WO2024200854A1

WO2024200854A1 - Allergen binding antibodies suitable for treating tree pollen allergies

Info

Publication number: WO2024200854A1
Application number: PCT/EP2024/058821
Authority: WO
Inventors: Peter Sejer Andersen; Lars Harder CHRISTENSEN; Niels Peter Christensen; Anders Lund
Original assignee: ALK Abello AS
Current assignee: ALK Abello AS
Priority date: 2023-03-31
Filing date: 2024-03-31
Publication date: 2024-10-03
Anticipated expiration: 2025-09-30

Abstract

Tree pollen allergy is a seasonal allergy caused by various airborne tree pollens. The allergy affects a significant percentage of the population, particularly in Europe and North America, and is characterized by symptoms such as sneezing, nasal congestion, runny nose, itchy and watery eyes, and throat irritation. The symptoms typically occur during the spring months when birch-, alder-, hazel- and oak trees release their pollen into the air. Tree pollen allergy is a type 1 hypersensitivity reaction mediated by immunoglobulin E (IgE) antibodies. The present invention relates to antibodies and antigen-binding fragments thereof that bind to one or more of tree pollen allergens; Bet v 1, Aln g 1, Cor a 1 and Que a 1, leading to inhibition of IgE mediated allergic reaction.

Description

ALLERGEN BINDING ANTIBODIES SUITABLE FOR TREATING TREE POLLEN ALLERGIES

FIELD OF THE INVENTION

The present invention is related to antibodies and antigen-binding fragments thereof that bind to one or more of the tree pollen allergens; Bet v 1, Ain g 1, Cor a 1 and Que a 1, therapeutic compositions comprising the antibodies, and methods of using the antibodies.

BACKGROUND OF THE INVENTION

Tree pollen allergy is a seasonal allergy caused by various airborne tree pollens. The allergy affects a significant percentage of the population, particularly in Europe and North America, and is characterized by symptoms such as sneezing, nasal congestion, runny nose, itchy and watery eyes, and throat irritation. The symptoms typically occur during the spring months when birch-, alder-, hazel- and oak trees release their pollen into the air. Tree pollen allergy is a type 1 hypersensitivity reaction mediated by immunoglobulin E (IgE) antibodies. IgE is produced by B cells in response to the presence of allergens, such as Bet v 1, which is the major allergen found in birch pollen (D'Amato et al., 2007).

Bet v 1 belongs to a family of proteins known as pathogenesis-related protein 10 (PR-10), which are found in various tree pollens and have been shown to induce cross- reactivity among different allergens. For instance, Que a 1, Ain g 1, and Cor a 1 are homologous allergens found in oak-, alder-, and hazelnut pollen, respectively, that share significant structural and immunological similarities with Bet v 1. Therefore, individuals allergic to one of the above mentioned allergen, may in addition, experience allergic reactions to the other cross- reactive allergens, which can complicate the diagnosis and management of the allergy (Asam et al. , 2015).

Tree pollen allergy symptoms are caused by crosslinking of tree pollen allergen specific IgE bound to the high-affinity epsilon receptor (FcsRI) on the surface of mast cells and basophils, which triggers the release of inflammatory mediators, such as histamine, leukotrienes, and cytokines. These mediators cause the characteristic symptoms of the allergy and may induce bronchial hyperresponsiveness and asthma in some individuals (Gould and Sutton, 2008).

The current treatment options for tree pollen allergy include avoidance of the allergen, pharmacotherapy, and allergen-specific immunotherapy (AIT). Avoidance of the allergen involves minimizing exposure to tree pollen through measures such as staying indoors during high pollen counts, wearing a mask, and keeping windows closed. Pharmacotherapy options include antihistamines, nasal corticosteroids, leukotriene receptor antagonists, and decongestants, which can alleviate the symptoms of the allergy but are not long-term disease-modifying. AIT is a disease-modifying treatment that involves the administration of gradually increasing doses of the allergen to induce immunological tolerance and reduce the severity of the allergy. AIT can be administered either subcutaneously or sublingually and has been shown to be effective in reducing the symptoms of tree pollen allergy and improving the quality of life of patients (Canonica et al., 2014).

Allergen specific antibodies have previously been proposed as a treatment for allergies, since they may be able to block the entry of allergenic molecules into the mucosal tissues or may bind the allergen before it has the opportunity to bind to the IgE bound to the high affinity receptor on mast cells or basophils, thus preventing the release of histamine and other inflammatory mediators from these cells.

Allergen specific antibodies for treatment of IgE mediated allergies has previously been described (US 5,670,626 and US 6,849,259), including antibodies for blocking of allergen binding to mast cells (US 2010/0034812)

The patent application WO 2018/222854 describes a treatment of birch allergy by using an antibody composition of two or more antibodies binding different epitopes on the same Bet v 1 allergen leading to a more efficient blocking of the allergen mediated cell activation. Only one of the Bet v 1 binding antibodies disclosed in WO 2018/222854 can also bind other tree pollen allergens Ain g 1, and Cor a 1. Thus, said antibody is cross reactive to the allergens, Ain g 1, and Cor a 1.

This strategy of combining antibodies specific for the same allergen has further been described in the patent applications WO 2013/166236 and WO 2018/234383, which relate to treating cat allergy by binding the allergen Fel d 1 and peanut allergy by binding the allergen Ara h 2, respectively.

However, the tree pollen season is not an isolated event but is an overlapping event where different tree pollen allergen described herein, peaks at different timepoints a year. Thus, aiming a treatment at a single allergen will not effectively treat the symptoms of patients being allergic to two or more of the PR-10 family allergens.

The present invention is directed towards overcoming the above-described problems. SUMMARY OF THE INVENTION

The present invention relates to an antibody or antigen-binding fragment thereof capable of binding a tree pollen allergen selected from the group consisting of Bet v 1, Ain g 1, Cor a 1 and Que a 1.

The present inventors have provided a selection of antibodies with the potential to treat pollen allergies caused by four important tree pollen allergens. As shown herein, the antibodies can individually bind to all the four allergens Bet v 1, Ain g 1, Cor a 1 and Que a 1 with good affinity and would in combination be able to outcompete the binding of these allergens to IgE antibodies in a human if administered in a therapeutically effective dose. As shown in example 2, a number ofindividual antibodies shown in table 1, such as the seven antibodies 2E02, 2C10, 2B04, 2E0, A07, BIO, and 2_10, were all able to bind all four tree pollen allergens (Bet v 1, Ain g 1, Cor a 1 and Que a 1), with higher affinity than prior art antibodies like REGN5713, REGN5714 and REGN5715 described in WO 2018/222854. Further as described in example 5, the combination of two or three antibodies showed an increased ability, compared to individual antibodies, to blocking the binding between the individual tree pollen allergens and IgE, leading to an inhibition of basophil activation, which as described earlier, is a key driver of allergy.

Accordingly, in a first aspect, the present invention relates to the antibodies 2E02, 2C10, 2B04, 2E0, A07, BIO, and 2_10 or antigen binding fragments thereof. Thus, a first aspect relates to an antibody or antigen-binding fragment thereof comprising three heavy chains CDRs (HCDR1, HCDR2 and HCDR3) and three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein;

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence contained within SEQ ID NO: 1, a HCDR2 amino acid sequence contained within SEQ ID NO: 1, a HCDR3 amino acid sequence contained within SEQ ID NO: 1, a LCDR1 amino acid sequence contained within SEQ ID NO: 2, a LCDR2 amino acid sequence contained within SEQ ID NO: 2 and a LCDR3 amino acid sequence contained within SEQ ID NO: 2; or

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence contained within SEQ ID NO: 21, a HCDR2 amino acid sequence contained within SEQ ID NO: 21, a HCDR3 amino acid sequence contained within SEQ ID NO: 21, a LCDR1 amino acid sequence contained within SEQ ID NO: 22, a LCDR2 amino acid sequence contained within SEQ ID NO: 22 and a LCDR3 amino acid sequence contained within SEQ ID NO: 22; or • the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence contained within SEQ ID NO: 41, a HCDR2 amino acid sequence contained within SEQ ID NO: 41, a HCDR3 amino acid sequence contained within SEQ ID NO: 41, a LCDR1 amino acid sequence contained within SEQ ID NO: 42, a LCDR2 amino acid sequence contained within SEQ ID NO: 42 and a LCDR3 amino acid sequence contained within SEQ ID NO: 42; or

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence contained within SEQ ID NO: 61, a HCDR2 amino acid sequence contained within SEQ ID NO: 61, a HCDR3 amino acid sequence contained within SEQ ID NO: 61, a LCDR1 amino acid sequence contained within SEQ ID NO: 62, a LCDR2 amino acid sequence contained within SEQ ID NO: 62 and a LCDR3 amino acid sequence contained within SEQ ID NO: 62; or

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence contained within SEQ ID NO: 81, a HCDR2 amino acid sequence contained within SEQ ID NO: 81, a HCDR3 amino acid sequence contained within SEQ ID NO: 81, a LCDR1 amino acid sequence contained within SEQ ID NO: 82, a LCDR2 amino acid sequence contained within SEQ ID NO: 82 and a LCDR3 amino acid sequence contained within SEQ ID NO: 82; or

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence contained within SEQ ID NO: 101, a HCDR2 amino acid sequence contained within SEQ ID NO: 101, a HCDR3 amino acid sequence contained within SEQ ID NO: 101, a LCDR1 amino acid sequence contained within SEQ ID NO: 102, a LCDR1 amino acid sequence contained within SEQ ID NO: 102 and a LCDR3 amino acid sequence contained within SEQ ID NO: 102; or

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence contained within SEQ ID NO: 121, a HCDR2 amino acid sequence contained within SEQ ID NO: 121, a HCDR3 amino acid sequence contained within SEQ ID NO: 121, a LCDR1 amino acid sequence contained within SEQ ID NO: 122, a LCDR2 amino acid sequence contained within SEQ ID NO: 122 and a LCDR3 amino acid sequence contained within SEQ ID NO: 122.

Each of the CDRs (HCDR1, HCDR2 and HCDR3, LCDR1, LCDR2 and LCDR3) as mentioned above may be determined according to the either IMGT, Kabat or Chothia method. By option, each of the CDR regions determined by either IMGT, Kabat or Chothia method may be subject to 1, 2, or 3 amino acid substitutions such as each HCDR1 may contain 1, 2, or 3 amino acid substitutions, each HCDR2 may contain 1, 2, or 3 amino acid substitutions, each HCDR3 may contain 1, 2, or 3 amino acid substitutions, each LCDR1 may contain 1, 2, or 3 amino acid substitutions, each LCDR2 may contain 1, 2, or 3 amino acid substitutions, and/or each LCDR3 may contain 1, 2, or 3 amino acid substitutions.

