound to FcεRI can cause degranulation of cells such as mast cells and lead to severe allergic reactions.

[0007] In recent years, the concept of specifically targeting IgE as a therapeutic concept has attracted widespread interest. For example, certain antibodies and antibody mimetics have been found to displace bound IgE from FcεRI and CD23. In other words, these antibodies would be able to disrupt the IgE:FcεRI complex (Balbino et al., 2018).

[0008] DARPins TM They are small and highly stable non-antibody protein scaffolds. The advantage of some anti-IgE DARPins is that they can not only neutralize free IgE, but also actively disrupt pre-formed IgE:FcεRI complexes by promoting dissociation mechanisms. Compared with the poorly displacing omalizumab sdAb, anti-IgE DARPins were 10,000 times more efficient than omalizumab sdAb in in vitro and ex vivo studies (Kim B et al., 2012 and Baumann, MJ 2010).

[0009] Biparatopic (bispecific) anti-IgE DARPins designed by linking two different anti-IgE DARPins (e.g., DARPin bi53_79) have been shown to be more effective than single epitope-binding DARPins (Eggel et al., 2014), and recently, Pennington et al. reported that a highly displacing, fast-acting anti-IgE DARPin molecule (KIH_E07_79) has the potential to treat anaphylaxis and rapidly desensitize allergic individuals without producing allergic activity (Pennington et al., 2021). The fast-acting anti-IgE DARPin (KIH_E07_79) is an IgG 1-Fc is fused to the biparatopic DARPin using a Knob-in-Hole mutation strategy and fused via a short peptide linker. International patent application WO2022 / 061240 discloses fast-acting anti-IgEDARPins with displacement activity.

[0010] Single domain antibodies (sdAbs), also known as sdAb 026 is a humanized sdAb of llama origin, described in international patent applications WO2012 / 175740 and WO2014 / 087010.

[0011] sdAb 026 has been shown to accelerate the dissociation of IgE from FcεRI and CD23 by binding to an epitope within the IgE-Fc domain that is similar to the CD23 binding site and that does not significantly overlap with the FcεRI binding site. sdAb 026 inhibits the interaction of IgE with FcεRI by trapping IgE-Fc in a closed conformation that mimics CD23 binding (Jabs et al., 2018). A bispecific sdAb targeting IgE and human serum albumin for plasma half-life extension (ALX-0962) has been reported to have a dual-mode anti-IgE effect by neutralizing soluble (free) IgE with greater potency than the omalizumab sdAb and by binding and displacing preformed IgE:FcεRI complexes on basophils (Rinaldi et al., 2014).

[0012] Further anti-IgE sdAbs are disclosed in patent applications WO2004 / 041867, WO2020 / 208177, CN113461823 and CN111875706.

[0013] The present inventors have addressed the unmet clinical need for new treatment and prevention regimens for allergic diseases, in particular by providing fast-acting anti-IgE constructs. [Summary of the invention]

[0014] The present inventors have found that by combining at least one single domain antibody (sdAb) having a moderate ability to displace IgE from its high affinity receptor with another sdAb that can bind to IgE but has no displacement activity or has low displacement activity, an efficient anti-IgE construct can be provided. This sdAb combination improves displacement activity, even when provided as a simple mixture of individual sdAbs. By using short peptide chains or Fc fusion proteins, efficient displacements can be provided by connecting two sdAbs. Examples 1 and 2 demonstrate methods for providing novel sdAbs having IgE binding activity or displacement activity, and their ability to displace IgE from their high affinity receptors alone or in combination with another sdAb was evaluated by using a simple ELISA assay (Example 6), and the results are listed in Table 15 of Example 6. It is noteworthy that, with very few exceptions, the combined sdAbs exhibited improved displacement activity compared to one or two sdAbs in the combination. Interestingly, the present inventors have improved the ability to displace IgE by incorporating the combined sdAbs into a sdAb having Figure 1 In a multispecific construct of the structure shown in , and using various linking parts to obtain high and rapid displacement activity, these linking parts may provide constraints to the free migration or direction of each of the two sdAbs to varying degrees. Importantly, the displacement activity can be simply addressed by using an ELISA assay, which is commonly known to the skilled person, so it is easy to assess whether an sdAb with IgE binding affinity can be used for the construct disclosed herein. In addition, the inventors have evaluated the allergic activity in vitro and in mouse models.

[0015] In a first aspect, the present invention relates to a multispecific construct comprising:

[0016] (a) a first single domain antibody (sdAb) which is capable of displacing bound IgE from FcεRI,

[0017] (b) a second sdAb, which binds to IgE,

[0018] (c) a moiety linking the first and second sdAbs,

[0019] in

[0020] the first and second sdAb bind to non-identical epitopes of IgE, and

[0021] The multispecific constructs may displace bound IgE from the IgE high affinity receptor (FcεRI) with improved displacement activity compared to the first or second sdAb.

[0022] In the first aspect of the invention, the multispecific construct may be an Fc fusion protein. Thus, part (c) may be the Fc of an immunoglobulin antibody, the Fc domain of which is linked directly or via a linker (simple peptide chain) to the first and second sdAb.

[0023] The second aspect relates to a novel monomeric sdAb that can bind to an IgE antibody, and which can be used in the multispecific construct of the first aspect. This sdAb may have displacement activity itself, or it may combine with another sdAb to promote displacement activity. Exemplary monomeric sdAbs (full-length amino acid sequences, camelid versions) are listed in Table 1, and Tables 2a to d show the complementary determining regions (CDRs), CDR1, CDR2, and CDR3 regions of each novel monomeric sdAb. A variety of methods can be applied to determine the CDR region, and the resulting CDR region may depend on the method. Tables 2a to d show the CDRs of novel sdAbs determined according to Aho, Kabat, Chotia, or IMGT schemes.

[0024] When the Aho method is used to determine the CDR regions, an exemplary monomeric sdAb may, in a second aspect, include a combination of CDR1, CDR2, and CDR3, each of which has SEQ ID NO: 6, 7 and 8; 10, 11 and 12; 14, 15 and 16; 18, 19 and 20; 22, 23 and 24; 26, 27 and 28; 30, 31 and 32; 34, 35 and 36; 38, 39 and 40; 42, 43 and 44; 46, 47 and 48; 50, 51 and 52; 54, 55 and 56; 58, 59 and 60; 62, 63 and 64; 66, 67 and 68; 70, 71 and 72; or 74, 75 and 76, wherein each CDR1 can contain 1, 2 or 3 amino acid substitutions, each CDR2 can contain 1, 2 or 3 amino acid substitutions and / or each CDR3 can contain 1, 2 or 3 amino acid substitutions.

[0025] A third aspect relates to a pharmaceutical composition comprising the multispecific construct of the first aspect and any embodiment thereof, or to a pharmaceutical composition comprising one or more monomeric sdAbs of the second aspect and any embodiment disclosed herein, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier and / or vehicle and / or diluent and / or excipient.

[0026] Yet another aspect relates to a method of treating or preventing an IgE-related disease or disorder, the method comprising administering an effective amount of (a) the multispecific construct of the first aspect of the invention and any embodiment thereof, (b) one or more monomeric sdAbs of the second aspect of the invention and any embodiment thereof disclosed herein, or (c) the pharmaceutical composition of the third aspect of the invention and any embodiment thereof disclosed herein.

[0027] Another aspect relates to the use of (a) the multispecific construct of the first aspect of the invention and any embodiment thereof, (b) one or more monomeric sdAbs of the second aspect of the invention and any embodiment thereof, or (c) the pharmaceutical composition of the third aspect of the invention and any embodiment thereof as a medicament, preferably for anti-IgE therapy, such as a medicament for treating or preventing IgE-related diseases or disorders.

[0028] Yet another aspect relates to the use of the multispecific construct of the first aspect of the invention and any embodiment thereof, one or more monomeric sdAbs of the second aspect of the invention and any embodiment thereof, or the pharmaceutical composition of the third aspect of the invention and any embodiment thereof disclosed therein in the manufacture of a medicament, e.g., a medicament for anti-IgE therapy or for treating or preventing IgE-related diseases or disorders.

Brief Description of the Figures

[0030] Figure 1 Various types of multispecific constructs of the invention are shown, including a summary of non-limiting examples of their components. (i) sdAb monomers, such as (a) or (b) as described herein. (ii) Multimeric constructs, comprising a first sdAb (a) and a second sdAb (b) (also referred to herein as a "dimer") fused by a portion (c) (which may be a peptide linker). (iii) Multimeric constructs, comprising a first sdAb (a) and a second sdAb (b) fused (optionally via a linker) to the same site of a portion (c) (which may be an Fc domain). (iv) Multimeric constructs similar to (iii), but wherein (a) and (b) are fused to opposite sites of a portion (c).

[0031] Figure 2Shown is the sdAb library selection for IgE-Fc Cε2-Cε4 and IgE-Fc Cε2-Cε4 / FcεRIα complex.(A) The library is incubated with 1 μM IgE-Fc Cε2-Cε4 and analyzed by FACS using two parameters (sdAb expression and IgE-Fc binding) before starting selection to control sdAb expression and IgE-Fc binding. The first round of selection is performed by MACS and 1 μM biotinylated IgE-Fc Cε2-Cε4.(B) The second round of selection is performed with 200nM IgE-Fc Cε2-Cε4 using 2 parameters (sdAb expression and IgE-Fc binding) by FACS. The positive group that expresses and IgE-Fc binding is selected in gate C. (C) Alternatively, a second round of selection was performed by FACS using 200 nM complex IgE-Fc Cε2-Cε4 / FcεRIα using 3 parameters (sdAb expression, IgE-Fc binding streptavidin, anti-FcεRIα). A large number of positive groups can be seen in both expression and IgE-Fc binding. When using anti-FcεRIα signal, the group can be distinguished among the 3 groups. Gate E was used to select potential replacement sdAbs in the IgE-Fc+ / FcεRIα- group.

[0032] Figure 3 : A relative epitope localization matrix is ​​shown, where the combined binding to the same IgE-Fc of different pairs of two sdAbs was evaluated. Dark grey indicates simultaneous binding of both sdAbs, thus binding to different epitopes of IgE-Fc, while white indicates no binding. Light grey indicates possible simultaneous binding of both sdAbs, but the result is inconclusive due to low affinity.

[0033] Figure 4 An example of a deep mutation scanning matrix of sdAb A1 is shown to improve binding affinity or displacement activity to IgE-Fc. Analysis of sequencing data of positive and negative groups. Values ​​are enrichment values ​​(enrichment = frequency of mutant after selection / frequency before selection), and "D" indicates that the mutant was depleted upon selection. The matrix is ​​colored in grayscale, proportional to the enrichment value. Mutations that improve A1 binding / activity are enriched in the A1 positive group and should be depleted or enriched <1 in the A1 negative group. Three target mutations that improve A1 binding to IgE-Fc are shown in the white box (T28R, P45Y, and V93E).

[0034] Figure 5 shows the inhibition of allergen-induced basophil activation after in vitro treatment. (A) Inhibition of allergen-induced basophil activation in PBMCs isolated from HDM allergic donors using monomeric sdAb A1 at a concentration of 1 μM (black dots: buffer, black triangles: sdAb A1, light grey squares: KIH-E07_79). (B) Inhibition of allergen-induced basophil activation in PBMCs isolated from HDM allergic donors using monomeric sdAb A2 or a mixture of two different monomeric sdAbs at a concentration of 1 μM (black dots: buffer, grey squares: A2, white dots: B1, crosses: B3, black triangles A1, white diamonds: A2+B1, white triangles: A2+B1, white triangles: A2+B2, black triangles: A2+B3). (C) shows the inhibition of allergen-induced or a-IgE-induced basophil activation in PBMCs isolated from HDM-allergic donor 83 using monomeric sdAb A2 or a mixture of two different monomeric sdAbs at a concentration of 1 μM (black dots: buffer, gray squares: A2, white dots: B1, crosses: B3, black triangles A1, white diamonds: A2+B1, white triangles: A2+B1, white triangles: A2+B2, black triangles: A2+B3). (D) shows the inhibition of allergen-induced or a-IgE-induced basophil activation in PBMCs isolated from HDM-allergic donor 83 (black dots: buffer, gray triangles CIgG4B1A1 (G 4 S) 1 10nM, light grey squares: CIgG4B1A1(G 4 S) 1 10000 nM). (E) shows the inhibition of allergen-induced or a-IgE-induced basophil activation in whole blood HDM allergic donors 220 (black dots: buffer, grey triangles CIgG4B1A1 (G 4 S) 1 50nM, light grey squares: CIgG4B1A1(G 4 S) 1 500 nM). (F) shows the inhibition of allergen-induced or a-IgE-induced activation of Birch allergic donor whole blood basophils 187 (black dots: buffer, grey triangles CIgG4B1A1 (G 4 S) 1 50nM, light grey squares: CIgG4B1A1(G 4 S) 1 500 nM). (Y axis: %CD63 positive basophils, X axis left: rDerp2 / a-IgE / Betv1 ng / ml).

[0035] FIG. 6 relates to the treatment of IgG4 Fc fusion constructs comprising two sdAbs A1 and B1, each sdAb bound to a peptide linker G 4 S(CIgG4B1A1(G 4 S)1 ) was linked to the C-terminus of the IgG4 Fc domain of Derp2, and inhibition / prevention in a mouse model of passive anaphylaxis. (A) Schematic diagram showing the experimental setup of the passive anaphylaxis assay performed: (i) mice expressing human FcεRIα chain were sensitized with a cocktail of three human anti-Derp2 antibodies, (ii) CIgG4B1A1 (G 4 S) 1 and (iii) challenged 4 h later by intraperitoneal injection of rDerp2. 4 S) 1 Body temperature was measured 1 hour after injection and 90 minutes at challenge. At termination, blood was collected to determine serum mast cell protease levels as an auxiliary readout of the severity of the allergic reaction. (B) Shows the results of injection of sterile PBS or different concentrations of CIgG4B1A1 (G 4 S) 1 (C) Shows the body temperature changes after challenge with rDerp2 and injection of sterile PBS or different concentrations of CIgG4B1A1 (G 4 S) 1 (D) Shows the average area under the curve value of the change in body temperature after challenge for each tested inhibitor concentration (Y axis: delta body temperature AUC; black dots: PBS, white squares: CIgG4B1A1 (G 4 S) 1 (E) Shows the mean serum mMCPT1 concentration for each tested inhibitor concentration 90 min after challenge (Y axis: delta body temperature AUC; black dots: PBS, white squares: CIgG4B1A1 (G 4 S) 1 ). Data are shown as SEM ± mean; *: P ≤ 0.05; **: P ≤ 0.01; : P ≤ 0,001; : P ≤ 0,0001 compared with PBS control.

[0036] Figure 7 shows the effects of three different IgG4 Fc fusion constructs combined with sdAb A1 in combination with D2, E1 or F4, each of which was linked to the C-terminus of the Fc domain of IgG4 with a peptide linker G 4Mouse model for the inhibition / prevention of systemic anaphylaxis by one or two iterations of S. (A) Body temperature changes after injection of sterile PBS or IgG4 Fc fusion constructs. (B) Body temperature changes after challenge with rDerp2, mean area under the curve values ​​for body temperature changes and mean serum mMCPT1 concentrations 90 minutes after challenge for each tested construct. Data are shown as SEM ± mean; *: P ≤ 0.05; **: P ≤ 0.01; : P ≤ 0,001 compared with PBS control (black dots: PBS, white squares: CIgG4D2A1 (G 4 S) 2 , white triangle: CIgG4E1A1(G 4 S) 2 , white diamond: CIgG4F4A1(G 4 S) 2 ), A+B left: Y axis is delta body temperature, x axis is time after challenge (in minutes).

[0037] Figure 8 shows allergen testing in a PSA mouse model and a PCA mouse model version using an IgG4 Fc fusion construct in which sdAbs A1 and B1 were linked to the N-terminus or C-terminus of the Fc domain of IgG4, respectively, with a peptide linker G 4 S(C / NIgG4B1A1(G 4 S) 3 ) for three iterations). (A) Body temperature changes after injection of sterile PBS or any inhibitor, and the average area under the temperature change curve. (B) Schematic diagram showing the experimental setup of the passive cutaneous anaphylaxis assay for allergen testing: (i) mice expressing human FcεRIα chain were sensitized intradermally with a mixture of three human anti-Derp2 antibodies, (ii) PBS or NIgG4B1A1 (G 4 S) 3 and Evan's Blue dye intravenous injection, (iii) terminated 30 minutes after injection for measurement of dye extravasation area and dye extraction from the skin. (C) The area of ​​dye extravasation into sensitized skin sections 30 minutes after inhibitor + dye injection, and the optical density of dye after extraction from skin sections. Data are shown as SEM ± mean; *: P ≤ 0.05; **: P ≤ 0,01 (dots: PBS, squares: CIgG4B1A1 (G 4 S) 3 , triangle: NIgG4B1A1(G 4 S) 3 ).

[0038] Figure 9 shows allergen and IgE displacement assessed by in vitro stimulation of human basophils from two independent allergic donors. CD63 activated basophils from heparinized whole blood were incubated with IgG4 Fc fusion constructs at concentrations ranging from 0.1 nM to 1 μM. Donor 1 Figure 9A and Donor 2 Figure 9B. (Panel A+D Y axis: % IgE displaced from basophils, Panel B+CY axis: % CD63+ cells, X axis: sdAb concentration (nM), black dots: CIgG4B1A1 (G 4 S) 1 , white square: CIgG4B1A1(G 4 S) 2 , white triangle: CIgG4B1A1(G 4 S) 3 , black triangles: CIgG4B1A1(RS), light grey squares: NIgG4B1A1(G 4 S), black cross: KIH-E07_79). [Detailed description of the invention]

[0040]

definition

[0041] The term "multi-specific construct" refers to a compound, molecule or complex that can bind to at least two different epitopes on IgE, particularly to an identical IgE antibody. Therefore, the wording multi-specific is intended to indicate that the construct can bind to multiple epitopes of an antigen, and is therefore paratopic. When the construct can bind to two different IgE epitopes, the construct can be named as a bispecific construct. In the present case, at least two different epitopes are preferably present outside the IgE antigen binding region, such as on IgE-Fc. It is therefore understood that in the present case, the multi-specific construct is not a construct that binds to several different antigens, but can bind to the same antigen (IgE) at several different surface exposure sites commonly found in the Fc portion of IgE. However, the multi-specific construct may be able to interfere with other biological targets. For example, a construct comprising an Fc domain that can bind or interfere with other targets beyond IgE can be mentioned.

[0042] The phrase "the multispecific construct can displace bound IgE from the IgE high affinity receptor (FcεRI receptor)" is intended to define that the multispecific construct can enhance the dissociation of IgE from FcεRI by, for example, promoting the dissociation mechanism, conformational changes and / or steric hindrance.

[0043] The terms "displacement activity" or "displacement effect" are interchangeable terms, all intended to define the ability of a compound (e.g., a monomeric sdAb, a combination sdAb, a linked sdAb, or a compound comprising an sdAb, such as a multispecific construct described herein) to displace IgE from its high affinity receptor FcεRI. As described in Example 6 herein, the ability of such test compounds to displace bound IgE from FcεRI can be assessed by using an ELISA-based IgE-FcεRIα displacement assay. In short, the assay is based on measuring the remaining IgE, which is not removed from the immobilized recombinant human FcεRIα preloaded with IgE after adding the test compound (e.g., an sdAb or multispecific construct described herein). The test results can be provided in the form of a percent displacement effect, calculated as a relative reduction in the signal compared to a control to which the test compound is not added, and the test compound may be administered at different concentrations. The concentration that provides a half-displacement effect can then be determined (the displacement effect is EC 50 , that is, EC 50 is the molar concentration of the test compound that dissociates 50% of the IgE preloaded to the FcεRIα receptor). In addition, displacement activity can be assessed by determining the percentage of IgE that is most dissociated from the IgE pool preloaded to the FcεRIα receptor (maximal displacement activity) and the extent to which the maximal effect is achieved (100% of the preloaded IgE is dissociated).

[0044] The displacement activity can also be assessed by other assay types, in vitro or ex vivo assays, and in vivo mouse models can be used to assess the displacement activity. This method is well known to those skilled in the art (Eggel et al., 2014, Pennington et al., 2021 and Jabs et al., 2018).

[0045] The substitution effect can be compared with a known substitution (control substitution), such as sdAb 026 mentioned in patent application WO2012 / 175740, herein named (A1) or the Darpin-based construct KIH_E7_79 (Pennington et al., 2021 and patent application WO2022 / 061240).

[0046] "Single domain antibodies" (sdAbs), which can be called "nanoantibodies", are antibody fragments in the form of monomeric variable antibody domains that can selectively bind to specific antigens. Traditionally, sdAbs are fragments of heavy chain-only antibodies that contain the VH domain of an antibody. sdAbs use only three complementary determining regions (CDR1, CDR2, and CDR3) to bind antigens, rather than the six regions present in traditional VH:VL antibodies. However, in some cases, sdAbs can also be derived from common antibodies such as IgG, but this approach requires amino acid substitutions to eliminate the natural dimerization of the VH and VL domains, which means that antigen affinity is usually affected. Typically, sdAbs are relatively short, such as 100 to 130 amino acid residues.

[0047] In addition, sdAbs are antibodies whose CDRs are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies that are naturally free of light chains, single domain antibodies derived from traditional 4-chain antibodies, engineered antibodies, and single domain scaffolds other than antibody derivatives. sdAbs may be from any species, including, but not limited to, mouse, human, camel, llama, goat, rabbit, and / or cow.

[0048] In some embodiments, sdAb used herein is derived from the variable domain of the naturally occurring heavy chain antibody without light chain. For the sake of clarity, this variable domain derived from only heavy chain antibody is called VHH domain, to be distinguished from the conventional VH of four-chain immunoglobulin. Here, the term sdAb can be exchanged with VHH antibody. When sdAb is a part of fusion protein / fusion construct, the term sdAb can be exchanged with VHH domain. Such VHH domain can be derived from the antibody produced in the animal that can produce only heavy chain antibody. For example, Camelidae species, such as camel, llama, dromedary, alpaca and camel. Other species except Camelidae may only produce heavy chain antibody, and such VHH is within the disclosed range. Therefore, in any construct described herein, wherein sdAb is a part of a larger construct, the term sdAb can be exchanged with the term VHH.

[0049] The binding region of an sdAb herein is intended to include one or more or all three complementarity determining regions (CDR1, CDR2 and CDR3).

[0050] The "complementarity determining region" or "CDR" is the variable part of an antibody, including sdAbs, and is therefore critical for diversity. Not all residues in a CDR may contribute to epitope binding. For example, cysteines in CDR1 and CDR3 of sdAbs are determining structure (Pellis et al., 2012).

[0051] As previously mentioned, sdAb and VHH are used interchangeably herein. In some embodiments, sdAb includes 3 CDRs and four framework regions (FRs), arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. In some embodiments, as long as VHH / sdAb substantially maintains the desired antigen binding and specificity, sdAb can be truncated at the N-terminus or C-terminus so that it only includes part of FR1 and / or FR4, or lacks one or two of these FRs.

[0052] As mentioned above, the main purpose of the CDR region is to define the region that is specific for the epitope. However, not all residues in the CDR may be responsible for epitope binding, but may only determine structural elements (Wilton E. et al., 2018, reference 6). In addition, the FR may not be entirely responsible for the structure, as FR residues adjacent to the CDR can affect binding.

[0053] Different methods can be applied to determine the locations of the different framework and CDR regions of a sdAb.

[0054] One method is the "Kabat numbering scheme" or "Kabat", first described by the American scientist Elvis Kabat. Here, the amino acid residues of the immunoglobulin single variable domains can be numbered according to the general numbering of VH domains given by Kabat et al. and applied to the VHH domains of camelids as described by Riechmann and Muyldemans (Riechmann and Muyldemans, 1999).

[0055] In the CDR determination according to Kabat, FR1 of VHH comprises amino acid residues at positions 1 to 30, CDR1 of VHH comprises amino acid residues at positions 31 to 35, FR2 of VHH comprises amino acid residues at positions 36 to 49, CDR2 of VHH comprises amino acid residues at positions 50 to 65, FR3 of VHH comprises amino acid residues at positions 66 to 94, CDR3 of VHH comprises amino acid residues at positions 95 to 102, and FR4 of VHH comprises amino acid residues at positions 103 to 113.

[0056] It should be noted that, as is well known in the art, the total number of amino acid residues in VH domains and VHH domains can vary and may not correspond to the total number of amino acid residues indicated by Kabat numbering. That is, one or more positions according to Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. The total number of amino acid residues in VH domains and VHH domains is usually between 110 and 120, and usually between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0057] Another way to identify CDR regions in VHH is through the Chothia numbering scheme. Here, the amino acid residues of the immunoglobulin single variable domain can be numbered using conservative amino acids, which always have the same position (Dondelinger M et al., 2018).

[0058] Another method for determining the CDR regions in VHH is by IMGT. Here, the amino acid residues of the immunoglobulin single variable domain can be numbered using conservative amino acids, which always have the same position. For example, cysteine ​​23, tryptophan 41, leucine 89, cysteine ​​104. The FR1 of VHH contains amino acid residues at positions 1 to 26, the CDR1 of VHH contains amino acid residues at positions 27 to 38, the FR2 of VHH contains amino acid residues at positions 39 to 55, the CDR2 of VHH contains amino acid residues at positions 56 to 65, the FR3 of VHH consists of amino acid residues at positions 66 to 104, the CDR3 of VHH contains amino acid residues at positions 105 to 117, and the FR4 of VHH contains amino acid residues at positions 118 and the rest of the sequence. The maximum length of the CDR is as described above. For shorter CDRs, gaps will occur (Lefranc et al., 2002).

