Displacers of IgE-FcERI

Abstract

Disclosed is a single-domain antibody-based construct that is useful for treating IgE-related disorders or conditions by promoting the dissociation of IgE from the high-affinity IgE receptor. Also disclosed are compositions comprising the construct. Further provided are nucleic acids and vectors, as well as methods of treating IgE-related disorders.

Description

Technical Field

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

Background Art

[0002] Allergic diseases are inflammatory disorders in which immunoglobulin E (IgE) antibodies play a key role. IgE recognizes allergens via its Fab region, but its effector functions are controlled through interactions of its 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 0.01 - 0.1 nM) at a site involving the Cε3 domain of IgE-Fc, providing a basis for long-term stability and a half-life of approximately 10 days on effector cells (Chang et al., 2000). IgE binds to CD23 with much lower affinity at a site involving both the Cε3 and Cε4 domains of IgE-Fc. Cross-linking of FcεRI-bound IgE by allergen binding causes activation and degranulation of effector cells (e.g., mast cells and basophils), leading to the release of histamine and other inflammatory mediators, and the synthesis of numerous cytokines and other factors that can generate an inflammatory response. IgE is also associated with CD23 present on cell types including B cells, macrophages, platelets, and epithelial cells, and plays several roles, for example, in the regulation of IgE synthesis, allergen transcytosis, transport of IgE:allergen immune complexes through the gut and airways, and promotion of antigen presentation on antigen-presenting cells.

[0003] Recent insights have revealed that due to the conformational flexibility of the Cε3 domain of IgE, IgE cannot bind to both types of receptors simultaneously (Holdom M., 2011, Dhaliwal et al., 2012). FcεRI binds to IgE only when the Cε3 domain adopts a so-called "open" structure, while CD23 binds to IgE only when the Cε3 domain adopts a so-called "closed" structure. This conformational selectivity has been further elucidated using engineered IgE-Fc Cε3-4 (IgE-Fc Cε3-4 335) with a disulfide bond at the position of Cys-335 that maintains IgE-Fc in the "closed" structure. FcεRI can bind to this construct only after the disulfide bond has been reduced (Wurzburg et al., 2012). Importantly, FcεRI stabilizes IgE in the open structure, thereby preventing the binding of CD23 to IgE. Conversely, the binding of CD23 to IgE stabilizes the closed structure, thereby preventing the binding of FcεRI to IgE. This makes the binding of both receptors to IgE mutually exclusive and prevents the overlap of the two pathways (Drinkwater et al., 2014).

[0004] IgE adopts a compact and bent three-dimensional structure, which was revealed from the crystal structure of IgE Fc Cε2-Cε4 (PDB: 2WQR), indicating that the Cε2 domain folds back in a state of extensive contact with the Cε3 domain and even the Cε4 domain. However, molecular dynamics simulations and biophysical studies have revealed a transiently extended three-dimensional structure in which the Cε2 domain "flips" from one side to the other. Furthermore, this energetically unfavorable extended three-dimensional structure has recently been stabilized by two anti-IgE Fab fragments, resulting in FcεRI inhibition and revealing a potential new mode of action for anti-IgE molecules (Drinkwater et al., 2014).

[0005] However, there is only one therapeutic anti-IgE antibody approved for the treatment of allergic conditions, including asthma, nasal polyps, and chronic spontaneous urticaria (CSU) [omalizumab, Xolair® (registered trademark) sold by Novartis / Genentech]. This is a humanized monoclonal IgG1 antibody that binds to the Cε3 domain of free IgE, thereby inhibiting the binding of IgE to both FcεRI and CD23. As a result, the binding of circulating IgE to both the FcεRI receptor and the CD23 receptor is impaired. Omalizumab exhibits several drawbacks, including the need for frequent administration (e.g., every 2-4 weeks), high-volume injections, immunogenicity, and the formation of immune complexes. Since omalizumab only addresses free IgE, treatment with omalizumab may not be able to effectively prevent all types of allergic reactions, such as anaphylactic reactions. It has been shown that even trace amounts of allergen-specific IgE bound to FcεRI can, for example, cause mast cell degranulation and lead to severe allergic reactions.

[0006] The concept of specifically targeting IgE as a therapeutic concept has received high interest in recent years. For example, certain antibodies and antibody mimetics have been found to be able to enhance the dissociation of bound IgE from FcεRI and CD23. In other words, these antibodies are thought to be able to disrupt the IgE:FcεRI complex (Balbino et al., 2018).

[0007] DARPin® is a scaffold of small, highly stable non-antibody proteins. Some anti-IgE DARPins have the advantage of not only neutralizing free IgE but also actively disrupting already formed IgE:FcεRI complexes through an accelerated dissociation mechanism. Compared to omalizumab with poor displacement activity, anti-IgE DARPins have been shown to be more than 10,000-fold more effective than omalizumab in both in vitro and ex vivo studies (Kim B et al., 2012 and Baumann, M.J. 2010).

[0008] Bispecific (dual-specificity) anti-IgE DARPins (e.g., DARPin bi53_79) engineered by linking two different anti-IgE DARPins have been shown to be far more effective than DARPins that bind to a single epitope (Eggel et al., 2014). Recently, Pennington et al. reported a fast-acting anti-IgE DARPin molecule (KIH_E07_79) with high dissociation enhancement that has the potential to treat anaphylaxis and rapidly desensitize allergic individuals without the risk of falling into anaphylaxis-inducing activity (Pennington et al. 2021). The fast-acting anti-IgE DARPin (KIH_E07_79) is IgG1-Fc fused to a bispecific DARPin via a short peptide linker using a knob into hole mutation strategy. International Patent Application WO2022 / 061240 discloses fast-acting anti-IgE DARPins with dissociation enhancement activity.

[0009] Single domain antibodies (sdAbs), also known as Nanobodies®, are the antigen-binding portions (VHH) of heavy chain antibodies present in camelid species and cartilaginous fish. SdAb026 is a humanized sdAb derived from llama described in International Patent Applications WO2012 / 175740 and WO2014 / 087010.

[0010] sdAb026 has been shown to promote 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 does not significantly overlap with the FcεRI-binding site. sdAb026 inhibits the interaction of IgE with FcεRI by capturing IgE-Fc in a closed conformation that mimics CD23 binding (Jabs et al., 2018). A bispecific sdAb (ALX-0962) targeting IgE and human serum albumin to obtain an extended plasma half-life neutralizes soluble (free) IgE with higher potency than omalizumab and has a dual-mode anti-IgE action by binding to and enhancing the dissociation of pre-formed IgE:FcεRI complexes on basophils (Rinaldi et al., 2014).

[0011] Further anti-IgE sdAbs are disclosed in patent applications 2004 / 041867, WO2020 / 208177, CN113461823 and CN111875706. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0012] The present inventors have addressed the unmet clinical needs for new treatment and prevention options for allergic diseases, and in particular provide an immediate-acting anti-IgE construct. MEANS FOR SOLVING THE PROBLEMS

[0013] The inventors have found that by combining at least one single domain antibody (sdAb) having a moderate ability to enhance the dissociation of IgE from the high affinity receptor with another sdAb that can bind to IgE but has no or low dissociation enhancing activity, a highly potent anti-IgE construct can be provided. Such a combination of sdAbs improved the dissociation enhancing activity even when provided as a simple mixture of the individual sdAbs. By either use as a short peptide chain or an Fc fusion protein, a highly potent displacer can be provided by linking two sdAbs. Methods for providing novel sdAbs with IgE binding activity or dissociation enhancing activity are shown in Examples 1 and 2, and the ability of the novel sdAbs to enhance the dissociation of IgE from its high affinity receptor, either alone or in combination with another sdAb, has been evaluated by the use of a simple ELISA assay (Example 6), and the results are listed in Table 15 of Example 6. In particular, with few exceptions, the combined sdABs exhibit improved dissociation enhancing activity compared to either or both of the two sdAbs in the combination. Interestingly, the inventors have been able to obtain high and rapid dissociation enhancing activity by incorporating the combined sdABs into a multispecific construct with the structural elements shown in FIG. 1 and by the use of various linker moieties that can impose various degrees of constraint on the free mobility or orientation of each of the two sdAbs. Importantly, the dissociation enhancing activity can be readily addressed by using an ELISA assay commonly known to those skilled in the art, and thus it can be easily evaluated whether an sdAb with IgE binding affinity can be used in the constructs disclosed herein. Furthermore, the inventors have evaluated the anaphylaxis-inducing activity both in vitro and in a mouse model.

[0014] In a first aspect, the invention provides a. a first single domain antibody (sdAb) capable of enhancing the dissociation of bound IgE from FcεRI, b. A second sdAb that binds to IgE, c. A moiety that links the first and second sdAbs comprising, - The first and second sdAbs bind to non-identical epitopes of IgE, - relates to a multispecific construct that can enhance the dissociation of bound IgE from the IgE high-affinity receptor (FcεRI) by dissociation-enhancing activity improved compared to the first or second sdAb.

[0015] In a 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 in which the Fc domain is directly linked to the first and second sdAbs or linked via a linker (a simple peptide chain).

[0016] A second aspect relates to a novel monomeric sdAb capable of binding to an IgE antibody and is suitable for use in the multispecific constructs of the first aspect. Such an sdAb can have dissociation-enhancing activity by itself or can promote dissociation-enhancing activity in combination with another sdAb. Exemplary monomeric sdAbs are listed in Table 1 (full-length amino acid sequence, camelid version), and Tables 2a-d show the regions of the respective complementarity-determining regions (CDRs), CDR1, CDR2, and CDR3 of the novel monomeric sdAbs. Several methods can be applied to determine the CDR regions, and the resulting CDR regions may vary depending on the method. Tables 2a-d show the CDRs of the novel sdAbs according to the determination schemes of Aho, Kabat, Chotia, or IMGT.

[0017] When using the Aho method to determine the CDR regions, exemplary monomeric sdAbs can, in a second aspect, include combinations of CDR1, CDR2, and CDR3 having SEQ ID NOs: 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 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.

[0018] A third aspect relates to a pharmaceutical composition comprising the multispecific construct of the first aspect disclosed herein and any of its embodiments, or a pharmaceutical composition comprising one or more monomeric sdAbs of the second aspect disclosed herein and any of its embodiments, the pharmaceutical composition comprising a pharmaceutically acceptable carrier and / or medium and / or diluent and / or excipient.

[0019] Yet another aspect is a method for treating or preventing an IgE-related disease or condition, the method comprising administering an effective amount of a) the multispecific construct of the first aspect of the invention disclosed herein and any of its embodiments, b) one or more monomeric sdAbs of the second aspect of the invention disclosed herein and any of its embodiments, or c) the pharmaceutical composition of the third aspect of the invention disclosed herein and any of its embodiments.

[0020] Yet another aspect relates to a) a multispecific construct of the first aspect of the invention disclosed herein and any of its embodiments, b) one or more monomeric sdAbs of the second aspect of the invention disclosed herein and any of its embodiments, or c) a pharmaceutical composition of the third aspect of the invention disclosed herein and any of its embodiments, for use as a medicament, preferably for use in anti-IgE therapy, for example for the treatment or prevention of IgE-related diseases or conditions.

[0021] Yet another aspect relates to the use of a multispecific construct of the first aspect of the invention disclosed herein and any of its embodiments, one or more monomeric sdAbs of the second aspect of the invention disclosed herein and any of its embodiments, or a pharmaceutical composition of the third aspect of the invention disclosed herein and any of its embodiments, in the manufacture of a medicament for use in anti-IgE therapy or for the treatment or prevention of IgE-related diseases or conditions, for example. BRIEF DESCRIPTION OF THE DRAWINGS

[0022]

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Mode for Carrying Out the Invention

[0023] Definition The term "multispecific construct" is intended to define a compound, molecule or complex that can bind to at least two distinct epitopes on IgE, particularly to the same IgE antibody. Thus, the use of the term multispecific is intended to indicate that the construct can bind to more than one epitope of an antigen and is thus paratopic. If a construct can bind to two distinct IgE epitopes, the construct may be termed a bispecific construct. In this context, the at least two distinct epitopes preferably are present outside the antigen-binding region of IgE, such as on IgE-Fc. Thus, in this context, a multispecific construct is understood not to bind to several different antigens, but rather to be able to bind to the same antigen (IgE) at several distinct surface-exposed sites that are normally found on the Fc portion of IgE. However, a multispecific construct can interfere with other biological targets. By way of example, constructs can be mentioned that contain an Fc domain that can bind to or interfere with targets other than IgE.

[0024] The phrase "a multispecific construct can enhance the dissociation of bound IgE from the IgE high-affinity receptor (FcεRI receptor)" is intended to define that a multispecific construct can enhance the dissociation of IgE from FcεRI, for example, by a facilitated dissociation mechanism, conformational change and / or steric hindrance.

[0025] The terms "dissociation enhancing activity" or "dissociation enhancing effect" are interchangeable terms, both intended to define the ability of a compound (e.g., any of the compounds including sdAbs such as monomeric sdAb, combined sdAB, linked sdAb, or multispecific constructs described herein) to enhance the dissociation of IgE from its high affinity receptor FcεRI. The ability of such a test compound to enhance the dissociation of bound IgE from FcεRI can be evaluated by use of the ELISA-based IgE-FcεRIα dissociation enhancement assay described in Example 6 herein. Briefly, the assay is based on measuring the residual IgE not removed from immobilized recombinant human FcεRIα preloaded with IgE when a test compound (e.g., an sdAb or multispecific construct described herein) is added. The test results can be provided as the percentage of the dissociation enhancing effect calculated as the relative decrease in signal compared to a control without the test compound added, and the test compound can be applied at various concentrations. Subsequently, the concentration resulting in a half effect of dissociation enhancement can be determined (the EC 50 , i.e., the EC 50 is the molar concentration of the test compound capable of dissociating 50% of the IgE preloaded on the FcεRIα receptor). In addition, the dissociation enhancing activity can be evaluated by determining the percentage of IgE that can be maximally dissociated from a pool of IgE preloaded on the FcεRIα receptor (maximum dissociation enhancing activity), and to what extent the maximum effect is achieved (100% of the preloaded IgE is dissociated).

[0026] The dissociation enhancing activity can also be evaluated by other assay types, in vitro or ex vivo assays, and an in vivo mouse model can be used for the evaluation of the dissociation enhancing activity. Such methods are known to those skilled in the art (Eggel et al., 2014, Pennington et al., 2021 and Jabs et al., 2018).

[0027] The dissociation enhancing effect is mentioned in patent application WO2012 / 175740 and can be compared with known displacer (comparative control displacer) such as sdAb026 named (A1) herein or Darpin-based construct KIH_E7_79 (in Pennington et al., 2021 and patent application WO2022 / 061240).

[0028] A "single domain antibody" (sdAb), sometimes also called a "nanobody", is an antibody fragment in the form of a single monomeric variable antibody domain that can selectively bind to a specific antigen. Conventionally, an sdAb is an antibody fragment of only the heavy chain containing the VH domain of the antibody. SdAb binds to an antigen using only three complementarity-determining regions (CDR1, CDR2, and CDR3), rather than the six complementarity-determining regions present in conventional VH:VL antibodies. However, sdAbs may in some cases be derived from common antibodies such as IgG, but such an approach requires the inactivation of the natural dimerization of the VH and VL domains by amino acid substitution, which means that antigen affinity is often impaired. Usually, sdAbs are relatively short, for example, 100 - 130 amino acid residues.

[0029] Furthermore, an sdAb is an antibody in which the CDR is part of a single-domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies that are naturally lacking in light chains, single-domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and scaffolds of single domains other than those derived from antibodies. SdAbs can be derived from any species including, but not limited to, mouse, human, camel, llama, goat, rabbit, and / or cow.

[0030] In some embodiments, the sdAb used herein is a variable domain derived from a naturally occurring heavy-chain antibody lacking a light chain. For clarity, such variable domains derived from antibodies consisting of only heavy chains are known as VHH domains to distinguish them from the conventional VH of the four-chain immunoglobulin. As used herein, the term sdAb is interchangeable with VHH antibody. When the sdAb is part of a fusion protein / fusion construct, the term sdAb may be interchangeable with the VHH domain. Such VHH domains can be derived from antibodies raised in animals capable of producing antibodies consisting of only heavy chains. Examples include species of the Camelidae family, such as camels, llamas, dromedaries, alpacas, and guanacos. In addition to Camelidae, other species may produce antibodies consisting of only heavy chains, and such VHHs are within the scope of the present disclosure. Thus, in any construct described herein, when the sdAb is part of a larger construct, the term sdAb is interchangeable with the term VHH.

[0031] The binding region of the sdAb herein is intended to include one or more or all of the three complementarity-determining regions (CDR1, CDR2, and CDR3).

[0032] "Complementarity-determining region" or "CDR" is the variable part of an antibody, including sdAb, and is thus extremely important for diversity. Not all residues within the CDR can be involved in epitope binding. For example, the cysteines in CDR1 and CDR3 of sdAb are crucial for the structure (Pellis et al., 2012).

[0033] As described above, sdAbs and VHHs are used interchangeably herein. In some embodiments, an sdAb comprises three CDRs and four framework regions (FRs), arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, so long as the VHH / sdAb substantially maintains the desired antigen binding and specificity, the sdAb can be truncated at the N-terminus or C-terminus, such that it may contain only partial FR1 and / or FR4, or may lack one or both of these FRs.

[0034] As described above, the primary purpose of the CDR regions is to define the regions that have specificity for an epitope. However, not all residues within a CDR can participate in epitope binding and may simply define structural elements (Wilton E. et al 2018 ref 6). Additionally, residues in the FR adjacent to the CDR can affect binding, such that the FR can, but is not limited to, participating in the structure.

[0035] A variety of methods can be applied to determine the positions of the various framework and CDR regions of an sdAb.

[0036] One method is the "Kabat numbering scheme" or "Kabat", first described by the American scientist Elvis Kabat. Herein, the amino acid residues of an immunoglobulin single variable domain are numbered according to the general numbering of the VH domain given by Kabat et al and can be applied to the VHH domain from camelids as described by Riechmann and Muyldemans (Riechmann and Muyldemans, 1999).

[0037] In the CDR determination by Kabat, FR1 of VHH contains amino acid residues at positions 1 to 30, CDR1 of VHH contains amino acid residues at positions 31 to 35, FR2 of VHH contains amino acids at positions 36 to 49, CDR2 of VHH contains amino acid residues at positions 50 to 65, FR3 of VHH contains amino acid residues at positions 66 to 94, CDR3 of VHH contains amino acid residues at positions 95 to 102, and FR4 of VHH contains amino acid residues at positions 103 to 113.

[0038] It should be noted that - as is well known in the art with respect to the VH domain and with respect to the VHH domain - the total number of amino acid residues in each CDR may vary and may not match the total number of amino acid residues indicated by the Kabat numbering. That is, one or more positions according to the 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 the Kabat numbering. This generally means that the numbering according to Kabat may or may not match the actual numbering of amino acid residues in the actual sequence. The total number of amino acid residues in the VH domain and the VHH domain is usually in the range from 110 to 120 and can often be between 112 and 115. However, it should be noted that smaller and longer sequences may also be appropriate for the purposes described herein.

[0039] Another method for determining the CDR regions of VHH is by the Chothia numbering scheme. In this specification, the amino acid residues of an immunoglobulin single variable domain can be numbered using conserved amino acids that always have the same positions (Dondelinger M et al., 2018).

[0040] A further method for determining the CDR regions of VHHs is that by IMGT. In the present specification, the amino acid residues of an immunoglobulin single variable domain can always be numbered using conserved amino acids having the same positions. Examples are 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 acids at positions 39 to 55, the CDR2 of VHH contains amino acid residues at positions 56 to 65, the FR3 of VHH contains 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 the amino acid residue at position 118 and the remaining sequence. The maximum length of the CDR is as defined above. For shorter CDRs, gaps are created (Lefranc et al., 2002).

[0041] Alternatively, the CDR regions of 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 this method, FR1 contains amino acid residues at positions 1 to 25, CDR1 contains amino acid residues at positions 26 to 35, FR2 contains amino acids at positions 36 to 49, CDR2 contains amino acid residues at positions 50 to 58, FR3 contains amino acid residues at positions 59 to 94, CDR3 contains amino acid residues at positions 95 to 102, and FR4 contains amino acid residues at positions 103 to 113.

[0042] A further method for determining the CDR regions of sdAbs is by using the Aho numbering scheme (Mitchelle & Colwell, 2017 and Honegger & Pluckthun et al., 2001).

[0043] Another alternative method for determining the CDR regions of VHHs uses the sdAb-DB described by Emily Wilton in 2018. This method is based on a series of sequence-based rules for identifying the CDRs of the light and heavy chains of conventional antibodies by Pantazes and Maranas (Wilton E et al 2018 Synthetic Biology) and has been adopted for sdAbs. In sdAb-DB, CDR1 starts 4 residues after the first cysteine, is 6 - 15 residues in length, and is followed by W. CDR2 starts 10 - 20 residues after the end of CDR1, following the sequence [I / L / V / M]-[G / A / S]. The length of CDR2 is 8 - 15 residues, and is followed by the sequence [Y / F / I / L / T / N / S / V / H]-X-X-X-[A / I / L / M / V]-[Q / K / R / A / E / G / L / T]. CDR3 is located 30 - 50 residues after the end of CDR2, following the sequence C-X-X. The length is 3 - 25 amino acids, and is followed by the sequence [W / A / E / F / H / K / L / Q / Y / G / S / R]-[G / S]-X-G-X-X-X-T-V-S. In all sequences, "X" can be any amino acid.