Alternatively worded the first aspect relates to an antibody or antigen-binding fragment thereof, wherein;

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence of SEQ ID NO: 3, a HCDR2 amino acid sequence of SEQ ID NO: 4, a HCDR3 amino acid sequence of SEQ ID NO: 5, a LCDR1 amino acid sequence of SEQ ID NO: 6, a LCDR2 amino acid sequence of SEQ ID NO: 7 and a LCDR3 amino acid sequence of SEQ ID NO: 8; or

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence of SEQ ID NO: 23, a HCDR2 amino acid sequence of SEQ ID NO: 24, a HCDR3 amino acid sequence of SEQ ID NO: 25, a LCDR1 amino acid sequence of SEQ ID NO: 26, a LCDR2 amino acid sequence of SEQ ID NO: 27 and a LCDR3 amino acid sequence of SEQ ID NO: 28; or

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence of SEQ ID NO: 43, a HCDR2 amino acid sequence of SEQ ID NO: 44, a HCDR3 amino acid sequence of SEQ ID NO: 45, a LCDR1 amino acid sequence of SEQ ID NO: 46, a LCDR2 amino acid sequence of SEQ ID NO: 47 and a LCDR3 amino acid sequence of SEQ ID NO: 48; or

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence of SEQ ID NO: 63, a HCDR2 amino acid sequence of SEQ ID NO: 64, a HCDR3 amino acid sequence of SEQ ID NO: 65, a LCDR1 amino acid sequence of SEQ ID NO: 66, a LCDR2 amino acid sequence of SEQ ID NO: 67 and a LCDR3 amino acid sequence of SEQ ID NO: 68; or

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence of SEQ ID NO: 83, a HCDR2 amino acid sequence of SEQ ID NO: 84, a HCDR3 amino acid sequence of SEQ ID NO: 85, a LCDR1 amino acid sequence of SEQ ID NO: 86, a LCDR2 amino acid sequence of SEQ ID NO: 87 and a LCDR3 amino acid sequence of SEQ ID NO: 88; or • the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence of SEQ ID NO: 103, a HCDR2 amino acid sequence of SEQ ID NO: 104, a HCDR3 amino acid sequence of SEQ ID NO: 105, a LCDR1 amino acid sequence of SEQ ID NO: 106, a LCDR2 amino acid sequence of SEQ ID NO: 107 and a LCDR3 amino acid sequence of SEQ ID NO: 108; or

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence of SEQ ID NO: 123, a HCDR2 amino acid sequence of SEQ ID NO: 124, a HCDR3 amino acid sequence of SEQ ID NO: 125, a LCDR1 amino acid sequence of SEQ ID NO: 126, a LCDR2 amino acid sequence of SEQ ID NO: 127 and a LCDR3 amino acid sequence of SEQ ID NO: 128, wherein each HCDR1 may contain 1, 2, or 3 amino acid substitutions, each HCDR2 may contain 1, 2, or 3 amino acid substitutions, each HCDR3 may contain 1, 2, or 3 amino acid substitutions, each LCDR1 may contain 1, 2, or 3 amino acid substitutions, each LCDR2 may contain 1, 2, or 3 amino acid substitutions, and/or each LCDR3 may contain 1, 2, or 3 amino acid substitutions.

Still, alternatively worded, the first aspect relates to an antibody or antigen-binding fragment thereof, wherein;

• the antibody or antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 1 and a LCVR having the amino acid sequence of SEQ ID NO: 2; or

• the antibody or antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 21 and a LCVR having the amino acid sequence of SEQ ID NO: 22; or

• the antibody or antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 41 and a LCVR having the amino acid sequence of SEQ ID NO: 42; or the antibody or antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 61 and a LCVR having the amino acid sequence of SEQ ID NO: 62; or the antibody or antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 81 and a LCVR having the amino acid sequence of SEQ ID NO: 82; or

• the antibody or antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 101 and a LCVR having the amino acid sequence of SEQ ID NO: 102; or

• the antibody or antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 121 and a LCVR having the amino acid sequence of SEQ ID NO: 122, wherein each HCVR may contain 1, 2, or 3 amino acid substitutions, and/or each LCVR may contain 1, 2, or 3 amino acid substitutions.

In a second aspect, the invention relates to combinations of two different antibodies or antigen-binding fragments thereof, such as a composition comprising at least two antibodies or antigen-binding fragment thereof, preferably the composition comprises three antibodies, such as at least three antibodies, optionally, the composition comprises four antibodies or antigen-binding fragments thereof, wherein the antibodies and antigen-binding fragments thereof are selected from the antibodies or antigen-binding fragments thereof described herein, such as particularly selected from the group the seven antibodies 2E02, 2C10, 2B04, 2E0, A07, BIO, and 2_10 or antigen-binding fragments thereof.

In a preferred embodiment, the composition comprises two antibodies or antigen-binding fragments thereof according to the invention.

In another preferred embodiment, the composition comprises three antibodies or antigenbinding fragments thereof according to the invention.

In a third aspect, the invention relates to a pharmaceutical composition comprising a therapeutic effective amount of one or more antibodies, or antigen-binding fragment thereof selected from the antibodies or antigen-binding fragment thereof defined herein together with one or more pharmaceutically acceptable excipients.

In a preferred embodiment thereof, the invention relates to a pharmaceutical composition comprising a therapeutic effective amount of three antibodies, or antigen-binding fragment thereof selected from the antibodies or antigen-binding fragment thereof defined herein together with one or more pharmaceutically acceptable excipients. LEGENDS TO THE FIGURE

Figure 1 shows epitope binning matrix of selected antibodies towards Bet v 1. A black square indicates no pair-wise binding of mAbs to Bet v 1. A white square indicates pair-wise binding of mAbs to Bet v 1. * = epitope bin representative of mAb 1_4 and 1_11. ** = epitope bin representative of mAb 1_5, 2_6, 2_7, 2_11, 2_13, 2_18.

Figure 2 shows an overview of the antibodies obtained as described in the examples. Each ring represents one of the antibodies in table 1. Overlapping rings illustrate that the antibodies are binding to overlapping epitopes on Bet v 1, whereas non-overlapping rings shows antibodies binding to distinct non-overlapping epitopes.

Figure 3 shows a sensorgram of sequential binding of three mAbs to Bet v 1. 0-60 s: baseline. 60-480 s: mAb A07 loaded to Protein A sensor tips. 480-510 s: baseline. 510-810 s: Blocking mAb. 810-840 s: baseline. 840-1140 s: Bet v 1. 1140-1200 s: baseline. 1200- 1500 s: mAb A07. 1500-1560 s: baseline. 1560-1860 s: mAb 2B04. 1860-1910 s: baseline. 1910-2210 s: mAb 2E02. 2210-2270 s: baseline.

Figure 4 shows inhibition of basophil activation induced by stimulation with nBet v 1. Mixes of mAbs containing 4 nM of each was preincubated with nBet v 1 at different concentrations shown at X-axis before adding to PBMC. Basophil activation was measured by flow cytometry as described in example 5- Activated basophils shown as percentage of CD63 positive cells at Y-axis. Data from 6 donors (Figure 4A-F) shown.

Figure 5 shows inhibition of basophil activation induced by stimulation with nAln g 1. Mixes of mAbs containing 4 nM of each was preincubated with nAln g 1 at different concentrations shown at X-axis before adding to PBMC.. Basophil activation was measured by flow cytometry as described in example 5- Activated basophils shown as percentage of CD63 positive cells at Y-axis. Data from 6 donors (Figure 5A-F) shown.

Figure 6 shows inhibition of basophil activation induced by stimulation with nCor a 1. Mixes of mAbs containing 4 nM of each was preincubated with nCor a 1 at different concentrations shown at X-axis before adding to PBMC.. Basophil activation was measured by flow cytometry as described in example 5- Activated basophils shown as percentage of CD63 positive cells at Y-axis. Data from 6 donors (Figure 6A-F) shown.

Figure 7 shows inhibition of basophil activation induced by stimulation with nQue a 1. Mixes of mAbs containing 4 nM of each was preincubated with nQue a 1 at different concentrations shown at X-axis before adding to PBMC. Basophil activation was measured by flow cytometry as described in example 5- Activated basophils shown as percentage of CD63 positive cells at Y-axis. Data from 6 donors (Figure 7A-F) shown.

Figure 8 shows inhibition of basophil activation induced by stimulation with nBet v 1, nAln g 1, nCor a 1, or nQue a 1. Allergen was preincubated with mAbs, before stimulation of PBMC. Basophil activation was measured by flow cytometry as described in example 5. Percentage of inhibition relative to activated basophils in absence of mAb shown at Y-axis.

Figure 9 shows inhibition of basophil activation induced by stimulation with nBet v 1, nAln g 1, nCor a 1, and nQue a 1. Allergen was preincubated with mAbs, before stimulation of PBMC. Basophil activation was measured by flow cytometry as described in example 5. Percentage of inhibition relative to activated basophils in absence of mAb shown at Y-axis.

DETAILED DISCLOSURE OF THE INVENTION

Definitions

Allergen

The term "allergen" in the context of the invention is a compound that is recognized by the immune system as foreign so that an immunoreaction to the allergen is evoked. In other words, it refers to molecules that are not present in the human body and may produce an abnormal immune response by IgE-mediated activation of mast cells and basophils triggering symptoms of allergy. The skilled person understands that in the context of the invention "autoantigens", i.e. molecules that are produced by the human body are not considered as allergen. Examples of allergens are reported by the World Health Organization and International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Subcommittee, which can be found on the web url: httD://alleraen.org/.

The term "antibody", as used herein, means any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g. , Bet v 1). The term "antibody", as used herein, is intended to refer to immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulphide bonds (i.e. , "full antibody molecules"), as well as multimers thereof (e.g. IgM) or antigen-binding fragments thereof. Each heavy chain is comprised of a heavy chain variable region ("HCVR" or "VH") and a heavy chain constant region (comprised of domains CHI, CH2 and CH3). Each light chain is comprised of a light chain variable region ("LCVR or "VL") and a light chain constant region (CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the invention, the FRs of the antibody (or antigen binding fragment thereof) may be identical to the human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

Antibody variant

An "antibody variant" is a protein derived from an antibody, which has the same binding specificity as an antibody, but which would not be a natural expression product in a mammal. As such, the term refers to various fragments of antibodies as well as artificial antibody analogue formats. Also, the term denotes antibody formats that are found in nature, but which are uncommon among mammals, such as heavy-chain antibodies and IgY found in birds and reptiles, but where CDRs from mammalian antibodies or combinatorically produced antibodies have been engineered into an antibody format from which it is not originally derived.

Antiqen-bindinq fragment

An "antigen-binding fragment" refer to one or more fragments of an antibody that retains the ability to specifically bind to an antigen.

Examples of antibody-binding fragments include, but are not limited to, a Fab fragment (a monovalent fragment consisting of the VL, VH, CL, and CHI domains), F(ab')2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region), a single chain Fv (scFv), a disulfide-linked Fv (dsFv), complementarity determining regions (CDRs), VL (light chain variable region), VH (heavy chain variable region), sdAbs (single-domain antibodies), and any combination of those or any other functional portion of an immunoglobulin peptide capable of binding to target antigen. Antibodies and antigenbinding portions thereof include domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, sdAbs (e.g. monovalent sdAbs, bivalent sdAbs, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains.