[0059] Alternatively, the CDR regions in sdAbs can be determined by using the AbM numbering described in Kontermann and Dubel (Eds. 2010, Antibody Engineering, vol 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51). According to the method, FR1 comprises amino acid residues at positions 1 to 25, CDR1 comprises amino acid residues at positions 26 to 35, FR2 consists of amino acids at positions 36 to 49, CDR2 consists of amino acid residues at positions 50 to 58, FR3 consists of amino acid residues at positions 59 to 94, CDR3 consists of amino acid residues at positions 95 to 102, and FR4 comprises amino acid residues at positions 103 to 113.

[0060] Another way to determine the CDR regions in sdAbs is to use the Aho numbering scheme (Mitchelle & Colwell, 2017 and Honegger & Pluckthun et al., 2001).

[0061] Another permutation method for determining CDR regions in VHH is to use sdAb-DB described by Emily Wilton in 2018. The method is built on a set of sequence-based rules used to identify CDRs in conventional antibody light and heavy chains by Pantazes and Maranas (Wilton E et al 2018, Synthetic Biology) and is used for sdAb. In sdAb-DB, CDR1 starts 4 residues after the first cysteine ​​and is 6 to 15 residues long, followed by a W. CDR2 starts 10 to 20 residues after the end of CDR1 and follows the sequence [I / L / V / M]-[G / A / S]. CDR2 is 8 to 15 residues long, followed by the sequence [Y / F / I / L / T / N / S / V / H]-XXX-[A / I / L / M / V]-[Q / K / R / A / E / G / L / T]. CDR3 is located 30 to 50 residues after the end of CDR2 and follows the sequence CXX. It is 3 to 25 amino acids long and is followed by the sequence [W / A / E / F / H / K / L / Q / Y / G / S / R]-[G / S]-XGXXXTVS. In all sequences, "X" can be any amino acid.

[0062] Substitution CDR definitions of interest include, but are not limited to, Ofran et al., Almagro JC (Ofran et al. 2001, Almagro JC 2004), each of which is specifically incorporated herein by reference.

[0063] An "antibody", also called an "immunoglobulin", is a protein that in humans consists of a pair of identical light and heavy chains, each of which contains a variable domain and a constant region, wherein the variable domain is responsible for the specific binding of the antibody to an antigen. In general, antibodies are classified into immunoglobulin isotypes IgA, IgD, IgE, IgG and IgM, which play different roles as antigen recognition agents in the immune system. The specificity of antibody antigen binding is determined by the variable regions in the variable domains, in particular by the complementarity determining regions (CDRs) in the variable domains. In general, antibodies are molecules that can be expressed in principle or in practice in an animal in its natural conformation, which means that the expressed antibody includes all the structural elements found in naturally occurring immunoglobulins.

[0064] "Antibody variants" are proteins derived from antibodies that have the same binding specificity as antibodies, but they are not naturally expressed in mammals. Therefore, the term refers to various antibody fragments and artificial antibody analogs. In addition, the term refers to antibody forms found in nature but not commonly found in mammals, such as heavy chain antibodies and IgY found in birds and reptiles, but the CDRs of antibodies from mammalian antibodies or antibodies produced in combination have been transformed into antibody forms that were not originally derived.

[0065] "Heavy chain only antibodies" are a form of antibodies that naturally occur in camelids (such as camels, dromedaries, llamas and alpacas) or cartilaginous fish (such as sharks, skates, rays, sawfish, etc.) and consist of only two heavy chains, while lacking two light chains.

[0066] The term "allergen" is used herein to describe the action of molecules that can activate effector cells such as basophils and mast cells to cause degranulation (histamine release) in the absence of allergens. An example is the multispecific construct NIgG4B1A1 (G 4 S) 1 or NIgG4B1A1(G 4 S) 3 , which was shown to activate basophils on its own in a mouse allergic response model (Example 9).

[0067] The term "IgE binding affinity" refers to the affinity of a test compound (e.g., sdAbs and multispecific constructs disclosed herein) for binding to free IgE. The ability of a test compound to bind to free IgE can be tested using biolayer interferometry (BLI), as described in Example 5 herein. Briefly, the assay measures the binding and dissociation of a test molecule to IgE or IgE-Fc. Based on these measurements, k can be determined. on , k off , k off / k on The ratio (=K D ). on is a constant that characterizes the rate at which the test molecule binds to IgE, while k off Indicates the speed at which the test molecule separates from IgE. k off / k on The ratio of yields the equilibrium dissociation constant K D . K D The lower the value, the higher the affinity of the test molecule for IgE. In an interesting embodiment, the monomeric sdAb exhibits improved IgE binding affinity (lower K) compared to a known IgE binder (control IgE binder), such as sdAb 026 (A1). D In other interesting embodiments, multispecific constructs (including dual sdAbs linked by a peptide linker) exhibit improved IgE binding affinity compared to DARPinKIH-E7_79 or constructs comprising two sdAbs 026 (A1) linked together by a peptide linker. It is contemplated that in other embodiments, the IgE binding affinity may be lower than the control. Typically, K in the nanomolar range D Values ​​may be desirable, for example particularly in the low nM affinity range, e.g. below 3 × 10 -10 M. Other IgE affinity binding assays may be used and are known to those skilled in the art.

[0068] The term "IgE-related disease or condition" refers to any disease or condition that would benefit from a reduction in receptor-bound IgE and / or a reduction in the level of free-circulating IgE. These conditions may also be referred to as IgE-mediated diseases or conditions. Examples of such specific diseases include allergic diseases, including type I allergic diseases, particularly allergic diseases with severe allergic symptoms (asthma, atopic dermatitis, urticaria) or allergic diseases in which allergic reactions develop rapidly (e.g. anaphylaxis). The term "allergic disease" is well known in the medical art. In particular, the term allergic disease refers to an allergic and / or atopic immune response to an antigen (e.g., an allergen), which results in allergic and / or atopic symptoms in the patient suffering from the allergic disease. Allergic diseases are often characterized by the production of antigen-specific IgE antibodies, which may be the result of IgE antibodies.

[0069] [Specific implementation methods of the present invention]

[0070]

sdAb / VHH domain

[0071] The present invention provides novel VHH domains (sdAbs) capable of binding IgE, which are suitable for use in the first and second aspects disclosed herein and in further aspects disclosed herein.

[0072] VHH domain can be expressed as monomer sdAb or be incorporated into larger construct (for example, become a part of polypeptide / protein construct / fusion protein). Two or more different VHH domains can be expressed as single monomer sdAb, which can be further connected to provide dimer or multimer sdAb to form multispecific construct. Two or more VHH domains can be incorporated into larger construct (for example, become a part of polypeptide / protein construct / fusion protein). sdAb may have displacement activity itself, or may be combined with another sdAb to promote displacement activity. Interested is sdAb, which can be combined in the multispecific construct described in this article, or combined in any other design providing required displacement activity.

[0073] Of interest are monomeric sdAbs that exhibit displacement activity themselves, and preferably have improved displacement activity (lower EC 50 Such sdAbs exhibiting displacement activity can be used as the first sdAb in the multispecific constructs described herein. In still interesting embodiments, the multispecific constructs (comprising two sdAbs connected by a peptide linker) exhibit similar or improved displacement activity (lower EC 50 and / or higher maximal effect), or with monomeric sdAb 026, dimeric sdAb 026 (two sdAb 026 connected by a peptide linker), or Fc fusion constructs with two sdAb 026 (bivalent constructs).

[0074] The monomeric sdAb (or alternatively "VHH domain") of the present invention can be obtained as an antibody fragment obtained from a camelid immunized with full-length human IgE or a portion of IgE, such as IgE-Fc Cε3-Cε4, IgE-Fc Cε2-Cε4, a mutant version of IgE-Fc Cε3-Cε4 (i.e., IgE-Fc Cε3-Cε4 335), wherein an additional cysteine ​​residue is incorporated at position 335 so as to establish an artificial disulfide bridge between the two to obtain IgE in a closed form, or any combination thereof. Such camelid monomeric sdAbs may be further affinity matured, humanized or otherwise sequence-varied to obtain sdAbs with desired properties.

[0075] Monomeric sdAbs can bind to IgE epitopes outside the variable domain of IgE, for example, in particular in the Fc region of IgE, such as in IgE-Fc Cε2-Cε4. Of interest are sdAbs that displace IgE from binding to FcεRI and / or have high binding affinity to IgE. However, it is not excluded that the monomeric sdAbs disclosed herein (e.g., the second sdAb) can bind to C in IgE. L Domain binding.

[0076] The IgE binding region of sdAb includes CDRs, which are specific regions where sdAbs usually bind to their targets. In a single sdAb, there are three CDRs, CDR1, CDR2, and CDR3, and one or more CDR combinations may be key to the activity of the sdAb. Individual CDR regions can be determined by different methods, including Aho, Kabat, or AbM described above.

[0077] New monomeric exemplary monomeric sdAb bodies capable of binding to IgE are shown in Table 1 and are named A2, B1, B2, B3, D1, D2, D3, E1, E2, E3, E4, F1, F2, F3, F4, F5, F6 and G1, respectively. As shown in the ELISA-based IgE-FcεRIα displacement assay, only the sdAbs named A2 and B1 showed displacement activity (Example 6, Table 15). A2 is a mutant of sdAb 026 (referred to herein as A1) and showed a lower EC than the control agent A1. 50 Compared with control A1, sdAb B1 exhibited a lower EC 50 15) also showed improved displacement activity under 10% to 20% sdAb, but could only displace 60% of the preloaded IgE (maximum displacement effect was achieved when IgE was 60% dissociated). Therefore, B1 can be considered more preferred than A1 and A2 for use as the first sdAb in the multispecific constructs described herein. As shown in Example 6 (Table 15), novel sdAbs with no or poor displacement activity (such as B1) can be successfully used as the second sdAb in the multispecific constructs herein to obtain compounds with high displacement activity.

[0078] Table 1 provides the full length amino acid sequences of the monomeric sdAbs (Camelidae versions) and information on the IgE constructs that have been used for immunization.

[0079] [Table 1: Full-length amino acid sequence of monomeric sdAb (Camelidae version)]

[0080]

[0081]

[0082] Thus, in some embodiments, the sdAb may comprise or consist of an amino acid sequence selected from any one of SEQ ID NOs: 5, 9, 13, 17, 21, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69 and 73. As such sdAbs may be subject to affinity maturation, humanization or other amino acid changes in the amino acid sequence, further embodiments relate to sdAbs having an amino acid sequence that is at least 80% identical, such as at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 17, 21, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69 and 73. In a preferred embodiment, the variation in the amino acid sequence is not in the CDR regions and is therefore only in the combined framework regions. Typically, the purpose of introducing such variants is affinity maturation and / or humanization of the sdAb. Thus, the novel sdAb may comprise or consist of an amino acid sequence selected from SEQ ID NO: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69 and 73, including affinity matured and / or humanized variants thereof.

[0083] Furthermore, novel sdAbs may be defined based on a single CDR region that may be derived from the entire sdAb amino acid sequence and may be identified by a different numbering scheme, such as, but not limited to, Kabat, Chothia, IMTG, or Aho.

[0084] Thus, in one embodiment, the sdAb comprises binding regions CDR1, CDR2 and CDR3, each comprising or consisting of an amino acid sequence determined according to the Kabat, Chothia, IMTG or Aho numbering scheme, the amino acid sequence being determined in an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 17, 21, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69 and 73, optionally wherein CDR1 comprises 1, 2 or 3 amino acid substitutions, CDR2 comprises 1, 2 or 3 amino acid substitutions and / or CDR3 comprises 1, 2 or 3 amino acid substitutions. Given that CDR1, CDR2 and CDR3 are directly determined by Kabat, Chothia, IMTH or Aho in the above sequences, substitutions may be incorporated.

[0085]

Specific CDR regions of novel sdAbs

[0086] [Table 2a: Amino acid sequences of CDR1, CRD2 and CDR3 of monomeric sdAb / VHH domains determined by Aho]

[0087]

[0088]

[0089] [Table 2b: Amino acid sequences of CDR1, CRD2 and CDR3 of monomeric sdAb / VHH domains determined by the Kabat method]

[0090]

[0091]

[0092] [Table 2c: Amino acid sequences of CDR1, CRD2 and CDR3 of monomeric sdAb / VHH domains determined by Chothia method]

[0093]

[0094]

[0095] [Table 2d: Amino acid sequences of CDR1, CRD2 and CDR3 of monomeric sdAb / VHH domains determined by the IMGT method]

[0096]

[0097] Exemplary monomers include a CDR1 having an amino acid sequence of SEQ ID NO: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70 or 74, wherein each amino acid sequence may have 1, 2 or 3 amino acid substitutions, and wherein the CDR regions are defined by Aho.

[0098] Exemplary monomers include a CDR2 having an amino acid sequence of SEQ ID NO: 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71 or 75, wherein each amino acid sequence may have 1, 2 or 3 amino acid substitutions, and wherein the CDR regions are defined by Aho.

[0099] Exemplary monomers include a CDR3 having an amino acid sequence of SEQ ID NO: 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72 or 76, wherein each amino acid sequence may have 1, 2 or 3 amino acid substitutions, and wherein the CDR regions are defined by Aho.

[0100] In alternative approaches, exemplary monomeric sdAbs A2, B1, B2, B3, D1, D2, D3, E1, E2, E3, E4, F1, F2, F3, F4, F5, F6 and G1 can have their specific CDR regions defined as determined by Kabat as shown in Table 2b, as determined by Chothia as shown in Table 2c, or as determined by IMGT as shown in Table 2d.

[0101] Here, sdAbs can be defined according to their binding regions CDR1, CDR2 and CDR3 defined in the VHH region defined in the parent version, for example, the Camelidae version. In any embodiment disclosed herein, each of CDR1, CDR2 and CDR3 can optionally undergo sequence variation, such as substitution, deletion or addition of 1, 2 or 3 amino acids. The purpose of introducing a variant may be to improve the IgE binding activity or displacement activity compared to a control IgE binder or displacement agent.

[0102] Thus, in all of the following embodiments, CDR1 may comprise 1, 2 or 3 amino acid substitutions, CDR2 may comprise 1, 2 or 3 amino acid substitutions, and CDR3 may comprise 1, 2 or 3 amino acid substitutions, such as 1 or 2 amino acid substitutions. The resulting variant sdAbs may be evaluated by determining binding affinity to IgE Fc or by determining substitution activity by an ELISA-based IgE-FcεRIα substitution assay. An ideal variant is one that has comparable activity to the parent sdAb or has improved IgE affinity or improved substitution activity.

[0103] More specifically, the sdAb comprises binding regions CDR1, CDR2 and CDR3, each of which has SEQ ID NO: 6, 7 and 8; 10, 11 and 12; 14, 15 and 16; 18, 19 and 20; 22, 23 and 24; 26, 27 and 28; 30, 31 and 32; 34, 35 and 36; 38, 39 and 40; 42, 43 and 44; 46, 47 and 48; 50, 51 and 52; 54, 55 and 56; 58, 59 and 60; 62, 63 and 64; 66, 67 and 68; 70, 71 and 72; or 74, 75 and 76, wherein each CDR is determined according to the Aho numbering scheme.

[0104] In another embodiment, the sdAb comprises binding regions CDR1, CDR2 and CDR3, each of which has SEQ ID NO: 132, 133 and 134; 135, 136 and 137; 138, 139 and 140; 141, 142 and 143; 144, 145 and 146; 147, 148 and 149; 150, 151 and 152; 153, 154 and 155; 156, 157 and 158; 159, 160 and 161; 162, 163 and 164; 165, 166 and 167; 168, 169 and 170; 171, 172 and 173; 174, 175 and 176; 177, 178 and 179; 180, 181 and 182; or 183, 184 and 185, wherein each CDR is determined according to the Kabat numbering scheme.

[0105] In a further embodiment, the sdAb comprises binding regions CDR1, CDR2 and CDR3, each of which has SEQ ID NO: 189, 190 and 191; 192, 193 and 194; 195, 196 and 197; 198, 199 and 200; 201, 202 and 203; 204, 205 and 206; 207, 208 and 209; 210, 211 and 212; 213, 214 and 215; 216, 217 and 218; 219, 220 and 221; 222, 223 and 224; 225, 226 and 227; 228, 229 and 230; 231, 232 and 233; 234, 235 and 236; 237, 238 and 239; or 240, 241 and 242, wherein each CDR is determined according to the Chothia numbering scheme.

[0106] In a further embodiment, the sdAb comprises binding regions CDR1, CDR2 and CDR3, each of which has SEQ ID NO: 245, 247 and 248; 249, 250 and 251; 252, 253 and 254; 255, 256 and 257; 258, 259 and 260; 261, 262 and 263; 264, 265 and 266; 267, 268 and 269; 270, 271 and 272; 273, 274 and 275; 276, 277 and 278; 279, 280 and 281; 282, 283 and 284; 285, 286 and 287; 288, 289 and 290; 291, 292 and 293; 294, 295 and 296; or 297, 298 and 299, wherein each CDR is determined according to the IMTG numbering scheme.

[0107] Monomeric sdAbs, alone or in combination, can enhance biological activity (e.g., displacement activity), improve drug potency (e.g., improved serum circulation half-life, improved biodistribution) or establish binding to other therapeutic targets besides IgE.

[0108] As previously mentioned, individual sdAbs A1, A2 and B1 are capable of displacing IgE from its high affinity receptor. Thus, interesting embodiments include those that can be used as the first sdAb in the multispecific constructs described herein.

[0109] According to an interesting embodiment, the sdAb has a binding region of A1 (as shown in Table 2a-d). Therefore, the sdAb may comprise binding regions CDR1, CDR2 and CDR3, each of which has SEQ ID NO:

[0110] 2, 3 and 4 (determined according to Aho);

[0111] 129, 130 and 131 (according to Kabat);

[0112] 186, 187 and 188 (as determined by Chothia); or

[0113] 243, 244 and 245 (as determined by IMGT).

[0114] According to another interesting embodiment, the sdAb has a binding region of A2. Thus, the sdAb may comprise binding regions CDR1, CDR2 and CDR3, each of which has SEQ ID NO:

[0115] 6, 7 and 8 (determined according to Aho);

[0116] 132, 133 and 134 (according to Kabat);

[0117] 189, 190 and 191 (as determined by Chothia); or

[0118] 246, 247 and 248 (as determined by IMGT).

[0119] According to another interesting embodiment, the sdAb has a binding region of B1. Thus, the sdAb may comprise binding regions CDR1, CDR2 and CDR3, each of which has SEQ ID NO:

[0120] 10, 11 and 12 (determined according to Aho);

[0121] 135, 136 and 137 (according to Kabat);

[0122] 192, 193 and 194 (as determined by Chothia); or

[0123] 249, 250 and 251 (as determined by IMGT).

[0124] In addition, as shown in Examples 6 and 7, monomeric sdAbs as described herein show increased displacement activity when, for example, two separate (unconnected) monomeric sdAbs are used in pairs. For example, when A1 and B1 are administered in combination, the ability to displace IgE from their receptors is higher than that of a single sdAb administered alone. It is noteworthy that by combining two sdAbs with separate displacement activity, improved displacement activity is also provided. The combination of a displacement sdAb and a non-displacement sdAb exhibits a displacement activity comparable to that of a combination of A1 and B1 (Example 6, Table 15 and Example 7, Table 16).

[0125] In further embodiments, any monomeric sdAb described herein can be combined in pairs to form two sdAbs or as three, for example four sdAbs, one of which exhibits displacement activity. In a preferred embodiment, the unidirectional antibodies are combined in pairs of two. However, any other non-IgE-bound sdAb can be combined with an IgE-bound sdAb to change biology or kinetics, such as combining with an albumin-bound sdAb to reduce the in vivo elimination rate.

[0126] Combinations of sdAbs and preferred embodiments are further described below.

[0127] [Modification of sdAb amino acid sequence]

[0128] Any amino acid sequence of CDR or FR outside CDR may undergo amino acid substitution, insertion or deletion to change biological activity, expression level, stability or other functional properties. In a preferred embodiment, amino acid changes are only incorporated outside the CDR region.

[0129] In some embodiments, the sdAb consists of an affinity matured human or humanized amino acid sequence. In further embodiments thereof, the amino acid sequence of the CDR regions of the Camelid sdAb may not be altered when affinity maturation or humanization of the Camelid sdAb is performed. In such embodiments, the sdAb consists of an affinity matured human or humanized amino acid sequence, but not in the CDR regions involved in binding to the target.

[0130] In some embodiments, another CDR (i.e., CDR1, CDR2 and / or CDR3) of the sdAb can be independently subjected to amino acid substitution, for example, by 1, 2, 3 or multiple amino acid residue substitutions. The amino acid substitutions in the CDRs may be conservative amino acid substitutions. "Conservative" amino acid substitutions are typically amino acid substitutions in which an amino acid residue is replaced by another amino acid residue with similar chemical structure and / or charge, which has little or substantially no effect on the function, activity or other biological properties of the resulting binding region, or the multispecific constructs described herein. Such conservative amino acid substitutions are well known in the art. For example, conservative substitutions are preferably substitutions in which an amino acid residue in the following groups (a) to (e) is replaced by another amino acid residue in the same group: (a) small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic non-polar residues: Met, Leu, Ile, Val and Cys; (e) aromatic residues: Phe, Tyr and Trp.

[0131] Particularly preferred conservative amino acid substitutions are as follows: Ala to Gly; Ala to Ser; Arg to Lys; Asn to Gln; Asn to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala; Gly to Pro; His to Asn; His to Gln; Ile to Leu, Ile to Val; Leu to Ile; Leu to Val; Lys to Arg; Lys to Gln; Lys to Glu; Met to Leu; Met to Tyr; Met to Ile; Phe to Met; Phe to Leu; Phe to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; Phe to Val; Phe to Ile and / or Phe or to Leu.

[0132] Conservative amino acid substitutions may also be made in non-CDR amino acid sequences.

[0133] In other embodiments, amino acid substitutions in the CDR amino acid sequence may be performed in order to provide sdAb with modified, for example, increased affinity for binding to IgE, which is displaced from FcεRI or combined with another target of interest. The amino acid sequence of CDR is generally more capable of determining target affinity than the amino acid sequence outside the CDR. Therefore, the sdAb described herein may also be affinity matured by introducing one or more changes in the amino acid sequence of one or more CDRs, which changes result in modification, such as compared to the first (parent) sdAb, and the resulting sdAb has an improved affinity for IgE. The method for sdAb affinity maturation may be prepared by methods known in the art, for example, as described in patent application WO2012 / 175740.

[0134] There may also be amino acid substitutions, insertions or deletions in one or more FRs outside of the CDRs. In particular, humanizing substitutions (i.e., replacing one or more amino acid residues in the amino acid sequence of a naturally occurring framework sequence with one or more amino acid residues in the same domain of a conventional human antibody at a corresponding position) may be performed. HH The framework region sequences of the sequences are identical to one or more closely related human V HH By comparing the corresponding framework sequences of the V sequences, potentially useful humanized substitutions can be determined, and then one or more potentially useful humanized substitutions (or combinations thereof) determined thereby can be introduced into the V HH sequence (in any manner known per se) and the humanized V HH The affinity of the sequence to the target (IgE binding), IgE displacement activity, stability, ease and level of expression, and / or other desired properties. Therefore, sdAbs can be partially humanized or fully humanized. Rossoti et al. (Rossoti et al., 2021) and Traian S. (Traian S., 2022) previously described methods for humanizing sdAbs.

[0135] Compared with the replacement in CDR, any amino acid replacement of the amino acid sequence outside CDR may generally make sdAb have less modified biological activity. However, according to the host organism used to express sdAb or the multi-specific construct described herein, any changes (such as deletions, insertions and / or replacements) of ssdAb amino acid sequence can also be designed to improve expression levels. For example, the design mode of these changes can be to remove one or more post-translational modification sites (such as one or more glycosylation sites), which will be within the capabilities of those skilled in the art. Alternatively, replacement or insertion can be designed to introduce one or more sites to connect functional groups, such as allowing the insertion of affinity tags (His tags) or for site-specific pegylation. By changing N-terminal glutamic acid (E) to aspartic acid (D), the possibility of N-terminal post-translational modification can be eliminated. Therefore, the amino acid difference may be that glutamic acid (Glu) is changed to aspartic acid (Asp) at position 1 (the position is determined according to Kabat numbering).

[0136] One embodiment illustrates how amino acid substitutions can alter the IgE binding affinity and / or displacement activity of the sdAbs described herein. Known monomeric sdAb 026 (designated herein as A1 (SEQ ID NO: 1 having binding regions CDR1, CDR2, and CDR3 having SEQ ID NOs: 2, 3, and 4, respectively, as determined by Aho) was optimized to improve IgE binding and displacement activity. The method for studying the effects of amino acid substitutions is described in Example 4. The resulting sdAb (A2) of SEQ ID NO: 5 having binding regions CDR1, CDR2, and CDR3 having SEQ ID NOs: 6, 7, and 8, respectively, showed a 2- to 4-fold improvement in displacement activity compared to sdAb A1, as determined by Aho. Notably, the improved sdAb A1 (i.e., sdAb A2) had amino acid substitutions in one of the three CDRs and outside of the CDRs.

[0137] In an alternative embodiment, the sdAb designated A1 (SEQ ID NO: 1) can be optimized to improve IgE binding and / or displacement activity by implementing one or more mutations selected from the list, which mutations are selected from: L11F, L11K, V12F, V12Y, V12G, Q13V, P14M, P14E, P14R, R19K, A23K, A23R, S25K, S25R, G26K, G26R, T28K, T28R, F29Y, G 30F, G30W, G30Y, G30D, G30H, G30K, G30K, G30R, K43R, P45W, P45Y, F68W, T69W, T69G, T69N, T6 9R, I70V, S71N, D73F, D73W, D73Y, D73M, D73I, D73L, D73V, D73A, D73G, D73S, D73T, D73T, D73A 4M, 48M, 48T, 48E, 48H, 48K, 48R, A75W, A75Y, A75P, A75M, A75G, A75S, A75N, A75K, A75R, N77Y, N77K, N77R, M78K, M78R, L79F, L79V, L79A, L79A, L79N, L79H, Q82W, Q82Y, M83I, T91A, T91G, T91Q, T91D, T91E, T91H, V93M, V93T, V93E, and L104M.