[0044] Alternative 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.

[0045] An "antibody", also referred to as an "immunoglobulin", is a protein in humans composed of an identical pair of light and heavy chains. The heavy and light chains each contain a variable domain and a constant region, and the variable domain is responsible for the specific binding of the antibody to an antigen. Generally, antibodies are grouped into immunoglobulin isotypes IgA, IgD, IgE, IgG, and IgM, each of which plays a different role as an antigen recognition agent in the immune system. The antigen-binding specificity of an antibody is determined by the variable region of the variable domain, particularly by the complementarity-determining regions (CDRs) of the variable domain. Generally, an antibody is a molecule that can be expressed in its native conformation in animals, in principle or in practice, which means that the expressed antibody contains all the structural elements found in naturally occurring immunoglobulins.

[0046] An "antibody variant" is a protein derived from an antibody that has the same binding specificity as the antibody but is not thought to be a natural expression product in mammals. Thus, this term refers to various fragments of an antibody as well as analog formats of artificial antibodies. This term also represents antibody formats that are naturally found in birds and reptiles, such as heavy-chain antibodies and IgY, which are rare among mammals, but in which the CDRs from mammalian antibodies or antibodies produced in combination have been engineered into antibody formats that are not originally derived from them.

[0047] A "heavy-chain only antibody" is an antibody format naturally found in camelids (e.g., camels, dromedaries, llamas, and alpacas) or cartilaginous fish (e.g., sharks, skates, rays, sawfish, etc.), and is composed of only two heavy chains lacking two light chains.

[0048] The term "anaphylaxis-inducing" is used herein to describe the effect of molecules that can activate effector cells such as basophils and mast cells in the absence of an allergen to cause degranulation (histamine release). Examples include the multispecific construct NIgG4B1A1(G4S)1 shown in Example 10, or NIgG4B1A1(G4S)3 which has been shown to activate basophils by itself in a mouse anaphylaxis model (Example 9).

[0049] The term "IgE binding affinity" is intended to designate the affinity with which a test compound (e.g., the sdAbs and multispecific constructs disclosed herein) binds to free IgE. The ability of a test compound to bind to free IgE can be tested by using the biolayer interferometry (BLI) described in Example 5 herein. Briefly, this assay measures the association and dissociation of the test molecule to IgE or alternatively to IgE-Fc. Based on these measurements, k on 、k off 、the ratio k off / k on (=K D ) can be determined. k on is a constant used to characterize how fast the test molecule binds to IgE, while k off characterizes how fast the test molecule dissociates from IgE. The ratio k off / k on results in the equilibrium dissociation constant K D . The lower the K D value, the higher the affinity of the test molecule for IgE. In an interesting embodiment, the monomeric sdAb has an improved IgE binding affinity (lower K D compared to known IgE binders such as sdAb026(A1) (comparative control IgE binder). DIn yet other interesting embodiments, the multispecific construct (comprising dual sdAbs linked by a peptide linker) exhibits improved IgE binding affinity compared to DARPin KIH-E7_79 or a construct comprising two sdAb026(A1) linked together by a peptide linker. In other embodiments, it is envisaged that the IgE binding affinity may be lower than the comparison control. Generally, K in the nanomolar range D Value, especially 3×10 -10 K in the low nM affinity range, such as less than M D Other IgE affinity binding assays can be used and are known to those of skill in the art.

[0050] The term "IgE-related disease or condition" is intended to encompass any disease or condition that would benefit from a reduction in receptor-bound IgE and / or a reduction in the level of circulating free IgE. Such 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, in particular allergic diseases with severe allergic symptoms (asthma, atopic dermatitis, urticaria) or allergic diseases in which the allergic response develops rapidly (e.g., anaphylaxis). The term "allergic disease" is known in the art of medicine. In particular, the term allergic disease is intended to be characterized by an allergic and / or atopic immune response to an antigen, such as an allergen, resulting in allergic and / or atopic symptoms in a patient suffering from the allergic disease. Allergic diseases are often characterized by the production of antigen-specific IgE antibodies, which may be a resultant biological effect of the IgE antibodies.

[0051] Specific embodiments of the present invention: SdAb / VHH domain The present invention provides novel VHH domains (sdAbs) capable of binding IgE, which are applicable for use in the first and second aspects disclosed herein, as well as further aspects disclosed herein.

[0052] The VHH domain may be expressed as a monomeric sdAb or incorporated into a larger construct (e.g., as part of a polypeptide / protein construct / fusion protein). Two or more different VHH domains may be expressed as individual monomeric sdAbs, and the monomeric sdAbs may be further linked to provide dimeric or multimeric sdAbs, which can form multispecific constructs. Two or more VHH domains may also be incorporated into a larger construct (e.g., as part of a polypeptide / protein construct / fusion protein). The sdAb may have dissociation enhancing activity by itself or may promote dissociation enhancing activity in combination with another sdAb. Of interest are sdAbs suitable for combination in the multispecific constructs described herein or alternatively in any other design that provides the desired dissociation enhancing activity.

[0053] Of interest are monomeric sdAbs that exhibit dissociation enhancing activity by themselves and preferably have improved dissociation enhancing activity (lower EC 50 and / or higher maximal effect) compared to a known displacer, e.g., sdAb026. Such sdAbs exhibiting dissociation enhancing activity can be used as the first sdAb in the multispecific constructs described herein. In a further interesting embodiment, the multispecific construct (including a dual sdAb linked by a peptide linker) has dissociation enhancing activity equivalent to or improved over DARPin KIH-E7_79 or alternatively monomeric sdAb026, dimeric sdAb026 (two sdAb026s linked via a peptide linker), or an Fc fusion construct having two sdAb026s (a bivalent construct) (lower EC 50and / or exhibit a higher maximum effect).

[0054] The monomeric sdAb (alternatively “VHH domain”) of the present invention can be obtained as a fragment of an antibody obtained from a camelid immunized with full-length human IgE or a part of said 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), in which case an artificial disulfide bridge is constructed between the two chains and an additional cysteine residue at position 335 is incorporated to obtain IgE or any combination thereof in a closed structure. Such monomeric sdAbs of camelids can undergo further affinity maturation, humanization or other sequence variations to obtain sdAbs with desired properties.

[0055] The monomeric sdAb can bind to an IgE epitope outside the variable domain of IgE, such as particularly within the Fc region of IgE, such as within IgE-Fc Cε2-Cε4. Of interest are sdAbs that enhance the dissociation of the binding of IgE to FcεRI and / or have a high binding affinity for IgE. However, it is not excluded that the monomeric sdAbs disclosed herein (e.g., the second sdAb) can bind to the C L domain of IgE.

[0056] The IgE-binding region of the sdAb contains CDRs, which are the specific regions where the sdAb normally binds to its target. A single sdAb has three CDRs, CDR1, CDR2 and CDR3, and one or more of the CDRs can be combined to serve as the key responsible for the activity of the sdAb. The individual CDR regions can be determined through various methods, including those described above such as Aho, Kabat or AbM.

[0057] Exemplary novel monomeric sdAbs that can bind 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 by an ELISA-based IgE-FcεRIα dissociation enhancement assay, only the sdAbs named A2 and B1 exhibited dissociation enhancement activity (Example 6, Table 15). A2 is a mutant of sdAb026 (named A1 herein) and exhibits lower EC 50 dissociation enhancement activity than the comparative control A1, while maintaining more than 90% of the maximum activity. SdAb B1 also exhibited improved dissociation enhancement activity even at an EC 50 state, but was only able to enhance the dissociation of 60% of the pre-loaded IgE (the maximum dissociation enhancement effect was obtained at 60% dissociation of IgE). Therefore, B1 can be considered less preferable 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 without or with poor dissociation enhancement activity (in the case of B1) can be successfully used as the second sdAb in the multispecific constructs herein to obtain compounds with high dissociation enhancement activity.

[0058] Table 1 provides the full-length amino acid sequences of the monomeric sdAbs (camelid version) along with information on the IgE constructs used for immunization.

[0059]

Table 1

[0060] Thus, in some embodiments, the sdAb can 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. Since such sdAbs may be subject to affinity maturation, humanization, or other amino acid changes of the amino acid sequence, further embodiments relate to sdAbs having an amino acid sequence that is at least 80%, 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, said mutations in the amino acid sequence are not present within the CDR regions and thus are present only within the combined framework regions. Typically, such mutations are introduced for the purpose of affinity maturing and / or humanizing the sdAb. Thus, the novel sdAbs can 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, including their affinity matured variants and / or humanized variants.

[0061] Furthermore, the novel sdAbs can be defined according to and are not limited to the individual CDR regions derivable from the entire sdAb amino acid sequence, which can be determined by different numbering schemes such as Kabat, Chothia, IMTG, or Aho.

[0062] Thus, in one embodiment, the sdAb comprises or consists of binding regions CDR1, CDR2, and CDR3 each having 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, determined according to any of the Kabat, Chothia, IMTG, or Aho numbering schemes, wherein CDR1 may contain 1, 2, or 3 amino acid substitutions, CDR2 may contain 1, 2, or 3 amino acid substitutions, and / or CDR3 may contain 1, 2, or 3 amino acid substitutions. The substitutions are intended to be incorporated in consideration of CDR1, CDR2, and CDR3 directly determined by any of Kabat, Chothia, IMTH, or Aho in said sequences.

[0063] Specific CDR regions of the novel sdAb.

[0064] [Table 2]

[0065] [Table 3]

[0066] [Table 4]

[0067] [Table 5]

[0068] Exemplary monomeric sdAbs include CDR1 having an amino acid sequence of SEQ ID NOs: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, or 74, each amino acid sequence can have 1, 2, or 3 amino acid substitutions, and the CDR regions are determined by Aho.

[0069] Exemplary monomeric sdAbs 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, each amino acid sequence can have 1, 2 or 3 amino acid substitutions, and the CDR regions are determined by Aho.

[0070] Exemplary monomeric sdAbs 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, each amino acid sequence can have 1, 2 or 3 amino acid substitutions, and the CDR regions are determined by Aho.

[0071] Alternatively, exemplary monomeric sdAbs A2, B1, B2, B3, D1, D2, D3, E1, E2, E3, E4, F1, F2, F3, F4, F5, F6 and G1 can be defined by their specific CDR regions determined by Kabat as shown in Table 2b, by Chothia as shown in Table 2c, or by IMGT as shown in Table 2d.

[0072] As used herein, sdAbs can be defined according to their binding region CDR1, CDR2 and CDR3 determined in the VHH region of the parental version, for example, the camelid version. In any of the embodiments disclosed herein, each CDR1, CDR2 and CDR3 can optionally undergo sequence variations such as substitution, deletion or addition of 1, 2 or 3 amino acids. The purpose of introducing the mutations can be to improve the binding activity or dissociation enhancing activity of IgE compared to a comparative control IgE binder or displacer.

[0073] Thus, in all of the following embodiments, CDR1 can contain 1, 2, or 3 amino acid substitutions, CDR2 can contain 1, 2, or 3 amino acid substitutions, CDR3 can contain 1, 2, or 3 amino acid substitutions, e.g., can contain 1 or 2 amino acid substitutions. The resulting variant sdAb can be evaluated by determining its binding affinity to IgE Fc or by determining its dissociation enhancing activity by an ELISA-based IgE-FcεRIα dissociation enhancement assay. Desired are variants that have an activity equivalent to the parental sdAb, or variants that have an improved IgE affinity or an improved dissociation enhancing activity.

[0074] More specifically, the sdAb comprises binding regions CDR1, CDR2, and CDR3 having SEQ ID NOs: 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, respectively, with each CDR determined according to the Aho numbering scheme.

[0075] In another embodiment, the sdAb comprises complementarity determining regions CDR1, CDR2 and CDR3 having respective SEQ ID NOs: 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, and each CDR is determined according to the Kabat numbering scheme.

[0076] In a further embodiment, the sdAb comprises complementarity determining regions CDR1, CDR2 and CDR3 having respective SEQ ID NOs: 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, and each CDR is determined according to the Chothia numbering scheme.

[0077] In yet a further embodiment, the sdAb comprises complementarity determining regions CDR1, CDR2, and CDR3 having SEQ ID NOs: 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, respectively, where each CDR is determined according to the IMTG numbering scheme.

[0078] The monomeric sdAbs, either alone or in combination, may be able to improve biological activity (e.g., dissociation enhancing activity), may be able to improve the properties of a drug (e.g., improve serum circulation half-life, improve biodistribution), or may also be able to establish binding to additional therapeutic targets other than IgE.

[0079] As described, the individual sdAbs A1, A2, and B1 were able to enhance the dissociation of IgE from its high affinity receptor. Thus, interesting embodiments include sdAbs that can be used as the first sdAb in the multispecific constructs described herein.

[0080] According to an interesting embodiment, the sdAb has the binding region of A1 (shown in Tables 2a-d). Thus, the sdAb has SEQ ID NOs 2, 3, and 4 determined according to Aho; 129, 130, and 131 determined according to Kabat; 186, 187, and 188 determined according to Chothia; or 243, 244, and 245 determined according to IMGT; It can include complementarity determining regions CDR1, CDR2 and CDR3 having

[0081] According to another interesting embodiment, the sdAb has the binding region of A2. Thus, the sdAb has, according to each of SEQ ID NOs 6, 7 and 8 determined according to Aho; 132, 133 and 134 determined according to Kabat; 189, 190 and 191 determined according to Chothia; or 246, 247 and 248 determined according to IMGT; It can include complementarity determining regions CDR1, CDR2 and CDR3 having

[0082] According to another interesting embodiment, the sdAb has the binding region of B1. Thus, the sdAb has, according to each of SEQ ID NOs 10, 11 and 12 determined according to Aho; 135, 136 and 137 determined according to Kabat; 192, 193 and 194 determined according to Chothia; or 249, 250 and 251 determined according to IMGT; It can include complementarity determining regions CDR1, CDR2 and CDR3 having

[0083] Furthermore, as seen in Examples 6 and 7, the monomeric sdAbs described herein show an improvement in the activity of enhancing dissociation when used, for example, in pairs of two individual (unlinked) monomeric sdAbs. For example, when A1 and B1 are administered in combination without being linked together, the ability to enhance the dissociation of IgE from its receptor is improved as compared to the individual sdAbs administered alone. In particular, combining two sdAbs both having individual dissociation-enhancing activity also results in improved dissociation-enhancing activity. A combination of a dissociation-enhancing sdAb and a sdAb that does not enhance dissociation exhibits a dissociation-enhancing activity equivalent to that of the combination of A1 and B1 (Examples 6, Table 15 and Example 7, Table 16).

[0084] In further embodiments, any of the monomeric sdAbs described herein can be combined in the form of a pair of two sdAbs, or as three, for example four sdAbs, and one of the sdAbs exhibits dissociation-enhancing activity. In a preferred embodiment, the sdAbs are combined in the form of two pairs. However, any other non-IgE-binding sdAb can be combined with an IgE-binding sdAb to modify biological properties or kinetics, for example, in combination with an sdAb that can bind to albumin, the excretion rate in vivo can be reduced.

[0085] Combinations of sdAbs and preferred embodiments will be further described below.

[0086] Modification of the amino acid sequence of sdAb Any amino acid sequence of the FR, either within or outside the CDR, can be subject to amino acid substitution, insertion, or deletion for the purpose of modifying biological activity, expression level, stability, or other functional properties. In a preferred embodiment, amino acid changes are incorporated only outside the CDR region.

[0087] 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 region of the camelid sdAb cannot be changed when affinity maturing or humanizing the camelid sdAb. In such embodiments, the sdAb is composed of an affinity matured human or humanized amino acid sequence, but not in the CDR region involved in binding to the target.

[0088] In some embodiments, one or more of the CDRs of the sdAb (i.e., CDR1, CDR2, and / or CDR3) can independently undergo amino acid substitutions, for example, by substitution of 1, 2, 3 or more amino acid residues. The amino acid substitutions in the CDRs can be conservative amino acid substitutions. A "conservative" amino acid substitution is generally an amino acid substitution that replaces an amino acid residue with another amino acid residue of similar chemical structure and / or charge, which has little or no substantial effect on the function, activity, or other biological properties of the binding region resulting from the sdAb or multispecific construct described herein. Such conservative amino acid substitutions are well known in the art. For example, conservative substitutions are preferably substitutions in which one amino acid residue within one of the following groups (a)-(e) is replaced by another amino acid residue within 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; and (e) aromatic residues: Phe, Tyr, and Trp.

[0089] 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 to Leu.

[0090] The conserved amino acid substitutions can also be made in the amino acid sequences outside the CDRs.

[0091] In other embodiments, the amino acid substitutions in the amino acid sequences of the CDRs can be carried out for the purpose of providing sdAbs with modified, e.g., improved, affinities for binding to IgE, enhancing dissociation of IgE from FcεRI, or binding to another target of interest. The amino acid sequences of the CDRs are generally more decisive with respect to target affinity than the amino acid sequences outside the CDRs. The amino acid sequences of the CDRs are generally far more decisive for target affinity than the amino acid sequences outside the CDRs. Thus, the sdAbs described herein can also be subjected to affinity maturation by introducing one or more changes into the amino acid sequences of one or more of the CDRs, which results in a modification, e.g., improvement, of the affinity of the resulting sdAb for IgE as compared to the first (parent) sdAb. Methods for affinity maturation of sdAbs can be prepared by methods known in the art, such as those described in patent application WO2012 / 175740.

[0092] There may also be amino acid substitutions, insertions or deletions in one or more FRs outside the CDRs. Specifically, humanizing substitutions can be made (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 present at the corresponding positions within the same domain from a conventional human antibody). Potentially useful humanizing substitutions can be identified by comparing the sequence of the framework region of the naturally occurring V HH sequence with the corresponding framework sequences of one or more closely related human V HH sequences, and then introducing one or more (or combinations thereof) of the potentially useful humanizing substitutions thus determined into the V HH sequence (in any manner known per se), resulting in a humanized V HHArrays can be tested for affinity to a target (IgE binding), IgE dissociation enhancing activity, stability, ease and level of expression, and / or other desired properties. Thus, the sdAb may be partially humanized or fully humanized. Methods for humanizing sdAbs have been previously described by Rossoti et al. (Rossoti et al., 2021) and Traian S (Traian S., 2022).

[0093] Any amino acid substitutions in the amino acid sequence outside the CDRs may typically result in sdAbs with a lower modified biological activity compared to substitutions in the CDRs. However, any changes (e.g., deletions, insertions, and / or substitutions) in the amino acid sequence of the sdAb can also be designed to improve the expression level depending on the host organism used to express the sdAbs or multispecific constructs described herein. For example, the changes can be designed within the capabilities of those skilled in the art in such a way that one or more sites of post-translational modification (e.g., one or more glycosylation sites) are removed. Alternatively, the substitutions or insertions can be designed to introduce one or more sites for attachment of functional groups, for example, to allow insertion of an affinity tag (His-tag) or site-specific PEGylation. The possibility of N-terminal post-translational modification can be eliminated by changing the N-terminal glutamic acid (E) to aspartic acid (D). Thus, the amino acid difference can be a change of glutamic acid (Glu) to aspartic acid (Asp) at position 1 (the position determined according to Kabat numbering).

[0094] One embodiment shows how amino acid substitutions can modify the IgE-binding affinity and / or dissociation enhancing activity of the sdAbs described herein. The known monomeric sdAb026 [designated herein as A1 (SEQ ID NO: 1 having the binding regions CDR1, CDR2, and CDR3 of SEQ ID NOs: 2, 3, and 4, respectively, determined according to Aho)] was optimized for improved IgE binding and dissociation enhancing activity. A method for investigating the impact of amino acid substitutions is described in Example 4. The resulting sdAb (A2) of SEQ ID NO: 5 having the binding regions CDR1, CDR2, and CDR3 of SEQ ID NOs: 6, 7, and 8, respectively, determined according to Aho, had an improved dissociation enhancing activity by 2- to 4-fold compared to sdAb A1. In particular, the improved sdAb A1 (i.e., sdAb A2) had amino acid substitutions in one of the three CDRs as well as outside the CDRs.

[0095] In an alternative embodiment, the sdAb designated A1 (SEQ ID NO: 1) can be optimized for improved IgE binding and / or dissociation enhancing activity by performing one or more mutations selected from the list consisting of 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, D73I, D73L, D73V, D73A, D73G, D73S, D73T, D73N, D73Q, D73E, D73H, D73K, D73R, 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.

[0096] Any of the novel sdAbs disclosed herein may be subject to evaluation of amino acid mutations similar to A1 for the purpose of improving IgE binding.

[0097] In one embodiment, sdAb B2 (SEQ ID NO: 13) can be optimized for improved IgE binding by introducing one or more mutations selected from the list consisting of V12T, A23Q, A23D, T28Q, T28H, D35M, D35A, D36V, Q39F, Q39W, Q39Y, Q39I, Q39V, Q39E, Q44P, D74P, D74I, S105I, S105N, S105D, and S105E.