The term "binding affinity" is meant to designate the affinity for a test compound (such as the antibodies or antigen-binding fragment thereof, disclosed herein) to bind to the specific allergen. The ability of a test compound to bind to the specific allergen may be tested by use of the bio-layer interferometry (BLI) as described herein. In short, this assay measures the association and the dissociation of the test molecule to a specific allergen. Based on these measurements, the k_on, k_Off , ratio k₀ff/k_0n (= KD> may be determined. k_on is a constant used to characterize how quickly the test molecule binds to the allergen, whereas k_orr characterizes how quickly the test molecule dissociate from the allergen. The ratio of k₀ff/k_0n results in the equilibrium dissociation constant KD. The lower the KD value the higher the affinity of the test molecule to the allergen. Generally, KD values in the sub-nanomolar range may be desirable, such as particularly in the sub-nM affinity range, such as below 1-lOnM. Other IgE affinity binding assays can be used and are known to the skilled person in the art.

Blockinq/neutralizinq antibody

A "blocking antibody" or a "neutralizing antibody", as used herein (or an "antibody that neutralizes allergen activity"), is intended to refer to an antibody, or an antigen binding portion thereof, who's binding to one of the allergens selected from the group consisting of Bet v 1, Ain g 1, Cor a 1 and Que a 1 results in inhibition of at least one biological activity of that allergen. For example, an antibody of the invention may aid in preventing the primary allergic response caused by Bet v 1. Alternatively, an antibody of the invention may demonstrate the ability to prevent a secondary allergic response caused by Bet v 1, or at least reduce, diminish, or inhibit one symptom of an allergic response caused by Bet v 1, including sneezing, coughing, an asthmatic condition, or an anaphylactic response caused by Bet v 1. This inhibition of the biological activity of Bet v 1 can be assessed by measuring one or more indicators of Bet v 1 biological activity by one or more of several standard in vitro or in vivo assays (such as a passive cutaneous anaphylaxis assay, as described herein) or other in vivo assays known in the art. Control a

The term "control antibodies" refers to antibodies used in the experimental part as comparators to the antibodies of the invention. In particular, three antibodies are used as control antibodies, which are REGN5713, REGN5714 and REGN5715 all described in WO 2018/222854. The VH and VL amino acid sequences for each of the comparator antibodies are listed below.

• REGN5713 (H4H 16992P)

O VH-Caamma4 (SEO ID NO 161) :

QVQLQESG PGLVKPSETLSLTCSVSGGSITNYFWTWI RQSPG KG LEWIGYIYYSGGTN YNPSLKSRVTISIDTSKNQFSLNMNSVTAADTAVYYCAGSYYYGVDVWGQGTTVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTV DKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

O VL-CKappa (SEQ ID NO 164) : EIVLTQSPATLSLSPGERATLSCRASQSIKSFLAWYRQKPGQAPRLLIYDASNRPTGIPA RFSGSGSGTDFTLTINSLESEDFAVYFCQQRNNWPFTFGPGTKVDIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

• REGN5714 (H4H 1738P2)

O VH-Caamma4 (SEO ID NO 162) :

EVQLVESGGDLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSFISDSSSN IYYADSVKGRFTISRDNAKKSLYLQMTSLRAEDTAVYYCAREAIGSTSFDNWGQGTLV TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK o VL-CKappa (SEO ID NO 165) :

EIVMTQSPATLSVSPGERATLSCRASQSVSSSLAWYQQKPGQAPRRLIYSASTRATGIP ARFSGSGSGTEFTLTISSLQSEDFAIYYCHQYNNWPLTFGGGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

• REGN5715 (H4H 16822P) O VH-Caamma4 (SEO ID NO 163) :

QVQLVQSGAEVKKPGASVKVSCKASGYTFISYNIFWVRQATGQGLDWMGWMNPFRN NAGYAQKFQGRVTVTWDTSISTAYMELSSLSSEDTAIYYCAREHGSSWGFFDYWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA PEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK o VL-CKappa (SEO ID NO 166) :

EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGI PDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. The term "epitope" also refers to a site on an antigen to which B and/or T cells respond. It also refers to a region of an antigen that is bound by an antibody. Epitopes may be either linear or conformational. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics. Epitopes may also be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. Full-lenqth a

The phrase "full-length antibody" when used herein, refers to an antibody (e.g., a parent or variant antibody) which contains all heavy and light chain constant and variable domains corresponding to those that are normally found in a wild-type antibody of that isotype.

Human-derived

The term "human-derived" in the context of the present invention means that at least the CDRs of the antibody are from a human antibody or a humanized transgenic animal, but the "human-derived" antibody may contain further elements that are different from the human antibody or the antibody from the humanized transgenic animal, where the CDRs originates from. Examples are parts or the complete framework of the heavy and/or light chain variable regions and/or the parts or the complete of the heavy and/or light chain constant region. For example, the variable regions, portions thereof or the CDRs may be from a human IgE antibody and grafted in a scaffold of an IgG or IgA antibody, such as a human IgG or IgA antibody. The peptide sequence of the human-derived antibody may be at least 60%, at least 65 %, at least 70%, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 % or at least 97 % identical to the sequence of the antibody extracted from the human.

Preferably, the peptide sequence of the human-derived antibody may be at least 90 %, at least 95 %, at least 97 %, at least 98 % or at least 99 % identical to the sequence of the antibody extracted from the human.

IMGT

The term "IMGT" in the context of the present invention, refers to a method for determining CDR regions. Here amino acid residues of an immunoglobulin single variable domain can be numbered using conserved amino acids, which always have the same position. For instance, Cysteine 23, Tryptophane 41, Leucine 89, Cysteine 104. FR1 of a VHH comprises the amino acids residues at position 1-26, CDR1 of a VHH comprises the amino acids residues at position 27-38, FR2 of a VHH comprises the amino acid position 39-55, CDR2 of a VHH comprises the amino acid residues of position 56-65, FR3 of a VHH comprises the amino acids residues at position 66-104, CDR3 of a VHH comprises the amino acid residues at position 105-117, and FR4 of a VHH comprises the amino acid residues at position 118 and the rest of the sequence. The maximum length of CDR is as defined above. For shorter CDRs, gaps are created (Lefranc et al., 2002).

The term "multi-specific antibody or multi-specific antigen-binding fragments" in the context of the present invention refers to antibodies or antigen-binding fragments that are able to bind at least two different epitopes on different antigens or on the same antigen.

Conventional monospecific antibodies, classically have two identical epitope binding sites (paratopes), one on each Fab region and are thus, only specific for one epitope (two paratopes and single specific). A multi-specific antibody or multi-specific antigen binding fragment has at least two different types of paratopes, meaning it can bind at least two different types of epitopes.

In particular, the multi-specific antibody may comprise two or more paratopes, wherein one or more paratopes may be identical so that all paratopes of the construct belongs to at least two different types of paratopes, hence the antibody has at least two specificities. For example, a multi-specific antibody may comprise four paratopes, wherein each two paratopes are identical, meaning has same specificity. Thus, the multi-specific antibody is bispecific and tetravalent. Accordingly, monospecific antibody refers to one or more paratopes having same specificity. Bispecific means an antibody having one, two, three etc. paratopes but in total having two specificities. For example, a bispecific antibody may have one paratope for each specificity. In another embodiment, the multi-specific antibody may have two or more paratopes for each of the specificities.

Thus, in one embodiment, the multi-specific antibody may be bispecific, trispecific or tetraspecific.

The term "paratope" refers to an antigen-binding (or epitope binding) site of an antibody.

Recombinant host cell

The phrase "recombinant host cell" (or "host cell"), as used herein, is intended to refer to a cell into which an expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Recombinant host cells include, for example, transfectomas, such as CHO cells, HEK-293 cells, PER.C6, NSO cells, and lymphocytic cells, and prokaryotic cells such as E. coli and other eukaryotic hosts such as plant cells and fungi.

effective amount

The phrase "therapeutically effective amount" is meant to define an amount that produces the desired effect for which it is administered. The exact amount will depend on the purpose of the treatment and will be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

Tree-pollen allergens.

The phrase "tree pollen allergen" is herein meant to at least include allergens from pollen of birch-, alder-, hazel- and/or oak trees. Among the pollen allergens from these trees, the most clinically relevant ones are the major allergens Bet v 1, Ain g 1, Cor a 1 and Que a 1. The prefix "n" before the name of an allergen, for example nBet v 1, is meant to designate that the allergen is in its natural occurring form, including isoforms and various natural occurring post-translational derivatisation (e.g. glycosylation). Natural allergens may be denoted by the prefix (n) to distinguish them from recombinant allergens, which are indicated by the prefix (r) before the allergen name (e.g., nBet v 1 versus rBet v 1) and the term "natural allergen" should be used to indicate any allergen purified from a natural source material.

Typically, the phrase "tree-pollen allergens", as used herein, is intended to refer to a group of allergens comprising Bet v 1, Ain g 1, Cor a 1 and Que a 1.

Vector

The phrase "vector," as used herein, is intended to refer to a nucleic acid molecule capable of inducing transcription a nucleic acid segment ligated into the vector. One type of vector is a "plasmid", which is in the form of a circular double stranded DNA loop. Another type of vector is a viral vector, wherein the nucleic acid segment may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (for instance bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (such as non-episomal mammalian vectors) may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the present invention is intended to include such other forms of expression vectors, such as viral vectors (such as replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

Short sequences not listed in the WIPO Standard 26 compliant sequence listing forming part of the description.

The following sequences are due to their short lengths not listed in the sequence listing :

Specific embodiments of the invention

Antibodies

According to a first aspect of the invention there is provided antibodies or antigen-binding fragments thereof capable of binding one or more tree pollen allergens selected from the group consisting of Bet v 1, Ain g 1, Cor a 1 and Que a 1. Thus, a single antibody may be specific to one, two, three of four of the allergens selected from the group consisting of Bet v 1, Ain g 1, Cor a 1 and Que a 1. Each of the antibodies described herein may alone or in combination of two, three, four or more antibodies be able to either promote clearance of one, two, three or any of the tree pollen allergens selected from the group consisting of Bet v 1, Ain g 1, Cor a 1 and Que a 1, or may be able to block the binding of one, two, three or any of the tree pollen allergens selected from the group consisting of Bet v 1, Ain g 1, Cor a 1 and Que a 1 to pre-formed IgE in a subject, thus leading to decreased activation of mast cells and basophils. In an interesting embodiment, the antibody, or antigen-binding fragments thereof, or combination of antibodies or combination of antigen-binding fragments thereof, may be able to reduce or prevent one or more symptoms in a subject allergic to or sensitized to one or more of the tree pollen allergens selected from the group consisting of Bet v 1, Ain g 1, Cor a 1 and Que a 1.

In preferred embodiments, at least the CDR regions are from a human antibody, such as an human IgE antibody, IgGl or IgG4 antibody. Therefore, the CDR region is meant to have 100% amino acid sequence alignment to the human antibody, where the CDR regions are originally detected. In further preferred embodiments the antibodies as disclosed herein are preferably with full length human IgG scaffold, such as IgGl or IgG4, but can be provided with any Fc part of a human antibody, such as IgE-Fc, IgG-Fc, IgA-Fc. The hinge region may comprise the mutation S228P to avoid Fab-arm exchange. Thus, antibodies as disclosed herein may be fully human antibodies, but optionally contains a few, such as one, two or three amino acid substitutions, deletions or additions to obtain antibodies with favourable properties, e.g., half-life extension. In alternative embodiments, the antibody may comprise only an antigen-binding portion, for example Fab.