[0138] Any of the novel sdAbs disclosed herein may be evaluated for amino acid mutations similar to those of A1 to improve IgE binding.

[0139] In one embodiment, sdAb B2 (SEQ ID NO: 13) can be optimized to improve IgE binding by implementing one or more mutations selected from the following list: V12T, A23Q, A23D, T28Q, T28H, D35M, D35A, D36V, Q39F, Q39W, Q39Y, Q39I, Q39V, Q39E, Q44P, D74P, D74I, S105I, S105N, S105D and S105E.

[0140] In another embodiment, sdAb B3 (SEQ ID NO: 17) can be optimized to improve IgE binding and / or displacement activity by implementing one or more mutations selected from the following list: S21E, A23D, A23E, S25D, W36I, V37W, V37Y, A40C, A40R, G44P, G44D, G44E, G44H, F45P, F45M, F45M, F45I, F45L, F45V, F45A, S45A, S45G, T58D, N59E, K65M, K65A, T69Q, S71I, N77G, R77H, 8M, R78V, R78E, Q82Y, N84D, N84E, K87L, K87G, K87C, K87S, K87N, K87D, K87E, K87H, P88D, T91A, T91G, T91N, T91Q, T91D, T91E, A97M, A97I, A97L, A97V, N105P, N105D, R108F, R108M, R108I, R108V, R108N, R108Q, and R108K.

[0141] As previously described, the parent CDR region may be subject to sequence changes, such as substitutions, deletions or additions of 1, 2 or 3 amino acids. The variant may have the same, improved or increased IgE binding activity or displacement activity compared to a control IgE binder or displacement agent. In a preferred embodiment, the displacement activity is improved or increased compared to sdAb A1 (SEQ ID NO: 1) or a multispecific construct comprising two sdAb A1s.

[0142] Multispecific constructs

[0143] sdAbs, such as monomeric sdAb / VHH domains, can be engineered into multimers, such as multispecific constructs, with the goal of improving biological activity (e.g., displacement activity), improving druggability (e.g., improved serum circulation half-life, improved biodistribution) or establishing binding to other therapeutic targets besides IgE.

[0144] In particular, two or more sdAbs can be combined to form a multimer, such as a multispecific construct, which can displace bound IgE from FcεRI. Preferably, such a multispecific construct can additionally limit, prevent or inhibit free IgE from binding to FcεRI. More preferably, such a construct also limits, prevents or inhibits free IgE from binding to CD23. Advantageously, by combining two or more sdAbs in a multimeric construct, in particular a multispecific construct, the construct may exhibit a better ability (i) to displace bound IgE from FcεRI compared to the monomeric sdAb present in the construct; (ii) to limit, prevent or inhibit free IgE from binding to FcεRI and / or (iii) to limit, prevent or inhibit free IgE from binding to CD23.

[0145] The multispecific construct consists of two or more sdAbs, at least one of which has displacement activity itself, and the two sdAbs are linked together by a linker, such as Figure 1 shown.

[0146] The multispecific construct comprises:

[0147] (a) a first single domain antibody (sdAb) which is capable of displacing bound IgE from FcεRI,

[0148] (b) a second sdAb, which binds to IgE,

[0149] (c) a moiety linking the first and second sdAbs,

[0150] in

[0151] The first and second sdAbs bind to non-identical epitopes of IgE, and

[0152] , the multispecific constructs can displace bound IgE in the IgE high affinity receptor (FcεRI) with improved displacement activity compared to the first or second sdAb.

[0153] If the present construct comprises at least one sdAb capable of displacing bound IgE, it is envisaged that the multispecific construct comprises: (a) a first single domain antibody (sdAb) which binds IgE, and (b) a second sdAb which binds IgE and may be capable of providing the desired displacement.

[0154] Preferably, both the first and second sdAbs bind to the same IgE antibody, e.g., simultaneously, to avoid binding of IgE to the IgE high affinity receptor, thereby preventing any degranulation of mast cells or basophils by the anti-IgE multispecific construct. Example 3 (Relative epitope mapping) describes a method for evaluating whether two sdAbs can bind to the same IgE molecule.

[0155] The binding site to IgE may be outside the variable domain of IgE. In some embodiments, it is preferred that at least one of the two sdAbs binds to the Fc region of IgE, while in other embodiments, it is preferred that both sdAbs bind to the Fc region of IgE. It is further emphasized that the multispecific construct may comprise at least one further IgE epitope that binds to the sdAb that binds to the first sdAb and the second sdAb. Preferably, the further sdAb binds to the same IgE molecule as the first and second sdAbs.

[0156] By implementing sdAbs with displacement activity into the described multispecific constructs, the displacement activity is increased compared to a single sdAb.

[0157] Since sdAbs are composed of VHH domains of camelid antibodies, the multispecific construct can be replaced by the following expression: the multispecific construct comprises: (a) a first VHH domain that binds to IgE and (b) a second VHH domain that binds to IgE. Preferably, both the first and second VHH domains bind to the same IgE antibody, for example, simultaneously bind to the same IgE antibody. The binding site to IgE may be located outside the variable domain of IgE. In some embodiments, it is preferred that at least one of the two VHH domains binds to the Fc region of IgE, while in other embodiments, it is preferred that both VHH domains bind to the Fc region of IgE. It is further emphasized that the multispecific construct may comprise at least one VHH domain that binds to another epitope of IgE with the epitope bound by the first VHH and the second VHH. Preferably, the further VHH domain binds to the same IgE molecule as the first and second VHH domains.

[0158] Therefore, in the most interesting embodiments, the multispecific construct comprises non-identical sdAbs, such as non-identical VHH domains. Thus, two sdAb / VHH domains may be able to bind to the same IgE antibody simultaneously, which can be assessed by relative epitope mapping.

[0159] In the following, any reference to sdAb is likely to be in accordance with the VHH domain.

[0160] In an embodiment of interest, the multispecific construct comprises a first sdAb and / or a second sdAb that, when bound to IgE bound to FcεRI, results in a reduction in the binding affinity between IgE and FcεRI. Thus, the multispecific construct comprises at least one sdAb, such as the first sdAb, that can displace bound IgE from FcεRI. Alternatively, neither sdAb has displacement activity, but the final multispecific construct may result in a reduction in the binding affinity between IgE and FcεRI after binding to IgE bound to FcεRI.

[0161] In further interesting embodiments, the multispecific construct comprises at least one further sdAb, e.g., a second sdAb, wherein the sdAb promotes an improvement or increase in the binding affinity of the multispecific construct to IgE compared to the IgE binding affinity of the first sdAb or the second sdAb. Thus, the second sdAb may increase the overall IgE binding affinity (avidity).

[0162] For example, the overall binding affinity (i.e., affinity) of the multispecific construct of IgE is increased, so that the sum of the observed binding affinities of the first sdAb and the second sdAb is increased. In addition, the displacement effect of the multispecific construct is increased, so that the sum of the observed displacement effects of any one of the first sdAb and the second sdAb is increased.

[0163] The displacement activity of a single sdAb or a multispecific construct can be assessed by an ELISA-based IgE-FcεRIα displacement assay based on measuring the remaining IgE that is not removed from immobilized recombinant human FcεRIα preloaded with IgE after the addition of a test compound (e.g., sdAb or multispecific construct) and determining the percentage of displacement activity as a relative reduction in signal compared to a control to which no test compound was added.

[0164] In the constructs disclosed herein, the first sdAb can be selected among sdAbs having displacement activity, wherein EC 50 In the nanomolar to molar range, for example, using EC 50 In the range of 100nM to 5000nM, for example, using EC 50 Above 200, 300, 400, 500 or 600 nM, but below 4000, 3000, 2000 or 1000 nM. Exemplary sdAbs meeting this criterion are sdAbA1, A2 or B1.

[0165] Additionally or alternatively, the displacement activity of the first sdAb is such that the maximum displacement activity is at least 80%, preferably at least 85%, 90% or 95%. Exemplary sdAbs meeting this criterion are sdAb A1 or A2.

[0166] Most preferably, the first sdAb has an EC in the range of 200 nM to 5000 nM. 50 The sdAb has a displacement activity of at least 100%, and a maximum displacement effect of at least 80%. An example sdAb meeting this criterion is sdAb A1 or A2.

[0167] For the multispecific constructs disclosed herein, the second sdAb can range from having no displacement activity (e.g., exhibiting lower displacement activity (higher EC) than the first sdAb (e.g., sdAb B2, B3, D1, D2, D3, E1, E2, E3, E4, F1, F2, F3, F4, F5, F6, and G1). 50 )), or the second sdAb may exhibit a maximal displacement effect of less than 75% (e.g., B1).

[0168] As previously described, the multispecific constructs improve the displacement activity of the individual sdAbs of the multispecific construct. The improved displacement activity may be such that the observed EC of the first sdAb or the second sdAb 50 or a mixture of the first and second sdAb resulting in an EC of the construct 50 Lower.

[0169] By way of example, the improved displacement activity of the multispecific construct allows for EC 50 In comparison, EC 50 Preferably, EC 50 At least 25, 30, 35, 40, 45, 50, 60, 70, 80, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, or 1000 times lower.

[0170] For example, the improved displacement activity of the multispecific constructs allows EC 50 Less than 200nM. EC 50 Preferably, it is less than 150, 100, 75, 50, 25, 20, 15, 10, 5, 4, 3, 2 or 1 nM.

[0171] As a substitution scheme or supplement, the improved substitution activity of the multispecific construct is such that the maximum substitution effect is at least 80%. Preferably, the maximum substitution effect is at least 85%, 90% or 95%, most preferably at least 95%.

[0172] In a preferred embodiment, the improved displacement activity of the multispecific construct is such that: 50 Below 100 nM, the maximum displacement is at least 80%, such as at least 85, 90 or 95%.

[0173] Typically, a multispecific construct may include as a first sdAb or a second sdAb, the sdAb comprising:

[0174] A CDR1 having the following amino acid sequence: SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70 or 74, determined according to Aho, wherein each amino acid sequence may have 1, 2 or 3 amino acid substitutions; and / or

[0175] A CDR2 having the following amino acid sequence: SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71 or 75, determined according to Aho, wherein each amino acid sequence may have 1, 2 or 3 amino acid substitutions; and / or

[0176] A CDR3 having the following amino acid sequence: SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72 or 76, as determined by Aho, wherein each amino acid sequence may have 1, 2 or 3 amino acid substitutions.

[0177] If it is found that not all three binding CDR regions are essential for binding, the first and second sdAbs can be constructed from a combination of the above CDR regions, but one of the three CDR regions may have been replaced by another sequence, e.g., subjected to humanization or other amino acid modification. In a preferred embodiment, the original CDR3 region of the Camelidae sdAb is maintained as a conserved sequence.

[0178] More specifically, a multispecific construct can include as a first or second sdAb comprising a binding region having a combination of CDR1, CDR2 and CDR3, wherein each of the CDR1, CDR2 and CDR3 has SEQ ID NO: 2, 3 and 4; 6, 7 and 8; 10, 11 and 12; 14, 15 and 16; 18, 19 and 20; 22, 23 and 24; 26, 27 and 28; 30, 31 and 32; 34, 35 and 36; 38, 39 and 40; 42, 43 and 44; 46, 47 and 48; 50, 51 and 52; 54, 55 and 56; 58, 59 and 60; 62, 63 and 64; 66, 67 and 68; 70, 71 and 72; or 74, 75 and 76, as determined by Aho.

[0179] The multispecific construct may comprise any combination of sdAb / VHH domains described herein, so long as the construct has displacement activity. In a preferred embodiment, the first sdAb itself has displacement activity, such as sdAb 026 (designated herein A1) or A2.

[0180] Thus, the multispecific construct may comprise binding regions from sdAb A1 or A2, and thus, the first sdAb of the construct may comprise binding regions CDR1, CDR2 and CDR3, each of which comprises or consists of an amino acid sequence determined according to the Kabat, Chothia, IMTG or Aho numbering scheme, the amino acid sequence being determined in an amino acid sequence selected from any one of the following: SEQ ID NO: 1 or 5, optionally, wherein CDR1 comprises 1, 2 or 3 amino acid substitutions, CDR2 comprises 1, 2 or 3 amino acid substitutions, and CDR3 comprises 1, 2 or 3 amino acid substitutions.

[0181] According to another definition, the first sdAb comprises a binding region, wherein the CDR1, CDR2 and CDR3 each have SEQ ID NOs: 2, 3 and 4; or 6, 7 and 8, wherein each CDR is identified according to the Aho numbering scheme.

[0182] The second sdAb can be any that has IgE binding activity in the presence of the first sdAb. An exemplary second sdAb comprises binding regions CDR1, CDR2, and CDR3, each of which comprises or consists of an amino acid sequence determined according to the Kabat, Chothia, IMTG, or Aho numbering scheme, the amino acid sequence being determined in an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 13, 17, 21, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, and 73, optionally wherein CDR1 comprises 1, 2, or 3 amino acid substitutions, CDR2 comprises 1, 2, or 3 amino acid substitutions, and CDR3 comprises 1, 2, or 3 amino acid substitutions.

[0183] In the replacement scheme, the second sdAb comprises binding regions CDR1, CDR2 and CDR3, each of which has SEQ ID NO: 10, 11 and 12; 14, 15 and 16; 18, 19 and 20; 22, 23 and 24; 26, 27 and 28; 30, 31 and 32; 34, 35 and 36; 38, 39 and 40; 42, 43 and 44; 46, 47 and 48; 50, 51 and 52; 54, 55 and 56; 58, 59 and 60; 62, 63 and 64; 66, 67 and 68; 70, 71 and 72; or 74, 75 and 76, wherein each CDR is determined according to the Aho numbering scheme.

[0184] Similarly, the second sdAb may comprise a binding region having a combination of CDR1, CDR2, and CDR3, wherein each of CDR1, CDR2, and CDR3 comprises SEQ ID NOs: 129, 130 and 131; 132, 133 and 134; 135, 136 and 137; 138, 139 and 140; 141, 142 and 143; 144, 145 and 146; 147, 148 and 149; 150, 151 and 152; 153, 154 and 155; 156, 157 and 158; 159, 160 and 161; 162, 163 and 164; 165, 166 and 167; 168, 169 and 170; 171, 172 and 173; 174, 175 and 176; 177, 178 and 179; 180, 181 and 182; or 183, 184 and 185, as determined by Kabat.

[0185] Similarly, the second sdAb may comprise a binding region having a combination of CDR1, CDR2, and CDR3, wherein each of CDR1, CDR2, and CDR3 comprises SEQ ID NOs: 186, 187 and 188; 189, 190 and 191; 192, 193 and 194; 195, 196 and 197; 198, 199 and 200; 201, 202 and 203; 204, 205 and 206; 207, 208 and 209; 210, 211 and 212; 213, 214 and 215; 216, 217 and 218; 219, 220 and 221; 222, 223 and 224; 225, 226 and 227; 228, 229 and 230; 231, 232 and 233; 234, 235 and 236; 237, 238 and 239; or 240, 241 and 242, as determined by Chothia.

[0186] Similarly, the second sdAb may comprise a binding region having a combination of CDR1, CDR2, and CDR3, wherein each of CDR1, CDR2, and CDR3 comprises SEQ ID NOs: 242, 243 and 244; 245, 247 and 248; 249, 250 and 251; 252, 253 and 254; 255, 256 and 257; 258, 259 and 260; 261, 262 and 263; 264, 265 and 266; 267, 268 and 269; 270, 271 and 272; 273, 274 and 275; 276, 277 and 278; 279, 280 and 281; 282, 283 and 284; 285, 286 and 287; 288, 289 and 290; 291, 292 and 293; 294, 295 and 296; or 297, 298 and 299, as determined by IMGT.

[0187] Thus, a first sdAb structured as described herein may preferably comprise any one of SEQ ID NOs: 1 or 2, and a second sdAb preferably comprises SEQ ID NOs: 5, 9, 13, 17, 21, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, and 73.

[0188] Wherein the first sdAb comprises the binding region of B1, the second sdAb may comprise the binding region of sdAb B2, B3, D1, D2, D3, E1, E2, E3, E4, F1, F2, F3, F4, F5, F6 or G1, respectively.

[0189] Thus, in still preferred embodiments, the first sdAb of the construct may comprise or consist of SEQ ID NO:

[0190] 1, 5 and 9, more preferably, the first sdAb of the construct may comprise or consist of SEQ ID NO: 1.

[0191] In particular, the first sdAb may be any one of sdAb A1, sdAb A2 or sdAb B1. Thus, the first sdAb may comprise or consist of an amino acid sequence selected from any one of SEQ ID NO: 1, 5 and 9, preferably the first sdAb may comprise or consist of the amino acid sequence SEQ ID NO: 1.

[0192] In addition to the above, sdAb A1 can also be defined by its three CDR regions, determined using different CDR calculation methods.

[0193] Thus, in a preferred embodiment, the first sdAb of the construct may comprise binding regions CDR1, CDR2 and CDR3, each of which has SEQ ID NO:

[0194] 2, 3 and 4 (determined according to Aho);

[0195] 129, 130 and 131 (according to Kabat);

[0196] 186, 187 and 188 (as determined by Chothia); or

[0197] 243, 244 and 245 (determined by IMGT),

[0198] Optionally, each CDR1 may contain 1, 2 or 3 amino acid substitutions, each CDR2 may contain 1, 2 or 3 amino acid substitutions, and each CDR3 may contain 1, 2 or 3 amino acid substitutions.

[0199] As mentioned above, in addition to A1, A2 may also be defined by its CDR regions.

[0200] Therefore, in a preferred embodiment, the first sdAb of the construct may comprise binding regions CDR1, CDR2 and CDR3, each of which has SEQ ID NO:

[0201] 6, 7 and 8 (determined according to Aho);

[0202] 132, 133 and 134 (according to Kabat);

[0203] 189, 190 and 191 (as determined by Chothia); or

[0204] 246, 247 and 248 (determined by IMGT),

[0205] Optionally, each CDR1 may contain 1, 2 or 3 amino acid substitutions, each CDR2 may contain 1, 2 or 3 amino acid substitutions, and each CDR3 may contain 1, 2 or 3 amino acid substitutions.

[0206] Furthermore, B1 may be defined by CDR regions in addition to A1 and A2.

[0207] Therefore, in a preferred embodiment, the first sdAb of the construct may comprise binding regions CDR1, CDR2 and CDR3, each of which has SEQ ID NO:

[0208] 10, 11 and 12 (determined according to Aho);

[0209] 135, 136 and 137 (according to Kabat);

[0210] 192, 193 and 194 (as determined by Chothia); or

[0211] 249, 250 and 251 (determined by IMGT),

[0212] Optionally, each CDR1 may contain 1, 2 or 3 amino acid substitutions, each CDR2 may contain 1, 2 or 3 amino acid substitutions, and each CDR3 may contain 1, 2 or 3 amino acid substitutions.

[0213] Alternatively, the first sdAb of the construct may comprise binding regions CDR1, CDR2 and CDR3, which comprise or consist of an amino acid sequence determined according to the Kabat, Chothia, IMTG or Aho numbering scheme, said amino acid sequence being determined in an amino acid sequence selected from any one of the following: SEQ ID NO: 1, 5 or 9, wherein each CDR1 may comprise 1, 2 or 3 amino acid substitutions, each CDR2 may comprise 1, 2 or 3 amino acid substitutions, and each CDR3 may comprise 1, 2 or 3 amino acid substitutions.

[0214] As the inventors further appreciate, multispecific constructs may exhibit allergenic activity, but allergenic activity may be affected by the assay concentration, diluent, and sensitivity of the assay method. Thus, preferred embodiments include as a first sdAb, sdAb A1, or a sdAb comprising a binding region of sdAb A1, and as a second sdAb, a sdAb selected from B1, D2, E1, and F4, or a binding region of a sdAb selected from B1, D2, E1, and F4.

[0215] Thus, in a multispecific construct, a first sdAb comprises binding regions CDR1, CDR2 and CDR3, each of which comprises or consists of an amino acid sequence determined according to the Kabat, Chothia, IMTG or Aho numbering scheme, which is determined in an amino acid sequence selected from the group consisting of: SEQ ID NO: 1 or 5, wherein CDR1 optionally comprises 1, 2 or 3 amino acid substitutions, CDR2 comprises 1, 2 or 3 amino acid substitutions, and CDR3 comprises 1, 2 or 3 amino acid substitutions; a second sdAb comprises binding regions CDR1, CDR2 and CDR3, each of which comprises or consists of an amino acid sequence determined according to the Kabat, Chothia, IMTG or Aho numbering scheme, which is determined in an amino acid sequence selected from the group consisting of: SEQ ID NO: 1 or 5. NO: 9, 25, 33, 61, wherein CDR1 comprises 1, 2 or 3 amino acid substitutions, CDR2 comprises 1, 2 or 3 amino acid substitutions, and CDR3 comprises 1, 2 or 3 amino acid substitutions.

[0216] In the replacement scheme, in the multispecific construct, the first sdAb comprises a binding region, wherein CDR1, CDR2 and CDR3 each have SEQ ID NO: 2, 3 and 4; or 6, 7 and 8, wherein each CDR is determined according to the Aho numbering scheme; the second sdAb comprises a binding region CDR1, CDR2 and CDR3, wherein each CDR1, CDR2 and CDR3 each have SEQ ID NO: 10, 11 and 12; 26, 27 and 28; 34, 35 and 36; or 62, 63 and 64, wherein each CDR is determined according to the Aho numbering scheme.

[0217] In a particularly interesting embodiment, the construct comprises the binding regions of sdAbs A1 and B1. Thus, the multispecific construct comprises a first sdAb comprising binding regions CDR1, CDR2 and CDR3, each of which comprises or consists of an amino acid sequence determined according to the Kabat, Chothia, IMTG or Aho numbering scheme, the amino acid sequence being determined in the following amino acid sequence: SEQ ID NO: 1, wherein CDR1 optionally comprises 1, 2 or 3 amino acid substitutions, CDR2 comprises 1, 2 or 3 amino acid substitutions, and CDR3 comprises 1, 2 or 3 amino acid substitutions; a second sdAb comprising binding regions CDR1, CDR2 and CDR3, each of which comprises or consists of an amino acid sequence determined according to the Kabat, Chothia, IMTG or Aho numbering scheme, the amino acid sequence being determined in the following amino acid sequence: SEQ ID NO: 1. NO: 9, optionally, wherein CDR1 comprises 1, 2 or 3 amino acid substitutions, CDR2 comprises 1, 2 or 3 amino acid substitutions, and CDR3 comprises 1, 2 or 3 amino acid substitutions.

[0218] In an alternative embodiment, the multispecific construct comprises a first sdAb, wherein the binding region comprises binding regions CDR1, CDR2 and CDR3, wherein each CDR1, CDR2 and CDR3 has SEQ ID NOs: 2, 3 and 4, respectively, wherein each CDR is determined according to the Aho numbering scheme; and a second sdAb comprises binding regions CDR1, CDR2 and CDR3, wherein each CDR1, CDR2 and CDR3 has SEQ ID NOs: 10, 11 and 12, respectively, wherein each CDR is determined according to the Aho numbering scheme.

[0219] When the second sdAb comprises a binding region from D2, CDR1, CDR2, and CDR3 may each have SEQ ID NOs:26, 27, and 28, and the sdAb may have SEQ ID NO:25.

[0220] When the second sdAb comprises a binding region from El, CDR1, CDR2, and CDR3 may each have SEQ ID NOs:34, 35, and 36, and the sdAb may have SEQ ID NO:33.

[0221] When the second sdAb comprises a binding region from F4, CDR1, CDR2, and CDR3 may each have SEQ ID NOs:62, 63, and 64, and the sdAb may have SEQ ID NO:61.

[0222] In a further embodiment, the first sdAb A1 or A2 can be replaced by further variants of A1, such as those described in WO2012175740A1: to be mentioned is the Camelidae sdAb 39D11 in WO2012 / 175740A1 (SEQ ID NO: 119 in WO2012 / 175740A1), the humanized versions of 39D11 sdAb = IGE009 (mutations: V5L, M77TK83R, Q108L), IGE010 (mutations: V5L, K83R, M77T, Q108L), IGE011 (mutations: V5L, M77L K83R, Q108L, W91Y) and IGE012 (mutations: V5L, M77L, K83R, Q108L, W91Y) have SEQ ID NOs: 120 to 123. Among them, IGE009 is reported to be the preferred one. The affinity matured versions of IGE009 in WO2012 / 175740A1 are IGE025 to IGE030, SEQ ID NOs: 127 to 133. It is reported that among the affinity matured IGE026 and IGE027, they are the preferred ones because their affinity is increased by 30 to 50 times after maturation. IGE026 was originally named A1 (SEQ ID NO: 1 in this application, SEQ ID NO: 128 in WO2012 / 175740A1). Here, SEQ ID NO: 316 for 39D11, SEQ ID NO: 317 for IGE009, SEQ ID NO: 318 for IGE010, SEQ ID NO: 319 for IGE011, and SEQ ID NO: 320 for IGE012.

[0223] Alternatively, the multispecific construct comprising the first replacement sdAb may comprise any sdAb capable of binding to IgE as the second sdAb, for example preferably capable of binding to IgE at another epitope than the first sdAb and having a high binding affinity in the nanomolar range. Such a second sdAb may be selected from any of the patent applications described in WO 2004 / 041867, WO 2012 / 175740, WO 2014 / 087010, WO 2020 / 208177, CN 113461823 and CN 111875706.