[0098] In another embodiment, sdAb B3 (SEQ ID NO: 17) can be optimized for improved IgE binding and / or enhanced dissociation activity by introducing one or more mutations selected from the list consisting of 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, R78M, 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.

[0099] As described above, the parental CDR regions can undergo sequence variations such as substitution, deletion, or addition of one, two, or three amino acids. The variant can have the same, improved, or enhanced IgE binding activity or dissociation enhancing activity as compared to the comparative control IgE binder or displacer. In a preferred embodiment, the dissociation enhancing activity is improved or enhanced as compared to sdAb A1 (SEQ ID NO: 1) or a multispecific construct comprising two sdAb A1s.

[0100] Multispecific construct SdAbs such as monomeric sdAb / VHH domains can be engineered into multimeric constructs such as multispecific constructs for the purpose of improving biological activity (e.g., dissociation enhancing activity), improving drugability (e.g., improving serum circulation half-life, improving biodistribution), or establishing binding to additional therapeutic targets other than IgE.

[0101] Specifically, two or more sdAbs can be combined to form a multimeric construct such as a multispecific construct, which can enhance the dissociation of bound IgE from FcεRI. Preferably, such a multispecific construct can additionally limit, prevent, or inhibit the binding of free IgE to FcεRI. Even more preferably, such a construct also limits, prevents, or inhibits the binding of free IgE to CD23. Advantageously, by combining two or more sdAbs in a multimeric construct, particularly a multispecific construct, the construct can exhibit: i) an improvement in the ability to enhance the dissociation of bound IgE from FcεRI as compared to the monomeric sdAbs present in the construct; ii) an improvement in the ability to limit, prevent, or inhibit the binding of free IgE to FcεRI, and / or iii) an improvement in the ability to limit, prevent, or inhibit the binding of free IgE to CD23.

[0102] The multispecific construct is composed of two or more sdAbs, at least one of which has dissociation enhancing activity by itself, and the two sdAbs are linked together via a linker as outlined in Figure 1.

[0103] The multispecific construct comprises a. A first single domain antibody (sdAb) capable of enhancing the dissociation of bound IgE from FcεRI, b. A second sdAb that binds to IgE, c. A moiety linking the first and second sdAbs and - the first and second sdAbs bind to non-identical epitopes of IgE, - the multispecific construct is capable of enhancing the dissociation of bound IgE from the IgE high affinity receptor (FcεRI) by virtue of improved dissociation enhancing activity compared to the first or second sdAb.

[0104] When the construct comprises at least one sdAb capable of enhancing the dissociation of bound IgE, it is assumed that a multispecific construct comprising a) a first single domain antibody (sdAb) that binds to IgE and b) a second sdAb that binds to IgE can effect the desired dissociation enhancement.

[0105] Preferably, both the first sdAb and the second sdAb bind to the same IgE antibody, for example simultaneously bind to the same IgE antibody, to avoid cross-linking of IgE bound to the IgE high affinity receptor, and thus prevent any degranulation of mast cells or basophils by the anti-IgE multispecific construct. A method for assessing that both sdAbs can bind to the same IgE molecule is described in Example 3 (Relative epitope mapping).

[0106] The binding site for 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, and 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 include at least one additional sdAb that binds to an IgE epitope different from any of the epitopes of IgE bound by the first sdAb and the second sdAb. Preferably, the additional sdAb binds to the same IgE molecule as the first and second sdAbs.

[0107] Implementing an sdAb having dissociation enhancement activity into the described multispecific construct improves the dissociation enhancement activity as compared to the individual sdAbs.

[0108] Since the sdAb is composed of the VHH domain of a camelid antibody, the multispecific construct can alternatively be expressed as follows. The multispecific construct includes a) a first VHH domain that binds to IgE, and b) a second VHH domain that binds to IgE. Preferably, both the first VHH domain and the second VHH domain bind to the same IgE antibody, for example, simultaneously to the same IgE antibody. The binding site for IgE may be 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, and 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 include at least one additional VHH domain that binds to an IgE epitope different from any of the epitopes of IgE bound by the first VHH and the second VHH. Preferably, the additional VHH domain binds to the same IgE molecule as the first and second VHH domains.

[0109] Thus, in the most interesting embodiments, the multispecific construct comprises non-identical sdAbs, such as non-identical VHH domains. Thus, the two sdAb / VHH domains can bind to the same IgE antibody simultaneously, as can be evaluated by relative epitope mapping.

[0110] Hereinafter, any reference to an sdAb may be considered to be consistent with a VHH domain.

[0111] In an interesting embodiment, the multispecific construct comprises a first sdAb and / or a second sdAb, which, when binding to IgE bound to FcεRI, causes a decrease in the binding affinity between IgE and FcεRI. Thus, the multispecific construct comprises at least one sdAb, such as a first sdAb, that can enhance the dissociation of bound IgE from FcεRI. Alternatively, neither of the two sdAbs need have dissociation-enhancing activity, but the final multispecific construct may cause a decrease in the binding affinity between IgE and FcεRI when binding to IgE bound to FcεRI.

[0112] In yet another interesting embodiment, the multispecific construct comprises at least one additional sdAb, such as a second sdAb, which in this case promotes an improvement or enhancement in the binding affinity (i.e., avidity) of the multispecific construct for IgE as compared to the IgE binding affinity of the first sdAb or the second sdAb. Thus, the second sdAb can increase the overall IgE binding affinity (avidity).

[0113] For example, the overall binding affinity (i.e., avidity) of the multispecific construct for IgE is improved, such that the sum of the binding affinities observed for either the first sdAb and the second sdAb is improved. Further, the dissociation-enhancing effect of the multispecific construct is improved, such that the sum of the dissociation-enhancing effects observed for either the first sdAb and the second sdAb is improved.

[0114] The dissociation enhancing activity of an individual sdAb or a multispecific construct can be evaluated by an ELISA-based IgE-FcεRIα dissociation enhancement assay, which measures the residual IgE not removed from immobilized recombinant human FcεRIα pre-loaded with IgE after the addition of a test compound (e.g., an sdAb or a multispecific construct), and determines the percentage of dissociation enhancing activity as the relative decrease in signal compared to a control without the test compound added.

[0115] In the constructs disclosed herein, the first sdAb can be selected from among sdAbs having dissociation enhancing activity in the state of EC in the nanomolar range to the upper limit of the molar range, for example, in the range of 100 nM to 5000 nM. 50 In the state of EC, for example, in the range of 100 nM to 5000 nM. 50 In the state of EC, for example, exceeding 200, 300, 400, 500 or 600 nM but less than 4000, 3000, 2000 or 1000 nM. 50 In the state of EC, it can be selected from among sdAbs having dissociation enhancing activity. Exemplary sdAbs that meet this criterion are sdAb A1, A2 or B1.

[0116] Furthermore or alternatively, the dissociation enhancing activity of the first sdAb is an activity such that the maximum dissociation enhancing activity is at least 80%, preferably at least 85%, 90%, or 95%. Exemplary sdAbs that meet this criterion are sdAb A1 or A2.

[0117] Most preferably, the first sdAb has dissociation enhancing activity in the state of EC in the range of 200 nM to 5000 nM, and the maximum dissociation enhancing effect is at least 80%. Exemplary sdAbs that meet this criterion are sdAb A1 or A2. 50 In the state of EC, it has dissociation enhancing activity, and the maximum dissociation enhancing effect is at least 80%. Exemplary sdAbs that meet this criterion are sdAb A1 or A2.

[0118] In the case of the multispecific constructs disclosed herein, the second sdAb may be selected from among sdAbs that do not exhibit dissociation enhancement activity as compared to the first sdAb (e.g., sdAb B2, B3, D1, D2, D3, E1, E2, E3, E4, F1, F2, F3, F4, F5, F6 and G1) [e.g., an sdAb exhibiting lower dissociation enhancement activity (higher EC 50 ), or the second sdAb may exhibit a maximum dissociation enhancement effect of less than 75% (e.g., B1).

[0119] As described, the multispecific construct has improved dissociation enhancement activity over the individual sdAbs of the multispecific construct. The improved dissociation enhancement activity is an activity such that the EC 50 for the construct is consequently lower than the EC 50 observed for either the first sdAb or the second sdAb, or a mixture of the first sdAb and the second sdAb.

[0120] As an example, the improved dissociation enhancement activity of the multispecific construct is an activity such that the EC 50 is at most one-twentieth lower than the EC 50 of the first sdAb. Preferably, the EC 50 is at most one twenty-fifth, one thirtieth, one thirty-fifth, one fortieth, one forty-fifth, one fiftieth, one sixtieth, one seventieth, one eightieth, one hundredth, one one-hundred-and-fiftieth, one two-hundredth, one two-hundred-and-fiftieth, one three-hundredth, one three-hundred-and-fiftieth, one four-hundredth, one five-hundredth, one six-hundredth, one seven-hundredth, one eight-hundredth, one nine-hundredth or one thousandth lower.

[0121] For example, the improved dissociation enhancement activity of the multispecific construct is an activity such that the EC 50 is less than 200 nM. Preferably, the EC 50 is less than 150, 100, 75, 50, 25, 20, 15, 10, 5, 4, 3, 2 or 1 nM.

[0122] Alternatively or in addition, the improved dissociation enhancing activity of the multispecific construct is an activity such that the maximum dissociation enhancing effect is at least 80%. Preferably, the maximum dissociation enhancing effect is at least 85%, 90% or 95%, most preferably at least 95%.

[0123] In a preferred embodiment, the improved dissociation enhancing activity of the multispecific construct is an activity such that the EC 50 is less than 100 nM and the maximum dissociation enhancement is at least 80%, such as at least 85%, 90% or 95%.

[0124] Overall, the multispecific construct can comprise, as the first sdAb or the second sdAb, an sdAb comprising a CDR1 having an amino acid sequence determined according to Aho, SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70 or 74, each amino acid sequence being able to have 1, 2 or 3 amino acid substitutions; and / or an sdAb comprising a CDR2 having an amino acid sequence determined according to Aho, SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71 or 75, each amino acid sequence being able to have 1, 2 or 3 amino acid substitutions; and / or an sdAb comprising a CDR3 having an amino acid sequence determined according to Aho, SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72 or 76, each amino acid sequence being able to have 1, 2 or 3 amino acid substitutions.

[0125] If it is found that not all three binding CDR regions are crucial for binding, the first and second sdAbs can be constructed from the aforementioned combinations of CDR regions, where one of the three CDR regions may be replaced by another sequence, for example, it may have been subject to humanization or other amino acid modifications. In a preferred embodiment, the original CDR3 region of the camelid sdAb is maintained as a conserved sequence.

[0126] More specifically, the multispecific construct can include an sdAb having a binding region with a combination of CDR1, CDR2, and CDR3 having, as the first or second sdAb, the respective SEQ ID NOs: 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 according to Aho.

[0127] As long as the multispecific construct has dissociation enhancing activity, the construct can include any combination of the sdAb / VHH domains described herein. In a preferred embodiment, the first sdAb, such as sdAb026 (designated as A1 herein) or A2, has dissociation enhancing activity by itself.

[0128] Accordingly, the multispecific construct can include a binding region from sdAb A1 or A2. In this way, the first sdAb of the construct can include or consist of binding regions CDR1, CDR2, and CDR3 having amino acid sequences determined according to any of the Kabat, Chothia, IMTG, or Aho numbering schemes in an amino acid sequence selected from any one of SEQ ID NO: 1 or 5, where 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.

[0129] Alternatively defined, the first sdAb includes binding regions CDR1, CDR2, and CDR3 having SEQ ID NO: 2, 3, and 4 or 6, 7, and 8, respectively, where each CDR is determined according to the Aho numbering scheme.

[0130] The second sdAb may be any that has IgE binding activity in the presence of the first sdAb. Exemplary second sdAbs include or consist of binding regions CDR1, CDR2, and CDR3 having amino acid sequences determined according to any of the Kabat, Chothia, IMTG, or Aho numbering schemes in an amino acid sequence selected from any one of SEQ ID NO: 9, 13, 17, 21, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, and 73, where 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.

[0131] Alternatively, the second sdAb comprises binding regions CDR1, CDR2, and CDR3 having respective SEQ ID NOs: 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, with each CDR determined according to the Aho numbering scheme.

[0132] Similarly, the second sdAb can comprise a binding region having a combination of CDR1, CDR2, and CDR3 determined according to Kabat, with respective 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.

[0133] Similarly, the second sdAb comprises binding regions CDR1, CDR2, and CDR3 having respective 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; determined according to Chothia. It can include a binding region having a combination of CDR1, CDR2, and CDR3 including 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.

[0134] Similarly, the second sdAb can include a binding region having a combination of CDR1, CDR2, and CDR3 determined according to IMGT, which are respectively SEQ ID NO: 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.

[0135] Thus, the first sdAb of the construct described herein can preferably comprise or consist of any one of SEQ ID NO: 1 or 2, and the second sdAb can preferably comprise or consist of any one of SEQ ID NO: 5, 9, 13, 17, 21, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69 and 73.

[0136] When the first sdAb includes the binding region of B1, the second sdAb can respectively include the binding regions of sdAb B2, B3, D1, D2, D3, E1, E2, E3, E4, F1, F2, F3, F4, F5, F6 or G1.

[0137] Thus, in a more preferred embodiment, the first sdAb of the construct can comprise or consist of any one of SEQ ID NOs: 1, 5, and 9, and more preferably, the first sdAb of the construct can comprise or consist of SEQ ID NO: 1.

[0138] Specifically, the first sdAb can be any one of sdAb A1, sdAb A2, or sdAb B1. Thus, the first sdAb can comprise or consist of an amino acid sequence selected from any one of SEQ ID NOs: 1, 5, and 9, and preferably, the first sdAb can comprise or consist of the amino acid sequence of SEQ ID NO: 1.

[0139] In addition to the above, SdAb A1 can also be defined by its three CDR regions determined using different CDR calculation methods.

[0140] Thus, in a preferred embodiment, the first sdAb of the construct is each SEQ ID NO 2, 3, and 4 determined according to Aho; 129, 130, and 131 determined according to Kabat; 186, 187, and 188 determined according to Chothia; or 243, 244, and 245 determined according to IMGT; and can comprise binding region CDR1, CDR2, and CDR3 having the same, and optionally, each CDR1 can contain 1, 2, or 3 amino acid substitutions, each CDR2 can contain 1, 2, or 3 amino acid substitutions, and each CDR3 can contain 1, 2, or 3 amino acid substitutions.

[0141] Similar to A1, A2 can also be defined by its CDR regions as described above.

[0142] Thus, in a preferred embodiment, the first sdAb of the construct is each SEQ ID NO 6, 7, and 8 determined according to Aho; 132, 133, and 134 determined according to Kabat; 189, 190, and 191 determined according to Chothia; or 246, 247, and 248 determined according to IMGT; and can include complementarity-determining regions CDR1, CDR2, and CDR3 having optionally, each CDR1 can contain 1, 2, or 3 amino acid substitutions, each CDR2 can contain 1, 2, or 3 amino acid substitutions, and each CDR3 can contain 1, 2, or 3 amino acid substitutions.

[0143] Furthermore, B1 can be defined by the CDR regions in addition to A1 and A2.

[0144] Thus, in a preferred embodiment, the first sdAb of the construct has complementarity-determining regions CDR1, CDR2, and CDR3 having 10, 11, and 12 determined according to Aho; 135, 136, and 137 determined according to Kabat; 192, 193, and 194 determined according to Chothia; or 249, 250, and 251 determined according to IMGT; and can include optionally, each CDR1 can contain 1, 2, or 3 amino acid substitutions, each CDR2 can contain 1, 2, or 3 amino acid substitutions, and each CDR3 can contain 1, 2, or 3 amino acid substitutions.

[0145] Alternatively defined, the first sdAb of the construct can include or consist of binding regions CDR1, CDR2, and CDR3 that include an amino acid sequence determined according to Kabat, Chothia, IMGT, or Aho in an amino acid sequence selected from any one of SEQ ID NOs: 1, 5, or 9, and optionally, each CDR1 can contain 1, 2, or 3 amino acid substitutions, each CDR2 can contain 1, 2, or 3 amino acid substitutions, and each CDR3 can contain 1, 2, or 3 amino acid substitutions.

[0146] As the inventors further recognize, the multispecific construct may exhibit anaphylaxis-inducing activity, however, the anaphylaxis-inducing activity may depend on the test concentration, diluent, and sensitivity of the test method. Accordingly, a preferred embodiment includes, as the first sdAb, sdAb A1 or an sdAb that includes the binding region of sdAb A1, and as the second sdAb, an sdAb selected from B1, D2, E1, and F4 or an sdAb that includes the binding region of an sdAb selected from B1, D2, E1, and F4.

[0147] Thus, in the multispecific construct, the first sdAb comprises or consists of binding regions CDR1, CDR2, and CDR3 each having an amino acid sequence determined according to any of the Kabat, Chothia, IMTG, or Aho numbering schemes in an amino acid sequence selected from any one of SEQ ID NO: 1 or 5, wherein 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; the second sdAb comprises or consists of binding regions CDR1, CDR2, and CDR3 each having an amino acid sequence determined according to any of the Kabat, Chothia, IMTG, or Aho numbering schemes in an amino acid sequence selected from any one of SEQ ID NO: 9, 25, 33, 61, wherein 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.

[0148] Alternatively, in the multispecific construct, the first sdAb comprises binding regions CDR1, CDR2, and CDR3 having each of SEQ ID NO: 2, 3, and 4; or 6, 7, and 8 and each CDR is determined according to the Aho numbering scheme; and the second sdAb comprises binding regions CDR1, CDR2, and CDR3 having each of SEQ ID NO: 10, 11, and 12; 26, 27, and 28; 34, 35, and 36; or 62, 63, and 64 and each CDR is determined according to the Aho numbering scheme.

[0149] In certain interesting embodiments, the construct comprises the binding regions of sdAb A1 and B1. Thus, the multispecific construct comprises a first sdAb comprising CDR1, CDR2, and CDR3 of a binding region that comprises or consists of an amino acid sequence determined according to any of the Kabat, Chothia, IMTG, or Aho numbering schemes in the amino acid sequence of SEQ ID NO: 1, wherein 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; a second sdAb comprising CDR1, CDR2, and CDR3 of a binding region that comprises or consists of an amino acid sequence determined according to any of the Kabat, Chothia, IMTG, or Aho numbering schemes in the amino acid sequence of SEQ ID NO: 9, wherein 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.

[0150] Alternatively, the multispecific construct comprises a first sdAb comprising CDR1, CDR2, and CDR3 of a binding region having SEQ ID NOs: 2, 3, and 4, respectively, wherein each CDR is determined according to the Aho numbering scheme; and a second sdAb comprising CDR1, CDR2, and CDR3 of a binding region having SEQ ID NOs: 10, 11, and 12, respectively, wherein each CDR is determined according to the Aho numbering scheme.

[0151] If the second sdAb comprises a binding region from D2, CDR1, CDR2, and CDR3 can have SEQ ID NOs: 26, 27, and 28, respectively, and the sdAb can have SEQ ID NO: 25.

[0152] When the second sdAb contains a binding region from E1, CDR1, CDR2, and CDR3 can have SEQ ID NOs: 34, 35, and 36 respectively, and the above sdAb can have SEQ ID NO: 33.

[0153] When the second sdAb contains a binding region from F4, CDR1, CDR2, and CDR3 can have SEQ ID NOs: 62, 63, and 64 respectively, and the above sdAb can have SEQ ID NO: 61.

[0154] In yet another embodiment, the first sdAb A1 or A2 can be replaced with a further variant of A1 such as those described in WO2012175740A1. It should be mentioned that the camelid sdAb39D11 (SEQ ID NO: 119 of WO2012 / 175740A1), 39D11 sdAb = IGE009 (having mutations: V5L, M77T K83R, Q108L), IGE010 (having mutations: V5L, K83R, M77T, Q108L), IGE011 (having mutations: V5L, M77L K83R, Q108L, W91Y), and IGE012 (having mutations: V5L, M77L, K83R, Q108L, W91Y) having SEQ ID NOs: 120 - 123 of WO2012 / 175740A1. Among these, IGE009 has been reported as preferred. The affinity - matured versions of IGE009 are IGE025 - IGE030 with SEQ ID NOs: 127 - 133 of WO2012 / 175740A1. Among the affinity - matured ones, IGE026 and IGE027 have been reported as preferred because their affinity is improved by 30 - 50 times after maturation. IGE026 is named A1 in this specification (having SEQ ID NO: 1 in this application and SEQ ID NO: 128 in WO2012 / 175740A1). In this specification, 39D11 has SEQ ID NO: 316, IGE009 has SEQ ID NO: 317, IGE010 has SEQ ID NO: 318, IGE011 has SEQ ID NO: 319, and IGE012 has SEQ ID NO: 320.

[0155] Alternatively, a multispecific construct comprising a first dissociation-enhancing sdAb can comprise, as a second sdAb, any sdAb capable of binding IgE, preferably any sdAb capable of binding IgE at a different epitope than, for example, the first sdAb and having a high binding affinity in the nanomolar range. Such a second sdAb can be selected from those described in any of patent applications WO2004 / 041867, WO2012 / 175740, WO2014 / 087010, WO2020 / 208177, CN113461823 and CN111875706.