In one embodiment, the antibody or antigen-binding fragment thereof is capable of binding to at least two of the allergens selected from the group consisting of Bet v 1, Ain g 1, Cor a 1 and Que a 1, such as binding to each of the allergens Bet v 1 and Ain g 1. Preferably, the antibody or antigen-binding fragment thereof is capable of binding at least three of the allergens selected from the group consisting of Bet v 1, Ain g 1, Cor a 1 and Que a 1, such as binding each of the allergens Bet v 1, Ain g 1 and Cor a 1, more preferably the antibody or antigen-binding fragment thereof can bind to each of the allergens Bet v 1, Ain g 1, Cor a 1 and Que a 1.

In another embodiment, the antibody or antigen-binding fragment thereof are capable of binding to the allergen

• Bet v 1 with an affinity of InM or below; and/or

• Ain g 1 with the affinity of InM or below; and/or

• Cor a 1 with the affinity of 300nM or below; and/or

• Que a 1 with the affinity of 300 nM or below.

The affinity may be determined by biolayer interferometry (BLI) assay.

In a preferred embodiment, the Fc part is a IgG4-Fc. The affinity of each antibody or antigen-binding fragment thereof is used to describe the strength of binding to the target (here for binding to a tree pollen allergen described herein) and may in a preferred embodiment be below 1000 nM, such as 300 nM, such as 100 nM, such as 10 nM, preferably 1 nM, more preferably 0.1 nM, even more preferably 0.01 nM.

The present invention relates to an antibody or antigen-binding fragment thereof capable of binding an allergen selected from the group consisting of Bet v 1, Ain g 1, Cor a 1 and Que a 1.

In one embodiment, the antibody or antigen-binding fragment thereof is capable of binding to at least two of the allergens selected from the group consisting of Bet v 1, Ain g 1, Cor a 1 and Que a 1, such as binding to each of the allergens Bet v 1 and Ain g 1. Preferably, the antibody or antigen-binding fragment thereof is capable of binding at least three of the allergens selected from the group consisting of Bet v 1, Ain g 1, Cor a 1 and Que a 1, such as binding each pf the allergens Bet v 1, Ain g 1 and Cor a 1, more preferably the antibody or antigen-binding fragment thereof can bind to each of the allergens Bet v 1, Ain g 1, Cor a 1 and Que a l.In another embodiment, the antibody or antigen-binding fragment thereof are capable of binding to the allergen

• Bet v 1 with an affinity of 1 nM or below; and/or

• Ain g 1 with the affinity of 1 nM below; and/or

• Cor a 1 with the affinity of 300 nM or below; and/or

• Que a 1 with the affinity of 300 nM or below.

The affinity may be determined by BLI assay.

In a further embodiment, the antibody or antigen-binding fragment thereof is capable of binding to the allergen Bet v 1 with an affinity 1000 nM or below, such as 300 nM or below, such as 100 nM or below, such as 10 nM or below.

In another embodiment, the antibody or antigen-binding fragment thereof is capable of binding to the allergen the Ain g 1 with the affinity 1000 nM or below, such as 300 nM or below, such as 100 nM or below, such as 10 nM or below. In another embodiment, the antibody or antigen-binding fragment thereof is capable of binding to the allergen the Cor a 1 with the affinity 1000 nM or below, such as 300 nM or below, such as 100 nM or below, such as 10 nM or below.

In another embodiment, the antibody or antigen-binding fragment thereof is capable of binding to the allergen the Que a 1 with the affinity 1000 nM or below, such as 300 nM or below, such as 100 nM or below, such as 1 OnM or below.

In yet another embodiment, the antibody or antigen-binding fragment thereof is capable of binding to each of the allergens the Bet v 1, Ain g 1, Cor a 1 and Que a 1 with the affinity lOOOnM or below, such as 300nM or below, such as lOOnM or below, such as lOnM or below.

In one embodiment, the antibody or antigen-binding fragment thereof are capable of binding to the allergen Bet v 1 with an affinity of InM or below.

In one embodiment, the antibody or antigen-binding fragment thereof are capable of binding to the allergen Ain g 1 with an affinity of InM or below.

In another embodiment, the antibody or antigen-binding fragment thereof are capable of binding to the allergen Cor a 1 with an affinity of 300nM or below.

In a further embodiment, the antibody or antigen-binding fragment thereof are capable of binding to the allergen Que a 1 with an affinity of 300nM or below.

As mentioned above the affinity may be determined by BLI.

The antibody or antigen-binding fragments thereof as described herein may be obtained from humans. The humans from which the antibody or antigen-binding fragment thereof is extracted may be selected from a human suffering from birch pollen allergy, hazel pollen allergy, alder pollen allergy and/or oak pollen allergy; a human sensitized to Bet v 1, Que a 1, Ain g 1 and/or Cor a 1, optionally the sensitized human is without clinically relevant allergy symptoms; or a human that have underwent allergen-specific immunotherapy against birch pollen allergy, hazel pollen allergy, alder pollen allergy and/or oak pollen allergy.

In a preferred embodiment, the antibodies of the present invention are therefore human antibodies. Thus, in one embodiment, the antibody is human-derived such as a human antibody and the antigen-binding fragment thereof derives from a human amino acid sequence.

In another embodiment, the antibody is a human antibody and the antigen-binding fragment thereof comprises a human amino acid sequence.

In some embodiments, the antibodies are derived from humans sensitised to Bet v 1 and/or Cor a 1 and/or Ain g 1 and/or Que a 1. In one embodiment, the antibodies are derived from humans sensitised to Bet v 1. In another embodiment, the antibodies are derived from humans sensitised to Cor a 1. In a further embodiment, the antibodies are derived from humans sensitised to Ain g 1. In yet another embodiment, the antibodies are derived from humans sensitised to Que a 1.

The allergen binding part of each antibody of the invention comprises six individual CDR regions, three in the heavy chain variable region (HCVR) and three in the light chain variable region (LCVR), which when combined are referred to as the "fab" domain. Further, the fab domain may be coupled to the constant FC region.

Non-limiting exemplary antibodies, defined by their fab region, are shown in table 1. Table 1 provides the full-length amino acid sequence of the individual HCVR and LCVR for each antibody, which in combination constitutes the fab region.

Table 1 shows examples of antibodies of the invention. Each antibody (mAb) is defined by a HCVR and a LCVR sequence.

Accordingly, one embodiment of the present invention, relates to an antibody or antigenbinding fragment thereof selected from the group consisting of 2E02, 2C10, 2B04, 2E07, A07, BIO, and 2_10, preferably selected from the group consisting of 2E02, 2C10, 2B04, and 2E07.

In embodiments of the first aspect of the invention, the antibody or antigen-binding fragment thereof is having a combination of HCVR and LCVR with the amino acid sequence of SEQ ID NO:

1 and 2 (HCVR and LCVR of antibody 2E02, respectively);

21 and 22 (HCVR and LCVR of antibody 2C10, respectively);

41 and 42 (HCVR and LCVR of antibody 2B04, respectively);

61 and 62 (HCVR and LCVR of antibody 2E07, respectively);

81 and 82 (HCVR and LCVR of antibody A07, respectively);

101 and 102 (HCVR and LCVR of antibody BIO, respectively); or

121 and 122 (HCVR and LCVR of antibody 2_10, respectively), wherein each of the amino acid sequences may have 1, 2, or 3 amino acid substitutions.

In further embodiments, each combination of HCVR and LCVR as set out in table 1, is linked to a FC domain, preferably an IgG-Fc domain, more preferably, an IgG4-Fc.

Since such an antibody may be subject to affinity maturation, humanization or other amino acid changes of the amino acid sequence, further embodiments relate to an antibody 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: 1 and 2;

21 and 22;

41 and 42;

61 and 62;

81 and 82;

101 and 102; or 121 and 122.

Thus, further embodiments of the first aspect of the invention, relates to an antibody or antigen-binding fragment thereof, wherein the antibody or fragment thereof comprises a HCVR and a LCVR, wherein,

• the antibody or antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 1 and a LCVR having the amino acid sequence of SEQ ID NO: 2 (HCVR and LCVR of antibody 2E02, respectively), wherein each of the amino acid sequences may have 1, 2, or 3 amino acid substitutions; or • the antibody or antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 21 and a LCVR having the amino acid sequence of SEQ ID NO: 22 (HCVR and LCVR of antibody 2C10, respectively), wherein each of the amino acid sequences may have 1, 2, or 3 amino acid substitutions; or

• The antibody or antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 41 and a LCVR having the amino acid sequence of SEQ ID NO: 42 (HCVR and LCVR of antibody 2B04, respectively), wherein each of the amino acid sequences may have 1, 2, or 3 amino acid substitutions; or

• The antibody or antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 61 and a LCVR having the amino acid sequence of SEQ ID NO: 62 (HCVR and LCVR of antibody 2E07, respectively), wherein each of the amino acid sequences may have 1, 2, or 3 amino acid substitutions; or

• The antibody or antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 81 and a LCVR having the amino acid sequence of SEQ ID NO: 82 (HCVR and LCVR of antibody A07, respectively), wherein each of the amino acid sequences may have 1, 2, or 3 amino acid substitutions; or

• The antibody or antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 101 and a LCVR having the amino acid sequence of SEQ ID NO: 102 (HCVR and LCVR of antibody B10, respectively), wherein each of the amino acid sequences may have 1, 2, or 3 amino acid substitutions; or

• The antibody or antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 121 and a LCVR having the amino acid sequence of SEQ ID NO: 122 (HCVR and LCVR of antibody 2_10, respectively), wherein each of the amino acid sequences may have 1, 2, or 3 amino acid substitutions.

• In a preferred embodiment the antibody or antigen-binding fragment thereof, comprises a HCVR and a LCVR as defined for an antibody selected from the group consisting of 2E02, 2C10, 2B04, 2E07 and A07.

Antibodies defined by CDR regions

The skilled person in the art, knows that each variable domain, including the HCVR and the LCVR in an antibody or antigen-binding region thereof, comprises three complementaritydetermining regions (CDRs), flanked by four framework regions (FR1-4). A binding region of an antibody herein is meant to include one or more or all the six complementarity-determining regions, with three on HCVR (HCDR1, HCDR2 and HCDR3) and three on LCVR (HCDR1, HCDR2 and HCDR3).

The main purpose of CDR regions is to define the regions having specificity to an epitope. However, not all residues in the CDRs are responsible for epitope binding, but merely determines structural elements (Wilton E. et al 2018 ref 6). In addition, the framework regions are not exclusively responsible for the structure. Residues of the FRs adjacent to CDRs can influence binding.

Different methods can be applied for determination of the position of the different frameworks and CDR regions of an antibody.

One method is the "Kabat numbering scheme" or "Kabat", first described by American scientist Elvis Kabat. Here amino acid residues of an immunoglobulin single variable domain can be numbered according to the general numbering of VH domains given by Kabat et al. (Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91).

It should be noted that - as is well known in the art - the total number of amino acid residues in each of the CDRs may vary and may not correspond to 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 for by the Kabat numbering).

This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. The total number of amino acid residues in a HCVR domain will usually be in the range of from 110 to 120, often between 112 and 115. It should however be noted that smaller and longer sequences may also be suitable for the purposes described herein.

Another method for determining CDR regions in HCVR and LCVR is by Chothia numbering scheme. Here amino acid residues of an immunoglobulin single variable domain can be numbered using conserved amino acids, which always have the same position (Dondelinger M et al. frontiers in immunology 2018).