[0224] [Peptide ligation of sdAb]

[0225] As previously mentioned, sdAb / VHH domains can be incorporated into larger constructs, such as multispecific constructs, in which two sdAb / VHH domains are combined in one construct. Such constructs may have the ability to bind to non-identical IgE epitopes of the same IgE. As shown in the Examples herein, multispecific constructs may comprise at least two different sdAb / VHH domains, which are linked together by part (c). In a preferred embodiment, the part is a peptide linker (see Figure 1 In still preferred embodiments, such constructs exhibit improved or increased displacement activity compared to the individual sdAbs present in the construct (Example 6, Table 15 and Example 7, Table 16).

[0226] Table 3 provides the full length amino acid sequences of exemplary multispecific constructs comprising at least two different sdAbs linked together by a portion consisting of a peptide linker. When determining improved displacement activity, two linked A1 sdAbs can be used as controls. As previously mentioned, at least one of the two sdAbs has displacement activity itself. Here, A1, A2 or B1 represent sdAbs with displacement activity.

[0227]

Table 3

[0228]

[0229] Thus, a multispecific construct can include a first and a second sdAb and a portion connecting the first and second sdAbs, wherein the portion is a peptide. Typically, a peptide is a short peptide having up to 30 amino acid residues. By way of example, a peptide can be composed of one or more glycine and serine amino acid modules according to formula (G n S) n , wherein n can be an integer from 1 to 6. Therefore, the peptide can be GS, G 2 S.G. 3 S, (G 2 S)2 G 4 S, (G 4 S) 2 , (G 4 S) 3 , (G 4 S) 4 etc., for example, a GS linker selected from SEQ ID NO: 88 to 91 or 452 to 454 or 457 (Table 5). Alternatively, the peptide may be an arginine-serine peptide. In addition, the linker may be EAAAK or (EAAAK) 2 , SEQ ID NO: 455 or 456.

[0230] [Table 5: Overview of peptide linkers]

[0231] Connectors SEQ ID NO sequence <![CDATA[(G 4 S)]]> 88 GGGGS <![CDATA[(G 4 S) 2 ]]> 89 GGGGSGGGGS <![CDATA[(G 4 S) 3 ]]> 90 GGGGSGGGGS GGGGS <![CDATA[(G 4 S) 4 ]]> 91 GGGGSGGGGS GGGGSGGGGS RS 92* RS GS 452 GS <![CDATA[G 2 S]]> 453 GGS <![CDATA[G 3 S]]> 454 GGGS EAAAK 455 EAAAK <![CDATA[(EAAAK) 2 ]]> 456 EAAAKEAAAK <![CDATA[G 2 SG 2 S]]> 457 GGSGGS

[0232] *SEQ ID NOs: 92, 452, and 453 are reported as the repeat sequences RSRS, GSGS, and GGSGGS, respectively, but the individual RS, GS, and GGS are usually sufficient.

[0233] In some embodiments, wherein the multispecific construct comprises the first and second sdAb, and the portion is a peptide, the multispecific construct may comprise one or more further sdAbs operably connected to the first or second sdAb. For example, one or more sdAbs may be connected to the first or second sdAb by a peptide, a polypeptide or a polysaccharide. In some embodiments, one or more additional sdAbs may also be able to bind IgE, preferably the same IgE molecule bound to the first and second sdAbs. In other embodiments, one or more additional sdAbs may be able to bind human serum albumin (HAS), which may extend the half-life of serum.

[0234] In the embodiments herein, the peptide linker can be selected from the group consisting of SEQ ID NOs: 88, 89, 90, 91, 92, 452, 453, 454, 455, 456 and 457. In other words, the peptide linker can be selected from the group consisting of RS, GS, GGS and the peptides of SEQ ID NOs: 88, 89, 90, 91, 454, 455, 456 and 457.

[0235] It will be appreciated that the moieties used to link two or more sdAbs may be independently selected from the group consisting of peptides, polypeptides, polymers and polysaccharides.

[0236] Moiety (c) may extend the serum half-life of the multispecific construct.The extended serum half-life may be determined relative to the serum half-life of the first and second sdAbs or a combination of two sdAbs (eg, wherein the sdAbs are linked to a peptide moiety).

[0237] Alternatively, or additionally, the moiety may be further multispecific in the construct by selecting a moiety (c) that has binding affinity for another biologically relevant target.

[0238] In general, there are several options for selecting a fusion partner for a monomeric sdAb or a multispecific construct for improved pharmacokinetic properties (Strohl W., 2015). For example:

[0239] (a) Fusion to native long half-life proteins or protein domains (e.g., Fc fusion, transferrin [Tf] fusion, or albumin fusion).

[0240] (b) Fusion to an inert polypeptide, such as XTEN (also called recombinant PEG or "rPEG"), a homologous amino acid polymer (HAP; HAPylation), a proline-alanine-serine polymer (PAS; PASylation), or an elastin-like peptide (ELP; ELPylation).

[0241] (c) Chemical coupling to recurring chemical moieties, such as conjugation to PEG (pegylation or hyaluronic acid).

[0242] (d) Significantly increasing the negative charge of a monomeric sdAb or multispecific construct by polysialylation; alternatively, by fusing a negatively charged, highly sialylated peptide (e.g., the carboxyl-terminal peptide [CTP] of the β chain of chorionic gonadotropin (CG)).

[0243] (e) Non-covalent binding via peptide linkers or protein binding domains to proteins that typically have a long half-life (such as HSA, human IgG, or possibly transferrin).

[0244] (f) Chemical coupling to long half-life proteins such as human IgG, Fc portion or HSA.

[0245] It is contemplated that one or more of options (a) to (f) may be incorporated into the multispecific constructs disclosed herein.

[0246]

Fc fusion of sdAb

[0247] Fc fusions between Fc domains and therapeutic agents are widely used to provide additional beneficial biological and pharmacological properties (Czajkowsky et al., 2012). The presence of the Fc domain significantly increases the plasma half-life, which prolongs the therapeutic activity due to its interaction with the salvaged neonatal Fc receptor (FcRn) and the slower renal clearance of larger molecules. The attached Fc domain also enables these molecules to interact with Fc receptors (FcRs) on immune cells. From a biophysical point of view, the independent folding of the Fc domain can improve the solubility and stability of partner molecules (e.g., sdAbs) in vitro and in vivo, while from a technical point of view, the Fc region allows for easy and economical purification by protein-G / A affinity chromatography during the manufacturing process.

[0248] Thus, in some embodiments, part (c) is a polypeptide which is a fragment of an antibody, in particular a fragment of a human antibody, selected from the group consisting of IgA, IgE, IgG and IgM, preferably a fragment of IgG. IgG fragments can be obtained from IgG 1 IgG 2 IgG 3 and IgG 4 The IgG fragment is preferably selected from any fragment of IgG 1 or IgG 4 , especially IgG 1 or IgG 4 The Fc domain of IgG H 1~C H 3, but IgG C may be preferred H 2~C H 3 (IgG-Fc region). In some embodiments, a short fragment of IgG, such as C H 1. C H 2 or C H Zone 3.

[0249] Typically, sdAbs can be independently fused or covalently coupled to the C-terminal domain of IgG-Fc. H 3 or C H 2, optionally also via a linker. Thus, sdAbs can be independently fused or covalently coupled to C H 2 N-terminal or C H 3, optionally through a linker. In a preferred embodiment, the IgG-Fc is IgG 4 -Fc, preferably human IgG 4 -Fc. In a further embodiment, wherein the two sdAbs are fused to the C-terminus or N-terminus of Fc via a peptide linker. In a further interesting embodiment, the two sdAbs are fused to the C-terminus of Fc via a peptide linker, as this appears to prevent allergenic activity.

[0250] Thus, a multispecific construct of interest is one in which

[0251] Both the first and second sdAbs are fused or covalently coupled to the C of IgG-Fc directly or via a linker. H 2,

[0252] Both the first and second sdAbs are fused or covalently coupled to the C of IgG-Fc directly or via a linker. H 3.

[0253] The first sdAb is fused or covalently coupled to the C of IgG-Fc directly or via a linker. H 2. The second sdAb is fused or covalently coupled to the C of IgG-Fc directly or through a linker. H 3, or

[0254] The second sdAb is fused or covalently coupled to the C of IgG-Fc directly or via a linker. H 2. The first sdAb is fused or covalently coupled to the C of IgG-Fc directly or through a linker. H 3.

[0255] In some more interesting embodiments, the first and second sdAbs are each bound to the C H 3 (C-terminus of IgG-Fc), or the first and second sdAbs are fused to the C-terminus of IgG-Fc, respectively. H 2 (the N-terminus of IgG-Fc), optionally via a linker. In a more interesting embodiment, the IgG Fc region is an IgG 4 -Fc region.

[0256] The first and second sdAbs may be fused to the antibody fragment using any suitable linker, such as a peptide of up to 30 amino acid residues.

[0257] By way of example, a flexible glycine-serine linker (GS linker) can be used, such as (G n S) n GS linker composed of one or more modules, wherein n can be an integer from 1 to 6. Therefore, the peptide can be GS, G 2 S.G. 3 S, (G 2 S) 2 G 4 S, (G 4 S) 2 , (G 4 S) 3 , (G 4 S) 4etc., for example, a GS linker selected from SEQ ID NO: 88 to 91 or 452 to 454 or 457 (Table 5). Alternatively, the peptide may be an arginine-serine (RS) peptide. In addition, the linker may be EAAAK or (EAAAK) 2 , SEQ ID NO: 455 or 456.

[0258] In one embodiment, the linker is a peptide selected from the group consisting of RS, GS, GGS and peptides having SEQ ID NOs: 88, 89, 90, 91, 454, 455, 456 and 457.

[0259] As previously described, antibody fragments (e.g., Fc regions) can be of any antibody type (e.g., IgG, IgE, IgM, IgD, and IgA), isotype (e.g., IgG1, IgG2, IgG3, IgG4, Ig A1, and Ig A2), or subclass, including engineered subclasses with altered Fc portions that can selectively provide reduced or enhanced effector cell activity or modifications in biodistribution, serum half-life, or excretion rate. Antibody fragments can be derived from any species. For example, the fragment is of human origin. Example effector functions include C1-q binding; GDC; Fc receptor binding; ADCC; ADCP; downregulation of cell surface receptors (e.g., B cell receptors), etc. Such effector functions generally require the Fc region to interact with a receptor, such as through FcyRI; FcyRIIA; FcyRIIBI; FcyRIIB2; FcyRIIIA; FcyRIIIB receptors and / or low affinity FcRn receptors. In addition, the Fc region may be a "dead" Fc, that is, an Fc that has been mutated to retain activity, for example, to extend serum half-life, but not activate high affinity Fc receptors. The Fc may also have reduced complement binding.

[0260] If it is desired not to have Fc interactions with receptors (e.g., FcγRIIB), the antibody fragment may be a CH1 immunoglobulin domain (e.g., IgG 1 -CH1 domain or IgG 4 -CH1 domain). Unlike the commonly used IgG-FC domain, they do not engage the inhibitory FcγRIIb receptor, do not compete with serum IgG for receptor binding, and their cytotoxic activity is independent of Fc glycosylation and FcγRIIIa polymorphism (Rozan et al., 2013).

[0261] In certain embodiments, the antibody fragment is derived from IgG4, such as IgG 4 -Fc. Examples of antibody fragments are listed in Table 6.

[0262] [Table 6: IgG-Fc list]

[0263] Fc constructs SEQ ID NO IgG4 complete hinge region 125 IgG4 truncated hinge region 126 IgG4 Fc Knob, truncated hinge region 433 IgG4 Fc-Hole, truncated hinge region 434 IgG4 Fc Knob, complete hinge region 435 IgG4 Fc-Hole, complete hinge region 436

[0264] Thus, when the construct is formed by the KIH structure Knob and Hole elements, part (c) may be selected from an IgG4 antibody fragment comprising or consisting of an amino acid sequence selected from any one of SEQ ID NOs: 125, 126 or 433, 434, 435 and 436. Preferably, the part (c) may comprise an IgG4 antibody fragment comprising or consisting of an amino acid sequence selected from any one of SEQ ID NOs: 125 and 126, optionally fused to a peptide linker. Preferably, the two heavy Fc parts are fused to the same peptide linker, respectively, so that when one Fc chain is fused to, for example, a GGS peptide, the other Fc chain is also fused to the GGS peptide.

[0265] Since the Ig-Fc part may contain different mutations, further embodiments thereof include that the IgG4 antibody fragment has an amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 125, 126, 433, 434, 435 and 436. Since IgG4 is unstable in vivo due to the phenomenon of "half antibody exchange", which means that it becomes bispecific (or in most cases a functional monomer). Therefore, when used for therapy, a single amino acid mutation may be introduced in the hinge region to prevent this dissociation - the so-called S228P mutation.

[0266] Another approach to prevent IgG4 instability in vivo may be to use IgG in the Knob-in-Hole (KIH) format. 4 -Fc scaffold, which might prevent this dissociation.

[0267] Another mutation implemented in the antibody fragment may be a mutation that enhances FcγRIIb (CD32b) binding, such as described in granted patent US8435517B2. It is preferably present at a k of less than about 100 nM. D Binds to FcγRIIb.

[0268] In addition, the Fc region may be a native sequence Fc region, comprising an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include a native sequence human IgG1 Fc region (non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region, and naturally occurring variants thereof or the Fc region may be a variant Fc region, the amino acid sequence being different from the amino acid sequence of the native sequence Fc region, the amino acid sequence being modified by at least one amino acid, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or the Fc region of the parent polypeptide, for example, at least 1 to about 10 amino acid substitutions in the native sequence Fc region or the Fc region of the parent polypeptide, and preferably about 1 to about 5 amino acid substitutions in the native sequence Fc region or the Fc region of the parent polypeptide. The variant Fc region herein may have at least about 80% amino acid sequence homology or sequence identity to a native sequence Fc region and / or to the Fc region of a parent antibody fragment, and most preferably at least about 90% homology thereto, more preferably at least about 95% homology thereto.

[0269] In one embodiment, the variant Fc sequence may include three amino acid substitutions in the CH2 region to reduce binding of FcγRI at EU index positions 234, 235 and 237 (Duncan et al., 1988). Two amino acid substitutions in the complement C1-q binding site at EU index positions 330 and 331 reduce complement fixation (Tao et al., 1993 and Canfield & Morrison, 1991). Replacement of IgG2 residues at positions 233 to 236 and IgG4 residues at positions 327, 330 and 331 in human IgG1 greatly reduced ADCC and CDC (Armour KL. et al., 1999 and Shields RL. et al., 2001). Other Fc variants are also possible, including but not limited to variants in which regions capable of forming disulfide bonds are deleted, or certain amino acid residues are eliminated at the N-terminus of the native Fc form or methionine residues are added thereto. Thus, one or more Fc portions of the molecule may include one or more mutations in the hinge region to eliminate disulfide bonds. In yet another embodiment, the hinge region of the Fc may be completely removed. In yet another embodiment, the molecule may comprise an Fc variant.

[0270] In addition, Fc variants can be constructed by replacing, deleting or adding amino acid residues to achieve complement binding or Fc receptor binding. The technology for preparing such sequence derivatives of immunoglobulin Fc fragments is disclosed in International Patent Publication Nos. WO 97 / 34631 and WO 96 / 32478. In addition, the Fc domain can be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0271] Antibody fragments, such as Fc portions, can also be modified for structural (eg, expression yield) reasons by replacing, deleting or inserting amino acid residues. Non-limiting examples of mutations that introduce expression yield are described below.

[0272] Antibody fragments can be modified to have native sugar chains, sugar chains increased compared to the native form, or sugar chains reduced compared to the native form, or may be non-glycosylated or deglycosylated. The increase, reduction, removal or other modifications of sugar chains can be achieved by methods common in the art, such as chemical methods, enzymatic methods or by expression in genetically engineered production cell lines. Such cell lines can include microorganisms, such as Pichia pastoris and mammalian cell lines, such as CHO cells, which naturally express glycosylation enzymes. In addition, microorganisms or cells can be modified to express glycosylation enzymes, or they can be made unable to express glycosylation enzymes. As an example of a cell engineered to have sialylation activity, the α-2,6-sialyltransferase 1 gene has been engineered into Chinese hamster ovary cells and Sf9 cells. Therefore, the constructs expressed by these engineered cells are sialylated by exogenous gene products. Another method for obtaining Fc molecules with modified amounts of sugar residues compared to multiple natural molecules includes separating the multiple molecules into glycosylated and non-glycosylated fractions, for example, using lectin affinity chromatography. The presence of a particular glycosylated portion has been shown to alter the function of immunoglobulins. For example, the removal of sugar chains from Fc molecules results in a sharp decrease in binding affinity to the C1-q portion of the first complement component C1, a decrease or loss of antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), and thus does not induce unnecessary immune responses in vivo. Other important modifications include sialylation and fucosylation: the presence of sialic acid in IgG is associated with anti-inflammatory activity, while the removal of fucose from IgG results in enhanced ADCC activity. The construct can have an Fc sequence with enhanced effector function, for example, by increasing their ability to bind to FcyRI and increasing ADCC activity. For example, fucose on the / V-linked glycan attached to the Asn-297 position of Fc sterically hinders the interaction of Fc with FcyRIIIA, and the removal of fucose by glycoengineering can increase the binding to FcyRIIIA, which means that the ADCC activity is >50-fold higher compared with the wild-type IgG1 control. Protein engineering has generated multiple variants through amino acid mutations in the Fc part of IgG1, which increase the affinity of Fc binding to FcyRIIIA. Notably, the triple alanine mutant S298A / E333A / K334A increased the binding to FcyRIIIA and ADCC function by 2-fold. The S239D / I332E (2X) and S239D / I332E / A330L (3X) variants showed significant increases in binding affinity to FcyRIIIA and enhanced ADCC capacity in vitro and in vivo. Other Fc variants identified by yeast display also showed improved binding to FcyRIIIA.See, for example, Liu et al., (2014) JBC 289(6):3571-90, hereby incorporated by reference.

[0273] The multispecific constructs described herein can be assembled in different ways.

[0274] In a non-limiting example, a multispecific construct comprises:

[0275] (i) the first sdAb is selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69 and 73, preferably SEQ ID NO: 1 or 5;

[0276] (ii) a second sdAb, different from the first sdAb, selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69 and 73, preferably selected from the group consisting of SEQ ID NOs: 9, 13, 17, 21, 25, 29, 33, 37 41, 45, 49, 53, 57, 61, 65, 69 and 73,

[0277] Among them, each sdAb is linked to the Fc part, e.g.

[0278] (iii) an Fc portion selected from SEQ ID NO: 125 or 126, or associated with a Knob portion selected from SEQ ID NO: 433 and 435 and a Hole portion selected from SEQ ID NO: 434 and 436, using a linker portion to link the sdAb to the Fc portion in the N or C terminus, such as

[0279] (iv) a linker moiety selected from the group consisting of SEQ ID NOs: 88-92 and 452-457.

[0280] As previously mentioned, the amino acid sequences of the first and second sdAbs may be subject to affinity maturation, humanization or other amino acid changes in the amino acid sequence, preferably outside of the CDR regions. Thus, the first sdAb may have at least 80%, such as at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sdAb selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69 and 73, and the second sdAb, different from the first sdAb, may have at least 80%, such as at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sdAb selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69 and 73. Likewise, the Fc portion may have any of the sequence variations described above under Fc-fused sdAbs.

[0281] In a preferred embodiment, the multispecific construct comprises: a first sdAb A1 having SEQ ID NO: 1 or having at least 80% sequence identity to SEQ ID NO: 1; a second sdAb B1 having SEQ ID NO: 9 or having at least 80% sequence identity to SEQ ID NO: 9, wherein each sdAb is connected to an Fc portion having SEQ ID NO: 125 or having at least 80% sequence identity to SEQ ID NO: 125 using a linker portion of sequence SEQ ID NO: 88.

[0282] In another preferred embodiment, the multispecific construct comprises: a first sdAb A1 having the sequence of SEQ ID NO: 1; a second sdAb B1 having the sequence of SEQ ID NO: 9, wherein each sdAb is connected to the Fc portion of sequence SEQ ID NO: 126 using a linker portion of sequence SEQ ID NO: 88.

[0283] In another non-limiting example, a multispecific construct comprises a first sdAb comprising CDR1, CDR2, and CDR3 regions determined using the Kabat, Chothia, IMGT, or Aho numbering scheme in an amino acid sequence selected from the group consisting of: SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, and 73; a second sdAb, which is different from the first sdAb, comprising CDR1, CDR2, and CDR3 regions determined using the Kabat, Chothia, IMGT, or Aho numbering scheme in an amino acid sequence selected from the group consisting of: SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, and 73, wherein each sdAb is numbered using a sequence selected from the group consisting of: SEQ ID NOs: The linker portion selected from the group consisting of NOs: 88 to 92 and 452 to 457 is linked to the Fc portion selected from SEQ ID NOs: 125 or 126.

[0284] In a preferred embodiment, the multispecific construct comprises: a first sdAb comprising CDR1, CDR2 and CDR3 regions determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the following A1 amino acid sequence: SEQ ID NO: 1; a second sdAb comprising CDR1, CDR2 and CDR3 regions determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the following B1 amino acid sequence: SEQ ID NO: 9, wherein each sdAb is connected to an Fc portion having a sequence of SEQ ID NO: 125 using a linker portion having a sequence of SEQ ID NO: 88.

[0285] In another preferred embodiment, the multispecific construct comprises: a first sdAb comprising CDR1, CDR2 and CDR3 regions determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the following A1 amino acid sequence: SEQ ID NO: 1; a second sdAb comprising CDR1, CDR2 and CDR3 regions determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the following B1 amino acid sequence: SEQ ID NO: 9, wherein each sdAb is connected to an Fc portion having a sequence of SEQ ID NO: 126 using a linker portion having a sequence of SEQ ID NO: 88.

[0286] In a further preferred embodiment, the multispecific construct comprises: a first sdAb comprising CDR1, CDR2 and CDR3 regions determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the following A1 amino acid sequence: SEQ ID NO: 1; a second sdAb comprising CDR1, CDR2 and CDR3 regions determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the following B1 amino acid sequence: SEQ ID NO: 25, wherein each sdAb is connected to an Fc portion having a sequence of SEQ ID NO: 125 using a linker portion having a sequence of SEQ ID NO: 88.

[0287] In a further preferred embodiment, the multispecific construct comprises: a first sdAb comprising CDR1, CDR2 and CDR3 regions determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the following A1 amino acid sequence: SEQ ID NO: 1; a second sdAb comprising CDR1, CDR2 and CDR3 regions determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the following B1 amino acid sequence: SEQ ID NO: 25, wherein each sdAb is connected to an Fc portion having a sequence of SEQ ID NO: 126 using a linker portion having a sequence of SEQ ID NO: 88.

[0288] In a further preferred embodiment, the multispecific construct comprises: a first sdAb comprising CDR1, CDR2 and CDR3 regions determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the following A1 amino acid sequence: SEQ ID NO: 1; a second sdAb comprising CDR1, CDR2 and CDR3 regions determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the following B1 amino acid sequence: SEQ ID NO: 33, wherein each sdAb is connected to an Fc portion having a sequence of SEQ ID NO: 125 using a linker portion having a sequence of SEQ ID NO: 88.

[0289] In a further preferred embodiment, the multispecific construct comprises: a first sdAb comprising CDR1, CDR2 and CDR3 regions determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the following A1 amino acid sequence: SEQ ID NO: 1; a second sdAb comprising CDR1, CDR2 and CDR3 regions determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the following B1 amino acid sequence: SEQ ID NO: 33, wherein each sdAb is connected to an Fc portion having a sequence of SEQ ID NO: 126 using a linker portion having a sequence of SEQ ID NO: 88.

[0290] In a further preferred embodiment, the multispecific construct comprises: a first sdAb comprising CDR1, CDR2 and CDR3 regions determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the following A1 amino acid sequence: SEQ ID NO: 1; a second sdAb comprising CDR1, CDR2 and CDR3 regions determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the following B1 amino acid sequence: SEQ ID NO: 61, wherein each sdAb is connected to an Fc portion having a sequence of SEQ ID NO: 125 using a linker portion having a sequence of SEQ ID NO: 88.

[0291] In a further preferred embodiment, the multispecific construct comprises: a first sdAb comprising CDR1, CDR2 and CDR3 regions determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the following A1 amino acid sequence: SEQ ID NO: 1; a second sdAb comprising CDR1, CDR2 and CDR3 regions determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the following B1 amino acid sequence: SEQ ID NO: 61, wherein each sdAb is connected to an Fc portion having a sequence of SEQ ID NO: 126 using a linker portion having a sequence of SEQ ID NO: 88.

[0292]

Knob into Hole Fc-Fusion

[0293] In some embodiments, the multispecific construct is expressed as a Knob into Hole (KIH) Fc-fusion. Thus, the multispecific constructs disclosed herein, wherein the first and second sdAbs are fused to IgG-Fc, optionally via a linker, can be generated as a Knob into Hole (KIH) Fc fusion construct.

[0294] KIH technology involves engineering the CH3 domain to create a “knob” or “hole” on each heavy chain to promote heterodimerization and can be used to assemble multispecific constructs.

[0295] In one embodiment, the Fc domain included in the multispecific construct is derived from a human Fc domain and comprises a mutation that induces heterodimerization. In some embodiments, such mutations include mutations referred to as "Knob" and "Hole" mutations. For example, amino acid modification is performed at Thr366 in the CH3 domain, and when replaced by a larger amino acid, such as Trp (T366W), it can be preferentially paired with a second CH3 domain, wherein the amino acids at Thr366, Leu368, and Tyr407 sites are modified to smaller amino acids, such as Ser, Ala, and Val (T366S / L368A / Y407V), respectively. In some embodiments, the "Knob" Fc domain comprises mutations S354C and T366W. In some embodiments, the "Hole" Fc domain comprises mutations T349C, T366S, L368A, and Y407V. Heterodimerization can be further stabilized by CH3 modification by introducing disulfide bonds, for example, changing Ser354 to Cys (S354C) and Y349 to Cys (Y349C) on the opposite CH3 domain (Carter et al., 2001). In some embodiments, the Fc domain used for heterodimerization comprises additional mutations, such as the mutation S354C on the first member of the heterodimeric Fc pair, which forms an asymmetric disulfide bond with the corresponding mutation Y349C on the second member of the heterodimeric Fc pair. In some embodiments, one member of the heterodimeric Fc pair comprises the modification H435R or H435K to avoid protein A binding while maintaining FcRn binding. In some embodiments, one member of the heterodimeric Fc pair comprises the modified H435R or H435K, while the second member of the heterodimeric Fc pair is unmodified at H435. In various embodiments, the HoleFc domain includes the modification H435R or H435K (in some cases, referred to as "Hole-R" when modified to H435R), while the KnobFc domain does not. In some cases, the Hole-R mutation improves the purification of heterodimers relative to the possible homodimer Hole-Fc domain.