[0156] Peptide linkage of sdAb As described, the sdAb / VHH domain can be incorporated into a larger construct, such as a multispecific construct in which two sdAb / VHH domains are combined in one construct. Such constructs can have the ability to bind to non-identical IgE epitopes of the same IgE. As shown by way of example herein, a multispecific construct can comprise at least two separate sdAb / VHH domains linked together by part c). In a preferred embodiment, the part is a peptide linker (as seen in Figure 1). In a more preferred embodiment, such a construct exhibits an improvement or enhancement of dissociation-enhancing activity compared to a single sdAb present in the construct (Example 6, Table 15 and Example 7, Table 16).

[0157] Table 3 provides the full-length amino acid sequences of exemplary multispecific constructs comprising at least two separate sdAbs linked together by a portion consisting of a peptide linker. The two A1 sdAbs linked together can be used as a control when determining the improvement of dissociation-enhancing activity. As described above, at least one of the two sdAbs has dissociation-enhancing activity by itself. Herein, A1, A2 or B1 represents an sdAb having dissociation-enhancing activity by itself.

[0158]

Table 6

[0159] Accordingly, the multispecific construct can include a first and a second sdAb and a portion that links the first and second sdAbs, where the portion is a peptide. Typically, the peptide is a short peptide having a maximum of 30 amino acid residues. By way of example, the peptide can consist of one or more modules of glycine and serine amino acids according to the formula (G n S) n (wherein n can be an integer from 1 to 6). Thus, the peptide can be a GS linker selected from, for example, SEQ ID NOs: 88-91 or 452-454 or 457 (Table 5), such as GS, G2S, G3S, (G2S)2, G4S, (G4S)2, (G4S)3, (G4S)4. Alternatively, the peptide can be an arginine-serine peptide. Further, the linker can be EAAAK or (EAAAK)2 of SEQ ID NO: 455 or 456.

[0160]

Table 7

[0161] In some embodiments, when the multispecific construct includes a first and a second sdAb and the portion is a peptide, the multispecific construct can include one or more additional sdAbs operably linked to either the first or the second sdAb. For example, one or more sdAbs can be linked to either the first or the second sdAb by a peptide, polypeptide, or polysaccharide. In some embodiments, one or more additional sdAbs can also bind to an IgE molecule, preferably the same IgE molecule to which the first and second sdAbs bind. In other embodiments, one or more additional sdAbs can bind to human serum albumin (HSA) that can extend the serum half-life.

[0162] In the embodiments of the present specification, 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 peptides having SEQ ID NOs: 88, 89, 90, 91, 454, 455, 456, and 457.

[0163] It will be understood that the moieties used to link two or more sdAbs can independently be selected from the group consisting of peptides, polypeptides, polymers, and polysaccharides.

[0164] Moiety c) can extend the serum half-life of the multispecific construct. The extended serum half-life can be determined compared to the respective serum half-lives of the first and second sdAbs or the combined serum half-life of the two sdAbs (for example, when the sdAbs are linked together with a peptide moiety).

[0165] Alternatively or additionally, the above moiety can add further multispecificity to the construct by selecting moiety c) having binding affinity for another biologically relevant target.

[0166] In general, there are several options for selecting a fusion partner for a monomeric sdAb or a multispecific construct to improve pharmacokinetic properties (Strohl W. 2015). For example: a. Fusion to a protein or protein domain with a long natural half-life [e.g., Fc fusion, transferrin (Tf) fusion, or albumin fusion]. b. Fusion to an inert polypeptide, such as XTEN (also known as recombinant PEG or "rPEG"), a homoamino acid polymer (HAP; HAPylation), a proline-alanine-serine polymer (PAS; PASylation), or an elastin-like peptide (ELP; ELPylation). c. Chemical conjugation to the repetitive chemical moiety, for example, to PEG (PEGylation or hyaluronic acid). d. By significantly increasing the negative charge of the monomeric sdAb or multispecific construct by polysialylation; or alternatively, by fusing a negatively charged and highly sialylated peptide [e.g., carboxy-terminal peptide (CTP; of the β-chain of chorionic gonadotropin (CG))]. e. Non-covalently binding to proteins such as HSA, human IgG, or optionally transferrin, which normally have a long half-life, via attachment with a peptide linker or protein binding domain. f. Chemical conjugation to proteins with a long half-life such as human IgG, the Fc portion or HSA.

[0167] It is contemplated that one or more of options a) - f) may be incorporated into the multispecific constructs disclosed herein.

[0168] Fc fusion of sdAb. Fc fusions between the Fc domain and therapeutic agents are widely used to confer additional beneficial biological and pharmacological properties (Czajkowsky et al. 2012). The presence of the Fc domain significantly improves plasma half-life, and due to the interaction with the salvage neonatal Fc receptor (FcRn) and slower renal clearance for larger molecules, the therapeutic activity is prolonged. The attached Fc domain enables these molecules to interact with Fc receptors (FcRs) found on immune cells. From a biophysical perspective, the Fc domain folds independently and can improve the solubility and stability of partner molecules (e.g., sdAb) both in vitro and in vivo, while from a technical perspective, the Fc region enables cost-effective purification by protein G / A affinity chromatography during the manufacturing process.

[0169] Thus, in some embodiments, part c) is a polypeptide that is a fragment of an antibody, particularly a human antibody fragment selected from the group consisting of, for example, IgA, IgE, IgG, and IgM, preferably a fragment of IgG. The fragment of IgG can be selected from any fragment of IgG1, IgG2, IgG3, and IgG4, and preferably the fragment of IgG is derived from IgG1 or IgG4, for example, particularly from the Fc domain of IgG1 or IgG4. The fragment may include C H 1-C H 3, but it may be preferable to use C H 2-C H 3 (IgG-Fc region). In some embodiments, it may be possible to select to use any of the regions of short fragments of IgG, such as C H 1, C H 2, or C H 3.

[0170] Typically, the sdAb may be independently fused or covalently linked, optionally via a linker, to C H 3 or C H 2 of IgG-Fc. Thus, the sdAb may be independently fused or covalently linked, optionally via a linker, to the N-terminus of C H 2 or to the C-terminus of C H 3. In a preferred embodiment, IgG-Fc is IgG4-Fc, preferably human IgG4-Fc. In yet another embodiment, both sdAbs are fused to the C-terminus or N-terminus of Fc through a peptide linker. In yet another interesting embodiment, both sdAbs are fused to the C-terminus of Fc through a peptide linker, as this is thought to protect against anaphylaxis-inducing activity.

[0171] Thus, the multispecific construct of interest is - The first and second sdAbs may both be fused or covalently linked, either directly or via a linker, to C H 2 of IgG-Fc, - The first and second sdAbs may both be fused or covalently bound either directly or via a linker to the C of IgG-Fc H 3, or - The first sdAb may be fused or covalently bound either directly or via a linker to the C of IgG-Fc H 2, and the second sdAb may be fused or covalently bound either directly or via a linker to the C of IgG-Fc H 3, or - The second sdAb may be fused or covalently bound either directly or via a linker to the C of IgG-Fc H 2, and the first sdAb may be fused or covalently bound either directly or via a linker to the C of IgG-Fc H 3.

[0172] In some more interesting embodiments, via an appropriate linker as appropriate, the first and second sdAbs are each fused to the C of IgG-Fc H 3 (the C-terminus of IgG-Fc), or the first and second sdAbs are each fused to the C of IgG-Fc H 2 (the N-terminus of IgG-Fc). In further interesting embodiments, the IgG Fc region is an IgG4-Fc region.

[0173] The first and second sdAbs can be fused to the antibody fragment by the use of any suitable linker, such as a peptide having up to 30 amino acid residues.

[0174] By way of example, a flexible glycine-serine linker (GS linker), such as, (G n S) nOne or more modules of (wherein n can be an integer from 1 to 6) can be used to form a GS linker. Thus, the peptide can be a GS linker selected from, for example, SEQ ID NOs: 88-91 or 452-454 or 457 (Table 5), such as GS, G2S, G3S, (G2S)2, G4S, (G4S)2, (G4S)3, (G4S)4. Alternatively, the peptide can be an arginine-serine (RS) peptide. Additionally, the linker can be EAAAK or (EAAAK)2 of SEQ ID NO: 455 or 456.

[0175] 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.

[0176] As described, the antibody fragment (e.g., Fc region) can be of any antibody type (e.g., IgG, IgE, IgM, IgD, and IgA), isotype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass, including engineered subclasses having a modified Fc portion that can appropriately provide for reduction or enhancement of effector cell activity or alteration of in vivo distribution, serum half-life, or excretion rate. The antibody fragment can be derived from any species. For example, the fragment can be of human origin. Exemplary effector functions include C1-q binding; CDC; Fc receptor binding; ADCC; ADCP; downregulation of cell surface receptors (e.g., B cell receptor), etc. Such effector functions generally require the Fc region to interact with receptors, for example, via FcγRI; FcγRIIA; FcγRIIB1; FcγRIIB2; FcγRIIIA; FcγRIIIB receptors, and / or the low-affinity FcRn receptor. Additionally, the Fc region can be a "dead" Fc, which is, for example, a Fc that has been mutagenized to retain activity in relation to, for example, extending serum half-life but does not activate high-affinity Fc receptors. The Fc may also have a reduced binding to complement.

[0177] When it is desirable for the antibody fragment not to participate in the interaction of Fc with a receptor, such as FcγRIIB, the antibody fragment can be a CH1 immunoglobulin domain (e.g., IgG1-CH1 domain or IgG4-CH1 domain). Unlike the conventionally used IgG-Fc domain, these domains do not participate in the inhibitory FcγRIIb receptor, do not compete with serum immunoglobulin G for receptor binding, and the cytotoxic activity of these domains is independent of Fc glycosylation and FcγRIIIa polymorphism (Rozan et al., 2013)

[0178] In certain embodiments, the antibody fragment is derived from IgG4, such as IgG4-Fc. Examples of antibody fragments are listed in Table 6.

[0179]

Table 8

[0180] Thus, when the construct is formed by the knob and hole elements of the KIH structure, part c) can be selected from IgG4 antibody fragments comprising or consisting of an amino acid sequence selected from any one of SEQ ID NOs: 125, 126 or alternatively 433, 434, 435, and 436. Preferably, part c) can comprise an IgG4 antibody fragment comprising or consisting of an amino acid sequence selected from any one of SEQ ID NOs: 125 and 126, and may be fused to a peptide linker. Preferably, both heavy chain Fc portions are fused to the same peptide linker respectively, and thus, if one Fc chain is fused to, for example, a GGS peptide, the other Fc chain is also fused to the GGS peptide.

[0181] Since the Ig-Fc portion can contain various mutations, further embodiments thereof include an IgG4 antibody fragment having 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 the amino acid sequence selected from the group consisting of SEQ ID NOs: 125, 126, 433, 434, 435 and 436. IgG4 is unstable in vivo due to the "half antibody exchange" phenomenon which results in bispecificity (or in most cases functional monomers). Thus, when used therapeutically, a single amino acid mutation can be introduced into the hinge region to prevent such dissociation - the so-called S228P mutation.

[0182] Another way to prevent the in vivo instability of IgG4 may be to apply the IgG4-Fc scaffold in the knob-into-hole (KIH) format, which can prevent the above-mentioned dissociation.

[0183] One of the further mutations implemented in the antibody fragment can be, for example, a mutation directed towards enhancing FcγRIIb (CD32b) engagement, as described in the granted patent US8435517B2. This mutation preferably binds to FcγRIIb with a K D less than about 100 nM.

[0184] Furthermore, the Fc region can be a wild-type sequence Fc region that includes an amino acid sequence identical to the amino acid sequence of the Fc region found in nature. The wild-type sequence human Fc region includes the wild-type sequence human IgG1 Fc region (non-A and A allotypes); the wild-type sequence human IgG2 Fc region; the wild-type sequence human IgG3 Fc region; and the wild-type sequence human IgG4 Fc region, as well as naturally occurring variants thereof, or the Fc region can be a variant Fc region that includes an amino acid sequence different from the amino acid sequence of the wild-type sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution, for example, from about 1 to about 10 amino acid substitutions, preferably from about 1 to about 5 amino acid substitutions, in the wild-type sequence Fc region or in the Fc region of the parental polypeptide, compared to the wild-type sequence Fc region or the Fc region of the parental polypeptide. The variant Fc regions herein can possess at least about 80% amino acid sequence homology or sequence identity with the wild-type sequence Fc region and / or the Fc region of the parental antibody fragment, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith.

[0185] In one embodiment, the variant Fc sequence can reduce FcγRI binding by including three amino acid substitutions in the CH2 region at positions 234, 235, and 237 of the EU index (Duncan et al., 1988). Complement binding is reduced by two amino acid substitutions at positions 330 and 331 of the complement C1-q binding site of the EU index (Tao et al., 1993 and Canfield & Morrison, 1991). Substitution of human IgG1 residues at positions 233-236 of IgG2 and at positions 327, 330, and 331 of IgG4 results in a significant reduction in ADCC and CDC (Armour KL. et al., 1999 and Shields RL. et al., 2001). Other Fc variants are possible and include, but are not limited to, those in which a region capable of forming disulfide bonds is deleted, or in which certain amino acid residues are deleted at the N-terminus of the native Fc form or a methionine residue is added thereto. Thus, one or more Fc portions of the molecule can include one or more mutations in the hinge region to eliminate disulfide bonds. In another embodiment, the Fc hinge region can be completely removed. In yet another embodiment, the molecule can include an Fc variant.

[0186] Furthermore, Fc variants can be constructed by substituting, deleting, or adding amino acid residues to cause complement binding or Fc receptor binding. Techniques for preparing such sequence derivatives of immunoglobulin Fc fragments are disclosed in International Patent Publications WO97 / 34631 and WO96 / 32478. In addition, the Fc domain can be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0187] Antibody fragments, such as the Fc portion, can also be modified for structural reasons (e.g., expression yield) by substituting amino acid residues, deleting them, or adding them. Non-limiting examples of mutations introduced for expression yield are described below.

[0188] The antibody fragment may be modified into a form having a natural-type sugar chain, a form having an increased sugar chain compared to the natural form, or a form having a decreased sugar chain compared to the natural form, or may be in a non-glycosylated form or a deglycosylated form. The increase, decrease, removal or other modification of the sugar chain can be carried out by general methods in the art such as chemical methods, enzymatic methods, or by expressing it in a genetically engineered production cell line. Such cell lines may include microorganisms that naturally express glycosylation enzymes, such as Pichia Pastoris, and mammalian cell lines, such as CHO cells. Furthermore, the microorganism or cell may be engineered to express a glycosylation enzyme, or may be made unable to express a glycosylation enzyme. As an example of a cell engineered to have a changed sialylation activity, the alpha-2,6-sialyltransferase 1 gene has been engineered into Chinese hamster ovary cells and sf9 cells. As a result, the constructs expressed by these engineered cells are sialylated by the exogenous gene product. A further method for obtaining an Fc molecule with an altered amount of sugar residues compared to a plurality of natural-type molecules includes, for example, using lectin affinity chromatography to separate the plurality of molecules into a glycosylated fraction and a non-glycosylated fraction. The presence of specific glycosylation moieties has been shown to alter the function of immunoglobulins. For example, removing the sugar chain from the Fc molecule results in a sharp decrease in the binding affinity for the C1-q portion of the first complement component C1, and a decrease or loss of antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), thereby not inducing an unnecessary immune response in vivo. Further important modifications include sialylation and fucosylation. The presence of sialic acid in IgG has been correlated with anti-inflammatory activity, while removing fucose from IgG leads to enhanced ADCC activity. The construct can have an Fc sequence with enhanced effector function, for example, by improving its binding ability to FcγRI and by improving ADCC activity.For example, the fucose attached to the N-linked glycan at Asn-297 of Fc sterically hinders the interaction of Fc with FcγRIIIA, and when the fucose is removed by sugar engineering, the binding to FcγRIIIA can be improved, which is converted to an ADCC activity more than 50-fold higher compared to the wild-type IgG1 control. Protein engineering has created multiple mutants that improve the affinity of Fc binding to FcγRIIIA through amino acid mutations in the Fc portion of IgG1. In particular, the triple alanine mutant S298A / E333A / K334A exhibits a two-fold improvement in binding to FcγRIIIA and ADCC function. Mutants of S239D / I332E(2×) and S239D / I332E / A330L(3×) have a significant improvement in binding affinity to FcγRIIIA and an enhancement of ADCC ability in vitro and in vivo. Other Fc mutants identified by yeast display also showed improved binding to FcγRIIIA. See, for example, Liu et al. (2014) JBC 289(6):3571 - 90, which is specifically incorporated herein by reference.

[0189] The multispecific constructs described herein can be assembled in a variety of ways.

[0190] In a non-limiting example, the multispecific construct is i. a 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 sdAbs of SEQ ID NO: 1 or 5; ii. a second sdAb different from the first sdAb and 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 sdAbs of SEQ ID NOs: 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, and 73 and each sdAb has an Fc portion, for example iii. An Fc portion selected from SEQ ID NO: 125 or 126, or an Fc portion associated by using the following linker portion that links an sdAb to the Fc portion at either the N-terminus or the C-terminus by a knob portion selected from SEQ ID NO: 433 and 435 and a hole portion selected from SEQ ID NO: 434 and 436, is linked thereto, for example, iv. The linker portion is selected from the group consisting of SEQ ID NO: 88-92 and 452-457.

[0191] As described above, the amino acid sequences of the first and second sdAbs may be subject to affinity maturation, humanization or other amino acid changes of the amino acid sequence, preferably outside the CDR regions. Thus, the first sdAb can have at least 80%, for example at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with 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, and the second sdAb, being different from the first sdAb, can have at least 80%, for example at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with 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. Similarly, the Fc portion may have any of the sequence variations described above under the Fc-fusion sdAb.

[0192] In a preferred embodiment, the multispecific construct comprises a first sdAb A1 having SEQ ID NO: 1 or at least 80% sequence identity with SEQ ID NO: 1, and a second sdAb B1 having SEQ ID NO: 9 or at least 80% sequence identity with SEQ ID NO: 9, and each sdAb is linked to an Fc portion having SEQ ID NO: 125 or at least 80% sequence identity with SEQ ID NO: 125 using a linker portion of the sequence of SEQ ID NO: 88.

[0193] In another preferred embodiment, the multispecific construct comprises a first sdAb A1 of the sequence of SEQ ID NO: 1 and a second sdAb B1 of the sequence of SEQ ID NO: 9, and each sdAb is linked to the Fc portion of the sequence of SEQ ID NO: 126 using the linker portion of the sequence of SEQ ID NO: 88.

[0194] In another non-limiting example, the multispecific construct comprises a first sdAb comprising regions of CDR1, CDR2, and CDR3 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, Unlike the first sdAb, a second sdAb comprising regions of CDR1, CDR2, and CDR3 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, and each sdAb is linked to an Fc portion selected from SEQ ID NO: 125 or 126 using a linker portion selected from the group consisting of SEQ ID NOs: 88 - 92 and 452 - 457.

[0195] In a preferred embodiment, the multispecific construct comprises a first sdAb comprising regions of CDR1, CDR2, and CDR3 determined using the Kabat, Chothia, IMGT, or Aho numbering scheme in the amino acid sequence of A1 of the sequence of SEQ ID NO: 1, and a second sdAb comprising regions of CDR1, CDR2, and CDR3 determined using the Kabat, Chothia, IMGT, or Aho numbering scheme in the amino acid sequence of B1 of the sequence of SEQ ID NO: 9, and each sdAb is linked to the Fc portion of the sequence of SEQ ID NO: 125 using the linker portion of the sequence of SEQ ID NO: 88.

[0196] In another preferred embodiment, the multispecific construct comprises a first sdAb comprising regions of CDR1, CDR2 and CDR3 determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the amino acid sequence of A1 of the sequence of SEQ ID NO: 1, and a second sdAb comprising regions of CDR1, CDR2 and CDR3 determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the amino acid sequence of B1 of the sequence of SEQ ID NO: 9, and each sdAb is linked to the Fc portion of the sequence of SEQ ID NO: 126 using the linker portion of the sequence of SEQ ID NO: 88.

[0197] In a more preferred embodiment, the multispecific construct comprises a first sdAb comprising regions of CDR1, CDR2 and CDR3 determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the amino acid sequence of A1 of the sequence of SEQ ID NO: 1, and a second sdAb comprising regions of CDR1, CDR2 and CDR3 determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the amino acid sequence of B1 of the sequence of SEQ ID NO: 25, and each sdAb is linked to the Fc portion of the sequence of SEQ ID NO: 125 using the linker portion of the sequence of SEQ ID NO: 88.

[0198] In a more preferred embodiment, the multispecific construct comprises a first sdAb comprising regions of CDR1, CDR2 and CDR3 determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the amino acid sequence of A1 of the sequence of SEQ ID NO: 1, and a second sdAb comprising regions of CDR1, CDR2 and CDR3 determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the amino acid sequence of B1 of the sequence of SEQ ID NO: 25, and each sdAb is linked to the Fc portion of the sequence of SEQ ID NO: 126 using the linker portion of the sequence of SEQ ID NO: 88.