A further method for determining CDR regions in HCVR and LCVR is by IMGT. Here amino acid residues of an immunoglobulin single variable domain can be numbered using conserved amino acids, which always have the same position. For instance, Cysteine 23, Tryptophane 41, Leucine 89, Cysteine 104. FR1 of a HCVR and LCVR comprises the amino acids residues at position 1-26, CDR1 of a HCVR and LCVR comprises the amino acids residues at position 27-38, FR2 of a HCVR and LCVR comprises the amino acid position 39-55, CDR2 of a HCVR and LCVR comprises the amino acid residues of position 56-65, FR3 of a HCVR and LCVR comprises the amino acids residues at position 66-104, CDR3 of a HCVR and LCVR comprises the amino acid residues at position 105-117, and FR4 of a HCVR and LCVR comprises the amino acid residues at position 118 and the rest of the sequence. The maximum length of CDR is as defined above. For shorter CDRs, gaps are created (Lefranc et al 2002 - Developmental and Comparative Immunology).

A further method for determining the CDR regions in an antibody is by using the Aho numbering scheme (Mitchelle & Colwell, Proteins, 2017 and Honegger & Pluckthun et al 2001).

Alternative CDR definitions of interest include, without limitation, those disclosed by Honegger, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool." J Mol Biol. 2001 ;309:657-670; Ofran et al. "Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B cell epitopes." J Immunol. 2008; 181 : 6230-6235; Almagro "Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires." J Mol Recognit. 2004; 17: 132-143; and Padlanet al. "Identification of specificitydetermining residues in antibodies." Faseb J. 1995;9: 133-139., each of which is herein specifically incorporated by reference.

As previously described, the combination of CDR regions in an antibody, determines the specificity. The six individual CDRs, three in each of HCVR and LCVR regions, can be determined through different methods, including IMGT, Kabat or Chothia as previous described.

Table 2a-c provides the amino acid sequence of the six CDR regions of each of the exemplary antibodies disclosed in table 1, determined by the method IMGT, Kabat and Chothia.

Table 2a shows the amino acid sequences of HCDRl, HCRD2, HCDR3, LCDR1, LCDR2 and LCDR3 of each fab region, determined by IMGT.

Table 2b shows the amino acid sequences of HCDR1, HCRD2, HCDR3, LCDR1, LCDR2 and LCDR3 of each fab region, determined by Kabat.

Table 2c shows the amino acid sequences of HCDR1, HCRD2, HCDR3, LCDR1, LCDR2 and LCDR3 of each fab region, determined by Chothia.

Thus, a first aspect of the invention also relates to antibodies defined by their CDR regions for example as determined by IMGT. One aspect thereof relates to an antibody or antigen- binding fragment thereof, wherein the antibody or fragment thereof comprises three heavy chains CDRs (HCDR1, HCDR2 and HCDR3) and three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein;

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence of SEQ ID NO: 3, a HCDR2 amino acid sequence of SEQ ID NO: 4, a HCDR3 amino acid sequence of SEQ ID NO: 5, a LCDR1 amino acid sequence of SEQ ID NO: 6, a LCDR2 amino acid sequence of SEQ ID NO: 7 and a LCDR3 amino acid sequence of SEQ ID NO: 8, wherein each of the amino acid sequences may have 1, 2, or 3 amino acid substitutions (CDRs of antibody 2E02); or

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence of SEQ ID NO: 23, a HCDR2 amino acid sequence of SEQ ID NO: 24, a HCDR3 amino acid sequence of SEQ ID NO: 25, a LCDR1 amino acid sequence of SEQ ID NO: 26, a LCDR2 amino acid sequence of SEQ ID NO: 27 and a LCDR3 amino acid sequence of SEQ ID NO: 28, wherein each of the amino acid sequences may have 1, 2, or 3 amino acid substitutions (CDRs of antibody 2C10); or

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence of SEQ ID NO: 43, a HCDR2 amino acid sequence of SEQ ID NO: 44, a HCDR3 amino acid sequence of SEQ ID NO: 45, a LCDR1 amino acid sequence of SEQ ID NO: 46, a LCDR2 amino acid sequence of SEQ ID NO: 47 and a LCDR3 amino acid sequence of SEQ ID NO: 48, wherein each of the amino acid sequences may have 1, 2, or 3 amino acid substitutions (CDRs of antibody 2B04); or

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence of SEQ ID NO: 63, a HCDR2 amino acid sequence of SEQ ID NO: 64, a HCDR3 amino acid sequence of SEQ ID NO: 65, a LCDR1 amino acid sequence of SEQ ID NO: 66, a LCDR2 amino acid sequence of SEQ ID NO: 67 and a LCDR3 amino acid sequence of SEQ ID NO: 68, wherein each of the amino acid sequences may have 1, 2, or 3 amino acid substitutions (CDRs of antibody 2E07); or

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence of SEQ ID NO: 83, a HCDR2 amino acid sequence of SEQ ID NO: 84, a HCDR3 amino acid sequence of SEQ ID NO: 85, a LCDR1 amino acid sequence of SEQ ID NO: 86, a LCDR2 amino acid sequence of SEQ ID NO: 87 and a LCDR3 amino acid sequence of SEQ ID NO: 88, wherein each of the amino acid sequences may have 1, 2, or 3 amino acid substitutions (CDRs of antibody A07); or • the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence of SEQ ID NO: 103, a HCDR2 amino acid sequence of SEQ ID NO: 104, a HCDR3 amino acid sequence of SEQ ID NO: 105, a LCDR1 amino acid sequence of SEQ ID NO: 106, a LCDR2 amino acid sequence of SEQ ID NO: 107 and a LCDR3 amino acid sequence of SEQ ID NO: 108, wherein each of the amino acid sequences may have 1, 2, or 3 amino acid substitutions (CDRs of antibody B10); or

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence of SEQ ID NO: 123, a HCDR2 amino acid sequence of SEQ ID NO: 124, a HCDR3 amino acid sequence of SEQ ID NO: 125, a LCDR1 amino acid sequence of SEQ ID NO: 126, a LCDR2 amino acid sequence of SEQ ID NO: 127 and a LCDR3 amino acid sequence of SEQ ID NO: 128, wherein each of the amino acid sequences may have 1, 2, or 3 amino acid substitutions (CDRs of antibody 2_10).

In one embodiment the antibody or antigen-binding fragment thereof comprises three heavy chains CDRs (HCDR1, HCDR2 and HCDR3) and three light chain CDRs (LCDR1, LCDR2 and LCDR3) as defined for an antibody selected from the group consisting of 2E02, 2C10, 2B04, 2E07 and A07.

CDRs as determined by HCVR and LCVR

Alternatively, the individual CDR regions may be derivable from a larger sequence, determined by different numbering schemes, such as, but not limited to Kabat, Chothia, or IMTG. Thus, in one embodiment, an antibody or antigen-binding fragment thereof, of the invention comprises a combination of complementarity determining region 1 (HCDR1), complementarity determining region 2 (HCDR2) and complementarity-determining region 3 (HCDR3) in the HCVR, and LCDR1, LCDR2 and LCDR3 in the LCVR, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprises or consists of an amino acid sequence as determined by Kabat, Chothia, or IMTH in the amino acid sequences selected from any one of SEQ ID NOs: 1, 2, 21, 22, 41, 42, 61, 62, 81, 82, 101, 102, 121 and 122. Since such sdAbs may be subject to affinity maturation, humanization or other amino acid changes of the amino acid sequence, further embodiments relate to a monomer sdAb 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: 1, 2, 21, 22, 41, 42, 61, 62, 81, 82, 101, 102, 121 and 122.

In another embodiment, said antibody or antigen-binding fragment thereof, of the invention comprises a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having an amino acid sequence as determined by Kabat, Chothia, IMTG or Aho in the amino acid sequences selected from any one of SEQ ID NOs: 1, 2, 21, 22, 41, 42, 61, 62, 81, 82, 101, 102, 121 and 122. Preferably, 1, 2, or 3 amino acids of each of the CDR regions determined by Kabat, Chothia, IMTG or Aho may be substituted to obtain a desired effect, for example change in affinity and/or lower immunogenicity.

Thus, alternatively worded, a first aspect of the invention, relates to an antibody or antigenbinding fragment thereof comprising three heavy chains CDRs (HCDR1, HCDR2 and HCDR3) and three light chain CDRs (LCDR1, LCDR2 and LCDR3) of an antibody selected from the group consisting of 2E02, 2C10, 2B04, 2E0, A07, BIO, and 2_10.

Thus, a first aspect relates also to an antibody or antigen-binding fragment thereof comprising three heavy chains CDRs (HCDR1, HCDR2 and HCDR3) and three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein;

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence contained within SEQ ID NO: 1, a HCDR2 amino acid sequence contained within SEQ ID NO: 1, a HCDR3 amino acid sequence contained within SEQ ID NO: 1, a LCDR1 amino acid sequence contained within SEQ ID NO: 2, a LCDR2 amino acid sequence contained within SEQ ID NO: 2 and a LCDR3 amino acid sequence contained within SEQ ID NO: 2 (antibody 2E02); or

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence contained within SEQ ID NO: 21, a HCDR2 amino acid sequence contained within SEQ ID NO: 21, a HCDR3 amino acid sequence contained within SEQ ID NO: 21, a LCDR1 amino acid sequence contained within SEQ ID NO: 22, a LCDR2 amino acid sequence contained within SEQ ID NO: 22 and a LCDR3 amino acid sequence contained within SEQ ID NO: 22 (antibody 2C10); or

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence contained within SEQ ID NO: 41, a HCDR2 amino acid sequence contained within SEQ ID NO: 41, a HCDR3 amino acid sequence contained within SEQ ID NO: 41, a LCDR1 amino acid sequence contained within SEQ ID NO: 42, a LCDR2 amino acid sequence contained within SEQ ID NO: 42 and a LCDR3 amino acid sequence contained within SEQ ID NO: 42 (antibody 2B04); or

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence contained within SEQ ID NO: 61, a HCDR2 amino acid sequence contained within SEQ ID NO: 61, a HCDR3 amino acid sequence contained within SEQ ID NO: 61, a LCDR1 amino acid sequence contained within SEQ ID NO: 62, a LCDR2 amino acid sequence contained within SEQ ID NO: 62 and a LCDR3 amino acid sequence contained within SEQ ID NO: 62 (antibody 2E07); or • the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence contained within SEQ ID NO: 81, a HCDR2 amino acid sequence contained within SEQ ID NO: 81, a HCDR3 amino acid sequence contained within SEQ ID NO: 81, a LCDR1 amino acid sequence contained within SEQ ID NO: 82, a LCDR2 amino acid sequence contained within SEQ ID NO: 82 and a LCDR3 amino acid sequence contained within SEQ ID NO: 82 (antibody A07); or

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence contained within SEQ ID NO: 101, a HCDR2 amino acid sequence contained within SEQ ID NO: 101, a HCDR3 amino acid sequence contained within SEQ ID NO: 101, a LCDR1 amino acid sequence contained within SEQ ID NO: 102, a LCDR1 amino acid sequence contained within SEQ ID NO: 102 and a LCDR3 amino acid sequence contained within SEQ ID NO: 102 (antibody B10); or

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence contained within SEQ ID NO: 121, a HCDR2 amino acid sequence contained within SEQ ID NO: 121, a HCDR3 amino acid sequence contained within SEQ ID NO: 121, a LCDR1 amino acid sequence contained within SEQ ID NO: 122, a LCDR2 amino acid sequence contained within SEQ ID NO: 122 and a LCDR3 amino acid sequence contained within SEQ ID NO: 122 (antibody 2_10).