[0296] In another embodiment, the Fc polypeptide in the heterodimer includes a mutation that changes the charge polarity on the Fc dimer interface, such that co-expression of electrostatically matched Fc chains supports favorable attractive interactions, thereby promoting the formation of desirable Fc heterodimers, while unfavorable repulsive charge interactions inhibit unwanted Fc homodimer formation (Gunescaran et al., 2010). When co-expressed in cells, the chains have the potential to bind to each other, but due to charge repulsion, the chains do not substantially bind to each other. Other strategies for producing heterodimeric Fc include mixing human IgG with IgA CH3 domain fragments to produce complementary CH3 heterodimers, referred to as SEED Fc.

[0297] Heterodimerization methods and variants also include methods and variants described in published international PCT application WO2014 / 145806, including "Knob and Hole" mutations (also known as "skew" variants), mutations associated with "electrostatic manipulation" or "charge pairs," and pI variants. Heterodimer variants also include any variants described in U.S. published application numbers. US2012 / 0149876 or US2018 / 011883.

[0298] In some embodiments, to promote heterodimerization, both polypeptides of the Fc heterodimer include paired or complementary amino acid modifications. Table 7 lists exemplary paired amino acid modifications of polypeptides in Fc fusions.

[0299] [Table 7: Examples of Fc mutations in the KIH format]

[0300]

[0301]

[0302] In some embodiments, the modification comprises introducing a knob into a first Fc polypeptide and a hole into a second Fc polypeptide, such that the knob is positioned in the hole to facilitate complexation of the first and second Fc-containing polypeptides. The amino acids targeted for substitution and / or modification to create a knob or hole in a polypeptide are typically interacting interface amino acids.

[0303] In embodiments herein, a multispecific construct comprising a first and a second sdAb fused to an IgG-Fc, optionally via a linker, can be generated as a Knob into Hole (KIH) Fc fusion construct. In some embodiments, the Knob portion of the KIH comprises an IgG having SEQ ID NO: 433 or 435. 4 -Fc. In a further embodiment, or in an alternative embodiment, the Hole portion of KIH comprises an IgG having SEQ ID NO: 434 or 436 4-Fc.

[0304] Table 8 provides a summary of the full-length amino acid sequences of the two separate portions of the Knob to Hole constructs disclosed herein.

[0305]

Table 8

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312] The multispecific constructs according to the present invention can be linked by fusing any Knob portion to any Hole portion shown in Table 8.

[0313] It is obvious to a skilled person that all structures described as "Knob" in Table 8 can be made into "Hole", and all structures described as "Hole" in Table 8 can be made into "Knob".

[0314] As described above, A1 fused to the Fc-Hole moiety can bind to any sdAb fused to the Fc Knob moiety, regardless of whether the two sdAbs are fused at the N-terminus or the C-terminus. Therefore, the Hole moiety with A1 can be selected from any one of SEQ ID NOs: 93-96, 113-116, 445, 447, 449 and 451.

[0315] Likewise, when A1 is fused to the Fc Knob portion, it can bind to any sdAb fused to the Fc-Hole portion, regardless of whether the two sdAbs are fused at the N-terminus or the C-terminus. Thus, the Knob portion with A1 can be selected from any one of SEQ ID NOs: 105 to 112.

[0316] Furthermore, any of the linkers mentioned in Table 8 can be used to fused two sdAbs to the Fc-Hole or Knob portion.

[0317] Thus, in one embodiment, the multispecific construct comprises: a Hole portion having an amino acid sequence selected from the group consisting of: SEQ ID NOs: 93, 94, 95, 96, 113, 114, 115, 116, 439, 441, 443, 445, 447, 449, and 451; and a Knob portion having an amino acid sequence selected from the group consisting of: SEQ ID NOs: 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 117, 118, 119, 120, 121, 122, 123, 124, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330 ,331,332,333,334,335,336,337,338,339,340,341,342,343,344,345,346,347,348,349,350,351,352,353,354,355,356,357,358,359,360,361,362,363,364 4,365,366,367,368,369,370,371,372,373,374,375,376,377,378,379,380,381,382,383,384,385,386,387,388,389,390,391,392、393、394、395、396、397、3 98, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 438, 440, 442, 444, 446, 448 and 450.

[0318] For all embodiments described herein, and embodiments related to VHH / sdAbs incorporated into Knob and / or Hole polypeptides, the VHH / sdAbs may be amino acid substituted, for example, for affinity maturation or for humanized sequences. In addition, CDR1 may contain 1, 2 or 3 amino acid substitutions, CDR2 may contain 1, 2 or 3 amino acid substitutions, and CDR3 may contain 1, 2 or 3 amino acid substitutions, for example, 1 or 2 amino acid substitutions. The resulting variant sdAbs may be evaluated by determining the binding affinity to IgE Fc or by determining the substitution activity by an ELISA-based IgE-FcεRIα substitution assay. An ideal variant is one that has comparable activity to the parent sdAb or has improved IgE affinity or improved substitution activity.

[0319] According to the present invention, it is preferred to combine the A1 Fc-Hole portion (with A1 us fused to the C-terminus) with any other sdAb, also fused to the C-terminus.

[0320] Thus, in a preferred embodiment, the multispecific construct comprises: a Hole portion having an amino acid sequence selected from the group consisting of: SEQ ID NOs: 93, 94, 95, 96, 439, 441, 443, 445, 447, 449 and 451, and a Knob portion having an amino acid sequence selected from the group consisting of: SEQ ID NOs: 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 321, 322, 323, 324, 329, 330, 331, 332, 337, 338, 339, 340, 345, 346, 347, 348, 353, 354, 355, 356, 361, 362, 363, 364 4,369,370,371,372,377,378,379,380,385,386,387,388,393,394,395,396,401,402,403,404,409,410,411,412,417,418,419,420,438,440,442,444,446,448 and 450.

[0321] Similar to above, preferably A1, N-terminally fused to the Fc-Hole moiety and any Stab, N-terminally fused to the FcKnob moiety.

[0322] Thus, in another preferred embodiment, the multispecific construct comprises: a Hole portion having an amino acid sequence selected from the group consisting of: SEQ ID NO: 113, 114, 115 and 116, and a Knob portion having an amino acid sequence selected from the group consisting of: 109, 110, 111, 112, 117, 118, 119, 120, 121, 122, 123, 124, 325, 326, 327, 328, 333, 334, 335, 336, 341, 342, 343, 344, 349, 350, 351, 352, 353, 354, 355 2, 357, 358, 359, 360, 365, 366, 367, 368, 373, 374, 375, 376, 381, 382, ​​383, 384, 389, 390, 391, 392, 397, 398, 399, 400, 405, 406, 407, 408, 413, 414, 415, 416, 421, 422, 423 and 424.

[0323] In addition, since different linkers can be used to connect sdAbs to the Fc Knob or Hole part, it is best to fuse the C-termini of both sdAbs to the Fc part using the same linker length.

[0324] Thus, in yet another embodiment, a multispecific construct according to the invention relates to a combination of a Hole portion and a Knob portion, each of which has SEQ ID NOs: 93 and 97; 94 and 98; 95 and 99; 96 and 100; 93 and 101; 94 and 102; 95 and 103; 96 and 104; 93 and 105; 94 and 106; 95 and 107; 96 and 108; 93 and 321; 94 and 322; 95 and 323; 96 and 324; 93 and 329; 94 and 330; 95 and 331; 96 and 332; 93 and 337; 94 and 338; 95 and 339; 96 and 340; 93 and 345; 94 and 346; 95 and 347; 96 and 348; 93 and 353; 94 and 354; 95 and 355; 96 and 356; 93 and 361; 94 and 362; 95 and 363; 96 and 364; 93 and 369; 94 and 370; 95 and 371; 96 and 372; 93 and 377; 94 and 378; 95 and 379; 96 and 380; 93 and 385; 94 and 386; 95 and 387; 96 and 388; 93 and 393; 94 and 394; 95 and 395; 96 and 396; 93 and 401; 94 and 402; 95 and 403; 96 and 404; 93 and 409; 94 and 410; 95 and 411; 96 and 412; 93 and 417; 94 and 418; 95 and 419; 96 and 420; 439 and 438; 441 and 440; 443 and 442; 445 and 444; 447 and 446; 449 and 448, or 451 and 450.

[0325] According to the above situation, different linkers can be used to connect the sdAb to the Fc Knob or Hole part. Preferably, the two sdAbs are fused to the Fc part at the N-terminus using the same linker length.

[0326] In a further embodiment, the multispecific construct according to the invention relates to a combination of a Hole portion and a Knob portion, each of which has SEQ ID NOs: 113 and 109; 114 and 110; 115 and 111; 116 and 112; 113 and 117; 114 and 118; 115 and 119; 116 and 120; 113 and 121; 114 and 122; 115 and 123; 116 and 124; 113 and 325; 114 and 326; 115 and 327; 116 and 328; 8; 113 and 333; 114 and 334; 115 and 335; 116 and 336; 113 and 341; 114 and 342; 115 and 343; 116 and 34 4; 113 and 349; 114 and 350; 115 and 351; 116 and 352; 113 and 357; 114 and 358; 115 and 359; 116 and 360 ;113 and 365; 114 and 366; 115 and 367; 116 and 368; 113 and 373; 114 and 374; 115 and 375; 116 and 376 ;113 and 381; 114 and 382; 115 and 383; 116 and 384; 113 and 389; 114 and 390; 115 and 391; 116 and 392; 113 and 397; 114 and 398; 115 and 399; 116 and 400; 113 and 405; 114 and 406; 115 and 407; 116 and 408; 113 and 413; 114 and 414; 115 and 415; 116 and 416; 113 and 421; 114 and 422; 115 and 423; or 116 and 424.

[0327] The multispecific construct comprising sdAbs B1 and A1 indeed showed very high displacement activity when tested in Example 6. Therefore, it is best to combine the A1 Hole part with the B1 Knob part, fused N-terminally to the Fc part.

[0328] Therefore, in a preferred embodiment, the multispecific construct according to the present invention involves a combination of a Hole part and a Knob part, wherein the Hole part and the Knob part each have SEQ ID NO: 113 and 121; 114 and 122; 115 and 123; or 116 and 124.

[0329] Similar to the above, the fusion of B1 and A1 C-termini with Fc also showed strong replacement activity.

[0330] Therefore, in a more preferred embodiment, the multispecific construct according to the present invention involves a combination of a Hole part and a Knob part, wherein the Hole part and the Knob part each have SEQ ID NO: 93 and 101; 94 and 102; 95 and 103; or 96 and 104.

[0331] As shown in Example 10, some constructs may be allergenic. Multispecific constructs are preferably non-allergenic. Therefore, as shown in Example 10, a construct comprising B1, A1 is used (G 4 S) 1 Linker and IgG 4 -Fc-linked, no allergens were shown.

[0332] Therefore, in a preferred embodiment, the multispecific construct according to the present invention involves a combination of a Hole part and a Knob part, said Hole part and Knob part having SEQ ID NO: 94 and 102, respectively.

[0333] Furthermore, when evaluated in mice, in Example 9, A1 conjugated to D2, E1 or F4 did not induce an allergic response in mice compared to B1A1 N-terminus linked to the Fc portion, meaning that these constructs are not allergenic.

[0334] Thus, in one embodiment, a multispecific construct according to the invention involves a combination of a Hole portion and a Knob portion, wherein the Hole portion and the Knob portion have SEQ ID NOs: 95 and 339, respectively.

[0335] In another embodiment, a multispecific construct according to the present invention involves a combination of a Hole portion and a Knob portion, wherein the Hole portion and the Knob portion have SEQ ID NOs: 95 and 347, respectively.

[0336] In a further embodiment, the multispecific construct according to the invention involves a combination of a Hole portion and a Knob portion, the Hole portion and the Knob portion having SEQ ID NO: 95 and 403, respectively.

[0337] In the above structures, A1 represents one of the two sdAbs in all examples.

[0338] Another non-limiting example is where A2 represents one of the two sdAbs and the sdAb listed in Table 1 represents the second sdAb. Thus, in one embodiment, the multispecific construct comprises: a Hole portion having an amino acid sequence selected from the group consisting of: SEQ ID NOs: 425, 426, 427, 428, 429, 430, 431 and 432; and a Knob portion having an amino acid sequence selected from the group consisting of: SEQ ID NOs: 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 117, 118, 119, 120, 121, 122, 123, 124, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330 ,331,332,333,334,335,336,337,338,339,340,341,342,343,344,345,346,347,348,349,350,351,352,353,354,355,356,357,358,359,360,361,362,363,364 4,365,366,367,368,369,370,371,372,373,374,375,376,377,378,379,380,381,382,383,384,385,386,387,388,389,390,391、392、393、394、395、396、397、3 98, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 438, 440, 442, 444, 446, 448 and 450.

[0339] The above list represents the Knob and Hole parts, where the sdAb is either N-terminally or C-terminally fused to the Fc part.

[0340] It is preferred that the Knob and Hole parts are fused to the same terminus, meaning both are N-terminal or C-terminal.

[0341] Thus, in one embodiment, the multispecific construct comprises: a Hole portion having an amino acid sequence selected from the group consisting of: SEQ ID NOs: 425, 426, 427, and 428; and a Knob portion having an amino acid sequence selected from the group consisting of: SEQ ID NOs: 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 321, 322, 323, 324, 329, 330, 331, 332, 337, 338, 339, 340, 345, 346, 347, 348, 353, 354, 355, 356, 361, 362, 363, 364, 4, 369, 370, 371, 372, 377, 378, 379, 380, 385, 386, 387, 388, 393, 394, 395, 396, 401, 402, 403, 404, 409, 410, 411, 412, 417, 418, 419, 420, 438, 440, 442, 444, 446, 448 and 450.

[0342] In a further embodiment, the multispecific construct comprises: a Hole portion having an amino acid sequence selected from the group consisting of SEQ ID NOs: 429, 430, 431 and 432; and a Knob portion having an amino acid sequence selected from the group consisting of SEQ ID NOs: 109, 110, 111, 112, 117, 118, 119, 120, 121, 122, 123, 124, 325, 326, 327, 328, 333, 334, 335, 336, 341, 342, 343, 344, 349, 350, 351, 352, 357, 358, 359, 360 0, 365, 366, 367, 368, 373, 374, 375, 376, 381, 382, ​​383, 384, 389, 390, 391, 392, 397, 398, 399, 400, 405, 406, 407, 408, 413, 414, 415, 416, 421, 422, 423, and 424.

[0343] In addition, the Knob and Hole parts contain different linkers for fusing the sdAb to the Fc part as shown in Table 8. Preferably, the sdAb fused to the Hole and Knob parts is fused to the Fc part using the same linker.

[0344] Thus, in another embodiment, a multispecific construct according to the invention relates to a combination of a Hole portion and a Knob portion, wherein the Hole portion and the Knob portion each have SEQ ID NOs: 425 and 97; 426 and 98; 427 and 99; 428 and 100; 425 and 101; 426 and 102; 427 and 103; 428 and 104; 425 and 105; 426 and 106; 427 and 107; 428 and 108; 425 and 321, 426 and 322, 427 and 323, 428 and 324, 425 and 329, 426 and 330, 427 and 331, 428 and 332, 425 and 337, 426 and 338, 427 and 339, 428 and 340, 425 and 345, 426 and 346, 427 and 347, 428 and 348, 425 and 353, 426 and 354, 427 and 355, 428 and 356, 425 and 361, 426 and 362, 427 and 363, 428 and 364, 425 and 369, 426 and 370, 427 and 371, 428 and 372, 425 and 377, 426 and 378, 427 and 379, 428 and 380, 425 and 385, 426 and 386, 427 and 387, 428 and 388, 425 and 393, 426 and 394, 427 and 395, 428 and 396, 425 and 401, 426 and 402, 427 and 403, 428 and 404, 425 and 409, 426 and 410, 427 and 411, 428 and 412, 425 and 417, 426 and 418, 427 and 419 or 428 and 420.

[0345] In a further embodiment, the multispecific construct according to the invention relates to a combination of a Hole portion and a Knob portion, wherein the Hole portion and the Knob portion each have SEQ ID NOs: 429 and 109; 430 and 110; 431 and 111; 432 and 112; 429 and 117; 430 and 118; 431 and 119; 432 and 120; 429 and 121; 430 and 122; 431 and 123; 432 and 124; 429 and 325, 430 and 326, 431 and 327, 432 and 328; 28, 429 and 333, 430 and 334, 431 and 335, 432 and 336, 429 and 341, 430 and 342, 431 and 343, 32 and 34 4, 429 and 349, 430 and 350, 431 and 351, 432 and 352, 429 and 357, 430 and 358, 431 and 359, 432 and 360 , 429 and 365, 430 and 366, 431 and 367, 432 and 368, 429 and 373, 430 and 374, 431 and 375, 432 and 376 , 429 and 381, 430 and 382, ​​431 and 383, 432 and 384, 429 and 389, 430 and 390, 431 and 391, 432 and 392 , 429 and 397, 430 and 398, 431 and 399, 432 and 400, 429 and 405, 430 and 406, 431 and 407, 432 and 408 , 429 and 413, 430 and 414, 431 and 415, 432 and 416, 429 and 421, 430 and 422, 431 and 423 or 432 and 424.

[0346] In a preferred embodiment, the multispecific construct according to the present invention involves a combination of a Hole portion and a Knob portion, wherein the Hole portion and the Knob portion have SEQ ID NOs: 429 and 121; 430 and 122; 431 and 123; or 432 and 124, respectively.

[0347] In a more preferred embodiment, the multispecific construct according to the present invention involves a combination of a Hole portion and a Knob portion, wherein the Hole portion and the Knob portion have SEQ ID NO: 425 and 101; 426 and 102; 427 and 103; or 428 and 104, respectively.

[0348] In a more preferred embodiment, the multispecific construct according to the present invention involves a combination of a Hole portion and a Knob portion, wherein the Hole portion and the Knob portion have SEQ ID NOs: 426 and 102, respectively.

[0349] Until now, A1 or A2 have been disruptive in themselves and have been part of multispecific constructs as the Hole moiety. Importantly, this construct can be designed as the first sdAb that has disruptive activity in itself and is a Knob moiety.

[0350] Therefore, in one embodiment, the multispecific construct comprises a Knob portion, whose amino acid sequence is selected from the group consisting of: SEQ ID NO: 105, 106, 107, 108; and a Hole portion, whose amino acid sequence is: SEQ ID NO: 437.

[0351] Preferably, the multispecific construct according to the invention has a combination of a Hole part and a Knob part having SEQ ID NO: 437 and 106, respectively.

[0352] In addition, multispecific constructs can be assembled without a linker between the sdAb and Fc parts, or by using linkers GS, G 2 S.G. 3 S, EAAAK or (EAAAK) 2 Assemble.

[0353] Therefore, in one embodiment, the multispecific construct comprises a Knob portion whose amino acid sequence is selected from the group consisting of: SEQ ID NO: 438, 440, 442, 444, 446, 448 and 450; and a Hole portion whose amino acid sequence is selected from the group consisting of: SEQ ID NO: 439, 441, 443, 445, 447, 449 and 451.

[0354] In a preferred embodiment, the multispecific construct according to the invention is associated with a combination of a Hole part and a Knob part having SEQ ID NOs: 439 and 438, respectively.

[0355] In another preferred embodiment, the multispecific construct according to the invention is associated with a combination of a Hole part and a Knob part having SEQ ID NOs: 441 and 440, respectively.

[0356] In a further preferred embodiment, the multispecific construct according to the invention is associated with a combination of a Hole part and a Knob part having SEQ ID NO: 443 and 442, respectively.

[0357] In yet another preferred embodiment, the multispecific construct according to the invention is associated with a combination of a Hole part and a Knob part having SEQ ID NOs: 445 and 444, respectively.

[0358] In yet another preferred embodiment, the multispecific construct according to the invention is associated with a combination of a Hole part and a Knob part having SEQ ID NO: 447 and 446, respectively.

[0359] In a more preferred embodiment, the multispecific construct according to the invention is associated with a combination of a Hole part and a Knob part having SEQ ID NO: 449 and 448, respectively.

[0360] In a more preferred embodiment, the multispecific construct according to the invention is associated with a combination of a Hole part and a Knob part having SEQ ID NOs: 451 and 450, respectively.

[0361] The Knob portion of the multispecific construct consists of three parts:

[0362] (i) an sdAb,

[0363] (ii) a peptide linker, and

[0364] (iii) an Fc portion, wherein the Fc portion is linked to the C-terminus or N-terminus of (ii).

[0365] wherein (i) is connected to the C-terminus or N-terminus of (iii) via (ii).

[0366] Furthermore, the Hole portion of the multispecific construct contains 3 parts;

[0367] (i) an sdAb that is different from the sdAb on the knob,

[0368] (ii) a peptide linker, and

[0369] (iii) an Fc portion,

[0370] wherein (i) is linked to the C-terminus or N-terminus of (iii) via (ii), and

[0371] The Fc portion is connected to other portions in the C-terminus and / or N-terminus.

[0372] The three parts (i), (ii) and (iii) of the Knob are gathered in one polypeptide, while the three parts of the Hole are gathered in another polypeptide. In addition, for the Knob part and the Hole part, (i) is fused with (ii); (ii) is fused with (iii).

[0373] In one embodiment, the Knob portion of the multispecific construct comprises three portions, (i), (ii) and (iii), wherein (i) is selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 33, 37, 41, 45, 49, 53, 57, 61 and 65, (ii) is selected from the group consisting of SEQ ID NOs: 88 to 92 and 452 to 457, and (iii) is selected from SEQ ID NOs: 433 and 435; and the Hole portion of the multispecific construct comprises three portions, (i), (ii) and (iii), wherein (i) is different from (i) in the Knob portion and is selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 33, 37, 41, 45, 49, 53, 57, 61 and 65, (ii) is selected from the group consisting of SEQ ID NOs: 88 to 92 and 452 to 457, and (iii) is selected from SEQ ID NOs: 433 and 435. The invention relates to a method for preparing the present invention which is preferably selected from the group consisting of SEQ ID NOs: 88-92 and 452-457, and (iii) selected from SEQ ID NOs: 434 and 436.

[0374] More specific examples related to KIH produced with sdAbs A1 and B1 are defined below.

[0375] In a preferred embodiment, the Knob portion of the multispecific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 1, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 433, and the Hole portion of the multispecific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 9, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 434.

[0376] In another preferred embodiment, the Knob part of the multispecific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 9, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 433, and the Hole part of the multispecific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 1, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 434.

[0377] In a further preferred embodiment, the Knob part of the multispecific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 1, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 435; the Hole part of the multispecific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 9, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 436.

[0378] In another preferred embodiment, the Knob part of the multispecific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 9, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 435; the Hole part of the multispecific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 1, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 436.

[0379] In a preferred embodiment, the Knob portion of the multispecific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 1, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 433, wherein (ii) is connected to the C-terminus of (iii); the Hole portion of the multispecific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 9, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 434, wherein (ii) is connected to the C-terminus of (iii).

[0380] In another preferred embodiment, the Knob part of the multi-specific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 9, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 433, wherein (ii) is connected to the C-terminus of (iii); the Hole part of the multi-specific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 1, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 434, wherein (ii) is connected to the C-terminus of (iii).

[0381] In a further preferred embodiment, the Knob part of the multi-specific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 1, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 435, wherein (ii) is connected to the C-terminus of (iii); the Hole part of the multi-specific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 9, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 436, wherein (ii) is connected to the C-terminus of (iii).

[0382] In a further preferred embodiment, the Knob part of the multi-specific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 9, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 435, wherein (ii) is connected to the C-terminus of (iii); the Hole part of the multi-specific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 1, (ii) is SEQ ID NO: 88, (iii) is SEQ ID NO: 436, wherein (ii) is connected to the C-terminus of (iii).

[0383] In a preferred embodiment, the Knob portion of the multispecific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 5, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 433; the Hole portion of the multispecific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 9, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 434.

[0384] In another preferred embodiment, the Knob part of the multispecific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 9, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 433; the Hole part of the multispecific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 5, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 434.

[0385] In a further preferred embodiment, the Knob part of the multispecific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 5, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 435; the Hole part of the multispecific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 9, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 436.

[0386] In a further preferred embodiment, the Knob part of the multi-specific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 9, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 435; the Hole part of the multi-specific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 5, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 436.

[0387] In a preferred embodiment, the Knob portion of the multi-specific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 5, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 433, wherein (ii) is connected to the C-terminus of (iii); the Hole portion of the multi-specific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 9, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 434, wherein (ii) is connected to the C-terminus of (iii).

[0388] In another preferred embodiment, the Knob part of the multi-specific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 9, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 433, wherein (ii) is connected to the C-terminus of (iii); the Hole part of the multi-specific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 5, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 434, wherein (ii) is connected to the C-terminus of (iii).

[0389] In a further preferred embodiment, the Knob part of the multi-specific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 5, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 435, wherein (ii) is connected to the C-terminus of (iii); the Hole part of the multi-specific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 9, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 436, wherein (ii) is connected to the C-terminus of (iii).