[0199] In a more preferred embodiment, the multispecific construct comprises a first sdAb comprising regions of CDR1, CDR2 and CDR3 determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the amino acid sequence of A1 of the sequence of SEQ ID NO: 1, and a second sdAb comprising regions of CDR1, CDR2 and CDR3 determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the amino acid sequence of B1 of the sequence of SEQ ID NO: 33, and each sdAb is linked to the Fc portion of the sequence of SEQ ID NO: 125 using the linker portion of the sequence of SEQ ID NO: 88.

[0200] In a more preferred embodiment, the multispecific construct comprises a first sdAb comprising regions of CDR1, CDR2 and CDR3 determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the amino acid sequence of A1 of the sequence of SEQ ID NO: 1, and a second sdAb comprising regions of CDR1, CDR2 and CDR3 determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the amino acid sequence of B1 of the sequence of SEQ ID NO: 33, and each sdAb is linked to the Fc portion of the sequence of SEQ ID NO: 126 using the linker portion of the sequence of SEQ ID NO: 88.

[0201] In a more preferred embodiment, the multispecific construct comprises a first sdAb comprising regions of CDR1, CDR2 and CDR3 determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the amino acid sequence of A1 of the sequence of SEQ ID NO: 1, and a second sdAb comprising regions of CDR1, CDR2 and CDR3 determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the amino acid sequence of B1 of the sequence of SEQ ID NO: 61, and each sdAb is linked to the Fc portion of the sequence of SEQ ID NO: 125 using the linker portion of the sequence of SEQ ID NO: 88.

[0202] In a further preferred embodiment, the multispecific construct comprises a first sdAb comprising regions of CDR1, CDR2 and CDR3 determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the amino acid sequence of A1 of the sequence of SEQ ID NO: 1, and a second sdAb comprising regions of CDR1, CDR2 and CDR3 determined using the Kabat, Chothia, IMGT or Aho numbering scheme in the amino acid sequence of B1 of the sequence of SEQ ID NO: 61, wherein each sdAb is linked to the Fc portion of the sequence of SEQ ID NO: 126 using the linker portion of the sequence of SEQ ID NO: 88.

[0203] Knob-into-hole Fc fusion In some embodiments, the multispecific construct is expressed as a knob-into-hole (KIH) Fc fusion. Thus, the multispecific constructs disclosed herein have the first and second sdAbs fused to IgG-fc via a linker as appropriate, and the multispecific construct can be generated as a knob-into-hole (KIH) Fc fusion construct.

[0204] The KIH technique involves engineering the CH3 domain to create a "knob" or "hole" in each heavy chain, which results in promoting heterodimerization and can be used to assemble multispecific constructs.

[0205] In one embodiment, the Fc domain included in the multispecific construct is derived from a human Fc domain and contains mutations that induce heterodimerization. In some embodiments, such mutations include mutations called "knob" mutations and "hole" mutations. For example, when an amino acid modification is made at Thr366 within the CH3 domain and Thr366 is replaced with a larger amino acid, such as Trp (T366W), the amino acids at the positions of Thr366, Leu368, and Tyr407 are changed to smaller amino acids, such as Ser, Ala, and Val (T366S / L368A / Y407V) respectively, and can preferentially form pairs with a second CH3 domain. In some embodiments, the "knob" Fc domain contains the mutations S354C and T366W. In some embodiments, the "hole" Fc domain contains the mutations T349C, T366S, L368A, and Y407V. Heterodimerization via modification of CH3 can be further stabilized by introducing disulfide bonds, for example, by changing Ser354 to Cys (S354C) and Y349 to Cys (Y349C) on opposite CH3 domains (reviewed in Carter et al., 2001). In some embodiments, the Fc domain used for heterodimerization contains 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 contains the modified form H435R or H435K to avoid protein A binding, while maintaining FcRn binding. In some embodiments, one member of the heterodimeric Fc pair contains the modified form H435R or H435K, while the second member of the heterodimeric Fc pair is not modified at H435. In various embodiments, the hole Fc domain contains the modified form H435R or H435K (in some cases, when the modified form is H435R, it is called "hole-R"), while the knob Fc domain does not contain them.In some cases, the purification of the heterodimer is improved by the hole-R mutation compared to the homodimeric hole Fc domain that may be present.

[0206] In another embodiment, the Fc polypeptide in the heterodimer contains mutations that change the charge polarity across the Fc dimer interface, such that co-expression of electrostatically matching Fc chains supports favorable attractive interactions, thereby promoting the formation of the desired Fc heterodimer, while unfavorable repulsive charge interactions inhibit the formation of undesired Fc homodimers (Gunescaran et al., 2010). When co-expressed intracellularly, the chains may bind to each other, but due to charge repulsion, the chains do not substantially bind to each other. Another strategy for making heterodimeric Fc is to mix human IgG with IgA CH3 domain segments to create complementary CH3 heterodimers called SEED Fc.

[0207] Heterodimerization methods and variants also include those described in the published international PCT application WO2014 / 145806, which include "knob and hole" mutations (also called "twisted" variants), mutations related to "electrostatic engineering" or "charge pairs", and pI variants. Heterodimeric variants also include any variants described in US Published Application Nos. US2012 / 0149876 or US2018 / 011883.

[0208] In some embodiments, to facilitate heterodimerization, both polypeptides of the Fc heterodimer contain paired or complementary amino acid modifications. Exemplary amino acid modifications in pairs of polypeptides in Fc fusions are listed in Table 7.

[0209]

Table 9

[0210] In some embodiments, the modification involves introducing a protrusion (knob) into the first Fc polypeptide and a cavity (hole) into the second Fc polypeptide, such that the protrusion is positioned within the cavity, facilitating the complexation of the first and second Fc-containing polypeptides. The amino acids targeted for substitution and / or modification to create a protrusion or cavity in the polypeptide are typically the interacting interface amino acids.

[0211] In embodiments herein, a multispecific construct comprising first and second sdAbs fused to IgG-fc via a linker as appropriate can be generated as a knob-into-hole (KIH) Fc fusion construct. In some of its embodiments, the knob portion of KIH comprises an IgG4-Fc having SEQ ID NO: 433 or 435. In further embodiments or alternatively, the hole portion of KIH comprises an IgG4-Fc having SEQ ID NO: 434 or 436.

[0212] Table 8 provides an overview of the full-length amino acid sequences of two individual parts of the knob-into-hole constructs disclosed herein.

[0213]

Table 10-1

Table 10-2

Table 10-3

Table 10-4

Table 10-5

Table 10-6

Table 10-7

Table 10-8

Table 10-9

Table 10-10

[0214] The multispecific constructs according to the present invention can associate by fusing any knob portion shown in Table 8 with any hole portion.

[0215] It is obvious to those skilled in the art that all constructs in Table 8 described as "knob" can, in addition, be made as "hole", and all constructs described as "hole" in Table 8 can be made as "knob".

[0216] As described above, A1 fused to the Fc-hole portion can be combined with any sdAb fused to the Fc-knob portion, regardless of whether the two sdAbs are fused at the N-terminus or the C-terminus. Therefore, the hole portion having A1 can be selected from any one of SEQ ID NOs: 93 to 96, 113 to 116, 445, 447, 449 and 451.

[0217] Similarly, when A1 is fused to the Fc-knob portion, A1 can be combined with any sdAb fused to the Fc-hole portion, regardless of whether the two sdAbs are fused at the N-terminus or the C-terminus. Therefore, the knob portion having A1 can be selected from any one of SEQ ID NOs: 105 to 112.

[0218] Furthermore, the two sdAbs can be fused to the Fc-hole portion or the Fc-knob portion using any one of the linkers described in Table 8.

[0219] Thus, in one embodiment, the multispecific construct comprises the whole part of the 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 the knob part of the 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, 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, 398, 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.

[0220] For all embodiments described herein and related to VHH / sdAb incorporated into the polypeptide of the knob and / or hole, such VHH / sdAb may undergo amino acid substitutions, for example, towards affinity maturation or towards humanization of the sequence. Further, CDR1 can contain 1, 2, or 3 amino acid substitutions, CDR2 can contain 1, 2, or 3 amino acid substitutions, and CDR3 can contain 1, 2, or 3 amino acid substitutions, for example, 1 or 2 amino acid substitutions. The resulting mutant sdAb can be evaluated by determining its binding affinity to IgE Fc or by determining its dissociation enhancement activity by an ELISA-based IgE-FcεRIα dissociation enhancement assay. Desired are mutants with activity equivalent to the parental sdAb or mutants with improved IgE affinity or improved dissociation enhancement activity.

[0221] According to the present invention, it is preferred to combine the A1 Fc-hole moiety with A1 fused at the C-terminus with any other sdAb similarly fused at the C-terminus.

[0222] Thus, in a preferred embodiment, the multispecific construct comprises a hole moiety of 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, as well as, It comprises a knob portion of 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, 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.

[0223] Similarly to the above, it is preferable to combine A1 fused at the N-terminus to the Fc-hole portion with any sdAb fused at the N-terminus to the Fc-knob portion.

[0224] Thus, in another preferred embodiment, the multispecific construct is SEQ ID NO 113, 114, 115 and 116, and it comprises a hole portion of 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, 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.

[0225] Furthermore, since various linkers can be used to attach the sdAb to the Fc-knob portion or the Fc-hole portion, it is preferable that both sdAbs are fused to the Fc portion at the C-terminus using the same linker length.

[0226] Thus, in yet another embodiment, the multispecific construct according to the invention has a combination of hole and knob portions having each of 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, and are associated.

[0227] According to the above, various linkers can be used to attach the sdAb to the Fc-knob or Fc-hole portion, and preferably both sdAbs are fused to the Fc portion at the N-terminus using the same linker length.

[0228] In yet a further embodiment, the multispecific construct according to the invention has each of SEQ ID NOs Combinations of hole portions and knob portions having 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; 113 and 333; 114 and 334; 115 and 335; 116 and 336; 113 and 341; 114 and 342; 115 and 343; 116 and 344; 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 are associated with each other having a combination.

[0229] The multispecific construct includes sdAb B1 and A1 which showed very high dissociation enhancing activity when tested in Example 6. Therefore, it is preferred to combine the A1 hole portion with the B1 knob portion fused to the Fc portion at the N-terminus.

[0230] Therefore, in a preferred embodiment, the multispecific construct according to the present invention has each sequence number 113 and 121; 114 and 122; 115 and 123; or 116 and 124 has a combined hole portion and knob portion and is associated therewith.

[0231] Similarly to the above, B1 combined with A1 fused at the C-terminus to the Fc portion also showed strong dissociation enhancing activity.

[0232] Thus, in a more preferred embodiment, the multispecific construct according to the invention has a combined hole portion and knob portion having respective SEQ ID NOs 93 and 101; 94 and 102; 95 and 103; or 96 and 104 has a combined hole portion and knob portion and is associated therewith.

[0233] As shown in Example 10, some constructs may have the risk of inducing anaphylaxis. The multispecific construct is preferably non-anaphylaxis-inducing. Thus, as seen in Example 10, the construct containing B1 and A1, linked to IgG4-Fc using a (G4S)1 linker, showed no anaphylactic potential at all.

[0234] Thus, in a preferred embodiment, the multispecific construct according to the invention has a combined hole portion and knob portion having respective SEQ ID NOs 94 and 102 and is associated therewith.

[0235] Furthermore, when evaluated in mice in Example 9, A1 combined with any of D2, E1 or F4 did not induce anaphylaxis in mice as compared to the B1A1 N-terminus linked to the Fc portion, which means that these constructs are not anaphylaxis-inducing.

[0236] Thus, in one embodiment, the multispecific construct according to the invention has a combined hole portion and knob portion having respective SEQ ID NOs 95 and 339 and is associated therewith.

[0237] In another embodiment, the multispecific construct according to the present invention has and is associated with a combination of a hole portion and a knob portion having SEQ ID NOs: 95 and 347, respectively.

[0238] In a further embodiment, the multispecific construct according to the present invention has and is associated with a combination of a hole portion and a knob portion having SEQ ID NOs: 95 and 403, respectively.

[0239] In the constructs described above, A1 represented one of the two sdAbs in all examples.

[0240] Another non-limiting example is a construct in which A2 represents one of the two sdAbs and the sdAbs listed in Table 1 represent the second sdAb. Thus, in one embodiment, the multispecific construct has a hole portion of an amino acid sequence selected from the group consisting of SEQ ID NOs: 425, 426, 427, 428, 429, 430, 431, and 432, and It comprises a knob portion of 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, 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, 398, 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.

[0241] The above list represents the knob portion and the hole portion, and the sdAb is fused to the Fc portion either at the N-terminus or the C-terminus.

[0242] The knob portion and the hole portion are preferably fused to the same terminus, which means that both are either at the N-terminus or the C-terminus.

[0243] Thus, in one embodiment, the multispecific construct comprises a hole portion of an amino acid sequence selected from the group consisting of SEQ ID NOs: 425, 426, 427 and 428, and, It comprises a knob portion of 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, 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.

[0244] In a further embodiment, the multispecific construct comprises a hole portion of an amino acid sequence selected from the group consisting of SEQ ID NOs: 429, 430, 431 and 432, and it comprises a knob portion of 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, 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.

[0245] Furthermore, as shown in Table 8, the knob portion and the hole portion comprise various linkers for fusing the sdAb to the Fc portion. The sdAb fused to the hole portion and the knob portion is preferably fused to the Fc portion using the same linker.

[0246] Thus, in another embodiment, the multispecific construct according to the invention has each SEQ ID NO Combinations of hole portions and knob portions having 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 are associated with each other having a combination of.

[0247] In yet a further embodiment, the multispecific construct according to the present invention has each sequence number Combinations of hole portions and knob portions having 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, 429 and 333, 430 and 334, 431 and 335, 432 and 336, 429 and 341, 430 and 342, 431 and 343, 32 and 344, 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 are associated and combined.

[0248] In a preferred embodiment, the multispecific construct according to the present invention is associated and combined with combinations of hole portions and knob portions having SEQ ID NOs: 429 and 121; 430 and 122; 431 and 123; or 432 and 124, respectively.

[0249] In a more preferred embodiment, the multispecific construct according to the present invention is associated and combined with combinations of hole portions and knob portions having SEQ ID NOs: 425 and 101; 426 and 102; 427 and 103; or 428 and 104, respectively.

[0250] In an even more preferred embodiment, the multispecific construct according to the invention has and is associated with a combination of a hole part and a knob part having SEQ ID NO: 426 and 102, respectively.

[0251] Heretofore, A1 or A2, which is destructive per se, was part of a multispecific construct as the hole part. Importantly, the construct can be designed such that the first sdAb having a destructive activity per se becomes the knob part.

[0252] Thus, in one embodiment, the multispecific construct has a knob part of an amino acid sequence selected from the group consisting of SEQ ID NO: 105, 106, 107, 108, and includes a hole part of the amino acid sequence of SEQ ID NO: 437.

[0253] Preferably, the multispecific construct according to the invention has and is associated with a combination of a hole part and a knob part having SEQ ID NO: 437 and 106, respectively.

[0254] Furthermore, the multispecific construct may be assembled without a linker between the sdAb and the Fc part, or by using linkers GS, G2S, G3S, EAAAK or (EAAAK)2.

[0255] Thus, in one embodiment, the multispecific construct has a knob part of an amino acid sequence selected from the group consisting of SEQ ID NO: 438, 440, 442, 444, 446, 448 and 450, and includes a hole part of an amino acid sequence selected from the group consisting of SEQ ID NO: 439, 441, 443, 445, 447, 449 and 451.

[0256] In a preferred embodiment, the multispecific construct according to the invention has and is associated with a combination of a hole part and a knob part having SEQ ID NO: 439 and SEQ ID NO: 438, respectively.

[0257] In another preferred embodiment, the multispecific construct according to the invention has and is associated with a combination of a hole part and a knob part having SEQ ID NO: 441 and SEQ ID NO: 440, respectively.

[0258] In a further preferred embodiment, the multispecific construct according to the invention has and is associated with a combination of a hole part and a knob part having SEQ ID NO: 443 and SEQ ID NO: 442, respectively.

[0259] In yet another preferred embodiment, the multispecific construct according to the invention has and is associated with a combination of a hole part and a knob part having SEQ ID NO: 445 and SEQ ID NO: 444, respectively.

[0260] In yet a further preferred embodiment, the multispecific construct according to the invention has and is associated with a combination of a hole part and a knob part having SEQ ID NO: 447 and SEQ ID NO: 446, respectively.

[0261] In a more preferred embodiment, the multispecific construct of the invention has and is associated with a combination of a hole part and a knob part having SEQ ID NO: 449 and SEQ ID NO: 448, respectively.

[0262] In an even more preferred embodiment, the multispecific construct of the invention has and is associated with a combination of a hole part and a knob part having SEQ ID NO: 451 and SEQ ID NO: 450, respectively.

[0263] The knob part of the multispecific construct consists of three parts: i) one sdAb, ii) one peptide linker, and iii) one Fc part linked to ii) at the C-terminus or N-terminus comprising i) is linked to the C-terminus or N-terminus of iii) via ii).

[0264] In addition, the whole part of the multispecific construct consists of three parts: i) One sdAb different from the sdAb present on the knob part, ii) One peptide linker, and iii) One Fc part, comprising i) is linked to the C-terminus or N-terminus of iii) via ii). Here, the Fc part is linked to other parts at the C-terminus and / or N-terminus. The three parts i), ii) and iii) of the knob are assembled into one polypeptide, while the three parts of the whole are assembled into another polypeptide. Furthermore, for both the knob part and the whole part, i) is fused with ii), and ii) is fused with iii).

[0265] In one embodiment, the knob part of the multispecific construct comprises three parts, i), ii) and iii), where i) is selected from the group consisting of SEQ ID NO: 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 NO: 88-92 and 452-457, and iii) is selected from SEQ ID NO: 434 and 436, and the whole part of the multispecific construct comprises three parts, i), ii) and iii), where i) is different from i) of the knob part and is selected from the group consisting of SEQ ID NO: 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 NO: 88-92 and 452-457, and iii) is selected from SEQ ID NO: 434 and 436.

[0266] More specific embodiments regarding the KIH prepared using sdAb A1 and B1 are defined below.

[0267] In a preferred embodiment, the knob portion of the multispecific construct comprises three parts, i), ii) and iii), where 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), where i) is SEQ ID NO: 9, ii) is SEQ ID NO: 88, and iii) is SEQ ID NO: 434.

[0268] In another preferred embodiment, the knob portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 9, 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), where i) is SEQ ID NO: 1, ii) is SEQ ID NO: 88, and iii) is SEQ ID NO: 434.

[0269] In a more preferred embodiment, the knob portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 1, ii) is SEQ ID NO: 88, and iii) is SEQ ID NO: 435, and, the hole portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 9, ii) is SEQ ID NO: 88, and iii) is SEQ ID NO: 436.

[0270] In yet a further preferred embodiment, the knob portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 9, ii) is SEQ ID NO: 88, and iii) is SEQ ID NO: 435, and, the hole portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 1, ii) is SEQ ID NO: 88, and iii) is SEQ ID NO: 436.

[0271] In a preferred embodiment, the knob portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 1, ii) is SEQ ID NO: 88, iii) is SEQ ID NO: 433, and ii) is linked to the C-terminus of iii). and the hole portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 9, ii) is SEQ ID NO: 88, iii) is SEQ ID NO: 434, and ii) is linked to the C-terminus of iii).

[0272] In another preferred embodiment, the knob portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 9, ii) is SEQ ID NO: 88, iii) is SEQ ID NO: 433, and ii) is linked to the C-terminus of iii). and the hole portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 1, ii) is SEQ ID NO: 88, iii) is SEQ ID NO: 434, and ii) is linked to the C-terminus of iii).

[0273] In a more preferred embodiment, the knob portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 1, ii) is SEQ ID NO: 88, iii) is SEQ ID NO: 435, and ii) is linked to the C-terminus of iii). and the hole portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 9, ii) is SEQ ID NO: 88, iii) is SEQ ID NO: 436, and ii) is linked to the C-terminus of iii).

[0274] In yet a further preferred embodiment, the knob portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 9, ii) is SEQ ID NO: 88, iii) is SEQ ID NO: 435, and ii) is linked to the C-terminus of iii). and The stalk portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 1, ii) is SEQ ID NO: 88, iii) is SEQ ID NO: 436, and ii) is linked to the C-terminus of iii).

[0275] In a preferred embodiment, the knob portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 5, ii) is SEQ ID NO: 88, and iii) is SEQ ID NO: 433, and the stalk portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 9, ii) is SEQ ID NO: 88, and iii) is SEQ ID NO: 434.

[0276] In another preferred embodiment, the knob portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 9, ii) is SEQ ID NO: 88, and iii) is SEQ ID NO: 433, and the stalk portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 5, ii) is SEQ ID NO: 88, and iii) is SEQ ID NO: 434.

[0277] In a further preferred embodiment, the knob portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 5, ii) is SEQ ID NO: 88, and iii) is SEQ ID NO: 435, and the stalk portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 9, ii) is SEQ ID NO: 88, and iii) is SEQ ID NO: 436.

[0278] In yet a further preferred embodiment, the knob portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 9, ii) is SEQ ID NO: 88, and iii) is SEQ ID NO: 435, and the hole portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 5, ii) is SEQ ID NO: 88, and iii) is SEQ ID NO: 436.