Each of the CDRs (HCDR1, HCDR2 and HCDR3, LCDR1, LCDR2 and LCDR3) as mentioned above may be determined according to the either IMGT, kabat or Chothia method.

By option, each of the CDR regions determined by either IMGT, Kabat or Chothia method may be subject to 1, 2, or 3 amino acid substitutions such as each HCDR1 may contain 1, 2, or 3 amino acid substitutions, each HCDR2 may contain 1, 2, or 3 amino acid substitutions, each HCDR3 may contain 1, 2, or 3 amino acid substitutions, each LCDR1 may contain 1, 2, or 3 amino acid substitutions, each LCDR2 may contain 1, 2, or 3 amino acid substitutions, and/or each LCDR3 may contain 1, 2, or 3 amino acid substitutions.

Preferred are an antibody or antigen-binding fragment thereof comprising three heavy chains CDRs (HCDR1, HCDR2 and HCDR3) and three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein the CDRs are from an antibody selected from the group consisting of 2E02, 2C10, 2B04, 2E0, A07, BIO, and 2_10.

Modifications of amino acid sequences of antibodies and antigen binding fragment thereof. Any amino acid sequence of CDR's or of the framework regions outside the CDR's, including the fab and Fc region, might be subject to amino acid substitutions, insertions, or deletions for the purpose of modifying biological activity, expression level, stability, or other functional properties. In preferred embodiments, the amino acid changes are only incorporated outside the CDR regions.

In some embodiments, an antibody of the invention consist(s) of affinity- matured, human or humanized amino acid sequences. In further embodiments thereof, the amino acid sequence of the CDR regions may not change upon performing affinity-maturation or humanization of the antibody. In such embodiments, the antibody consist(s) of affinity-matured, human or humanized amino acid sequences, but not in the CDR regions involved in the binding to the target.

In some embodiments, one more of the CDR's of an HCVR or LCVR (i.e. CDR1, CDR2 and/or CDR3) may independently be subject to amino acid substitution, such as by 1, 2, 3, or more amino acid residue substitutions. The amino acid substitution in the CDR's may be conservative amino acid substitution. "Conservative" amino acid substitutions are generally amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and/or charge, which provides little or essentially no influence on the function, activity, or other biological properties of the resulting binding region of an antibody described herein. Such conservative amino acid substitutions are well known in the art. For example, conservative substitutions preferably are substitutions in which one amino acid residue within the following groups (a) - (e) is substituted by another amino acid residue within the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gin; (c) polar, positively charged residues; His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Vai and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

Particularly preferred conservative amino acid substitutions are as follows: Ala into Gly; Ala into Ser; Arg into Lys; Asn into Gin; Asn into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala; Gly into Pro; His into Asn; His into Gin; He into Leu, He into Vai; Leu into He; Leu into Vai; Lys into Arg; Lys into Gin; Lys into Glu; Met into Leu; Met into Tyr; Met into He; Phe into Met; Phe into Leu; Phe into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; Phe into Vai; Phe into He and/or Phe or into Leu.

Conservative amino acid substitutions may also be made in the non-CDR amino acid sequence. In other embodiments, amino acid substitutions in the amino acid sequence of CDR's might be performed with the purpose to provide an antibody or antigen-binding fragment thereof with modified, such as increased, affinity for the target. The amino acid sequences of the CDR's are typically more determining for the target affinity than the amino acid sequences outside the CDR's. Accordingly, the antibody or antigen-binding fragment thereof described herein can also be subjected to affinity maturation by introducing one or more alterations in the amino acid sequence of one or more CDRs, which alterations result in a modified such as an improved affinity of the resulting antibody or antigen-binding fragment thereof for the target as compared to the first (parent) antibody or antigen-binding fragment thereof. Methods for affinity-maturation of antibody or antigen-binding fragment thereof are known in the art and trivial for the skilled person.

There might also be amino acid substitutions, insertions or deletions made in one or more of the framework regions outside the CDR's.

Any amino acid substitution in the amino acid sequence outside the CDR's may typically provide antibody or antigen-binding fragment thereof with less modified biological activity compared to substitutions in the CDRs. However, any changes in the amino acid sequence of an antibody or antigen-binding fragment thereof (such as deletions, insertions and/or substitutions) may also be designed to improve the expression level depending on the host organism used to express the antibody or antigen-binding fragment thereof described herein. For example, the changes may be designed in such a way that one or more sites for post- translational modification (such as one or more glycosylation sites) are removed, as will be within the ability of the person skilled in the art. Alternatively, substitutions or insertions may be designed to introduce one or more sites for attachment of functional groups, for example to allow for insertion of affinity tags (His-tags) or for site-specific PEgylation. The possibility of post-translational modification of the N-terminus can be eliminated by changing the N- terminal Glutamic acid (E) into an Aspartic acid (D). Thus, an amino acid difference may be change of Glutamic acid (Glu) at position 1 (said position determined according to Kabat numbering) into an Aspartic acid (Asp).

As mentioned, the parent CDR regions may be subject to sequence variation, such as 1 2 or 3 amino acid substitution, deletion or addition. The variant may have the same, improved or increased allergen binding activity.

Mutations

The fully human monoclonal antibodies that specifically bind to Bet v 1, Que a 1, Ain g 1 and/or Cor a 1, as disclosed herein, may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The present invention includes antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as "germline mutations"). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof.

The FC region of the antibody

As mentioned, the antibody fragment (e.g., Fc region) of the antibodies discloses herein, can be that of any antibody type (e.g., IgG, IgE, IgM, IgD, and IgA), isotype (e.g., IgGl, lgG2, lgG3, lgG4, Ig Al and Ig A2) or subclass, including engineered subclasses with altered Fc portions that optionally may provide for reduced or enhanced effector cell activity or modification in bio-distribution, serum half-life or excretion rates. The antibody fragment can be derived from any animal species, but preferred is the Fc of human origin. Exemplary effector functions include Cl-q binding; GDC; 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 a receptor, e.g. via the FcyRI; FcyRIIA; FcyRIIBI; FcyRIIB2; FcyRIIIA; FcyRIIIB receptors, and/or the low affinity FcRn receptor. In addition, the Fc-region can be a "dead" Fc, which is one that has been mutagenized to retain activity with respect to, for example, prolonging serum half-life, but which does not activate a high affinity Fc receptor. An Fc may also have decreased binding to complement.

Where it is desirable not to engage Fc interaction with receptors, e.g. FcyRIIB, the antibody fragment may be a CHI immunoglobulin domains (such as IgGl-CHl domain or a IgG4-CHl domain). Unlike conventionally used IgG-FC domains, they do not engage inhibitory FcyRIIb receptor, do not compete with serum immunoglobulins G for receptor binding, and their cytotoxic activity is independent of Fc glycosylation and FcyRIIIa polymorphism (Rozan et al., 2013)

In certain embodiments, the antibody fragment is from IgG4, such as IgG4-Fcm such as human IgG4-Fc Thus, the Fc part of the antibody of the invention, may be selected from an IgG4 antibody fragment.

IgG4 is unstable in vivo because of the phenomena "half antibody exchange" meaning that it becomes bispecific (or functional monomeric in most cases). Therefore, when used therapeutically, a single amino acid mutation might be introduced in the hinge region to prevent this dissociation - the so called S228P mutation.

Another way of preventing the instability of IgG4 in vivo may be to apply the IgG4-Fc scaffold in the knobs-into-holes (KIH) format, which may prevent this dissociation.

Thus, in one embodiment, the Fc part is a IgG4 Fc.

In a further embodiment, the IgG4 Fc comprises the mutation S228P.

Further the Fc-region can be a native-sequence Fc region comprising an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native-sequence human Fc regions include a native-sequence human IgGl Fc region (non-A and A allotypes); nativesequence human lgG2 Fc region; native-sequence human lgG3 Fc region; and nativesequence human lgG4 Fc region, as well as naturally occurring variants thereof or the Fc- region can be a variant Fc region comprises an amino acid sequence that differs from that of a native-sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to a native-sequence Fc region or to the Fc region of a parent polypeptide, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native-sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein may possess at least about 80% amino acid sequence homology or sequence identity with a native-sequence Fc region and/or with an Fc region of a parent antibody fragment, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith.

In one embodiment, variant Fc sequences may include three amino acid substitutions in the CH2 region to reduce FcyRI binding at EU index positions 234, 235, and 237 (see Duncan et al., (1988) Nature 332: 563). Two amino acid substitutions in the complement Cl-q binding site at EU index positions 330 and 331 reduce complement fixation (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173: 1483 (1991)).

Substitution into human IgGl of lgG2 residues at positions 233-236 and lgG4 residues at positions 327, 330 and 331 greatly reduces ADCC and CDC (see, for example, Armour KL. et ai., 1999 Eur J Immunol. 29(8) :2613- 24; and Shields RL. et al., 2001, J Biol Chem. 276(9) :6591 -604). Other Fc variants are possible, including without limitation one in which a region capable of forming a disulfide bond is deleted, or in which certain amino acid residues are eliminated at the N-terminal end of a native Fc form or a methionine residue is added thereto. Thus, one or more Fc portions of the molecule can comprise one or more mutations in the hinge region to eliminate disulfide bonding. In yet another embodiment, the hinge region of an Fc can be removed entirely. In still another embodiment, the molecule can comprise an Fc variant.

Further, an Fc variant can be constructed by substituting, deleting, or adding amino acid residues to effect complement binding or Fc receptor binding. Techniques of preparing such sequence derivatives of the immunoglobulin Fc fragment are disclosed in International Patent Publication Nos. WO 97/34631 and WO 96/32478. In addition, the Fc domain may be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like.

The antibody fragment, e.g., Fc part, may also be modified for constructional (e.g., expression yield) reasons by substituting, deleting, or inserting amino acid residues. Nonlimiting examples of mutations introduced to expression yield is described in the following.

The antibody fragment may be modified to be in a form having native sugar chains, increased sugar chains compared to a native form or decreased sugar chains compared to the native form or may be in an aglycosylated or deglycosylated form. The increase, decrease, removal or other modification of the sugar chains may be achieved by methods common in the art, such as a chemical method, an enzymatic method or by expressing it in a genetically engineered production cell line. Such cell lines can include microorganisms, e.g., Pichia Pastoris, and mammalians cell line, e.g. CHO cells, that naturally express glycosylating enzymes. Further, microorganisms or cells can be engineered to express glycosylating enzymes or can be rendered unable to express glycosylation enzymes. As one example of a cell engineered to have altered sialylation activity, the alpha-2, 6-sialyltransferase 1 gene has been engineered into Chinese Hamster Ovary cells and into Sf9 cells. Constructs expressed by these engineered cells are thus sia lylated by the exogenous gene product. A further method for obtaining Fc molecules having a modified amount of sugar residues compared to a plurality of native molecules includes separating said plurality of molecules into glycosylated and non-glycosylated fractions, for example, using lectin affinity chromatography. The presence of particularly glycosylation moieties has been shown to alter the function of Immunoglobulins. For example, the removal of sugar chains from an Fc molecule results in a sharp decrease in binding affinity to the Cl-q part of the first complement component Cl and a decrease or loss in antibody-dependent cell- mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), thereby not inducing unnecessary immune responses in vivo. Additional important modifications include sialylation and fucosylation: the presence of sialic acid in IgG has been correlated with antiinflammatory activity, whereas removal of fucose from the IgG leads to enhanced ADCC activity.