[0390] In a further preferred embodiment, the Knob part of the multi-specific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 9, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 435, wherein (ii) is connected to the C-terminus of (iii); the Hole part of the multi-specific construct comprises three parts, (i), (ii) and (iii), wherein (i) is SEQ ID NO: 5, (ii) is SEQ ID NO: 88, and (iii) is SEQ ID NO: 436, wherein (ii) is connected to the C-terminus of (iii).

[0391] [Other linking parts (polymers, polysaccharides)]

[0392] In some embodiments, group c) of the multispecific construct is a polymer, in particular composed of several repeating units of the monomer ethylene glycol (C 2 H 6 O 2 ). These polymers, called polyethylene glycol (PEG), are synthetically produced and can be linear or branched, with terminal groups being either standard hydroxyl or methoxy (denoted as mPEG). The conjugation of PEG moieties to peptides and proteins is a well-established and effective method for improving their pharmacokinetic properties, such as plasma / serum half-life. PEGylation can also alter the hydrophilicity of a peptide or protein and may render the PEGylated monomeric sdAb or the multispecific constructs described herein less susceptible to renal clearance and protease degradation, and may also reduce immunogenicity.

[0393] Other polymers of interest may include poly(N-vinylpyrrolidone) (PVP), polyglycerol (PG), poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA), polyoxazoline (POZ), poly[oligo(ethylene glycol)methyl methacrylate] (POEGMA), and poly(2-methacryloyloxyethylphosphorylcholine) (PMPC).

[0394] In still other embodiments, portion (c) of the multispecific construct is a polysaccharide, such as natural and semisynthetic polysaccharides, including O- and N-linked oligosaccharides, dextran, hydroxyethyl starch (HES), polysialic acid and hyaluronic acid, and unstructured protein polymers, such as isotype amino acid polymers, elastin-like polypeptides, XTENs (a class of unstructured biodegradable protein polymers developed by Amunix for increasing the half-life of therapeutic peptides and proteins for gene fusion or chemical coupling. XTEN polymers are considered non-immunogenic polypeptides composed of six hydrophilic, chemically stable amino acids A, E, G, P, S and T) and PAS (a proline-alanine-serine polymer).

[0395] [Drug composition]

[0396] In a further aspect, the present invention relates to a pharmaceutical composition comprising a multispecific construct or one or more monomeric sdAbs disclosed herein, and a pharmaceutically acceptable carrier and / or vehicle and / or diluent and / or excipient.

[0397] The pharmaceutical composition may be in liquid, semi-solid or solid dosage form (eg lyophilisate). The composition / dosage form may be formulated for various routes of administration, preferably for administration by injection, such as intramuscular injection.

[0398]

Used as a therapeutic agent

[0399] The multispecific constructs, one or more monomeric sdAbs, or pharmaceutical compositions disclosed herein can be used to displace IgE pre-bound to its high affinity receptor in a subject in need thereof.

[0400] Therefore, further aspects relate to methods for treating or preventing IgE-related diseases or conditions, the methods comprising administering an effective dose of a multispecific construct, one or more monomeric sdAbs, or a pharmaceutical composition disclosed herein. The effective dose is expected to be a single dose in the range of 10 to 300 mg, which can be administered once daily, weekly, biweekly, or monthly.

[0401] In other words, further aspects relate to the multispecific construct, one or more monomeric sdAbs, or a pharmaceutical composition for use as a medicament, preferably for use in anti-IgE therapy, such as for the treatment or prevention of an IgE related disease or disorder.

[0402] Still further aspects of the permuted wording relate to the multispecific construct, one or more monomeric sdAbs, or a pharmaceutical composition in the manufacture of a medicament, such as a medicament for anti-IgE therapy or for the treatment or prevention of an IgE-related disease or disorder.

[0403] In an interesting embodiment, the IgE-related disease or disorder is selected from any one example of an allergic disease, which is allergic asthma, allergic rhinitis (including seasonal allergic rhinitis and perennial allergic rhinitis), nasal polyps, atopic dermatitis, conjunctivitis, anaphylaxis, croup, food allergies, allergies to non-food related substances, including venom from insects, wasps, bees or spiders, and therapeutic drugs, including antibiotics.

[0404] Other IgE-related diseases suitable for treatment include other hyper-IgE syndromes, allergic bronchopulmonary aspergillosis and other aspergillosis-related conditions, idiopathic allergic reactions, anaphylaxis, bullous pemphigoid, pemphigus vulgaris, urticaria, such as chronic urticaria, chronic spontaneous urticaria, nasal polyposis, chronic sinusitis, mastocytosis and other mast cell diseases.

[0405] There are also some IgE-related diseases that may be treated by the constructs disclosed herein that may be the same as those proposed for Omalizumab (Incorvaia et al., 2014).

[0406] The multispecific construct, one or more monomeric sdAbs or the pharmaceutical composition can be administered to a subject in need thereof, particularly a human subject. [Example]

[0407] [Example 1: Immunization and library generation]

[0408] The aim of this study was to generate a sdAb library from camelids immunized with IgE-Fc.

[0409] Materials and methods

[0410]

Animal Immunization

[0411] Single domain antibodies were produced by immunizing camelids. Immunizations were performed by different companies (Capralogics (Hardwick, USA), Eurogentec (Seraing, Belgium) and Preclinics (Potsdam, Germany)) using llamas and alpacas. Animals were immunized with human IgE or human IgE according to the immunization schemes shown in Tables 9 to 12. IgE-Fc used Cε2-Cε4 or Cε3-Cε4. The protein was mixed with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) before injection.

[0412] Table 9: Immunization schedule and tissue collection of llama RAY

[0413] day Antigen type Antigen amount Tissue collection 0 IgE 100 μg 15–20 mL of blood 21 IgE 100 μg 42 IgE 100 μg 52 25mL 63 IgE 100 μg 73 500mL

[0414] Table 10: Immunization schedule and tissue collection of llama TOM

[0415] day Antigen type Antigen amount Tissue collection 0 IgE-Fc Cε2-Cε4 100 μg 15–20 mL of blood 21 IgE-Fc Cε2-Cε4 100 μg 42 IgE-Fc Cε2-Cε4 100 μg 52 25mL 63 IgE-Fc Cε2-Cε4 100 μg 73 500mL

[0416] Table 11: Immunization schedule and tissue collection for llama MAX and alpaca ZOE

[0417] day Antigen type Antigen amount Tissue collection 0 IgE-Fc Cε2-Cε4 100 μg 3–4 mL of blood 14 IgE-Fc Cε2-Cε4 100 μg 28 IgE-Fc Cε2-Cε4 100 μg 3–4 mL of blood 35 IgE-Fc Cε2-Cε4 100 μg 43 300mL

[0418]

Library Generation

[0419] By using PBMCs were purified from blood isolated from animals using a Cytiva gradient and sdAb libraries were constructed from each animal. RNA was extracted from purified PBMC using RNA kit (Macherey-Nagel). cDNA was synthesized from RNA using Superscript III First-Strand kit (Invitrogen). sdAb genes were amplified from cDNA by PCR using primers 1 and 2 (IgG2) and primers 1 and 3 (IgG3). Using the amplified sdAb genes, the library was constructed into a yeast display plasmid called pNT by homologous recombination in Saccharomyces cerevisiae EBY100 (ATCC).

[0420] The library was generated using the following primers:

[0421] Primer 1:

[0422]

[0423] Primer 2:

[0424]

[0425] Primer 3:

[0426]

[0427]

result

[0428] For each immunized animal, >10 7 Yeast libraries from 10 transformed cells were obtained. Quality control of the libraries by sequencing colony PCR showed that all libraries contained 50-90% of the sdAbs in the correct reading frame.

[0429] [Example 2: Selection of sdAbs that bind to IgE-Fc]

[0430] The aim of this study was to select sdAbs that bind to IgE-Fc.

[0431] Materials and methods

[0432] In order to select sdAbs that can bind to IgE-Fc, the starting culture of the library obtained in Example 1 was inoculated with 10 times the library size (>10 8 The culture was cultured in 1 L of glucose minimal medium (SD-CAA) containing 10 cells and incubated overnight at 30°C and 180 rpm. The next day, the culture was passaged to an OD of 600 =0.2-0.3, and cultured at 30°C and 180 rpm until reaching the early exponential phase (OD 600 =0.8~1.0). Cells were pelleted by centrifugation and resuspended in galactose minimal medium (SG-CAA) to induce yeast cell surface display of sdAb (sdAb gene under GAL1 promoter). The culture was incubated overnight at 20°C and 180rpm. The next day, cells were pelleted by centrifugation and resuspended in PBS 5% BSA. The first round of selection of cells for their ability to bind IgE-Fc Cε2-Cε4 was performed by magnetic activated cell sorting (MACS) and / or fluorescence activated cell sorting (FACS) for biotinylated IgE-Fc Cε2-Cε4.

[0433] MACS: 200 μL of streptavidin Dynabeads were quantified using a magnet (Dynal). TM MyOne TM C1 (Invitrogen) for 2×10 8 First, negative selection was performed after positive selection with biotinylated IgE-Fc Cε2-Cε4: cells were incubated with 1 μM biotinylated IgE-Fc Cε2-Cε4 for 1 h, washed with PBS 5% BSA, and incubated with 100 μL streptavidin Dynabeads. TM MyOne TM C1 (Invitrogen) for 30 min, washed with PBS 5% BSA and sorted using a magnet (Dynal).

[0434] FACS: 3 × 10 7 Cells were incubated with 1 μM biotinylated IgE-Fc Cε2-Cε4 in PBS 1% BSA for 1 hour. Cells were washed with PBS 1% BSA and incubated with anti-HA PE / Cy7 (BioLegend) and streptavidin PerCP / Cy5.5 (BioLegend) at a ratio of 1:100 for 15 minutes. Cells were washed with PBS 1% BSA and sorted using a SH800 cell sorter (SONY).

[0435] The sorted cells from MACS and FACS were combined and cultured in SD-CAA. Subsequent rounds of selection were performed for biotinylated IgE-Fc Cε2-Cε4, biotinylated IgE-Fc Cε3-Cε4 335, or biotinylated IgE-Fc Cε2-Cε4 / FcεRIα complex, with the same settings as the first round of selection. Multiple rounds of selection were repeated with decreasing target concentrations (1000nM, 200nM, 50nM, 10nM) until one or several positive populations were visible, requiring 2 to 4 rounds of selection depending on the library.

[0436] After the selection process is completed, positive cells are cultured on SD-CAA plates and the CFU are subsequently sequenced by Sanger sequencing to identify the sdAb of interest.

[0437]

result

[0438] The immune library generated by llama RAY (Table 9) was incubated with 1 μM biotinylated IgE-Fc Cε2-Cε4 and analyzed by FACS using two parameters (sdAb expression and IgE-Fc binding) before starting the selection. Here, a large population positive for sdAb expression can be seen, and within this range, a small population positive for IgE-Fc binding can be observed ( Figure 2 A). The first round of screening was performed by MACS on 1 μM biotinylated IgE-Fc Cε2-Cε4. During the first round of selection, <1% of the cells were positively selected. The positive populations were subjected to a second round of selection by FACS on 200 nM biotinylated IgE-Fc Cε2-Cε4 or 200 nM complex biotinylated IgE-Fc Cε2-Cε4:FcεRIα. Round 2 for IgE-Fc Cε2-Cε4 showed two populations positive for sdAb expression and IgE-Fc binding. These two populations were sorted in gate C and together they accounted for 18.31% of the cells ( Figure 2 B). Cultivation from round 2 ( Figure 2 B, cells selected in gate C) and sdAbs were identified by Sanger sequencing. Ten colonies were sequenced from the population and revealed two dominant sdAbs, B1 and B3 (Table 1).

[0439] The above method was repeated for all generated libraries. Table 1 on page 15 lists the obtained sdAbs and shows the full-length amino acid sequence of each hit. Tables 2a to d show the CDRs of each hit determined by different methods. [Example 3: Expression of single domain antibodies]

[0440] The aim of this study was to express sdAb monomers and dimers using different types of linkers.

[0441] Materials and methods

[0442] The target sdAb monomers (Table 1) were amplified from the pNT plasmid by PCR and identified in Example 2 and recloned in the pET22b plasmid by SLiCE cloning reaction. Therefore, the pelB signal sequence and C-terminal His / HA tag were added to the sdAb, allowing secretion into the periplasmic space and subsequent purification and detection. The sdAb was expressed in Escherichia coli (E. coli) Rosetta (DE3) cells using 2YT medium. Expression was induced in the early exponential phase with 0.5 mM IPTG and maintained at 20°C to 25°C overnight. In the first step, the sdAb was purified from the supernatant or supernatant + periplasmic extract by IMAC using a HisTrap excel column (Cytiva), and in the second step, a HiLoad16-600 was used. SEC was performed on a 75pg (Cytiva) column. The size and purity of each sdAb were evaluated on a 15% acrylamide SDS-PAGE. The immunoreactivity of the sdAb to immobilized IgE-Fc Cε2-4 and IgE-Fc Cε3-4 was then analyzed by ELISA. IgE-Fc was coated overnight in Nunc MaxiSorp plates (ThermoFisher) at a concentration of 2μg / mL. After blocking, sdAb diluted 1:10 in TBS was added and incubated for 3 hours at room temperature. Mouse anti-HA antibody (ThermoFisher) and conjugated anti-mouse IgG AP (Sigma Aldrich) were used for detection.

[0443] After expression, relative epitope mapping (binding) of each individual sdAb monomer relative to different sdAbs was performed by using the Octet RED96e system (FortéBIO). Here, biotinylated IgE Fc (manufactured by ALK in Denmark) was immobilized on a SAX (high-precision streptavidin) sensor (FortéBIO) to a maximum of 1 nm using a kinetic buffer (PBS, 0.02% Tween20, 0.1% BSA) as a diluent. The first sdAb (100nM) was then incubated with the IgE Fc-loaded streptavidin sensor for 300 seconds, followed by the second sdAb (100nM) incubated with the sensor for 300 seconds. The method was performed for all sdAb combinations. The data was analyzed and a matrix was generated in the Octet Data Analysis HT 11.1 software (FortéBIO) using the epitope sorting function. [Constructs containing two sdAbs connected by a peptide linker]

[0444] Constructs containing two sdAbs were made with sdAbs that were able to bind to IgE-Fc simultaneously as detected by the relative epitope mapping above.

[0445] A construct containing two sdAb ("dimer") genes was constructed by PCR. First, a construct containing (G 4 S) 4 Each ssdAb was amplified with primers for the adapter. The sdAbs were then assembled by a second PCR to form "dimers" before cloning. Expression and purification were performed as described for sdAb monomers. Purification of "dimers" was performed as described for monomers.

[0446] Construct containing two sdAbs linked by IgG-Fc

[0447] Another approach to combining two different sdAbs is through Fc fusion ( Figure 1 ) Here, IgG 4 -Fc as an example. 4 -Fc-fused sdAb heterodimerization is through IgG 4 - "Knob into Hole" mutation of the Fc domain (Carter et al., 1998) and peptide linkers, such as sdAb and IgG 4 -Fc between RS or (G 4 S) n Connector( Figure 1 ). Encoding the first sdAb fused to the "Knob" IgG 4 -Fc (mutated in S354C or T366W) ( Figure 1 A) and a second sdAb with a "Hole" IgG4 Fc (mutated in T349C, T366S, L368A, or Y407V) ( Figure 1 B) plasmid was co-transfected in human embryonic kidney cells (Expi293). After 6 days, the expression supernatant was collected, centrifuged and filtered. The bispecific IgG4 antibody construct was purified using a Protein A column. After the supernatant, the column was washed with 10CV of 20mM sodium phosphate buffer pH 7, and the bound protein was eluted with 0.1M citric acid pH 3. The eluted fraction was immediately neutralized with 1M Tris-HCl pH 9. In addition, the eluted fraction was applied to a size exclusion chromatography (SEC) column with PBS as an electrophoresis buffer. The eluted fractions were combined, concentrated and sterile filtered through a 0.2 μm syringe filter.

[0448]

result

[0449] Selected monomeric sdAbs (Table 1) and A1 (control sdAb 026) were cloned in pET22b plasmid. All genes were expressed by E. coli Rosetta (DE3) with different expression yields depending on the sdAb (1-10 mg / L culture). The sdAb size and purity were controlled on 15% acrylamide SDS-PAGE and Coomassie blue staining (data not shown). As shown by monomeric sdAb B1, the size of the target compound was within the expected range of 15 kDa to 20 kDa (data not shown). After the first step of purification, additional compounds or aggregates of approximately 130 kDa can be seen above the sdAb band. The second step of purification can remove these contaminants and show highly pure monomers (data not shown).

[0450] The binding of sdAb monomers to IgE-Fc was verified by ELISA. All sdAbs showed immunoreactivity to IgE-Fc Cε2-4 (Table 13). However, only 4 monomeric sdAbs (A1, D3, F6 and G1) showed immunoreactivity to IgE-Fc Cε3-4 (Table 13). This indicates that most selected sdAbs bind to IgE-Fc via the Cε2 domain.

[0451] [Table 13: ELISA evaluation of sdAb binding to IgE-Fc Cε2-4 and IgE-Fc Cε3-4]

[0452] sdAb IgE-Fc Cε2-4 IgE-Fc Cε3-4 A1 yes yes B1 yes no B2 yes no B3 yes no D1 yes no D2 yes no D3 yes yes E1 yes no E2 yes no E3 yes no E4 yes no F1 yes no F3 yes no F4 yes no F5 yes no F6 yes yes G1 yes yes

[0453] Relative epitope mapping of the expressed sdAbs showed that most monomeric sdAbs bound to IgE antibodies simultaneously with A1, thereby binding to another IgE-binding epitope other than A1 ( Figure 3 ). Here, sdAbs B3, B1, D1, and D2 share a similar epitope that is different from the epitope of B2 or A1. This sdAb can be used in combination with A1, A2, or other sdAbs with different binding sites at the IgE site to change the overall anti-IgE activity of one sdAb. For example, to improve the dissociation of receptor-bound IgE.

[0454] Epitope mapping was used to determine sdAb combinations for dimerization, both with and without IgG4 fusion.

[0455] cloned and expressed by (G 4 S) 4 Various examples of linker-fused sdAb "dimers" (Table 3). 4 S) 4) as an example. After Coomassie blue staining, the elution of the first purification showed a visible transparent band between 35 kDa and 45 kDa of the expected size on SDS-PAGE. However, many contaminant compounds of different sizes were also shown. The second step of purification allows the removal of these contaminants and improves the purity of the sdAb "dimer" (data not shown).

[0456] For IgG 4 -Fc-fused sdAb heterodimerization, Table 8 shows the "dimer" (sdAb, IgG 4 -Other examples of combinations of Fc and linkers).

[0457]

in conclusion

[0458] Expression and purification of monomeric and dimeric single domain antibodies and IgG 4 -Fc fused sdAb, showing high purity and expected size in SDS-PAGE analysis.

[0459] [Example 4: Single domain antibody mutation]

[0460] The aim of this study was to investigate the effect of single point mutations on sdAb A1 to generate mutants with improved displacement activity.

[0461] Materials and methods

[0462] Starting from A1 (SEQ ID NO: 1), two libraries were constructed. Library 1 covers amino acids 11 to 61 of A1, and library 2 covers amino acids 62 to 112. Each library was generated by SOE PCR, using an oligonucleotide pool from Integrated DNA Technologies to introduce an NNK codon at each position. The resulting DNA libraries contain all possible single amino acid mutants at positions 11 to 61 of library 1 and 62 to 112 of library 2. To evaluate the impact of these mutations, the libraries were cloned into a yeast display plasmid named pNT by homologous recombination in Saccharomyces cerevisiae EBY100 (ATCC) following the procedure described by (Benatuil et al., 2010).

[0463] Each library was cultured, induced for expression and stained using three color staining as described in Example 2. The concentrations corresponding to the K on yeast of the sdAb were analyzed by FACS as described in Example 2. D A single round of selection (determined by titration) was performed with biotinylated IgE: FcεRIα complexes. Two populations were selected: a negative population (anti-HA+, streptavidin-) and a positive population (anti-HA+, streptavidin+, anti-FcεRIα-).

[0464] The selected population was cultured in SD-CAA medium at 30°C. Plasmids were extracted from the unselected library, negative population, and positive population using the Zymoprep yeast plasmid miniprep II kit (Zymo Research). The sdAb gene of the extracted plasmid was amplified by PCR. The purified PCR product was sent to Deeptope (France) for deep sequencing using Illumina technology and data analysis.

[0465] The mutations were inserted into A1 by site-directed mutagenesis PCR directly on pET22b containing the A1 gene. The A1 triple mutant was expressed and purified as previously described in Example 2.

[0466]

result

[0467] sdAb A1 displayed on yeast shows the K of biotinylated IgE:FcεRIα complex D is 0.345nM (data not shown). Therefore, the A1 DMS library was selected at this concentration to monitor the effect of mutations on binding. A negative sdAb group expressed on yeast but not binding to biotinylated IgE was selected (data not shown). A positive sdAb group expressed on yeast, binding to biotinylated IgE, but negative for FcεRIα was selected (data not shown). The sdAbs of the pre-selection library, negative group, and positive group were sequenced using deep sequencing, and the frequency of each nanobody in the library before and after selection was determined. Therefore, the effect of each single mutation on A1 can be evaluated by calculating the enrichment score (enrichment = frequency of mutants after selection / frequency before selection) ( Figure 4 ). Mutations with high enrichment scores in the negative population are detrimental to the activity of A1 and are likely to be depleted in the positive population, such as mutation P45K. Mutations with high enrichment scores in the positive population and depleted in the negative population (such as mutation V93E) will increase the affinity of A1 for IgE-Fc.

[0468] Using the deep mutational scanning data, three mutations (T28R, P45Y and V93E) that were enriched in the positive population and depleted in the negative population were selected to construct a mutant version of sdAb A1, named A2. A2 was expressed, purified and its binding to IgE-Fc was confirmed as previously described in Example 2.

[0469] Other mutations were also enriched in the positive population and therefore, like the three selected mutations above, would increase the affinity of A1 for IgE-Fc. The following mutations can be used alone or in combination to construct mutant versions of A1: L11F, L11K, V12F, V12Y, V12G, Q13V, P14M, P14E, P14R, R19K, A23K, A23R, S25K, S25R, G26K, G26R, T28K, T28R, F29Y, G30F, G30W, G30Y, G30D, G30H, G30K, G30K, G30R, K43R, P45W, P45Y, F68W, T69W, T69G, T69N, T69R, I70V, S71N, D73F, D73W, D73Y, D73M, D 73I, D73L, D73V, D73A, D73G, D73S, D73T, D73N, D73Q, D73E, D73H, D73K, D73R, A75W, A75Y, A75P, A75M, A75G, A75S, A75N, A75K, A75R, N77Y, N 77K, N77R, M78K, M78R, L79F, L79V, L79A, L79A, L79N, L79H, Q82W, Q82Y, M83I, T91A, T91G, T91Q, T91D, T91E, T91H, V93M, V93T, V93E, L104M.

[0470] The above approach also applies to sdAbs B2 and B3, the following mutations are also enriched in the positive population and will therefore improve affinity for IgE-Fc. These mutations can be used alone or in combination to construct mutant versions of B2 or B3, respectively.

[0471] B2: V12T, A23Q, A23D, T28Q, T28H, D35M, D35A, D36V, Q39F, Q39W, Q39Y, Q39I, Q39V, Q39E, Q44P, D74P, D74I, S105I, S105N, S105D, and S105E.

[0472] B3: S21E, A23D, A23E, S25D, W36I, V37W, V37Y, A40C, A40R, G44P, G44D, G44E, G44H, F45P, F45M, F45M, F45I, F45L, F45V, F45A, S45A, S45G, T58D, N59E, K65M, K65A, T65A, T65G9Q, S71I, N77G, R78M, R78V, R7 8E, Q82Y, N84D, N84E, K87L, K87G, K87C, K87S, K87N, K87D, K87E, K87H, P88D, T91A, T91G, T91N, T91Q, T91D, T91E, A97M, A97I, A97L, A97V, N105P, N105D, R108F, R108M, R108I, R108V, R108N, R108Q, and R108K.

[0473]

in conclusion

[0474] Using deep mutational scanning, all single mutations of sdAbs A1, B2 and B3 were evaluated for their effect on the interaction with IgE-Fc / FcεRIa. A series of mutations that could improve the binding affinity of A1, B2 and B3 to IgE-Fc could be generated. In addition, a triple mutant of A1, named A2, has been generated, successfully expressed and purified.

[0475] [Example 5: Affinity determination]

[0476] The aim of this study was to test the affinity of the sdAbs obtained in the previous example for IgE-Fc.

[0477] Materials and methods

[0478] The affinity of monomeric, dimeric sdAbs and multispecific constructs to IgE-Fc was tested on the OctetRED96e system (FortéBIO) using kinetic buffer (PBS, 0.02% Tween20, 0.1% BSA) as diluent. Biotinylated IgE Fc (made in-house at ALK, Denmark) was immobilized on a SAX (high-precision streptavidin) sensor (FortéBIO) with a maximum of 1 nm. Binding (240 s) and dissociation (600 s) of serially diluted (from 10 nM to 0.37 nM) analytes were measured. The data were subtracted from a blank reference sensor exposed to the same series of analyte dilutions, aligned, and analyzed using a 1:1 fitting model in the Octet Data Analysis HT 11.1 software (FortéBIO).

[0479]

result

[0480] The affinity of five individual sdAbs to IgE Fc was measured and compared to A1 (Table 14). on is a constant used to characterize the binding rate of sdAb to IgE, while k off Indicates the speed of dissociation of sdAb from IgE. off / k on The ratio yields the equilibrium dissociation constant K D . K D The lower the value, the higher the affinity of sdAb for IgE. 5 ~10 6 K on Value (M -1 s -1 ) and 10 -3 K off Rate (s -1 ) binds to the antigen, resulting in a binding event in the low nM affinity range. Here, B2 and B3 exhibit the highest affinity for IgE, which is consistent with the low nM affinity range (~1×10 -10 M) was comparable to A1. B1 and D2 showed lower affinity (6-8×10 -8 M).