[0279] In a preferred embodiment, the knob portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 5, ii) is SEQ ID NO: 88, and iii) is SEQ ID NO: 433, and ii) is linked to the C-terminus of iii), and the hole portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 9, ii) is SEQ ID NO: 88, and iii) is SEQ ID NO: 434, and ii) is linked to the C-terminus of iii).

[0280] In another preferred embodiment, the knob portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 9, ii) is SEQ ID NO: 88, and iii) is SEQ ID NO: 433, and ii) is linked to the C-terminus of iii), and the hole portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 5, ii) is SEQ ID NO: 88, and iii) is SEQ ID NO: 434, and ii) is linked to the C-terminus of iii).

[0281] In a further preferred embodiment, the knob portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 5, ii) is SEQ ID NO: 88, and iii) is SEQ ID NO: 435, and ii) is linked to the C-terminus of iii), and The stalk portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 9, ii) is SEQ ID NO: 88, iii) is SEQ ID NO: 436, and ii) is linked to the C-terminus of iii).

[0282] In yet a further preferred embodiment, the knob portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 9, ii) is SEQ ID NO: 88, iii) is SEQ ID NO: 435, and ii) is linked to the C-terminus of iii), and The stalk portion of the multispecific construct comprises three parts, i), ii) and iii), where i) is SEQ ID NO: 5, ii) is SEQ ID NO: 88, iii) is SEQ ID NO: 436, and ii) is linked to the C-terminus of iii).

[0283] Other linking moieties (polymers, polysaccharides) In some embodiments, part c) of the multispecific construct is a polymer, for example, a polymer consisting of several repeating units of the monomer ethylene glycol (C2H6O2). Such polymers are called polyethylene glycols (PEGs), are synthetically produced, can be either linear or branched, and their end groups can be either standard hydroxy groups or methoxy groups (denoted as mPEG). The attachment 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 the peptide or protein, and the PEGylated monomeric sdAbs or multispecific constructs described herein can be less susceptible to renal clearance as well as proteolytic degradation and can also reduce immunogenicity.

[0284] Other polymers for the purpose 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-methacryloyloxyethyl phosphorylcholine) (PMPC).

[0285] In yet other embodiments, part c) of the multispecific construct is polysaccharides such as O-linked and N-linked oligosaccharides, dextran, hydroxyethyl starch (HES), poly sialic acid, and hyaluronic acid, natural and semi-synthetic polysaccharides, as well as unstructured protein polymers such as homoamino acid polymers, elastin-like polypeptides, XTEN (a class of unstructured biodegradable protein polymers developed by Amunix to improve the half-life of therapeutically conjugated or chemically conjugated peptides and proteins. XTEN polymers are considered non-immunogenic polypeptides composed of six hydrophilic and chemically stable amino acids A, E, G, P, S, and T.), and PAS (proline-alanine-serine polymer).

[0286] Pharmaceutical composition In a further aspect, the 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.

[0287] The pharmaceutical composition may be in liquid, semi-solid, or solid dosage form (e.g., lyophilized). The composition / dosage form can be formulated for various routes of administration, preferably for administration by injection such as intramuscular injection.

[0288] Use as a therapeutic agent The multispecific constructs, one or more monomeric sdAbs, or pharmaceutical compositions disclosed herein can be used to enhance the dissociation of IgE pre-bound to its high-affinity receptor in vivo in a subject in need thereof.

[0289] Accordingly, a further aspect relates to a method for treating or preventing an IgE-related disease or condition, the method comprising administering an effective dose of a multispecific construct, one or more monomeric sdAbs, or pharmaceutical composition disclosed herein. The effective dose is predicted to be a single dose in the range of 10 - 300 mg and can be administered once daily, once weekly, once every two weeks, or once monthly.

[0290] In other words, a further aspect relates to a multispecific construct, one or more monomeric sdAbs, or pharmaceutical composition for use as a medicament, preferably for use in anti-IgE therapy, for example for the treatment or prevention of an IgE-related disease or condition.

[0291] Putting it yet another way, a further aspect relates to a multispecific construct, one or more monomeric sdAbs, or pharmaceutical composition in the manufacture of a medicament, for example a medicament for use in anti-IgE therapy or for the treatment or prevention of an IgE-related disease or condition.

[0292] In an interesting embodiment, the IgE-related disease or disorder is selected from any one of the examples of allergic diseases, which are allergic asthma, allergic rhinitis (including seasonal allergic rhinitis and perennial allergic rhinitis), nasal polyps, atopic dermatitis, conjunctivitis, anaphylaxis, urticaria, food allergy, allergy to non-food related substances including venom from insects, wasps, bees or spiders, and allergy to therapeutic agents including antibiotics.

[0293] Other IgE-related diseases suitable for treatment include other hyper-IgE syndromes, allergic bronchopulmonary aspergillosis and other aspergillosis-related conditions, idiopathic anaphylaxis, anaphylaxis, bullous pemphigoid, pemphigus vulgaris, urticaria, for example, chronic urticaria, chronic idiopathic urticaria, nasal polyposis, chronic rhinosinusitis, mastocytosis and other mast cell disorders.

[0294] Still other IgE-related diseases that can be treated by the constructs disclosed herein may be the same as those suggested for omalizumab (Incorvaia et al., 2014).

[0295] Multispecific constructs, one or more monomeric sdAbs, or pharmaceutical compositions can be administered to a subject in need thereof, particularly a human subject. [Example 1]

[0296] Immunization and library generation The aim of this study was to generate a library of sdAbs from camels immunized with IgE-Fc.

[0297] Materials and methods Immunization of animals: Single-domain antibodies were produced by immunization of camels. Immunization was performed by different companies [Capralogics (Hardwick, USA), Eurogentec (Seraing, Belgium), and Preclinics (Potsdam, Germany)] using both llamas and alpacas. The animals were immunized with either human IgE or human IgE- according to the immunization protocols shown in Tables 9 - 12. For IgE-Fc, either Cε2 - Cε4 or Cε3 - Cε4 was used. The protein was mixed with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) before injection.

[0298]

Table 11

[0299]

Table 12

[0300]

Table 13

[0301] Library generation: sdAb libraries were constructed from each animal by purifying PBMCs from blood isolated from the animals using a Ficoll® (Cytiva) gradient. RNA was extracted from the purified PBMCs using a NucleoSpin® RNA kit (Macherey-Nagel). cDNA was synthesized from the RNA using a Superscript III First-Strand kit (Invitrogen). The sdAb gene was amplified from the cDNA by PCR using primer 1 and 2 (IgG2) and primer 1 and 3 (IgG3). The amplified sdAb gene was used to incorporate the library into a yeast display plasmid designated pNT by homologous recombination in Saccharomyces cerevisiae EBY100 (ATCC).

[0302] The library was generated using the following primers. Primer 1: taagATGCAGTTACTTCGCTGTTTTTCAATATTTTCTGTTATTGCTAGCGTTTTAGCAGCTGGAGATGTGCAGCTGCAGGAGTCTGGRGGAGG Primer 2: CCTCCACCGCCagcgtagtctggaacgtcgtatgggtatctaccttcaaTACTAGTGGGGTCTTCGCTGTGGTGCG Primer 3: CCTCCACCGCCagcgtagtctggaacgtcgtatgggtatctaccttcaaTACTAGTAGGTTGTGGTTTTGGTGTCTTGGG

[0303] Result 10 7 For each animal immunized with a yeast library of hyper-transformed cells, a yeast library of hyper-transformed cells is obtained. Quality control of the library by sequencing of colony PCR shows that all libraries contain 50 - 90% sdAb in the correct reading frame. [Example 2]

[0304] Selection of sdAb that binds to IgE-Fc The purpose of this study was to select sdAb that can bind to IgE-Fc.

[0305] Materials and methods To select sdAb that can bind to IgE-Fc, the starter culture of the library obtained in Example 1 was inoculated with 10× library size (10 8 hyper-transformed cells) in 1 L of glucose minimal medium (SD-CAA) and incubated overnight at 30 °C and 180 rpm. The next day, the culture was passaged until OD 600 = 0.2 - 0.3, and the culture was in the early exponential phase (OD 600The cells were cultured at 30 °C and 180 rpm until the optical density at 600 nm ([OD.sub.600]) reached 0.8-1.0. The cells were pelleted by centrifugation and resuspended in synthetic minimal medium containing galactose (SG-CAA) to induce display of the sdAb on the yeast cell surface (sdAb gene under the GAL1 promoter). The culture was incubated overnight at 20 °C and 180 rpm. The next day, the cells were pelleted by centrifugation and resuspended in PBS containing 5% BSA. The first round of selection for cells capable of binding IgE-Fc Cε2-Cε4 was performed by either magnetic activated cell sorting (MACS) and / or fluorescent activated cell sorting (FACS) against biotinylated IgE-Fc Cε2-Cε4.

[0306] MACS: 2×10 8 For MACS, 2×10.sup.6 cells were negatively selected against 200 μL of Streptavidin Dynabeads™ MyOne™ C1 (Invitrogen) using a magnet (Dynal). First, positive selection was performed followed by negative selection using biotinylated IgE-Fc Cε2-Cε4. The cells were incubated with 1 μM biotinylated IgE-Fc Cε2-Cε4 for 1 hour, washed with PBS containing 5% BSA, incubated with 100 μL of Streptavidin Dynabeads™ MyOne™ C1 (Invitrogen) for 30 minutes, washed with PBS containing 5% BSA, and sorted using a magnet (Dynal).

[0307] FACS: 3×l0 7The cells were incubated with 1 μM biotinylated IgE-Fc Cε2-Cε4 in PBS 1% BSA for 1 hour. The cells were washed with PBS 1% BSA and incubated with both anti-HA PE / Cy7 (BioLegend) and streptavidin PerCP / Cy5.5 (BioLegend) at 1:100 for 15 minutes. The cells were washed with PBS 1% BSA and sorted using a SH800 cell sorter (SONY).

[0308] Cells sorted from both MACS and FACS were pooled and cultured in SD-CAA. Subsequent rounds of selection were performed against biotinylated IgE-Fc Cε2-Cε4, biotinylated IgE-Fc Cε3-Cε4 335, or biotinylated IgE-Fc Cε2-Cε4 / FcεRIα complex using the same settings as the first round of selection. The rounds of selection were repeated while decreasing the target concentration (1000 nM, 200 nM, 50 nM, 10 nM) until one or more positive populations were visible, although depending on the library, 2 - 4 rounds of selection were required.

[0309] When the selection process was complete, the positive cells were cultured on SD-CAA plates and subsequently the CFUs were sequenced by Sanger sequencing to identify the sdAb of interest.

[0310] Results The immune library generated from 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 selection was initiated. Here, a large population of sdAb-expressing positives was visually identified, and a small population of IgE-Fc-binding positives could be observed within this (Figure 2A). The first round of selection was performed by MACS against 1 μM biotinylated IgE-Fc Cε2-Cε4. During the first round of selection, less than 1% of the cells were positively selected. The positive population was picked for the second round of selection performed by FACS against 200 nM biotinylated IgE-Fc Cε2-Cε4 or 200 nM of the complex biotinylated IgE-Fc Cε2-Cε4:FcεRIα. Round 2 against IgE-Fc Cε2-Cε4 shows two populations that are positive for sdAb expression and IgE-Fc binding. These two populations were sorted at gate C and together represent 18.31% of the cells (Figure 2B). The cells selected from round 2 (Figure 2B, gate C) were cultured, and the sdAbs were identified by Sanger sequencing. Ten colonies from the population were sequenced, and these colonies showed two dominant sdAbs, B1 and B3 (Table 1).

[0311] The above method was repeated for all libraries generated. The resulting sdAbs are listed in Table 1 on page 15, showing the full-length amino acid sequences of each hit, and the CDRs of each hit determined by different methods are shown in Tables 2a-d. [Example 3]

[0312] Expression of single-domain antibodies The aim of this study was to express both sdAb monomers and dimers using different types of linkers.

[0313] Materials and methods The SdAb monomer identified in the objective (Table 1) and Example 2 was amplified from the pNT plasmid by PCR and recloned into the pET22b plasmid by the SLiCE cloning reaction. Thereby, the pelB signal sequence and the C-terminal his / HA tag were added to the sdAb, resulting in secretion into the periplasmic space followed by purification and detection. The SdAb was expressed in Escherichia coli Rosetta(DE3) cells using 2YT medium. Expression was induced in the early exponential phase using 0.5 mM IPTG and carried out overnight at 20 °C to 25 °C. Purification of the sdAb from the supernatant or supernatant + periplasmic extract was performed by IMAC using a HisTrap excel column (Cytiva) as the first step and by SEC using a Hiload 16-600 superdex® 75 pg (Cytiva) column as the second step. The size and purity of each sdAb were evaluated by 15% acrylamide SDS-PAGE. Subsequently, the immunoreactivity of the sdAb against immobilized IgE-Fc Cε2-4 and IgE-Fc Cε3-4 was analyzed by ELISA. IgE-Fc was coated overnight at 2 μg / mL on Nunc MaxiSorp plates (ThermoFisher). After blocking, the sdAb was added at a 1:10 dilution in TBS and incubated at room temperature for 3 hours. Detection was performed using a mouse anti-HA antibody (ThermoFisher) and an anti-mouse IgG AP-conjugated (Sigma A1drich).

[0314] Following expression, relative epitope mapping (binding) was performed for each individual sdAb monomer compared to different sdAbs using the Octet RED96e system (ForteBIO). In this specification, biotinylated IgE Fc (manufactured in-house at ALK, Denmark) was immobilized onto SAX (high-precision streptavidin) sensors (ForteBIO) up to 1 nm using kinetic buffer (PBS, 0.02% Tween20, 0.1% BSA) as a diluent. Subsequently, the first sdAb (100 nM) was incubated with the IgE Fc-loaded streptavidin sensor for 300 seconds, and then the second sdAb (100 nM) was incubated with the sensor for 300 seconds. This method was performed for all combinations of sdAbs. Data was analyzed in Octet Data Analysis HT 11.1 software (ForteBIO) using the epitope binning function to generate a matrix.

[0315] Constructs containing two sdAbs linked by a peptide linker Using sdAbs that can simultaneously bind to IgE-Fc detected by the above relative epitope mapping, constructs containing two sdAbs were prepared.

[0316] Constructs containing two sdAb ("dimer") genes were constructed by PCR. First, each sdAb was amplified using primers containing a (G4S)4 linker. Subsequently, the sdAbs were assembled by a second-step PCR to form a "dimer" prior to cloning. Expression and purification were performed as described for the sdAb monomer. Purification of the "dimer" was carried out as described for the monomer.

[0317] Constructs containing two sdAbs linked by IgG-Fc Another way to combine two separate sdAbs is through Fc fusion (Figure 1). In this specification, IgG4-Fc is used as an example. The heterodimerization of sdAbs fused to IgG4-Fc is achieved using the "knob-into-hole" mutation of the IgG4 Fc domain (Carter et al., 1998), and an RS or (G4S) n peptide linker such as between the sdAb and IgG4-Fc (Figure 1). A plasmid encoding the first sdAb (mutated to S354C or T366W) fused to the "knob" IgG4-Fc (Figure 1A) and the second sdAb (mutated to T349C, T366S, L368A or Y407V) fused to the "hole" IgG4 Fc (Figure 1B) is co-transfected into human embryonic kidney cells (Expi293). The expression supernatant is collected after 6 days, centrifuged, and filtered. The bispecific IgG4 antibody construct is purified using a protein A column. After loading the supernatant, the column is washed with 10 CV of 20 mM sodium phosphate buffer pH 7, and the bound protein is eluted with 0.1 M citric acid pH 3. The eluted fractions are immediately neutralized with 1 M Tris-HCl pH 9. Further, the eluted fractions are loaded onto a size exclusion chromatography (SEC) column using PBS as the running buffer. The eluted fractions are combined, concentrated, and sterile filtered through a 0.2 μm syringe filter.

[0318] Results The selected monomeric sdAbs (Table 1) and A1 (comparative control sdAb026) were cloned into the pET22b plasmid. All were expressed by Escherichia coli Rosetta(DE3), but the yields of expression differed depending on the sdAb (1 - 10 mg / 1 L of culture). The size and purity of the sdAbs were controlled by 15% acrylamide SDS-PAGE and Coomassie blue staining (data not shown). As shown for monomeric sdAb B1, the size of the target compound was within the expected range between 15 kDa and 20 kDa (data not shown). After the first stage of purification, extra compounds or aggregates around 130 kDa were visually observed above the sdAb band. The second stage of purification made it possible to remove this contaminant and show a high-purity monomer (data not shown).

[0319] The sdAb monomers that bind towards IgE-Fc were verified by ELISA. All sdAbs showed immunoreactivity against IgE-Fc Cε2-4 (Table 13). However, only four monomeric sdAbs (A1, D3, F6 and Gl) showed immunoreactivity against IgE-Fc Cε3-4 (Table 13). This indicates that most of the selected sdAbs bind to IgE-Fc through the Cε2 domain.

[0320]

Table 14

[0321] Relative epitope mapping of the expressed sdAbs showed that most monomeric sdAbs bind to the IgE antibody simultaneously with A1 and thus bind to another IgE-binding epitope other than A1 (Figure 3). In the present specification, sdAbs B3, B1, D1 and D2 share similar epitopes different from the epitopes of B2 or A1. Such sdAbs can be used in combination with any of A1, A2 or other sdAbs having different binding sites to IgE to modify the overall anti-IgE activity of one sdAb. For example, it is to improve the dissociation of IgE from the receptor binding.

[0322] Epitope mapping was used to determine combinations of sdAbs for dimerization, with or without IgG4 fusion.

[0323] Various examples of sdAb "dimers" fused through a (G4S)4 linker were cloned and expressed (Table 3). As an example, a dimer formed by N-terminal B1 and C-terminal A1 [B1A1(G4S)4] is shown. From the elution of the first purification, a distinct band is visualized at the expected size between 35 kDa and 45 kDa on SDS-PAGE after Coomassie blue staining. However, a number of contaminating compounds of various sizes are also presented. The second stage of purification can remove such contaminants and refine the purity of the sdAb "dimer" (data not shown).

[0324] Regarding the heterodimerization of sdAbs fused to IgG4-Fc, Table 8 shows other examples of "dimers" (combinations of sdAb, IgG4-Fc, and linker).

[0325] Conclusion Both monomeric and dimeric single-domain antibodies as well as IgG4-Fc fusion sdAbs were expressed and purified, resulting in high purity and having the sizes predicted by SDS-PAGE analysis. [Example 4]

[0326] Mutation of Single-Domain Antibodies The aim of this study was to investigate the effect of single-point mutations on sdAb A1 and generate mutants with improved dissociation-enhancing activity.

[0327] Materials and Methods Two libraries were constructed from A1 (Accession No. 1). Library 1 covered the amino acids of A1 from position 11 to position 61, and Library 2 covered positions 62 - 112. Each library was generated by SOE PCR using oligopools from Integrated DNA Technologies, and the NNK codon was introduced at each position. The resulting DNA libraries contained all possible single amino acid mutants at positions 11 - 61 of Library 1 and positions 62 - 112 of Library 2. To evaluate the effects of these mutations, the libraries were cloned into a yeast display plasmid designated pNT by homologous recombination in the budding yeast EBY100 (ATCC) according to the procedure described by (Benatuil et al., 2010).

[0328] Each library was cultured, expression was induced, and stained using triple staining as described in Example 2. The single round of selection by FACS for the biotinylated IgE:FcεRIα complex at a concentration corresponding to K D for sdAb in yeast (determined by titration) was performed as described in Example 2. Two populations were selected: a negative population (anti - HA +, streptavidin -) and a positive population (anti - HA +, streptavidin +, anti - FcεRIα -).

[0329] The selected populations were cultured at 30 °C in SD - CAA medium. Plasmids were extracted separately from the non - selected library, the negative population, and the positive population using the Zymoprep Yeast Plasmid Miniprep II kit (Zymo Research). The sdAb gene from the extracted plasmids was amplified by PCR. The purified PCR products were sent to Deeptope (France) for deep sequencing and data analysis using Illumina technology.

[0330] 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.

[0331] result SdAb A1 displayed on yeast had a K of 0.345 nM for the biotinylated IgE:FcεRIα complex. D ) (data not shown). Selection of the A1 DMS library was therefore performed at this concentration to monitor the effect of mutations on binding. A negative population of sdAbs expressed on yeast but not binding biotinylated IgE was selected (data not shown). A positive population of sdAbs expressed on yeast and binding biotinylated IgE but negative for FcεRIα was selected (data not shown). The sdAbs from the pre-selection library, the negative and positive populations were sequenced using deep sequencing to determine the frequency of each nanobody in the library before and after selection. Thus, the effect of each single mutation on A1 can be assessed by calculating the enrichment score (enrichment = frequency of mutation after selection / frequency before selection) (Figure 4). Mutations with a high enrichment score in the negative population are deleterious to the activity of A1 and will most likely be removed in the positive population, such as the mutation P45K. Mutations with a high enrichment score in the positive population but removed in the negative population, such as the mutation V93E, will improve A1 affinity for IgE-Fc.