Constructs can have an Fc sequence with enhanced effector functions, e.g., by increasing their binding capacities to FcyRI and increasing ADCC activity. For example, fucose attached to the /V-linked glycan at Asn-297 of Fc sterica lly hinders the interaction of Fc with FcyRIIIA, and removal of fucose by glyco-engineering can increase the binding to FcyRIIIA, which translates into >50-fold higher ADCC activity compared with wild type IgG 1 controls. Protein engineering, through amino acid mutations in the Fc portion of IgGl, has generated multiple variants that increase the affinity of Fc binding to FcyRIIIA. Notably, the triple alanine mutant S298A/E333A/K334A displays 2-fold increase binding to FcyRIIIA and ADCC function. S239D/I332E (2X) and S239D/I332E/A330L (3X) variants have a significant increase in binding affinity to FcyRIIIA and augmentation of ADCC capacity in vitro and in vivo. Other Fc variants identified by yeast display also showed the improved binding to FcyRIIIA. See, for example Liu et al. (2014) JBC 289(6) : 3571 -90, herein specifically incorporated by reference.

As mentioned, an antibody can be engineered to provide multi-specificity for more different epitopes. Therefore, in some embodiments of the invention, the antibody is multispecific. A multi-specific antibody may comprise two or more antigen-binding fragments selected among antigen-binding fragments disclosed herein.

of antibodies of the second

According to a second aspect of the invention, the antibodies or antigen-binding fragments thereof as disclosed herein can be combined in a composition comprising two or more antibodies.

In one embodiment of the invention the composition comprises antibodies, wherein each antibody or antigen-binding fragment are able to simultaneous bind the same Bet v 1 molecule.

When combining different antibodies, it is key to select antibodies that can bind simultaneously to the target allergen, e.g., Bet v 1 and/or any of the three other tree pollen allergens. For example, the distant epitope binding can be verified by epitope binning studies by BLI. Further, the antibodies may be selected on their ability to bind all the target allergens, such as all the four tree pollen allergens mentioned herein. In one embodiment of the invention the composition comprises antibodies or antigen-binding fragments able to simultaneous bind the same Bet v 1 molecule. In one embodiment the composition comprises antibodies or antigen-binding fragments able to simultaneous bind the same Que a 1 molecule. In one embodiment of the invention the composition comprises antibodies or antigen-binding fragments able to simultaneous bind the same Ain g 1 molecule. In one embodiment the composition comprises antibodies or antigen-binding fragments able to simultaneous bind the same Cor a 1 molecule.

In a preferred embodiment of the invention, the composition comprises antibodies or antigen-binding fragments able to simultaneous bind the same Bet v 1 and/or, Cor a 1, and/or Ain g 1 and/or Que a 1.

As seen in figure 2, the antibodies of table 1 can be divided into groups depending on the epitope they bind on the allergen. When a group of antibodies bind distant epitopes on the same target allergen (meaning non-overlapping circles on figure 2), they have the potential to bind the target allergen simultaneously and then shield the allergen from binding to IgE, leading to a decreased allergic reaction.

In an example, antibodies 2B04, A07 and 2E02 (marked with bold and dotted circles in figure 2) does not bind any overlapping epitopes, illustrated by the circles being separated on figure 2.

That, in addition goes for the antibodies 2B04, A07 and 2C10, which in figure 2, illustrated by bold dotted circles are completely separated. Further described in example 3.

The number of antibodies able to block one or more of the target allergens from being bound to IgE can be tested in a BLI assay. Typically, combination of three antibodies would be sufficient to obtain an effective blocking of IgE. However, occasionally two different antibodies could provide the desired effect.

Thus, a second aspect of the present invention relates to a composition comprising two, such as at least two antibodies or antigen-binding fragments thereof. Typically, the composition comprises three, such as at least three antibodies or antigen-binding fragments thereof, wherein the antibodies are selected from the antibodies or antigen-binding fragments thereof as defined herein.

In one embodiment, the composition comprises four different antibodies or antigen-binding fragment thereof, such as five different antibodies or antigen-binding fragments thereof as defined herein. In preferred embodiments, the composition comprises two or three antibodies. As mentioned, the combination of antibodies should be able to prevent IgE from a human with a tree pollen allergy to bind to a tree pollen allergen. Preferably, the combination should at least provide satisfactorily blocking effect against Bet v 1. More preferred the combination should provide blocking effect against one or more, preferably all the tree pollen allergen selected from the group consisting of Bet v 1, Ain g 1, Cor a 1 and Que a 1. Typically, the blocking effect can be determined by conducting basophil activation testing with basophils from tree pollen allergic individuals and to show reduced binding of IgE to either or all the four tree pollen allergens.

In some embodiments, the composition comprises two antibodies or antigen-binding fragments thereof having antigen-binding regions, such as CDRs contained in the antibodies selected from the group consisting of the antibodies 2B04, A07 and 2E02 or from the group consisting of the antibodies 2B04, A07 and 2C10. In preferred embodiments, the composition comprises two antibodies or antigen-binding fragments thereof antigen-binding regions, such as CDRs contained in the antibodies selected from the group consisting of the antibodies 2B04, A07 and 2E02 or from the group consisting of the antibodies 2B04, A07 and 2C10.

Further, as seen in example 5, the combination of two antibodies is able to provide high inhibition of allergen binding to IgE and thus, basophil activation. It is also revealed that the antibody A07 provides high blocking activity in the basophil activation testing. Therefore, compositions described herein may at least comprise an antibody or antigen-binding fragment thereof having antigen-binding regions, such as CDRs contained in the antibody A07.

Thus, in one embodiment, the composition comprises a combination of a first antibody and a second antibody, wherein,

• the first antibody comprises HCVR with the amino acid sequence of SEQ ID NO: 1 and a LCVR with the amino acid sequence of SEQ ID NO: 2 and the second antibody comprises HCVR with the amino acid sequence of SEQ ID NO: 41 and a LCVR with the amino acid sequence of SEQ ID NO: 42; or

• the first antibody comprises HCVR with the amino acid sequence of SEQ ID NO: 1 and a LCVR with the amino acid sequence of SEQ ID NO: 2 and the second antibody comprises HCVR with the amino acid sequence of SEQ ID NO: 81 and a LCVR with the amino acid sequence of SEQ ID NO: 82; or • the first antibody comprises HCVR with the amino acid sequence of SEQ ID NO: 21 and a LCVR with the amino acid sequence of SEQ ID NO: 22 and the second antibody comprises HCVR with the amino acid sequence of SEQ ID NO: 41 and a LCVR with the amino acid sequence of SEQ ID NO: 42; or

• the first antibody comprises HCVR with the amino acid sequence of SEQ ID NO: 21 and a LCVR with the amino acid sequence of SEQ ID NO: 22 and the second antibody comprises HCVR with the amino acid sequence of SEQ ID NO: 81 and a LCVR with the amino acid sequence of SEQ ID NO: 82; or

• the first antibody comprises HCVR with the amino acid sequence of SEQ ID NO: 41 and a LCVR with the amino acid sequence of SEQ ID NO: 42 and the second antibody comprises HCVR with the amino acid sequence of SEQ ID NO: 81 and a LCVR with the amino acid sequence of SEQ ID NO: 82.

The antibodies may be defined according to their CDR regions, as previous described. In the following embodiments, the HCDR as well as the LCDR regions have been determined by IMGT.

Therefore, in other embodiments, the composition comprises a combination of a first antibody and a second antibody each comprising three heavy chains CDRs (HCDR1, HCDR2 and HCDR3) and three light chain CDRs (LCDR1, LCDR2 and LCDR3)wherein,

• the first antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 1 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 2, and the second antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 41 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 42; or

• the first antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 1 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 2, and the second antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 81 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 82; or the first antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 21 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 22, and the second antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 41 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 42; or

• the first antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 21 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 22 and the second antibody comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 81 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 82; or

• the first antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 41 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 42, and the second antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 81 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 82.

In another embodiment, the composition comprises a combination of three antibodies, such as a composition comprising a combination of a first antibody, a second antibody and a third antibody, wherein,

• the first antibody comprises HCVR with the amino acid sequence of SEQ ID NO: 81 and a LCVR with the amino acid sequence of SEQ ID NO: 82;

• the second antibody comprises HCVR with the amino acid sequence of SEQ ID NO: 41 and a LCVR with the amino acid sequence of SEQ ID NO: 42; and

• the third antibody comprises HCVR with the amino acid sequence of SEQ ID NO: 1 and a LCVR with the amino acid sequence of SEQ ID NO: 2.

In a further embodiment, the composition comprises three antibodies wherein,

• the third antibody comprises HCVR with the amino acid sequence of SEQ ID NO: 121 and a LCVR with the amino acid sequence of SEQ ID NO: 122. In yet another embodiment, the composition comprises three antibodies wherein,

• the third antibody comprises HCVR with the amino acid sequence of SEQ ID NO: 21 and a LCVR with the amino acid sequence of SEQ ID NO: 22.

In yet another embodiment, the composition comprises three antibodies wherein,

• the first antibody comprises HCVR with the amino acid sequence of SEQ ID NO: 101 and a LCVR with the amino acid sequence of SEQ ID NO: 102;

In yet another embodiment, the composition comprises three antibodies wherein,

• the third antibody comprises HCVR with the amino acid sequence of SEQ ID NO: 121 and a LCVR with the amino acid sequence of SEQ ID NO: 122.

In yet another embodiment, the composition comprises three antibodies wherein,

The antibodies may be defined according to their CDR regions, as previous described. Thus, in one embodiment, the composition comprises three antibodies wherein, • the first antibody comprises a HCDR1, HCDR2 and HCDR3 with the amino acid sequence of SEQ ID NO: 83, 84 and 85, respectively and a LCDR1, LCDR2 and LCDR 3 with the amino acid sequence of SEQ ID NO: 86, 87 and 88, respectively;

• the second antibody comprises a HCDR1, HCDR2 and HCDR3 with the amino acid sequence of SEQ ID NO: 43, 44 and 45 respectively, and a LCDR1, LCDR2 and LCDR 3 with the amino acid sequence of SEQ ID NO: 46, 47 and 48, respectively; and

• the third antibody comprises a HCDR1, HCDR2 and HCDR3 with the amino acid sequence of SEQ ID NO: 3, 4 and 5, respectively, and a LCDR1, LCDR2 and LCDR 3 with the amino acid sequence of SEQ ID NO: 6, 7 and 8, respectively.