[0481] [Table 14: Affinity of sdAbs determined using biomembrane interferometry (BLI, Octet) and global 1:1 fitting.

[0482] sdAb constructs <![CDATA[k on (M -1 s -1 )]]> <![CDATA[k off (s -1 )]]> <![CDATA[K D (M)]]> A1 <![CDATA[1.0×10 6 ]]> <![CDATA[2.0×10 -4 ]]> <![CDATA[1.9×10 -10 ]]> B1 <![CDATA[7.6×10 5 ]]> <![CDATA[4.9×10 -2 ]]> <![CDATA[6.5×10 -8 ]]> B2 <![CDATA[7.0×10 5 ]]> <![CDATA[7.8×10 -5 ]]> <![CDATA[1.1×10 -10 ]]> B3 <![CDATA[8.8×10 5 ]]> <![CDATA[2.5×10 -4 ]]> <![CDATA[2.8×10 -10 ]]> D1 <![CDATA[1.4×10 5 ]]> <![CDATA[4.5×10 -4 ]]> <![CDATA[3.2×10 -9 ]]> D2 <![CDATA[2.4×10 5 ]]> <![CDATA[2.0×10 -2 ]]> <![CDATA[8.1×10 -8 ]]>

[0483] The affinity of additional sdAbs described herein to IgE-Fc will be measured by the same procedure.

[0484]

in conclusion

[0485] Selected, non-optimized sdAbs showed high affinity for IgE in the high to low nM range comparable to A1.

[0486] [Example 6: Displacement effect of single domain antibodies measured by ELISA]

[0487]

Target

[0488] The goal of this example was to evaluate the ability of sdAbs to displace the binding between human IgE and the high affinity human IgE receptor (FcεRIα) using a biochemical ELISA assay.

[0489] Materials and methods

[0490] The displacement effect was tested using the IgE-FcεRIα displacement ELISA assay. Microtiter plates (Thermo Scientific Nunc, Roskilde, Denmark) were coated with 100 μl of streptavidin, 5 μg / ml (Thermo Scientific, Waltham, MA) for 18 h at 4°C and then washed with 300 μl of wash buffer (PBS containing 0.05% Tween 20). 2000 plate washer (Molecular Devices, San Jose, CA). The remaining binding sites were blocked with 250 μl blocking buffer (PBS containing 2% BSA) for 2 hours and washed with 300 μl washing buffer. Subsequently, the bound streptavidin was loaded with 100 μl (0.1 μg / ml) recombinant human FcεRIα (biotinylated by avi tag site-specifically, made in-house, ALK, Denmark) for 1 hour and the plate was washed twice with 300 μl wash buffer. The bound receptors were then loaded with 100 μl recombinant human IgE (made in-house, ALK, Denmark), 13 ng / ml (pre-titrated to achieve an OD in the range of 1.5 to 2.5 without replacement components 450 signal) for 1 hour and then washed three times with 300 μl of wash buffer.

[0491]

Replacement steps

[0492] 100 μl of sdAb diluted in a concentration series (pM to μM range) or blocking buffer alone as a non-displacement negative control was added to the ELISA wells and incubated for 1 hour. Any IgE displaced from the receptor by the sdAb was washed away by washing three times with 300 μl of wash buffer.

[0493] Remaining undisplaced IgE was detected by adding 100 μl HRP-conjugated anti-human lambda light chain (Bethyl Laboratories, Montgomery, Tx), diluted 1:20.000 in blocking buffer for 1 hour, followed by three washes of 300 μl. 100 μl TMB One (Kementec, Taastrup, Denmark) was then added to the wells and incubated for 15 minutes, and the reaction was stopped by adding 100 μl 1N sulfuric acid.

[0494] Finally, OD was measured on a Glomax Discover microplate reader (Promega, Madison, Wis.). 450 The absorbance at .

[0495] Results were analyzed using GraphPad Prism version 9.3.0 (GraphPad Software, San Diego, CA), and the percent displacement effect was determined as the relative decrease in signal compared to the control without added sdAb. 50 Values ​​and maximum effects were determined from the graphs listed in Table 15 (not shown).

[0496] All types of sdAbs, including monomers and dimers, and sdAb-Fc fusion complexes can be tested using this procedure.

[0497]

result

[0498] Table 1 lists the sdAbs tested in the IgE-FcεRIα displacement ELISA assay as monomers, or in combination pairs constructed as dimers or multispecific constructs. sdAbs B1, B2, B3, D1, D2, E1, E2, E3, E4, F1, F2, F3, F4, F5, F6 bind to A1 and to IgG 4 -Fc fusion (eg Figure 1 IgG 4 -Fc fusion to the N-terminus or C-terminus in sdAb and IgG 4 Different linkers were used between them. In addition, sdAb A2 was combined with B1 in the same manner. Table 15 shows the results of all tested sdAbs and constructs and Fc fusion constructs.

[0499] The ability of various sdAbs to displace IgE from the receptor (FcεRIα) was assessed by using an ELISA-based IgE-FcεRIα displacement assay. The percent displacement effect was calculated as the relative reduction in signal compared to the control without added sdAb. The EC 50 Values ​​and maximum effects (Table 15).

[0500] [Table 15: EC of monomeric sdAbs and multispecific constructs 50 and the replacement effect of the "maximum effect"]

[0501]

[0502]

[0503]

[0504]

[0505]

[0506]

[0507]

[0508] Overall, displacement activity was observed for only a few monomeric sdAbs: A1, A2, and B1. Notably, this displacement activity could be greatly increased by pairing sdAbs with dispersed activity with sdAbs that bind to different epitopes on IgE-Fc, either as a simple mixture of two sdAbs, dimers, or fused to IgG-Fc.

[0509] The most potent constructs were identified among the multimeric constructs, which appeared to be much more potent than the sdAb monomers. The most potent multimeric constructs displayed EC values ​​in the subnanomolar to single-digit nanomolar range. 50 values, and had a maximum effect of >95% at the highest tested concentration. In contrast, the control construct KIH_E07_79 was also very effective in the assay (EC 50 =0.6 nM and maximal effect >95%). If the monomeric sdAbs of the multimeric constructs bound different IgE epitopes, they were considered multispecific constructs.

[0510] In some cases, the relative orientation of the two linked sdAb entities within the sdAb dimer has a significant effect, for example, B3A1(G 4 S) 4 Reverse A1B3(G 4 S) 4 (EC 50 =190 nM) is much more efficient (EC 50 =0.3 nM). Nevertheless, there are some examples where relative orientation does not seem to be important, e.g., D2A1 (G 4 S) 4 With A1D2(G 4 S) 4 Display EC in the same range 50 values ​​(8 nM and 10 nM, respectively).

[0511] sdAb A1 and additional sdAb monomer mixtures (but not linked together - see sdAb mixtures, (Table 15)) showed synergistic effects, as in most cases the EC 50 The values ​​were much lower than either of the two sdAbs alone. Nevertheless, none of these combinations was as effective as the most potent linked sdAb dimer.

[0512] A2 displaces IgE from receptors more efficiently than A1 (EC 50 250nM and 800nM respectively).

[0513]

in conclusion

[0514] Several linked sdAb dimers and sdAb-IgG4Fc fusion constructs showed EC in the subnanomolar to single-digit nanomolar range 50 value, and can displace >95% of IgE from the receptor.

[0515] [Example 7: Cellular evaluation of sdAb displacement activity using the RBL assay]

[0516] The aim of this study was to evaluate the ability of sdAbs to displace binding of human IgE to the high affinity human IgE receptor (FcεRIα) expressed in a cellular system.

[0517] Materials and methods

[0518] The ability of sdAbs to displace IgE from the receptor was tested using the RBL assay.

[0519] Rat basophilic leukemia cells (RBL SX-38) were cultured in horizontal TC flasks with complete MEM / cMEM: MEM (Gibco 11095-080) + 100 mM hot sodium phosphate (Gibco 11360-+70) + 15% FCS + penicillin / streptomycin (Lonza DE17-602E) + 1 mg / ml G418 (Gibco 10131-027). For IgE sensitization, cells were detached from TC flasks after washing out proteins with PBS before adding trypsin-Versene (LONZA Product No. 17-161E). After incubation at 37°C for 5 minutes, cells were seeded in 200 μl cMEM and incubated for 10 min. 5 cells / well in a 96-well plate (Nunc167008). The cells were incubated overnight (5% CO 2 , 37°C).

[0520] After removing the supernatant, RBL SX-38 were sensitized with IgE: a mixture of three Derp2-specific recombinant human IgE clones was used: H10, H12, and P4E (Christensen et al., 2008). 100 μl of cMEM containing 1 μg / ml recombinant human IgE was added to each well and incubated at 4 °C for 2 h. 2 Incubator (5% CO 2 The cells were incubated in 4% paraformaldehyde (5% paraformaldehyde) and 4% paraformaldehyde (5% paraformaldehyde) for 2 hours at 37° C. Non-sensitized control wells were incubated with cMEM for comparison only.

[0521] Displacement of bound IgE: 50 μl / well of sdAb or control was added to sensitized RBL SX-38 cells. Peptide-linked and IgG-Fc fused sdAbs were tested at 1 μM concentration and 10-fold dilutions, while individual sdAbs were tested at 1 μM concentration. In addition, mixtures of sdAb A1 and individual sdAbs were tested at 1 μM each. Cells were incubated in CO 2 Incubator (5% CO 2 , 37°C) for 1 hour.

[0522] FACS staining of surface-bound IgE: Cells from the RBL assay were washed twice with FACS staining buffer (BD FACS flow + 0.5% BSA), centrifuged at 500×g for 5 minutes, and the cell pellet was stained with a-FcεR1 FITC (Invitrogen 11-5899-42) and anti-IgE APC (Invitrogen 17-6886-42). The cells were incubated on ice for 30 minutes, then washed with FACS staining buffer and then fixed with 200 μl cell fix (BD 340181) / well at room temperature for 15 minutes. After further washing, the cells were analyzed on a flow cytometer: Cytek Aurora with a plate loader. FACS data were analyzed using SpectroFlo software, and relevant cells were identified and gated based on scatter and FcεR1 expression. The measured IgE mean fluorescence readings were analyzed using GraphPad Prism version 9.3.0 (GraphPad Software, San Diego, CA), and the percent displacement effect was determined as the relative decrease in signal compared to the control without added sdAb. 50 The concentration of sdAb that resulted in 50% inhibition relative to the control without added sdAb was determined from dose-response plots of IgE expression (mean fluorescence) using nonlinear curve fitting of log-transformed data in GraphPad Prism.

[0523]

result

[0524] The displacement activity of the sdAbs of interest (Table 16) was tested in the RBL assay using rat basophilic leukemia cells (RBL-SX38). The sdAbs were tested in different combinations, either as sdAb monomers or as sdAb dimers (bispecific or bivalent) linked together, with and without an Fc domain, or in combination with two individual sdAb monomers added in the same setup. The displacement effect is shown in (Table 16), where EC 50 The maximum effect of the linked sdAb and the effects of the remaining sdAbs (comparison of A1 and KIH_E7_79) were 1 μM.

[0525] sdAb conjugated to A1 or A2 (alone) using different linkers to IgG 4 -Fc fusion (eg Figure 1 Table 16 shows the results of the test constructs.

[0526] The activity of each test construct was expressed as EC 50 and the form of maximum displacement activity is shown.

[0527] Compared with the monospecific bivalent A1A1(G 4 S) 4 Compared to the constructs, all linked sdAb dimers showed efficient displacement of IgE from FcεR1 on RBL-SX38 cells (Table 16). The 11 most potent dimers completely displaced IgE, EC 50 2 nM or less: B1A1(G 4 S) 4 , A1B1(G 4 S) 4 , A1B2(G 4 S) 4 , B2A1(G 4 S) 4 , A1B3(G 4 S) 4 , A1E1(G 4 S) 4 , A1E4(G 4 S) 4 , F3A1(G 4 S) 4 , F4A1(G 4 S) 4 , A1F6(G 4 S) 4 , F6A1(G 4 S) 4 (Table 16).

[0528] The data suggest that the order of attachment plays a role, as D2A1(G 4 S) 4 The efficacy is lower than that of A1D2(G 4 S)4,EC 50 ~78nMvs~4nM, B2A1(G 4 S) 4 With A1B2(G 4 S) 4 So too, E.C. 50 ~6nM vs ~1nM.

[0529] In all cases, mixing sdAb monomer A1 with a different sdAb monomer (non-linked) improved displacement, being most effective for mixing B1 and B2, with an effect of -100% using 1 [mu]M sdAb (Table 16).

[0530] Single sdAb displacement showed that at 1 μM, B1 displaced IgE more efficiently than A1.

[0531] For multispecific constructs, in which two sdAbs were linked to one Fc domain, most of the tested constructs showed potent displacement activity compared to a single unlinked sdAb.

[0532] [Table 16: sdAb displacement data tested with RBL-SX36 cells (monomers and dimers and combinations)]

[0533]

[0534]

[0535]

[0536]

[0537]

[0538]

[0539] [Example 8: Inhibition of effector cell degranulation]

[0540] The aim was to evaluate whether sdAbs could inhibit effector cell degranulation upon allergen challenge.

[0541] Materials and methods

[0542] PBMCs were isolated from heparinized whole blood of allergic donors by the method of Lymfoprep (Fresenius Kabi 1114547) and Leucoseptubes (Greiner 227290). After separation, cells were washed and resuspended in RPMI, HSA: (RPMI1640 (Gibco 72400-021) + 0.5% HSA (Sigma A1653)) to 1 / 16 of the original blood volume and incubated at 37°C for 1 hour with different concentrations for replacement of sdAB constructs. Pre-incubated cells were washed in RPMI, HSA and resuspended in RPMI, HSA (RD peprotec cat 200-03) supplemented with 2ng / ml of IL-3 to 1 / 8 of the blood volume.

[0543] IgE-replaced cells and controls were used to activate basophils with a-IgE or allergens diluted in RPMI + 0.5% HSA and stimulated with different concentrations of rDerp2, rBetv1 or Derp extract (ALK in-house), depending on donor sensitization and anti-IgE (Dako A0094), 1000-100-10 ng / ml.

[0544] In CO 2 Incubator (5% CO 2 After incubation for 1 hour at 37°C, the reaction was terminated by adding BD FACS flow + 0.5% BSA + 10 mM EDTA.

[0545] FACS staining of activated cells: Wash cells twice with FACS staining buffer (BD FACS flow + 0.5% BSA), centrifuge at 500 × g for 5 minutes, and stain the cell pellet with a-CD63 FIC (BD92467), a-CD203c APC (e-bioscience, 324610), and CD123 PE (BD034345). The cells were incubated on ice for 30 minutes, then washed with FACS staining buffer, and then fixed with 200 μl cell fixation (BD 340181) / well at room temperature for 15 minutes. After further washing, the cells were analyzed on a flow cytometer: Cytek Aurora with a plate loader. In order to compare with the IgE replacement efficiency, a-IgE APC (ebioscience 17-6986) and FcεR1 Cra1 BV605 (BD 747785) were combined with CD123 PE to stain the cell samples not used for activation assays for IgE, as described above. Data were analyzed using SpectroFlo software and basophils were identified as cells with low side scatter and high CD203c+CD123 expression. The % of basophils with high CD63 expression was depicted in graphs as the percentage of activated basophils using Graph Prisma.

[0546] The above method was further used in a more physiological setting. Here, whole heparinized blood was used instead of PBMCs for direct 1+1 incubation with sdAb construct dilutions. After washing, blood was reconstituted with RPMI, HSA and IL-3 were added and assayed as described for PBMCs.

[0547]

result

[0548] Treatment of basophils with sdAb resulted in reduced activation of basophils after stimulation with allergens or a-IgE ( Figure 5A-C ).

[0549] The dose response of rDerp2-induced activation in HDM-allergic donors was reduced -10-fold following treatment with 1 [mu]M sdAb A1, with cells showing a 16% displacement effect as measured by IgE expression (Table 17).

[0550] When sdAb A2 was used in combination with single sdAb B1 or B2, effective treatment was observed, while no effect was observed when sdAb A2 was used in combination with B3, where activation was comparable to that of A2 alone (Fig. 5B-C). These findings mirror the effect of displacement on IgE surface expression (Table 17), where sdAb A2 combined with B1 or B2 showed ∼100% effect, while A2 combined with B3 and A2 alone showed comparable displacement effect ∼70%.

[0551] Multispecific construct (CIgG4B1A1(G 4 S) 1 The constructs showed no direct cell activation by reducing basophil activation in PBMC (Fig. 5D) and whole blood (Fig. 5E-F) in a dose-dependent manner over the concentration range of 10-1000 nM (Fig. 5D-F).

[0552]

in conclusion

[0553] In conclusion, the cellular evaluation of sdAb-mediated IgE displacement showed that efficient displacement of IgE from the FcεR1 receptor located on the cell surface can be obtained by combining the displacing sdAbs A1 or A2 with some of the other tested sdAbs or as a linked sdAb.

[0554] The displacement of IgE was demonstrated by RBL-SX38 cells sensitized with recombinant IgE.

[0555] Furthermore, we demonstrated the displacement of IgE from human basophils from allergic donors and its effect on subsequent challenge of basophils with allergen or anti-IgE.

[0556] [Example 9: Evaluation of the ability of mouse models to prevent allergic reactions]

[0557] The purpose of this example is to evaluate the ability of sdAb to prevent allergic reactions in vivo.

[0558] Materials and methods

[0559] B6.Cg-Fcer1a tm1KntTg(FCER1A)1Bhk / J mice were originally obtained from Jackson Laboratories and then bred in-house for use in passive systemic anaphylaxis (PSA) or passive cutaneous anaphylaxis (PCA) models. For the PSA model, mice were sensitized by intraperitoneal injection of a mixture of three Derp2-specific recombinant human IgE clones: H10, H12, P4E (Christensen et al., 2008), 200 μl per mouse, each Ab at 16.67 μg / ml in PBS. After 24 hours, mice were intraperitoneally injected with 200 μl of sterile PBS or different concentrations (150 μM, 10 μM, 2 μM, 0.4 μM and 0.08 μM in PBS) of the sdAb CIgG4B1A1 (G 4 S) 1 , and allergen challenge was performed 4 hours later (200 μl of 0.05 μg / ml rDerp2 (ALK in-house) was injected intraperitoneally). Core body temperature was measured directly before inhibitor injection or allergen challenge using a temperature transponder (IPTT-300, implanted subcutaneously at least one day before the start of the experiment), every 5 minutes thereafter for 1 hour or until serum was collected 90 minutes after challenge (BD Microtainer SST tubes, 365968). Serum mouse mast cell protease 1 (mMCPT1) concentrations ( Fig. 6A ). For the PCA model, mice were sensitized with the same three anti-Derp2 antibodies, but 20 μl of each antibody was injected intradermally at 2.5 μg / ml. Typically, mice will be injected with inhibitors intradermally or intraperitoneally the next day, and rDerp2 (10 μg per mouse, 100 μl) will be added to a 1% Evan's Blue (Sigma-Aldrich, E2129) sterile NaCl solution 6 hours before intravenous challenge. However, for allergenicity testing, inhibitors were injected intradermally and Evan's Blue solution without rDerp2 was injected intravenously at the same time. Half an hour after dye injection, mice were killed and the dorsal dye extravasation area was measured. The dorsal skin was then harvested and the dye was extracted using formamide (Merck, S4117). The optical density of the extracted dye was measured at 600 nm (Figure 8B).

[0560]

result

[0561] CIgG4B1A1(G 4 S) 1 Prevented allergic reactions in the PSA model in a dose-dependent manner. Although mice showed no change in core body temperature after injection of the inhibitor alone (Figure 6B), treatment with the lowest concentration of CIgG4B1A1 (G 4S) 1 Animals treated with CIgG4B1A1 (0.08 μM) showed similar responses to control mice (PBS injection) in terms of decreased body temperature and increased serum mMCPT1. Mice were partially protected after treatment with the 0.4 μM dose and were not significantly protected after treatment with the two highest doses of CIgG4B1A1 (G 4 S) 1 Treatment almost completely prevented allergic reactions (Fig. 6C-E). 4 S) 2 、CIgG4E1A1(G 4 S) 2 and CIgG4F4A1(G 4 S) 1 It does not contain allergens ( Fig. 7A ), but can disrupt allergic reactions with varying degrees of efficiency ( Figure 7B On the other hand, during the PSA model, the N-linked construct NIgG4B1A1 (G 4 S) 3 It induced a decrease in core body temperature in mice ( FIG. 8A ) and induced dye extravasation into sensitized skin areas in the PCA model for allergen testing ( FIG. 8C ).

[0562]

in conclusion

[0563] In a mouse passive anaphylaxis model, sdAb CIgG4B1A1 (G 4 S) 1 、CIgG4D2A1(G 4 S) 2 、CIgG4E1A1(G 4 S) 2 and CIgG4F4A1(G 4 S) 1 It can prevent allergic reactions in the body.

[0564] [Example 10: Evaluation of potential allergen constructs]

[0565] The aim of this study was to evaluate the ability of sdAbs alone or as part of a construct to activate basophils.

[0566] Materials and methods

[0567] PBMCs isolated from heparinized whole blood (as described in Example 8) or from allergic donors were incubated with various concentrations of sdAb constructs diluted in RPMI, 5% HSA in CO 2 Incubator (5% CO 2The reaction was terminated by adding BD FACS+0.5% BSA+10 mM EDTA, and the cells were FACS stained to measure CD63 and IgE expression using the same methods and reagents as described in Example 8.

[0568]

result

[0569] As shown in Figure 9, the displacement activity of different constructs at increasing concentrations is shown (similar to the setup in Example 7). In addition, Figures 9A+C show examples of the percentage of basophil activation triggered by different concentrations of the same construct. The overall construct fused at the N-terminus showed high activation in the above interval, while the allergenicity of the C-terminal fused construct was lower. In addition, as shown in Figures 9A+C, the linker of the C-terminal fused construct also affected the allergenic activity. The construct CIgG4B1A1 (G 4 S) 1 , showed no allergic activity at any concentration in any donor. On the other hand, construct NIgG4B1A1 (G 4 S) 1 The tests were performed in a similar number of donors and indeed showed allergenic activity in all of them. Interestingly, the compared KIH_E7_79 did show allergenic activity. In some donors KIH_E7_79 activated only at high concentrations, while in other donors activation was also observed at lower concentrations. The remaining constructs listed in Table 15 showed high variability in allergenic activity between different donors and within the same donor, which means that no clear conclusions can be drawn from the assay alone, but can be confirmed in mice, as shown in Example 9.

[0570]

in conclusion

[0571] Overall, this example demonstrates that N-terminally linked multispecific constructs generally activate basophils and therefore have allergenic activity, whereas most C-terminally linked ones do not, depending on the linker. Importantly, when tested in more than 200 donors, CIgG4B1A1 (G 4 S) 1 No allergic activity was shown. 4 S) 1 In contrast, the compared KIH_E7_79 did show a high degree of allergenic activity. This setup is a simple way to test whether a particular construct has allergenic activity. Allergenic activity may be confirmed in a mouse model.

[0572] [Example 11: Humanization of sdAb]

[0573]

Target

[0574] Humanization of therapeutic antibodies derived from animal immunization is often required to minimize the risk of immunogenicity in humans, which can lead to potentially harmful and severe side effects and reduce antibody efficacy. Therefore, the aim of this study was to humanize the sdAb described here.

[0575] Materials and methods

[0576] The CDRs and frameworks of the sdAbs used for humanization were determined as described herein, where for each sdAb, three individual CDR regions (CDR1-3) and four framework regions (FR1-4) are given.

[0577] Each of the 4 framework regions (FR1-FR4) was initially fully humanized by identifying the nearest human germline VH sequences and replacing these sequences with camelid framework portions. This would provide a 100% degree of humanization.

[0578] The next step is to test the functionality of the humanized sdAb to determine whether the original function is retained.

[0579] If the adjusted sequence is not affected by humanization, or if an acceptable decrease in function is observed (this could be a reduction in antigen binding or a less effective substitution), the humanization procedure is terminated and the final degree of humanization is 100%.

[0580] On the other hand, if an unacceptable function is affected at a 100% degree of humanization, the sdAb is subjected to a "reverse mutation procedure" with the goal of reducing the degree of humanization by reintroducing the original camelid amino acid sequences one by one into a single framework. After reintroduction of the original camelid amino acid sequences, the function of the sdAb is determined. When the function lost due to humanization is reestablished after the reverse mutation procedure, the degree of humanization is recalculated and the humanization procedure is terminated. The final degree of humanization (0 to 100%) depends on the impact on the function of the humanized structure and cannot be predicted.

[0581]

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Claims

1. A multispecific construct comprising: (a) a first single domain antibody (sdAb) which is capable of displacing bound IgE from FcεRI, (b) a second sdAb, which binds to IgE, (c) a moiety linking the first and second sdAbs, in The first and second sdAbs bind to non-identical epitopes of IgE, and The multispecific construct can displace bound IgE in the IgE high affinity receptor (FcεRI) with improved displacement activity compared to the first or second sdAb.

2. The multispecific construct according to claim 1, wherein The second sdAb is capable of binding IgE-Fc.

3. A multispecific construct according to any one of the preceding claims, wherein: The displacement activity was assessed by an ELISA-based IgE-FcεRIα disruption assay based on measuring the remaining IgE that was not removed in immobilized recombinant human FcεRIα preloaded with IgE after the addition of sdAbs or multispecific constructs and determining the percentage of displacement activity as the relative reduction in signal compared to controls without added test compound.

4. A multispecific construct according to any one of the preceding claims, wherein: The displacement activity of the first sdAb allows EC 50 In the range of 100nM~5000nM.