[0332] Using Deep Mutational Scanning data, three mutations (T28R, P45Y and V93E) that were enriched in the positive population and eliminated in the negative population were selected to construct a mutant version of sdAb A1, named A2. A2 was expressed and purified as previously described in Example 2, and its binding towards IgE-Fc was confirmed.

[0333] Other mutations are similarly enriched in the positive population and thus, like the three selected mutations above, improve the A1 affinity for IgE-Fc. The following mutations can be used, either 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, D73I, D73L, D73V, D73A, D73G, D73S, D73T, D73N, D73Q, D73E, D73H, D73K, D73R, 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.

[0334] The above method is similarly applicable to sdAb B2 and B3, and the following mutations are similarly enriched in the positive population and thus improve the affinity for IgE-Fc. Such mutations can be used, either alone or in combination, to construct mutant versions of B2 or B3 respectively. B2: V12T, A23Q, A23D, T28Q, T28H, D35M, D35A, D36V, Q39F, Q39W, Q39Y, Q39I, Q39V, Q39E, Q44P, D74P, D74I, S105I, S105N, S105D and S105E 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, T69Q, S71I, N77G, R78M, 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

[0335] Conclusion Using deep mutagenesis scanning, the effects of all single mutations of sdAbs A1, B2 and B3 on their interaction with IgE-Fc / FcεRIa were evaluated. A list of mutations that could improve the binding affinity of A1, B2, B3 to IgE-Fc could be created. Furthermore, a triple mutant of A1 named A2 was generated and successfully expressed and purified. [Example 5]

[0336] Determination of Affinity The purpose of this study was to test the affinity of the sdAbs obtained in the previous examples for IgE-Fc.

[0337] Materials and Methods The affinity of the monomer, dimer sdAb, and multispecific construct for IgE-Fc was tested using kinetic buffer (PBS, 0.02% Tween 20, 0.1% BSA) as a diluent on an Octet RED96e system (ForteBIO). Biotinylated IgE Fc (produced in-house at ALK, Denmark) was immobilized on SAX (high-precision streptavidin) sensors (ForteBIO) up to a maximum of 1 nM. Association (240 seconds) and dissociation (600 seconds) were measured for the analytes in serial dilutions (from 10 nM to 0.37 nM). The data were subtracted from the input of a blank reference sensor exposed to the same serial analyte dilutions, aligned, and analyzed using the Octet Data Analysis HT 11.1 software (ForteBIO) with a 1:1 fitting model.

[0338] Results The affinity for IgE Fc was measured for five individual sdAbs and compared to A1 (Table 14). k on is a constant used to characterize how fast the sdAb binds to IgE, while k off characterizes how fast the sdAb dissociates from IgE. The ratio of k off / k on yields the equilibrium dissociation constant K D . The lower the K D value, the higher the affinity of the sdAb for IgE. Typically, sdAbs bind their antigens with k on 10 5 ~10 6 (M -1 s -1 ) and k off dissociation rates of 10 -3 (s -1 ), resulting in binding events in the low nM affinity range. Here, B2 and B3 showed the highest affinity towards IgE, which is equivalent to A1 in the low nM affinity range (approximately 1×10 -10 M). B1 and D2 showed lower affinities (6 - 8×10 -8 M).

[0339]

Table 15

[0340] The affinity of the additional sdAbs described in this specification for IgE-Fc is measured by the same procedure.

[0341] Conclusion The selected non-optimized sdAbs show high affinity for IgE comparable to A1 in the high nM to low nM range. [Example 6]

[0342] Dissociation enhancement effect of single domain antibodies measured by ELISA Objective The objective of this example was to evaluate the ability of sdAbs to enhance the dissociation of the binding between human IgE and the high-affinity human IgE receptor (FcεRIα) using a biochemical ELISA assay.

[0343] Materials and methods The dissociation enhancing effect was tested using an IgE-FcεRIα dissociation-enhanced ELISA assay. Maxisorp® microtiter plates (Thermo Scientific Nunc, Roskilde, Denmark) were coated with 100 μl of streptavidin (Thermo Scientific, Waltham, MA) at 5 μg / ml at 4 °C for 18 h, followed by washing with 300 μl of wash buffer (PBS containing 0.05% Tween 20) using an AquaMax® 2000 plate washer (Molecular Devices, San Jose, CA). Residual binding sites were blocked with 250 μl of blocking buffer (PBS containing 2% BSA) for 2 h and washed with 300 μl of wash buffer. Subsequently, 100 μl (0.1 μg / ml) of recombinant human FcεRIα (site-specifically biotinylated via an avi-tag, in-house production, ALK, Horsholm, Denmark) was loaded onto the immobilized streptavidin for 1 h, and the plate was washed twice with 300 μl of wash buffer. Next, 100 μl of recombinant human IgE (in-house production, ALK, Horsholm, Denmark) at 13 ng / ml, (pre-titrated to reach an OD 450 450 signal in the range of 1.5 - 2.5 in the absence of the dissociation enhancing component) was loaded for 1 h, followed by washing three times with 300 μl of wash buffer.

[0344] Dissociation enhancement step: 100 μl of sdAb diluted in a concentration series (range pM - μM) or only blocking buffer as a negative control that does not enhance dissociation was added to the ELISA wells and incubated for 1 h. Any IgE whose dissociation from the receptor was enhanced by the sdAb was washed away by washing three times with 300 μl of wash buffer.

[0345] The remaining IgE that was not enhanced in dissociation was detected by adding 100 μl of HRP-conjugated anti-human lambda light chain (Bethyl Laboratories, Montgomery, Tx) diluted 1:20,000 in blocking buffer for 1 hour, followed by washing 3 times with 300 μl. Then, 100 μl of TMB One (Kementec, Taastrup, Denmark) was added to the wells, incubated for 15 minutes, and the reaction was stopped by adding 100 μl of 1N sulfuric acid.

[0346] Finally, the absorbance at OD 450 was measured with a Glomax Discover plate reader (Promega, Madison, Wi).

[0347] Results were analyzed using GraphPad Prism ver. 9.3.0 (GraphPad Software, San Diego, CA), and the percent dissociation enhancement effect was determined as the relative decrease in signal compared to the sdAb-free control. EC 50 values and maximum effects were determined from the graphs (not shown) listed in Table 15.

[0348] All types of both monomeric and dimeric sdAbs as well as sdAb-Fc fusion complexes can be tested using this procedure.

[0349] Results Listed in Table 1 are sdAbs tested in an IgE-FcεRIα dissociation-enhancing ELISA assay as either monomers or pairs of combinations constructed as dimers or multispecific constructs. The sdAbs of B1, B2, B3, D1, D2, E1, E2, E3, E4, Fl, F2, F3, F4, F5, F6 were combined with A1 and fused to IgG4-Fc (as seen in Figure 1). The IgG4-Fc fusion was either at the N-terminus or C-terminus using various linkers between the sdAb and IgG4. Additionally, sdAb A2 was combined with B1 in the same manner. Table 15 shows the results of all the sdAbs and constructs tested as well as the Fc fusion constructs.

[0350] The ability of various sdAbs to enhance the dissociation from the IgE receptor (FcεRIα) was evaluated by use of an ELISA-based IgE-FcεRIα dissociation-enhancing assay. The percent dissociation-enhancing effect was calculated as the relative decrease in signal compared to an sdAb-free control. EC 50 values and the maximum effect were determined (Table 15).

[0351]

Table 16

[0352] Overall, it was observed that only a few monomers sdAb:A1, A2 and B1 had dissociation enhancing activity. In particular, this dissociation enhancing activity could be significantly improved by pairing an sdAb with dissociation enhancing activity with another sdAb that could bind to a separate epitope on IgE-Fc, either as a simple mixture of two sdAbs, a dimer or a construct fused to IgG-Fc.

[0353] The most effective constructs were identified from among the multimeric constructs, which appeared to be much more effective than the sdAb monomers. The most effective multimeric constructs showed EC 50 values in the sub-nanomolar to single-digit nanomolar range, with a maximum effect of over 95% at the highest test concentration. In comparison, the control construct KIH_E07_79 was also very potent in this assay (EC 50 = 0.6 nM and maximum effect over 95%). As long as the monomeric sdAbs of the multimeric constructs bind to separate IgE epitopes, they are considered multispecific constructs.

[0354] The relative orientation of the two linked sdAb entities within the sdAb dimer had a significant effect in some cases. For example, B3A1(G4S)4 was much more effective than the reverse orientation A1B3(G4S)4 (EC 50 = 190 nM) (EC50 = 0.3 nM). However, there were also examples where the relative orientation did not seem to be as important. For example, D2A1(G4S)4 vs A1D2(G4S)4 showed EC 50 values in the same range (8 nM and 10 nM, respectively).

[0355] A mixture of sdAb A1 and additional sdAb monomers [but not linked together - see sdAb mixture, (Table 15)] showed a synergistic effect, which in most cases had an EC 50This is because the value was significantly lower than that of any two sdAbs alone. Nevertheless, none of these mixtures were as effective as the most potent linked sdAb dimer.

[0356] A2 was more effective than A1 in enhancing the dissociation of IgE from the receptor (ECs of 250 nM and 800 nM, respectively). 50 )

[0357] Conclusion Some of the linked sdAb dimers and sdAb-IgG4 Fc fusion constructs showed EC values in the sub-nanomolar to single-digit nanomolar range and were able to enhance the dissociation of more than 95% of IgE from the receptor. 50 [Example 7]

[0358] Cellular evaluation of sdAb dissociation-enhancing activity using the RBL assay The aim of this study was to evaluate the ability of sdAbs to enhance the dissociation of the binding between human IgE expressed in a cell line and the high-affinity human IgE receptor (FcεRIα).

[0359] Materials and methods The ability of sdAbs to enhance the dissociation of IgE from the receptor was tested using the RBL assay.

[0360] Rat basophilic leukemia cells (RBL SX-38) were cultured in complete MEM / cMEM:MEM (Gibco 11095-080) + 100 mM Na-pyruvate (Gibco 11360-+70) + 15% FCS + pen / strep (Lonza DE17-602E) + G418 (Gibco 10131-027) at 1 mg / ml in a horizontal TC culture flask. For IgE sensitization, the cells were washed with PBS to remove proteins, detached from the TC culture flask, and then trypsin-Versene (LONZA cat no 17-161E) was added. After incubation at 37 °C for 5 minutes, 10 5Cells were seeded at 200 μl of cMEM per well. The cells were incubated overnight (5% CO2, 37 °C).

[0361] Sensitization of RBL SX-38 with IgE was performed after removing the supernatant. A mixture of three Der p 2-specific recombinant human IgE clones: H10, H12, P4E (Christensen et al. 2008) was used. 100 μl of cMEM containing 1 μg / ml of recombinant human IgE was added to each well and incubated in a CO2 incubator (5% CO2, 37 °C) for 2 hours. Wells for non-sensitized controls were incubated with cMEM only for comparison.

[0362] Dissociation enhancement of bound IgE: To the sensitized RBL SX-38 cells, SdAb or a comparative control was added. SdAbs with peptide linkage and IgG-Fc fusion were tested at a concentration of 1 μM and its 10-fold dilution, while single sdAbs were tested at a concentration of 1 μM. In addition, a mixture of sdAb A1 and individual single sdAbs was also tested using 1 μM of each. The cells were incubated in a CO2 incubator (5% CO2, 37 °C) for 1 hour.

[0363] FACS staining of surface-bound IgE: Cells from the RBL assay were washed twice separately with FACS staining buffer (BD FACS Flow + 0.5% BSA) by centrifugation at 500×g for 5 minutes, and the cell pellet was stained with α-FcεR1 FITC (Invitrogen 11-5899-42) and anti-IgE APC (Invitrogen 17-6886-42). The cells were incubated on ice for 30 minutes, followed by washing with FACS staining buffer and then fixed with 200 μl / well of cell fixative (BD 340181) for 15 minutes at room temperature. After further washing, the cells were analyzed on a flow cytometer: Cytek Aurora equipped 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 mean fluorescence reading of IgE for the assay was analyzed using GraphPad Prism ver. 9.3.0 (GraphPad Software, San Diego, CA), and the percent dissociation enhancement effect was determined as the relative decrease in signal compared to the sdAb-free control. The maximum dissociation enhancement effect and EC 50 (the sdAb concentration that results in 50% inhibition compared to the sdAb-free control) was determined from the dose-response graph of IgE expression (mean fluorescence) using non-linear curve fitting of logarithmically transformed data in GraphPad Prism.

[0364] Results The sdAbs of interest (Table 16) were tested for dissociation enhancement activity in the RBL assay using rat basophilic leukemia cells (RBL-SX38). The sdAbs were tested in various combinations as either sdAb monomers, sdAb dimers (bispecific or bivalent) linked together regardless of the presence or absence of the Fc domain, or combinations of two individual sdAb monomers added in the same setting. The dissociation enhancement effect was determined for the EC 50 and maximum effect (A1 and KIH_E7_79 for comparison), and the effects for the remaining sdAbs were listed together with 1 μM (Table 16).

[0365] The sdAb is (individually) combined with A1 or A2 and is fused to IgG4-Fc at either the N-terminus or C-terminus using various linkers (described in Tables 8 and 15), as schematically seen in Figure 1. Table 16 shows the results of the constructs tested.

[0366] The activity of each construct tested is shown in the form of EC 50 and maximum dissociation enhancement activity.

[0367] All linked sdAb dimers showed effective dissociation enhancement of IgE from FcER1 on RBL-SX38 cells, in contrast to the monospecific bivalent A1A1(G4S)4 construct (Table 16). The 11 most effective dimers completely enhanced the dissociation of IgE at an EC 50 of 2 nM or less: B1A1(G4S)4, A1B1(G4S)4, A1B2(G4S)4, B2A1(G4S)4, A1B3(G4S)4, A1E1(G4S)4, A1E4(G4S)4, F3A1(G4S)4, F4A1(G4S)4, A1F6(G4S)4, F6A1(G4S)4 (Table 16).

[0368] The data shows that D2A1(G4S)4 was less potent than A1D2(G4S)4 with an EC50 of approximately 78 nM vs approximately 4 nM, and the same was true for B2A1(G4S)4 vs B2A1(G4S)4 with an EC 50 of approximately 6 nM vs approximately 1 nM, indicating that the order of linkage plays a role.

[0369] Mixtures of the sdAb monomer A1 with various sdAb monomers (unlinked) improved dissociation enhancement in all cases, and were most effective with mixtures with B1 and B2, with an effect of approximately 100% using 1 μM sdAb (Table 16).

[0370] Dissociation enhancement by a single sdAb shows that B1 enhances the dissociation of IgE more effectively than A1 at 1 μM.

[0371] In the case of a multispecific construct in which two sdAbs are linked to an Fc domain, most of the constructs tested showed effective dissociation-enhancing activity compared to the individual unlinked sdAbs.

[0372]

Table 17

[0373] Inhibition of effector cell degranulation. The aim is to evaluate whether an sdAb can inhibit effector cell degranulation during an allergen challenge.

[0374] Materials and methods PBMCs were separated from heparinized whole blood from allergic donors by the method using Lymfoprep (Fresenius Kabi 1114547) and Leucoseptubes (Greiner 227290). After separation, the cells were washed and resuspended in RPMI, HSA: [RPMI 1640 (Gibco 72400-021) + 0.5% HSA (Sigma A1653)] to 1 / 16 of the original blood volume and used for dissociation enhancement with the sdAB construct by incubating at 37°C for 1 hour at various concentrations. The pre-incubated cells were washed with RPMI, HSA and resuspended in RPMI, HSA supplemented with 2 ng / mL of IL-3 (RD peprotec cat 200-03) to 1 / 8 of the blood volume.

[0375] Cells with enhanced IgE dissociation and controls were used for basophil activation with a-IgE or allergen diluted in RPMI + 0.5% HSA, and were stimulated with different concentrations of rDerp2, rBetv1 or Derp extract (in-house at ALK), 1000 - 100 - 10 ng / mL, depending on donor sensitization and anti-IgE (Dako A0094).

[0376] Following a 1-hour incubation in a CO2 incubator (5% CO2, 37 °C), the reaction was stopped by the addition of BD FACS flow + 0.5% BSA + 10 mM EDTA.

[0377] FACS staining of activated cells: Cells were washed twice with FACS staining buffer (BD FACS flow + 0.5% BSA) by centrifugation at 500 × G for 5 minutes, and the cell pellet was stained with a-CD63 FIC (BD92467), a-CD203c APC (e-bioscience, 324610), CD123 PE (BD034345). Cells were incubated on ice for 30 minutes, followed by washing with FACS staining buffer, and then fixed with 200 μl / well of cell fixative (BD340181) at room temperature for 15 minutes. After further washing, the cells were analyzed on a flow cytometer: Cytek Aurora equipped with a plate loader. When comparing with the IgE dissociation enhancement efficiency, samples of cells not used in the activation assay were stained for IgE using a-IgE APC (ebioscience 17-6986) and FcER1 Cra1 BV605 (BD747785) combined with CD123 PE 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 percentage of basophils with high CD63 expression was graphed as the % of activated basophils using Graph Prisma.

[0378] The above method was further used in a more physiological setting. In this specification, heparinized whole blood was used instead of PBMCs for 1+1 direct incubations with dilutions of the sdAb construct. After washing, the blood was reconstituted with RPMI supplemented with IL-3 and HSA, and the assay was performed as described for PBMCs.

[0379] Results Treatment of basophils with sdAbs results in a decrease in basophil activation upon stimulation with allergen or a-IgE (Figure 5A-C).

[0380] The dose-response induction of activation by rDerp2 in donors with HDM allergy was reduced to approximately one tenth after treatment with 1 μM sdAb A1, and cells showing a 16% dissociation enhancement effect as measured by IgE expression were seen (Table 17).

[0381] Effective treatment was observed when sdAb A2 was combined with a single sdAb B1 or B2, while no effect was observed when sdAb A2 was combined with B3, in which case the activation was equivalent to that of A2 alone (Figure 5B-C). These findings reflect the effect of dissociation enhancement on IgE surface expression (Table 17), where sdAb A2 combined with B1 or B2 shows an effect of approximately 100%, while A2 combined with B3 and A2 alone show an equivalent dissociation enhancement effect of approximately 70%.

[0382] The multispecific construct [CIgG4B1A1(G4S)1 was able to decrease the activation of basophils in PBMCs (Figure 5D) and whole blood (Figure 5E-F) in a dose-dependent manner demonstrated at concentrations in the range of 10 - 1000 nM (Figure 5D-F), although the construct did not show direct cell activation.

[0383] Conclusion In conclusion, what the cellular evaluation of sdAb-mediated IgE dissociation enhancement reveals is that effective enhancement of IgE dissociation from the FcER1 receptor located on the cell surface can be obtained by using dissociation-enhancing sdAbs A1 or A2 in combination with some of the other tested sdAbs, or by using them as conjugated sdAbs.

[0384] Enhanced dissociation of IgE was demonstrated using RBL-SX38 cells sensitized with recombinant IgE.

[0385] In addition, enhanced dissociation of IgE from human basophils derived from allergic donors and the impact of this enhanced dissociation on subsequent challenges of basophils with either an allergen or anti-IgE were similarly demonstrated. [Example 9]

[0386] Evaluation of the ability to prevent anaphylaxis in a mouse model The purpose of this example is to evaluate the ability of sdAbs to prevent anaphylaxis in vivo.

[0387] Materials and methods B6.Cg-Fcer1a tm1KntTg(FCER1A)1Bhk / J mice were originally obtained from the Jackson Laboratory and then bred in-house for use in models of passive systemic anaphylaxis (PSA) or passive cutaneous anaphylaxis (PCA). For the PSA model, mice were sensitized by intraperitoneal (i.p.) injection of 200 μl per mouse of a mixture of three Der p 2-specific recombinant human IgE clones: H10, H12, P4E (Christensen et al. 2008) (containing 16.67 μg / ml of each Ab in PBS). Twenty-four hours later, mice were injected i.p. with 200 μl of sterile PBS or sdAb CIgG4B1A1(G4S)1 at various concentrations (150 μM, 10 μM, 2 μM, 0.4 μM, and 0.08 μM in PBS), followed 4 hours later by an allergen challenge [i.p. injection of 200 μl of 0.05 μg / ml rDer p 2 (ALK in-house)]. Deep body temperature was measured every 5 minutes using a temperature transponder (IPTT-300, implanted subcutaneously at least 1 day before the start of the experiment) until just prior to inhibitor injection or allergen challenge and until 1 hour after that or 90 minutes after the challenge for serum collection (BD Microtainer SST tubes, 365968). The mast cell protease 1 (mMCPT1) concentration in mouse serum was measured using an MCPT-1 (mMCP-1) Mouse Uncoated ELISA kit (Invitrogen, 88-7503-88) according to the manufacturer's recommendations (Figure 6A). For the PCA model, mice were sensitized intradermally with 20 μl of the same three anti-Der p 2 antibodies but at 2.5 μg / ml each. Normally, inhibitors were injected either intradermally or i.p. into the mice 6 hours before intravenous challenge with rDer p 2 (10 μg per mouse, 100 μl) in a 1% Evans blue (Sigma-Aldrich, E2129) solution contained in sterile NaCl solution the following day. However, for the anaphylatoxin test, the inhibitor was injected intradermally and an Evans blue solution without rDer p 2 was injected i.v. simultaneously. Mice were sacrificed 30 minutes after dye injection and the extravascular leakage area of the dye on the back was measured.Next, dorsal skin was harvested for pigment extraction using formamide (Merck, S4117). The optical density of the extracted pigment was measured at 600 nm (Figure 8B).