5. The multispecific construct according to any one of the preceding claims, wherein: The displacement activity of the first sdAb is such that the maximum displacement activity is at least 80%.

6. A multispecific construct according to any one of the preceding claims, wherein: The displacement activity of the first sdAb allows EC 50 The range is 100 nM to 5000 nM, and the maximum displacement effect is at least 80%.

7. A multispecific construct according to any one of the preceding claims, wherein: The second sdAb No displacement activity, or With a higher EC compared to the first sdAb 50 has a lower displacement activity, or Has a maximum displacement effect of less than 75%.

8. A multispecific construct according to any one of the preceding claims, wherein: The improved displacement activity of the multispecific constructs resulted in an observed EC of either the first sdAb or the second sdAb or a mixture of the first and second sdAbs. 50 The EC of the construct resulted in 50 Lower.

9. The multispecific construct according to any one of the preceding claims, wherein: The improved displacement activity of the multispecific constructs allows EC 50 EC with the first sdAb 50 It is at least 20 times lower than before.

10. The multispecific construct according to any one of the preceding claims, wherein: The improved displacement activity of the multispecific constructs allows EC 50 Less than 100nM.

11. A multispecific construct according to any one of the preceding claims, wherein: The improved displacement activity of the multispecific constructs is such that the maximum displacement effect is at least 80%.

12. The multispecific construct according to any one of the preceding claims, wherein: The improved displacement activity of the multispecific constructs allows EC 50 Below 100 nM and with a maximum displacement of at least 80%.

13. A multispecific construct according to any one of the preceding claims, in, The first sdAb comprises binding regions CDR1, CDR2 and CDR3, each of which comprises or consists of an amino acid sequence determined according to the Kabat, Chothia, IMTG or Aho numbering scheme, the amino acid sequence being determined in an amino acid sequence selected from any one of the following: SEQ ID NO: 1 or 5, Optionally, wherein CDR1 contains 1, 2 or 3 amino acid substitutions, CDR2 contains 1, 2 or 3 amino acid substitutions, and CDR3 contains 1, 2 or 3 amino acid substitutions.

14. The multispecific construct according to any one of the preceding claims, in, The first sdAb comprises binding regions CDR1, CDR2 and CDR3, each of which has SEQ ID NO: 2, 3 and 4; or 6, 7 and 8, Each CDR is identified according to the Aho numbering scheme. Optionally, wherein CDR1 contains 1, 2 or 3 amino acid substitutions, CDR2 contains 1, 2 or 3 amino acid substitutions, and CDR3 contains 1, 2 or 3 amino acid substitutions.

15. The multispecific construct according to any one of the preceding claims, in, The second sdAb comprises binding regions CDR1, CDR2 and CDR3, each of which comprises or consists of an amino acid sequence determined according to the Kabat, Chothia, IMTG or Aho numbering scheme, the amino acid sequence being determined in an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 13, 17, 21, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69 and 73, Optionally, wherein CDR1 contains 1, 2 or 3 amino acid substitutions, CDR2 contains 1, 2 or 3 amino acid substitutions, and CDR3 contains 1, 2 or 3 amino acid substitutions.

16. A multispecific construct according to any one of the preceding claims, in, The second sdAb comprises binding regions CDR1, CDR2 and CDR3, each of which has SEQ ID NO: 10, 11 and 12; 14, 15 and 16; 18, 19 and 20; 22, 23 and 24; 26, 27 and 28; 30, 31 and 32; 34, 35 and 36; 38, 39 and 40; 42, 43 and 44; 46, 47 and 48; 50, 51 and 52; 54, 55 and 56; 58, 59 and 60; 62, 63 and 64; 66, 67 and 68; 70, 71 and 72; or 74, 75 and 76, Each CDR is identified according to the Aho numbering scheme. Optionally, wherein CDR1 contains 1, 2 or 3 amino acid substitutions, CDR2 contains 1, 2 or 3 amino acid substitutions, and CDR3 contains 1, 2 or 3 amino acid substitutions.

17. A multispecific construct according to any one of the preceding claims, wherein (a) the first sdAb comprises binding regions CDR1, CDR2 and CDR3, each of which comprises or consists of an amino acid sequence determined according to the Kabat, Chothia, IMTG or Aho numbering scheme, the amino acid sequence being determined in an amino acid sequence selected from the group consisting of: SEQ ID NO: 1 or 5, Optionally, wherein CDR1 contains 1, 2 or 3 amino acid substitutions, CDR2 contains 1, 2 or 3 amino acid substitutions, and CDR3 contains 1, 2 or 3 amino acid substitutions; and (b) the second sdAb comprises binding regions CDR1, CDR2 and CDR3, each of which comprises or consists of an amino acid sequence determined according to the Kabat, Chothia, IMTG or Aho numbering scheme, the amino acid sequence being determined in an amino acid sequence selected from the group consisting of: SEQ ID NO: 9, 25, 33, 61, Optionally, wherein CDR1 contains 1, 2 or 3 amino acid substitutions, CDR2 contains 1, 2 or 3 amino acid substitutions, and CDR3 contains 1, 2 or 3 amino acid substitutions.

18. A multispecific construct according to any one of the preceding claims, wherein (a) The first sdAb comprises binding regions CDR1, CDR2 and CDR3, each of which has SEQ ID NO: 2, 3 and 4; or 6, 7 and 8, in, Each CDR is identified according to the Aho numbering scheme. Optionally, wherein CDR1 contains 1, 2 or 3 amino acid substitutions, CDR2 contains 1, 2 or 3 amino acid substitutions, and CDR3 contains 1, 2 or 3 amino acid substitutions; and (b) the second sdAb comprises binding regions CDR1, CDR2 and CDR3, each of which has SEQ ID NO: 10, 11 and 12; 26, 27 and 28; 34, 35 and 36; or 62, 63 and 64, Each CDR is identified according to the Aho numbering scheme. Optionally, wherein CDR1 contains 1, 2 or 3 amino acid substitutions, CDR2 contains 1, 2 or 3 amino acid substitutions, and CDR3 contains 1, 2 or 3 amino acid substitutions.

19. A multispecific construct according to any one of the preceding claims, wherein (a) the first sdAb comprises binding regions CDR1, CDR2 and CDR3, each of which comprises or consists of an amino acid sequence determined according to the Kabat, Chothia, IMTG or Aho numbering scheme, the amino acid sequence being determined in the following amino acid sequence: SEQ ID NO: 1, Optionally, wherein CDR1 contains 1, 2 or 3 amino acid substitutions, CDR2 contains 1, 2 or 3 amino acid substitutions, and CDR3 contains 1, 2 or 3 amino acid substitutions; and (b) the second sdAb comprises binding regions CDR1, CDR2 and CDR3, each of which comprises or consists of an amino acid sequence determined according to the Kabat, Chothia, IMTG or Aho numbering scheme, the amino acid sequence being determined in the following amino acid sequence: SEQ ID NO: 9, Optionally, wherein CDR1 contains 1, 2 or 3 amino acid substitutions, CDR2 contains 1, 2 or 3 amino acid substitutions, and CDR3 contains 1, 2 or 3 amino acid substitutions.

20. The multispecific construct according to any one of the preceding claims, wherein (a) the first sdAb comprises binding regions CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 have SEQ ID NOs: 2, 3 and 4, respectively, in, Each CDR is identified according to the Aho numbering scheme. Optionally, wherein CDR1 contains 1, 2 or 3 amino acid substitutions, CDR2 contains 1, 2 or 3 amino acid substitutions, and CDR3 contains 1, 2 or 3 amino acid substitutions; and (b) the second sdAb comprises binding regions CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 have SEQ ID NOs: 10, 11 and 12, respectively, Each CDR is identified according to the Aho numbering scheme. Optionally, wherein CDR1 contains 1, 2 or 3 amino acid substitutions, CDR2 contains 1, 2 or 3 amino acid substitutions, and CDR3 contains 1, 2 or 3 amino acid substitutions.

21. The multispecific construct of any one of claims 1 to 18, wherein (a) the first sdAb comprises binding regions CDR1, CDR2 and CDR3, each of which comprises or consists of an amino acid sequence determined according to the Kabat, Chothia, IMTG or Aho numbering scheme, the amino acid sequence being determined in the following amino acid sequence: SEQ ID NO: 1, Optionally, wherein CDR1 contains 1, 2 or 3 amino acid substitutions, CDR2 contains 1, 2 or 3 amino acid substitutions, and CDR3 contains 1, 2 or 3 amino acid substitutions; and (b) the second sdAb comprises binding regions CDR1, CDR2 and CDR3, each of which comprises or consists of an amino acid sequence determined according to the Kabat, Chothia, IMTG or Aho numbering scheme, the amino acid sequence being determined in the following amino acid sequence: SEQ ID NO: 25, Optionally, wherein CDR1 contains 1, 2 or 3 amino acid substitutions, CDR2 contains 1, 2 or 3 amino acid substitutions, and CDR3 contains 1, 2 or 3 amino acid substitutions.

22. The multispecific construct of any one of claims 1 to 18 and 21, wherein: (a) the first sdAb comprises binding regions CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 have SEQ ID NOs: 2, 3 and 4, respectively, Each CDR is identified according to the Aho numbering scheme. Optionally, wherein CDR1 contains 1, 2 or 3 amino acid substitutions, CDR2 contains 1, 2 or 3 amino acid substitutions, and CDR3 contains 1, 2 or 3 amino acid substitutions; and (b) the second sdAb comprises binding regions CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 have SEQ ID NOs: 26, 27 and 28, respectively, Each CDR is identified according to the Aho numbering scheme. Optionally, wherein CDR1 contains 1, 2 or 3 amino acid substitutions, CDR2 contains 1, 2 or 3 amino acid substitutions, and CDR3 contains 1, 2 or 3 amino acid substitutions.

23. The multispecific construct of any one of claims 1 to 18, wherein (a) the first sdAb comprises binding regions CDR1, CDR2 and CDR3, each of which comprises or consists of an amino acid sequence determined according to the Kabat, Chothia, IMTG or Aho numbering scheme, the amino acid sequence being determined in the following amino acid sequence: SEQ ID NO: 1, Optionally, wherein CDR1 contains 1, 2 or 3 amino acid substitutions, CDR2 contains 1, 2 or 3 amino acid substitutions, and CDR3 contains 1, 2 or 3 amino acid substitutions; and (b) the second sdAb comprises binding regions CDR1, CDR2 and CDR3, each of which comprises or consists of an amino acid sequence determined according to the Kabat, Chothia, IMTG or Aho numbering scheme, the amino acid sequence being determined in the following amino acid sequence: SEQ ID NO: 33, Optionally, wherein CDR1 contains 1, 2 or 3 amino acid substitutions, CDR2 contains 1, 2 or 3 amino acid substitutions, and CDR3 contains 1, 2 or 3 amino acid substitutions.

24. The multispecific construct of any one of claims 1 to 18 and 23, wherein: (a) the first sdAb comprises binding regions CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 have SEQ ID NOs: 2, 3 and 4, respectively, Each CDR is identified according to the Aho numbering scheme. Optionally, wherein CDR1 contains 1, 2 or 3 amino acid substitutions, CDR2 contains 1, 2 or 3 amino acid substitutions, and CDR3 contains 1, 2 or 3 amino acid substitutions; and (b) the second sdAb comprises binding regions CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 have SEQ ID NOs: 34, 35 and 36, respectively, Each CDR is identified according to the Aho numbering scheme. Optionally, wherein CDR1 contains 1, 2 or 3 amino acid substitutions, CDR2 contains 1, 2 or 3 amino acid substitutions, and CDR3 contains 1, 2 or 3 amino acid substitutions.

25. The multispecific construct of any one of claims 1 to 18, wherein: (a) the first sdAb comprises binding regions CDR1, CDR2 and CDR3, each of which comprises or consists of an amino acid sequence determined according to the Kabat, Chothia, IMTG or Aho numbering scheme, the amino acid sequence being determined in the following amino acid sequence: SEQ ID NO: 1, Optionally, wherein CDR1 contains 1, 2 or 3 amino acid substitutions, CDR2 contains 1, 2 or 3 amino acid substitutions, and CDR3 contains 1, 2 or 3 amino acid substitutions; and (b) the second sdAb comprises binding regions CDR1, CDR2 and CDR3, each of which comprises or consists of an amino acid sequence determined according to the Kabat, Chothia, IMTG or Aho numbering scheme, the amino acid sequence being determined in the following amino acid sequence: SEQ ID NO: 61, Optionally, wherein CDR1 contains 1, 2 or 3 amino acid substitutions, CDR2 contains 1, 2 or 3 amino acid substitutions, and CDR3 contains 1, 2 or 3 amino acid substitutions.

26. The multispecific construct of any one of claims 1 to 18 and 25, wherein: (a) the first sdAb comprises binding regions CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 have SEQ ID NOs: 2, 3 and 4, respectively, Each CDR is identified according to the Aho numbering scheme. Optionally, wherein CDR1 contains 1, 2 or 3 amino acid substitutions, CDR2 contains 1, 2 or 3 amino acid substitutions, and CDR3 contains 1, 2 or 3 amino acid substitutions; and (b) the second sdAb comprises binding regions CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 have SEQ ID NOs: 62, 63 and 64, respectively, Each CDR is identified according to the Aho numbering scheme. Optionally, wherein CDR1 contains 1, 2 or 3 amino acid substitutions, CDR2 contains 1, 2 or 3 amino acid substitutions, and CDR3 contains 1, 2 or 3 amino acid substitutions.

27. The multispecific construct of any one of claims 13 to 26, wherein: 1, 2 or 3 amino acid substitutions in CDR1, CDR2 and CDR3 are conservative substitutions.

28. The multispecific construct of any one of claims 13 to 27, wherein: One, two or three amino acid substitutions in CDR1, CDR2 and CDR3 are conservative substitutions selected from the group consisting of Ala to Gly; Ala to Ser; Arg to Lys; Asn to Gln; Asn to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala; Gly to Pro; His to Asn; His to Gln; Ile to Leu, Ile to Val; Leu to Ile; Leu to Val; Lys to Arg; Lys to Gln; Lys to Glu; Met to Leu; Met to Tyr; Met to Ile; Phe to Met; Phe to Leu; Phe to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; Phe to Val; Phe to Ile; and Phe or to Leu.

29. The multispecific construct according to any one of the preceding claims, wherein: The moiety is a peptide, polypeptide, polymer or polysaccharide.

30. The multispecific construct according to any one of the preceding claims, wherein: The moieties are peptides having up to 30 amino acid residues.

31. A multispecific construct according to any one of the preceding claims, in, The part is from the molecular formula (G n S) n The peptides selected from the group consisting of peptides, Wherein, n can be an integer from 1 to 6; arginine-serine peptide; peptides EAAAK and (EAAAK)2.

32. The multispecific construct according to any one of the preceding claims, wherein: The portion is a peptide selected from the group consisting of RS, GS, GGS and peptides having SEQ ID NOs: 88, 89, 90, 91, 454, 455, 456 and 457.

33. A multispecific construct according to any one of the preceding claims, wherein: The portion is a polypeptide that is or comprises a fragment of an antibody selected from the group consisting of IgA, IgE, IgG and IgM.

34. The multispecific construct of claim 33, wherein: The polypeptide is or comprises a fragment of an IgG antibody selected from IgG1, IgG2, IgG3 and IgG4.

35. The multispecific construct according to claim 33 or 34, in, The antibody fragment is the Fc portion of the antibody. Wherein, the first sdAb and the second sdAb are optionally independently fused or covalently coupled to the N-terminus or C-terminus of the Fc portion via a linker.

36. The multispecific construct according to claims 33 to 35, in, The fragment of the antibody is an IgG-Fc region selected from IgG1-Fc, IgG2-Fc, IgG3-Fc and IgG4-Fc, and Wherein, the first sdAb and the second sdAb are optionally independently fused or covalently coupled to the C of IgG-Fc via a linker. H 3 or C H 2,.

37. The multispecific construct of claim 36, wherein The first and second sdAbs are fused or covalently coupled directly or via a linker to the C of IgG-Fc. H 2, The first and second sdAbs are fused or covalently coupled directly or via a linker to the C of IgG-Fc. H 3. The first sdAb is fused or covalently coupled to the C of IgG-Fc directly or via a linker. H 2, and the second sdAb is fused or covalently coupled to the C of IgG-Fc directly or through a linker H 3, or The second sdAb is fused or covalently coupled to the C of IgG-Fc directly or via a linker. H 2, and the first sdAb is fused or covalently coupled to the C of IgG-Fc directly or through a linker H 3.

38. The multispecific construct of claim 36 or 37, in, The first and second sdAbs were respectively bound to the C H 3 Fusion, or The first and second sdAbs are respectively bound to the C H 2 fused, optionally via a linker.

39. The multispecific construct of any one of claims 36 to 38, wherein: The IgG-Fc region is an IgG4-Fc region.

40. The multispecific construct of any one of claims 36 to 39, wherein: The linker is a peptide having up to 30 amino acid residues.

41. The multispecific construct according to any one of claims 36 to 40, in, The linker is a molecular formula (G n S) n The peptides selected from the group consisting of peptides, Wherein, n can be an integer from 1 to 6; arginine-serine peptide; peptides EAAAK and (EAAAK)2.

42. The multispecific construct of any one of claims 36 to 41, wherein: The linker is a peptide selected from the group consisting of RS, GS, GGS and peptides having SEQ ID NOs: 88, 89, 90, 91, 454, 455, 456 and 457.

43. The multispecific construct of any one of claims 33 to 42, wherein: The antibody fragment is derived from a human antibody, particularly human IgG4-Fc.

44. The multispecific construct of any one of claims 33 to 43, wherein: IgG4-Fc has the amino acid sequence of SEQ ID NO: 125 or 126.

45. A multispecific construct according to any one of the preceding claims, wherein: The first sdAb or the second sdAb or both sdAbs consist of affinity matured human or humanized amino acid sequences in non-CDR regions.

46. ​​A multispecific construct according to any one of the preceding claims, wherein: When the first and second sdAbs are fused to IgG-Fc, optionally via a linker, the multispecific construct can be generated as a Knob in Hole (KIH) Fc fusion construct.

47. The multispecific construct of claim 46, wherein: The Knob portion of KIH comprises IgG4-Fc having SEQ ID NO: 433 or 435.

48. The multispecific construct of any one of claims 46 or 47, wherein: The hole portion of KIH comprises IgG4-Fc having SEQ ID NO: 434 or 436.

49. The multispecific construct of claim 46, wherein The hole portion of KIH comprises the first sdAb defined in claims 19 to 26, and The hole portion is selected from any one of SEQ ID NOs: 93-96, 113-116, 445, 447, 449 and 451.

50. The multispecific construct of claim 46 or 49, wherein The Knob portion of KIH comprises a second sdAb as defined in claim 19 or 20, and The Knob portion is selected from any one of SEQ ID NOs: 101-104, 121-124, 438, 440, 442, 444, 446, 448 and 450.

51. The multispecific construct of claim 46 or 49, wherein: The Knob portion of KIH comprises a second sdAb as defined in claim 21 or 22, and The Knob portion is selected from any one of SEQ ID NOs: 338-344.

52. The multispecific construct of claim 46 or 49, wherein: The Knob portion of KIH comprises a second sdAb as defined in claim 23 or 24, and The Knob portion is selected from any one of SEQ ID NOs: 345-352.

53. The multispecific construct of claim 46 or 49, wherein: The Knob portion of KIH comprises a second sdAb as defined in claim 25 or 26, and The Knob portion is selected from any one of SEQ ID NOs: 401 to 406.

54. The multispecific construct of claim 40, wherein The Knob portion of KIH comprises the first sdAb defined in claims 19 to 26, and The Knob portion is selected from any one of SEQ ID NOs: 105-112.

55. The multispecific construct of claim 40 or 54, wherein: The Hole portion of KIH comprises a second sdAb as defined in claim 19 or 20, and The hole portion has SEQ ID NO:

437.

56. The multispecific construct of any one of claims 40 to 55, wherein: The Hole portion and the Knob portion comprise the same peptide linker.

57. The multispecific construct of any one of claims 40 to 56, wherein: The Hole and Knob parts contain sdAb fused to the C-terminus or N-terminus of IgG4-Fc via a linker.

58. The multispecific construct of claim 40, wherein: The Knob into Hole construct consists of two polypeptides, each of which has SEQ ID NO: 93 and 101; 94 and 102; 95 and 103; 96 and 104; 113 and 121; 114 and 122; 115 and 123; 116 and 124; 439 and 438; 441 and 440; 443 and 442; 445 and 444; 447 and 446; 449 and 448; 451 and 450; or 106 and 437.

59. The multispecific construct of claim 40, wherein: The Knob into Hole construct consists of two polypeptides having SEQ ID NOs: 94 and 102, respectively.

60. The multispecific construct of any one of claims 40 to 59, wherein: The amino acid sequences of the sdAb in the Hole and Knob parts were humanized.

61. The multispecific construct according to any one of claims 40 to 61, in, The sdAb amino acid sequences of the hole and knob portions have amino acid substitutions in the amino acid sequences outside the CDR regions. Wherein, each framework of the sdAb amino acid sequence contains at most 3 amino acid substitutions.

62. A monomeric sdAb capable of binding to IgE, comprising binding regions CDR1, CDR2 and CDR3, each of which comprises or consists of an amino acid sequence determined according to the Kabat, Chothia, IMTG or Aho numbering scheme, the amino acid sequence being determined in an amino acid sequence selected from any one of the following: SEQ ID NO: 5, 9, 13, 17, 21, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69 and 73, Optionally, wherein CDR1 contains 1, 2 or 3 amino acid substitutions, CDR2 contains 1, 2 or 3 amino acid substitutions, and CDR3 contains 1, 2 or 3 amino acid substitutions.

63. The monomeric sdAb of claim 62, comprising binding regions CDR1, CDR2 and CDR3, each of which has SEQ ID NO: 6, 7 and 8; 10, 11 and 12; 14, 15 and 16; 18, 19 and 20; 22, 23 and 24; 26, 27 and 28; 30, 31 and 32; 34, 35 and 36; 38, 39 and 40; 42, 43 and 44; 46, 47 and 48; 50, 51 and 52; 54, 55 and 56; 58, 59 and 60; 62, 63 and 64; 66, 67 and 68; 70, 71 and 72; or 74, 75 and 76, Each CDR is identified according to the Aho numbering scheme. Optionally, wherein CDR1 contains 1, 2 or 3 amino acid substitutions, CDR2 contains 1, 2 or 3 amino acid substitutions, and CDR3 contains 1, 2 or 3 amino acid substitutions.

64. The monomeric sdAb of claim 62 or 63, wherein 1, 2 or 3 amino acid substitutions in CDR1, CDR2 and CDR3 are conservative substitutions.

65. The monomeric sdAb of claims 62-64, wherein one, two or three amino acid substitutions in CDR1, CDR2 and CDR3 are conservative substitutions selected from the group consisting of: Ala to Gly; Ala to Ser; Arg to Lys; Asn to Gln; Asn to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala; Gly to Pro; His to Asn; His to G ln; Ile to Leu, Ile to Val; Leu to Ile; Leu to Val; Lys to Arg; Lys to Gin; Lys to Glu; Met to Leu; Met to Tyr; Met to Ile; Phe to Met; Phe to Leu; Phe to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; Phe to Val; Phe to Ile; and Phe or to Leu.

66. The monomeric sdAb of any one of claims 62 to 65, comprising or consisting of an amino acid sequence selected from any one of SEQ ID NOs: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69 and 73 or a humanized amino acid sequence thereof.

67. A polypeptide, a sequence portion of which comprises one or more monomeric sdAbs according to any one of claims 62 to 66.

68. A pharmaceutical composition comprising a multispecific construct according to any one of claims 1 to 61, one or more monomeric sdAbs according to any one of claims 62 to 66, or a polypeptide according to claim 67, wherein The composition further comprises a pharmaceutically acceptable carrier and / or vehicle and / or diluent and / or excipient.

69. The pharmaceutical composition according to claim 68, wherein The composition is in liquid, semisolid or solid dosage form.

70. The pharmaceutical composition according to claim 68 or 69, wherein The composition is formulated for administration by injection, such as intramuscular injection.

71. A method for treating or preventing an IgE-related disease or condition, the method comprising administering an effective amount of The multispecific construct according to any one of claims 1 to 61, or one or more monomeric sdAbs according to any one of claims 62 to 66, and The polypeptide according to claim 67, or A pharmaceutical composition according to any one of claims 68 to 70.

72. A multispecific construct according to any one of claims 1 to 61 or one or more monomeric sdAbs according to any one of claims 62 to 66, or a polypeptide according to claim 67 or a pharmaceutical composition according to any one of claims 68 to 70 for use as a medicament, preferably for anti-IgE therapy, e.g., for the treatment or prevention of an IgE-related disease or disorder.

73. Use of the following substances in the manufacture of medicines: The multispecific construct according to any one of claims 1 to 61, or One or more monomeric sdAbs according to any one of claims 62 to 66, or The polypeptide according to claim 67, or The pharmaceutical composition according to any one of claims 68 to 70, The drug is, for example, a drug for anti-IgE therapy or for treating or preventing IgE-related diseases or disorders.

74. The method of claim 71, the use of the multispecific construct of claim 72, or the use of claim 73, wherein The IgE-related disease or disorder is any one selected from the following: Allergic asthma, allergic rhinitis (including seasonal allergic rhinitis and perennial allergic rhinitis), nasal polyps, atopic dermatitis, conjunctivitis, anaphylaxis, laryngitis laryngosacchariosis, food allergy, allergy to non-food related substances (including venom of insects, wasps, bees or spiders), allergy to therapeutic drugs including antibiotics, allergic bronchopulmonary aspergillosis and other aspergillosis-related conditions, anaphylaxis, bullous pemphigoid, pemphigus vulgaris, urticaria (including chronic urticaria, chronic spontaneous urticaria), nasal polyposis, chronic sinusitis and mastocytosis.

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