[0388] Results Anaphylaxis was prevented in a dose-dependent manner in the PSA model by CIgG4B1A1(G4S)1. Mice showed no change in core body temperature after injection of the inhibitor alone (Figure 6B), but animals treated with the lowest concentration of CIgG4B1A1(G4S)1 (0.08 μM) responded similarly to control group (PBS-injected) mice with respect to both a decrease in body temperature and an increase in serum mMCPT1. After treatment with the 0.4 μM dose, mice were partially protected, and anaphylaxis was almost completely prevented after treatment with the two highest doses of CIgG4B1A1(G4S)1 (Figure 6C-E). Similarly, constructs of CIgG4D2A1(G4S)2, CIgG4E1A1(G4S)2, and CIgG4F4A1(G4S)1 were not anaphylaxis-inducing per se (Figure 7A), but were able to interfere with anaphylaxis with varying efficiencies (Figure 7B). On the other hand, the N-linked construct NIgG4B1A1(G4S)3 induced a decrease in the core body temperature of mice during the PSA model procedure (Figure 8A) and induced extravasation of pigment into the sensitized skin area in the PCA model of the anaphylaxis-provoking test (Figure 8C).

[0389] Conclusion The sdAbs of CIgG4B1A1(G4S)1, CIgG4D2A1(G4S)2, CIgG4E1A1(G4S)2, and CIgG4F4A1(G4S)1 were able to prevent anaphylaxis in vivo in a murine passive systemic anaphylaxis model. [Example 10]

[0390] Evaluation of potentially anaphylaxis-inducing constructs. The aim of this study was to evaluate the ability of sdAbs, alone or as part of a construct, to activate basophils.

[0391] Materials and Methods PBMC isolated from heparinized whole blood (described in Example 8) or heparinized whole blood from allergic donors were incubated with various concentrations of sdAb constructs diluted in RPMI, 5% HSA in a CO2 incubator (5% CO2, 37°C) for 1 hour. The reaction was stopped by the addition of BD FACS Flow + 0.5% BSA + 10 mM EDTA, and FACS staining of the cells to measure CD63 expression and IgE expression was performed using the same methods and reagents as described in Example 8.

[0392] Results An example is shown in Figure 9, demonstrating the dissociation-enhancing activity of various constructs upon increasing concentration (similar setup as in Example 7). Further, Figure 9A+C shows examples of percentages regarding basophil activation induced by different concentrations of the same construct. The entire construct fused at the N-terminus showed high activation at the aforementioned intervals, while the construct fused at the C-terminus was less anaphylaxis-inducing. Additionally, as seen in Figure 9A+C, the linker of the construct fused at the C-terminus similarly affected the anaphylaxis-inducing activity. The construct CIgG4B1A1(G4S)1 was tested on cells isolated from over 20 different donors, and none of the donors showed any anaphylaxis-inducing activity at any concentration. On the other hand, the construct NIgG4B1A1(G4S)1 was tested on a similar number of donors, and all donors showed anaphylaxis-inducing activity. Interestingly, the comparative control KIH_E7_79 showed anaphylaxis-inducing activity. In some donors, KIH_E7_79 was activated only at high concentrations, while in other donors, activation was also seen at lower concentrations. The remaining constructs listed in Table 15 showed high variability in terms of anaphylaxis-inducing activity between different donors, but also within the same donor, meaning that clear conclusions cannot be drawn from this assay alone but can be confirmed in mice as seen in Example 9.

[0393] Conclusion Overall, this example shows that N-terminally linked multispecific constructs generally activate basophils and thus have anaphylactic activity, while most of the C-terminally linked constructs, depending on the linker, did not have anaphylactic activity. Importantly, CIgG4B1A1(G4S)1 showed no anaphylactic activity when tested in over 200 donors. The comparative control KIH_E7_79 showed a high degree of anaphylactic activity compared to CIgG4B1A1(G4S)1. This setting is an easy and simple way to test whether a particular construct may have anaphylactic activity. Anaphylactic activity can be confirmed in a mouse model. [Example 11]

[0394] Humanization of sdAb Objective Humanizing therapeutic antibodies derived from animal immunization is often required to minimize the risk of immunogenicity in humans, which can potentially be harmful, cause severe side effects, and reduce the effectiveness of the antibody. Therefore, the objective of this study is to humanize the sdAbs described herein.

[0395] Materials and methods The CDRs and frameworks of the sdAbs for humanization are determined as described herein, and each sdAb is provided with three individual CDR regions (CDR1-3) and four framework regions (FR1-4).

[0396] Each of the four framework regions (FR1-FR4) is first fully humanized by identifying the closest human germline VH sequences and replacing these sequences with the camelid framework sections. This results in a 100% degree of humanization.

[0397] The next step is to test the functionality of the humanized sdAb as to whether its original functionality is maintained.

[0398] If the adjusted sequence is not affected by humanization or if an acceptable decrease in functionality is observed (which may be a decrease in antigen binding or a decrease in the dissociation enhancement effect), the humanization procedure is terminated and the final degree of humanization is 100%.

[0399] On the other hand, if functionality is unacceptably affected at 100% humanization, the sdAb is subjected to a "reverse mutation procedure" in order to reduce the degree of humanization by reintroducing the original camelid amino acid sequence one by one into individual frames. Following the reintroduction of the original camelid amino acid sequence, the functionality of the sdAb is determined. The degree of humanization is recalculated and the humanization procedure is terminated. If the loss of functionality due to humanization is restored upon undergoing the reverse mutation procedure, the degree of humanization is recalculated and the humanization procedure is terminated. The final degree of humanization (0 - 100%) depends on the effect on the functionality of the humanized construct and cannot be predicted.

[0400] List of references JPEG2025523583000037.jpg211170JPEG2025523583000038.jpg243170JPEG2025523583000039.jpg236170JPEG2025523583000040.jpg74170

Claims

1. d. A first single domain antibody (sdAb) capable of enhancing the dissociation of bound IgE from FcεRI, e. A second sdAb that binds to IgE, f. A moiety linking the first and second sdAbs comprising, - the first and second sdAbs bind to non-identical epitopes of IgE, - a multispecific construct that can enhance the dissociation of bound IgE from the IgE high affinity receptor (FcεRI) by an improved dissociation enhancing activity compared to the first or second sdAb.

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

3. The dissociation enhancing activity is evaluated by an ELISA-based IgE-FcεRIα disruption assay that measures the residual IgE not removed from immobilized recombinant human FcεRIα pre-loaded with IgE after the addition of the sdAb or multispecific construct, and determines the percentage of dissociation enhancing activity as the relative decrease in signal compared to a control without the test compound added. The multispecific construct according to any one of the preceding claims.

4. The dissociation enhancing activity of the first sdAb is such that the EC ５０ is an activity in the range of 100 nM to 5000 nM, the multispecific construct according to any one of the preceding claims.

5. The dissociation enhancing activity of the first sdAb is an activity such that the maximum dissociation enhancing activity is at least 80%. The multispecific construct according to any one of the preceding claims.

6. The dissociation enhancing activity of the first sdAb is such that EC ５０ is in the range of 100 nM to 5000 nM and the maximum dissociation enhancing effect is at least 80%, the multispecific construct according to any one of the preceding claims.

7. The second sdAb has no dissociation enhancement activity, or has a lower dissociation enhancement activity in a state with a higher EC compared to the first sdAb, or has a maximum dissociation enhancement effect of less than 75%, the multispecific construct according to any one of the preceding claims. ５０ ​

8. Improved dissociation-enhancing activity of the multispecific construct is the EC observed for either the first sdAb or the second sdAb, or a mixture of the first sdAb and the second sdAb ５０ such that the EC for the construct is consequently lower ５０ The multispecific construct according to any one of the preceding claims, which is an activity of this nature

9. The improved dissociation enhancing activity of the multispecific construct is EC ５０ is the EC of the first sdAb ５０ The multispecific construct according to any one of the preceding claims, which has an activity such that it is reduced to 1 / 20 or less compared to

10. The improved dissociation-enhancing activity of the multispecific construct is EC ５０ The multispecific construct according to any one of the preceding claims, which has an activity such that EC is less than 100 nM.

11. The improved dissociation enhancing activity of the multispecific construct is an activity such that the maximum dissociation enhancing effect is at least 80%. The multispecific construct according to any one of the preceding claims.

12. The improved dissociation enhancing activity of the multispecific construct is such that the EC ５０ is less than 100 nM and the maximum dissociation enhancement is at least 80%, the multispecific construct according to any one of the preceding claims.

13. The first sdAb comprises or consists of complementarity determining regions (CDRs) CDR1, CDR2 and CDR3 each having an amino acid sequence determined according to any of the Kabat, Chothia, IMTG or Aho numbering schemes in an amino acid sequence selected from any one of SEQ ID NO: 1 or 5, wherein 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. The multispecific construct according to any one of the preceding claims.

14. The first sdAb comprises complementarity-determining regions (CDRs) CDR1, CDR2 and CDR3 having respective SEQ ID NOs 2, 3 and 4; or 6, 7 and 8, each CDR being determined according to the Aho numbering scheme, 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, the multispecific construct according to any one of the preceding claims. **Claim 15** The second sdAb comprises complementarity-determining regions (CDRs) CDR1, CDR2 and CDR3 each containing or consisting of an amino acid sequence determined according to any of the Kabat, Chothia, IMTG or Aho numbering schemes in an amino acid sequence selected from any one of SEQ ID NOs 9, 13, 17, 21, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69 and 73, 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, the multispecific construct according to any one of the preceding claims. **Claim 16** The second sdAb comprises complementarity-determining regions (CDRs) CDR1, CDR2 and CDR3 having respective SEQ ID NOs 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 being determined according to the Aho numbering scheme, 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, the multispecific construct according to any one of the preceding claims. **Claim 17** a) The first sdAb comprises or consists of binding regions CDR1, CDR2, and CDR3 each containing an amino acid sequence determined according to any one of the Kabat, Chothia, IMTG, or Aho numbering schemes in an amino acid sequence selected from any one of SEQ ID NO: 1 or 5, wherein 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; b) The second sdAb comprises or consists of binding regions CDR1, CDR2, and CDR3 each containing an amino acid sequence determined according to any one of the Kabat, Chothia, IMTG, or Aho numbering schemes in an amino acid sequence selected from any one of SEQ ID NO: 9, 25, 33, 61, wherein 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, the multispecific construct according to any one of the preceding claims. **Claim 18** a) The first sdAb comprises binding regions CDR1, CDR2, and CDR3 having respective SEQ ID NOs 2, 3, and 4; or 6, 7, and 8, each CDR being determined according to the Aho numbering scheme, wherein 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; b) The second sdAb comprises binding regions CDR1, CDR2, and CDR3 having respective SEQ ID NOs 10, 11, and 12; 26, 27, and 28; 34, 35, and 36; or 62, 63, and 64, each CDR being determined according to the Aho numbering scheme, wherein 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, the multispecific construct according to any one of the preceding claims. **Claim 19** a) The first sdAb comprises or consists of binding regions CDR1, CDR2, and CDR3, each of which comprises an amino acid sequence determined according to any one of the Kabat, Chothia, IMTG, or Aho numbering schemes in the amino acid sequence of SEQ ID NO:

1. 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; b) The second sdAb comprises or consists of binding regions CDR1, CDR2, and CDR3, each of which comprises an amino acid sequence determined according to any one of the Kabat, Chothia, IMTG, or Aho numbering schemes in the amino acid sequence of SEQ ID NO:

9. 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. The multispecific construct according to any one of the preceding claims. **Claim 20** a) The first sdAb comprises binding regions CDR1, CDR2, and CDR3 having SEQ ID NOs: 2, 3, and 4, respectively. Each CDR is determined according to the Aho numbering scheme. 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; b) The second sdAb comprises binding regions CDR1, CDR2, and CDR3 having SEQ ID NOs: 10, 11, and 12, respectively. Each CDR is determined according to the Aho numbering scheme. 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. The multispecific construct according to any one of the preceding claims. **Claim 21** a) The first sdAb comprises or consists of complementarity-determining regions CDR1, CDR2, and CDR3 each containing an amino acid sequence determined according to any of the Kabat, Chothia, IMGT, or Aho numbering schemes in the amino acid sequence of SEQ ID NO: 1, wherein 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; b) The second sdAb comprises or consists of complementarity-determining regions CDR1, CDR2, and CDR3 each containing an amino acid sequence determined according to any of the Kabat, Chothia, IMGT, or Aho numbering schemes in the amino acid sequence of SEQ ID NO: 25, wherein 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. The multispecific construct according to any one of claims 1 to 18. **Claim 22** a) The first sdAb comprises complementarity-determining regions CDR1, CDR2, and CDR3 having SEQ ID NOs: 2, 3, and 4, respectively, each CDR being determined according to the Aho numbering scheme, wherein 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; b) The second sdAb comprises complementarity-determining regions CDR1, CDR2, and CDR3 having SEQ ID NOs: 26, 27, and 28, respectively, each CDR being determined according to the Aho numbering scheme, wherein 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. The multispecific construct according to any one of claims 1 to 18 and 21. **Claim 23** a) The first sdAb comprises or consists of binding regions CDR1, CDR2, and CDR3, each of which comprises an amino acid sequence determined according to any one of the Kabat, Chothia, IMGT, or Aho numbering schemes in the amino acid sequence of SEQ ID NO: 1, and 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; b) The second sdAb comprises or consists of binding regions CDR1, CDR2, and CDR3, each of which comprises an amino acid sequence determined according to any one of the Kabat, Chothia, IMGT, or Aho numbering schemes in the amino acid sequence of SEQ ID NO: 33, and 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. The multispecific construct according to any one of claims 1 to 18. **Claim 24** a) The first sdAb comprises binding regions CDR1, CDR2, and CDR3 having SEQ ID NOs: 2, 3, and 4, respectively, each CDR being determined according to the Aho numbering scheme, and 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; b) The second sdAb comprises binding regions CDR1, CDR2, and CDR3 having SEQ ID NOs: 34, 35, and 36, respectively, each CDR being determined according to the Aho numbering scheme, and 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. The multispecific construct according to any one of claims 1 to 18 and 23. **Claim 25** a) The first sdAb comprises or consists of binding regions CDR1, CDR2, and CDR3 each containing an amino acid sequence determined according to any one of the Kabat, Chothia, IMTG, or Aho numbering schemes in the amino acid sequence of SEQ ID NO: 1, wherein 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; b) The second sdAb comprises or consists of binding regions CDR1, CDR2, and CDR3 each containing an amino acid sequence determined according to any one of the Kabat, Chothia, IMTG, or Aho numbering schemes in the amino acid sequence of SEQ ID NO: 61, wherein 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. The multispecific construct according to any one of claims 1 to 18. **Claim 26** a) The first sdAb comprises binding regions CDR1, CDR2, and CDR3 having SEQ ID NOs: 2, 3, and 4 respectively, each CDR being determined according to the Aho numbering scheme, wherein 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; b) The second sdAb comprises binding regions CDR1, CDR2, and CDR3 having SEQ ID NOs: 62, 63, and 64 respectively, each CDR being determined according to the Aho numbering scheme, wherein 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. The multispecific construct according to any one of claims 1 to 18 and 25. **Claim 27** The multispecific construct according to any one of claims 13 to 26, wherein the 1, 2, or 3 amino acid substitutions in CDR1, CDR2, and CDR3 are conservative substitutions. **Claim 28** 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 to Leu, the multispecific construct according to any one of claims 13 to 27.

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 moiety is a peptide having a maximum of 30 amino acid residues.

31. a moiety having the formula (G ｎ S) ｎ (wherein n can be an integer from 1 to 6); an arginine-serine peptide; a peptide EAAAAK and (EAAAAK) ２ a peptide selected from the group consisting of: a multispecific construct according to any one of the preceding claims

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

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

35. The multispecific construct according to claim 33 or 34, wherein the fragment of the antibody is the Fc portion of the antibody, and the first sdAb and the second sdAb are independently fused or covalently linked to the N-terminus or C-terminus of the Fc portion via an optional linker.

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

37. - The first and second sdAbs may both be fused or covalently bound directly to or via a linker to the C Ｈ 2 of IgG-Fc, - The first and second sdAbs may both be fused or covalently bound directly or via a linker to the C Ｈ 3 of IgG-Fc, - The first sdAb is fused or covalently bound directly or via a linker to the C of IgG-Fc Ｈ 2, and the second sdAb is fused or covalently bound directly or via a linker to the C of IgG-Fc Ｈ 3, or - The second sdAb is fused or covalently linked directly or via a linker to the C of IgG-Fc, and the first sdAb is fused or covalently linked directly or via a linker to the C of IgG-Fc. Ｈ 2, or the multispecific construct according to claim 36, wherein the first sdAb is fused or covalently linked directly or via a linker to the C of IgG-Fc. Ｈ 3.

38. Via a suitable linker, the first and second sdAbs are each fused to the C of IgG-Fc Ｈ 3, or the first and second sdAbs are each fused to the C of IgG-Fc Ｈ 2, the multispecific construct according to claim 36 or 37.

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

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

41. A linker is a peptide having the formula (G ｎ S) ｎ wherein n can be an integer from 1 to 6); an arginine-serine peptide; a peptide EAAAAK and (EAAAAK) ２ A multispecific construct according to any one of claims 36 to 40, which is a peptide selected from the group consisting of

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

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

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

46. The multispecific construct according to any one of the preceding claims, which can be produced as a knobs-into-holes (KIH) Fc fusion construct when the first and second sdAbs are fused to IgG-fc via an appropriate linker.

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

48. The multispecific construct according to claim 46 or 47, wherein the hole portion of the KIH comprises an IgG4-Fc having SEQ ID NO: 434 or 436.

49. The multispecific construct according to claim 46, wherein the hole portion of the 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 to 96, 113 to 116, 445, 447, 449 and 451.

50. The knob portion of KIH comprises the second sdAb 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, the multispecific construct according to claim 46 or 49.

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

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

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

54. 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, the multispecific construct according to claim 40.

55. The shaft portion of KIH comprises the second sdAb defined in claim 19 or 20, and the shaft portion has SEQ ID NO: 437, the multispecific construct according to claim 40 or 54.

56. The multispecific construct according to any one of claims 40 to 55, wherein the shaft portion and the knob portion comprise the same peptide linker.

57. The multispecific construct according to any one of claims 40 to 56, wherein the shaft portion and the knob portion comprise an sdAb fused to either the C-terminus or the N-terminus of IgG4-Fc via a linker.

58. The knob-into-hole construct consists of two polypeptides having array numbers 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 respectively, the multispecific construct according to claim 40.

59. The knob-into-hole construct consists of two polypeptides having array numbers 94 and 102 respectively, the multispecific construct according to claim 40.

60. The multispecific construct according to any one of claims 40 to 59, wherein the sdAb amino acid sequences of the hole and knob portions are humanized.

61. The multispecific construct according to claims 40 to 61, wherein the sdAb amino acid sequences of the hole and knob portions have amino acid substitutions in the amino acid sequences outside the CDR regions, and each frame of the sdAb amino acid sequence contains a maximum of three amino acid substitutions.

62. A monomeric sdAb capable of binding to IgE, comprising or consisting of binding regions CDR1, CDR2 and CDR3 each containing an amino acid sequence determined according to any of the Kabat, Chothia, IMTG or Aho numbering schemes in 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, wherein 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, monomeric sdAb.

63. comprising complementarity-determining regions CDR1, CDR2, and CDR3 having sequence numbers 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 respectively, each CDR being determined according to the Aho numbering scheme, wherein 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, the monomeric sdAb according to claim 62. **Claim 64** The monomeric sdAb according to claim 62 or 63, wherein the 1, 2, or 3 amino acid substitutions in CDR1, CDR2, and CDR3 are conservative substitutions. **Claim 65** The monomeric sdAb according to any one of claims 62 to 64, wherein the 1, 2, or 3 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 to Leu. **Claim 66** The monomeric sdAb according to any one of claims 62 to 65, comprising or consisting of an amino acid sequence selected from any one of sequence numbers 5, 9, 13, 17, 21, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, and 73 or a humanized version thereof. **Claim 67** A polypeptide comprising one or more monomeric sdAbs according to any one of claims 62 to 66 as part of its sequence.

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, further comprising a pharmaceutically acceptable carrier and / or vehicle and / or diluent and / or excipient.

69. The pharmaceutical composition according to claim 68, which is in liquid, semi-solid or solid dosage form.

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

71. A method for the treatment or prevention of IgE-related diseases or conditions, comprising administering an effective amount of 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, a polypeptide according to claim 67, or a pharmaceutical composition according to any one of claims 68 to 70.

72. For use as a medicament, preferably for use in anti-IgE therapy, for example for the treatment or prevention of IgE-related diseases or conditions, 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.

73. Use of 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, in the manufacture of a medicament, for example for use in anti-IgE therapy or for the treatment or prevention of IgE-related diseases or conditions.

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

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