In a further embodiment, the composition comprises three antibodies wherein,

• the first antibody comprises a HCDR1, HCDR2 and HCDR3 with the amino acid sequence of SEQ ID NO: 83, 84 and 85, respectively and a LCDR1, LCDR2 and LCDR 3 with the amino acid sequence of SEQ ID NO: 86, 87 and 88, respectively;

• the second antibody comprises a HCDR1, HCDR2 and HCDR3 with the amino acid sequence of SEQ ID NO: 43, 44 and 45, respectively and a LCDR1, LCDR2 and LCDR 3 with the amino acid sequence of SEQ ID NO: 46, 47 and 48, respectively; and

• the third antibody comprises a HCDR1, HCDR2 and HCDR3 with the amino acid sequence of SEQ ID NO: 123, 124 and 125, respectively and a LCDR1, LCDR2 and LCDR 3 with the amino acid sequence of SEQ ID NO: 126, 127 and 128, respectively.

In yet another embodiment, the composition comprises three antibodies wherein,

• the third antibody comprises a HCDR1, HCDR2 and HCDR3 with the amino acid sequence of SEQ ID NO: 23, 24 and 25, respectively and a LCDR1, LCDR2 and LCDR 3 with the amino acid sequence of SEQ ID NO: 26, 27 and 28, respectively.

In yet another embodiment, the composition comprises three antibodies wherein, the first antibody comprises a HCDR1, HCDR2 and HCDR3 with the amino acid sequence of SEQ ID NO: 103, 104 and 105, respectively and a LCDR1, LCDR2 and LCDR 3 with the amino acid sequence of SEQ ID NO: 106, 107 and 108, respectively; • the second antibody comprises a HCDR1, HCDR2 and HCDR3 with the amino acid sequence of SEQ ID NO: 43, 44 and 45, respectively and a LCDR1, LCDR2 and LCDR 3 with the amino acid sequence of SEQ ID NO: 46, 47 and 48, respectively; and

• the third antibody comprises a HCDR1, HCDR2 and HCDR3 with the amino acid sequence of SEQ ID NO: 3, 4 and 5, respectively and a LCDR1, LCDR2 and LCDR 3 with the amino acid sequence of SEQ ID NO: 6, 7 and 8, respectively.

In yet another embodiment, the composition comprises three antibodies wherein,

• the first antibody comprises a HCDR1, HCDR2 and HCDR3 with the amino acid sequence of SEQ ID NO: 103, 104 and 105, respectively and a LCDR1, LCDR2 and LCDR 3 with the amino acid sequence of SEQ ID NO: 106, 107 and 108, respectively;

• the second antibody comprises a HCDR1, HCDR2 and HCDR3 with the amino acid sequence of SEQ ID NO: 43, 44 and 45, respectively and a LCDR1, LCDR2 and LCDR 3 with the amino acid sequence of SEQ ID NO: 46, 47 and 48 respectively; and

In yet another embodiment, the composition comprises three antibodies wherein,

Alternatively defined, compositions herein comprises a first antibody, a second antibody and a third antibody each comprising three heavy chains CDRs (HCDR1, HCDR2 and HCDR3) and three light chain CDRs (LCDR1, LCDR2 and LCDR3)wherein,

• the first antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 1 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 2, the second antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 41 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 42, and the third antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 81 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 82; or

• the first antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 1 and a LCDR1, LCDR2 AND LCDR3 with the amino acid sequence contained within SEQ ID NO: 2, the second antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 41 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 42 and the third antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 101 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 102; or

• the first antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 21 and a LCDR1, LCDR2 and LCDR3 with the amino acid sequence contained within SEQ ID NO: 22, the second antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 41 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 42 and the third antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 81 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 82; or

• the first antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 21 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 22, the second antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 41 and comprises LCDR1, LCDR2 and LCDR3 with the amino acid sequence of SEQ ID NO: 42 and the third antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 101 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 102; or the first antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 41 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 42, the second antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 101 and a LCDR1, LCDR2 AND LCDR3 with the amino acid sequence contained within SEQ ID NO: 102 and the third antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 121 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 122.

In further embodiments, the composition, comprises:

• an antibody or antigen-binding fragment thereof comprising a HCVR having an amino acid sequence of SEQ ID NO: 1 and a LCVR having an amino acid sequence of SEQ ID NO: 2; and

• one or more isolated human monoclonal antibodies or antigen-binding fragments thereof able to bind one or more of the allergens Bet v 1, Ain g 1, Cor a 1 and Que a 1.

In another embodiment, the composition comprises,

• an antibody or antigen-binding fragment thereof comprising a HCVR having an amino acid sequence of SEQ ID NO: 21 and a LCVR having an amino acid sequence of SEQ ID NO: 22.

In a further embodiment, the composition comprises,

• an antibody or antigen-binding fragment thereof comprising a HCVR having an amino acid sequence of SEQ ID NO: 1 and a LCVR having an amino acid sequence of SEQ ID NO: 2;

• an antibody or antigen-binding fragment thereof comprising a HCVR having an amino acid sequence of SEQ ID NO: 21 and a LCVR having an amino acid sequence of SEQ ID NO: 22; and

• an antibody or antigen-binding fragment thereof comprising a HCVR having an amino acid sequence of SEQ ID NO: 41 and a LCVR having an amino acid sequence of SEQ ID NO: 42. Pharmaceutical

A third aspect of the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of one or more antibodies, or antigen-binding fragments thereof, as defined herein, together with one or more pharmaceutically acceptable excipients. Thus, antibodies of the first aspect of the invention or antibody combinations of the second aspect of the invention can be formulated into a pharmaceutical composition, wherein the pharmaceutical composition comprises antibodies including multi-specific antibodies of the first aspect of the invention or antibody combinations of the second aspect of the invention and one or more pharmaceutically acceptable excipients.

Typically, the pharmaceutical composition is formulated for being administered by injection, such as by s.c. or i.m. injection.

The antibodies of the invention are able to bind to anyone of the different allergens as descried herein. Further, the different antibodies are able to bind to the individual allergen simultaneous, as they bind to non-overlapping epitopes, as described in the example section. Thus, by binding the different allergens, the individual antibodies can block binding of IgE to that particular allergen. By combining binding of the different antibodies, more of the allergen can be covered leading to an increase blocking of IgE.

Thus, in one embodiment, the antibody or antigen-binding fragment thereof according to the invention can reduce or inhibiting the binding of an IgE antibody to Bet v 1 and/or Cor a 1, and/or Ain g 1 and/or Que a 1.

As IgE is the direct link between the allergen and an allergic reaction, through activation of mast cells and basophils, inhibiting IgE binding to the allergen is an effective prevention of cellular activation, which can be used in treatment of allergy.

Thus, a fourth aspect of the present invention relates to a method of preventing or reducing mast cell degranulation and/or blocking basophil activation associated with Bet v 1 and/or Cor a 1, and/or Ain g 1 and/or Que a 1 sensitization, the method comprising administering an antibody or antigen-binding fragment thereof according to a first aspect, a combination of antibodies according to a second aspect or pharmaceutical composition according to a third aspect to a subject in need thereof. Further, a fourth aspect of the present invention relates to a method of treating a tree pollen allergy, the method comprising administering an antibody or antigen-binding fragment thereof according to a first aspect, a combination of antibodies according to a second aspect or a pharmaceutical composition according to a third aspect to a subject in need thereof.

Still further, a fourth aspect of the present invention relates to an antibody or antigenbinding fragment thereof according to a first aspect, a combination of antibodies according to a second aspect or a pharmaceutical composition according to a third aspect for use in a method of treating a tree pollen allergy to a subject in need thereof.

Still further, a fourth aspect of the present invention relates to the use of an antibody or antigen-binding fragment thereof according to a first aspect, a combination of antibodies according to a second aspect or a pharmaceutical composition according to a third aspect for the preparation of a medicament for use in a method of treating a tree pollen allergy to a subject in need thereof.

It should be understood that the tree pollen allergy may be selected from the group consisting of allergy to birch pollen, alder pollen, hazel pollen, and oak pollen. Also, it may be understood that the tree pollen allergy may be associated with allergy or sensitization towards one or more of the allergens Bet v 1, Cor a 1, Ain g 1 and Que a 1.

The subject in need thereof may have a sensitivity to Bet v 1. That is, the patient may have detectable IgE antibodies towards Bet v 1, for example above 0.7 kll IgE in blood.

Another embodiment relates to a method for treating a subject who demonstrates a sensitivity to Ain g 1, the method comprises administering one or more antibodies according to the invention, the antibody composition or the pharmaceutical composition as described herein.

One embodiment relates to a method for treating a subject who demonstrates a sensitivity to Cor a 1, the method comprises administering one or more antibodies according to the invention, the antibody composition or the pharmaceutical composition as described herein.

One embodiment relates to a method for treating a subject who demonstrates a sensitivity to

Que a 1, the method comprises administering one or more antibodies according to the invention, the antibody composition or the pharmaceutical composition as described herein.

In preferred embodiments, the subject is a human.

The antibodies or antigen-binding fragments as described herein can be obtained in several different ways. One option is using host cell having an expression vector comprising a nucleic acid molecule encoding the antibody or antigen-binding fragment thereon as described herein.

In this respect, the person skilled in the art will readily appreciate that the polynucleotides encoding at least the variable domain of the light and/or heavy chain may encode the variable domains of both immunoglobulin chains or only one. Likewise, said polynucleotides may be under the control of the same promoter or may be separately controlled for expression.

Furthermore, the present invention relates to vectors, particularly plasmids, cosmids, viruses and bacteriophages used conventionally in genetic engineering that comprise a polynucleotide encoding the antigen or preferably a variable domain of an immunoglobulin chain of an antibody of the invention; optionally in combination with a polynucleotide of the invention that encodes the variable domain of the other immunoglobulin chain of the antibody of the invention. Preferably, said vector is an expression vector and/or a gene transfer or targeting vector. Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno- associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of the polynucleotides or vector of the invention into targeted cell population. Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors.

The present invention furthermore relates to host cells transformed with a polynucleotide or vector of the invention. Said host cell may be a prokaryotic or eukaryotic cell. The polynucleotide or vector of the invention which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained extrachromosomally. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal or human cell. Preferred fungal cells are, for example, those of the genus Saccharomyces, in particular those of the species S. cerevisiae. The term "prokaryotic" is meant to include all bacteria which can be transformed or transfected with a DNA or RNA molecules for the expression of an antibody of the invention or the corresponding immunoglobulin chains. Prokaryotic hosts may include gram negative as well as gram positive bacteria such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. The term "eukaryotic" is meant to include yeast, higher plant, insect and preferably mammalian cells, most preferably HEK293, NSO and CHO cells. A further aspect refers to a method for preparing an anti-allergen antibody or allergenbinding fragment thereof, comprising of culturing the cell comprising a polynucleotide encoding the antibody or binding fragment thereof as described herein and isolating the antibody or allergen binding fragment thereof from the cell or culture medium of the cell.

In a further embodiment, the present invention relates to a method for the production of an antibody or a binding fragment thereof, said method comprising

(a) culturing a cell as described herein; and

(b) isolating said antibody or binding fragment thereof from the culture.

The transformed hosts can be grown in fermentors and cultured according to techniques known in the art to achieve optimal cell growth. Once expressed, the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms of the present invention, can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like; see, Scopes, "Protein Purification", Springer Verlag, N.Y. (1982). The antibody or its corresponding immunoglobulin chain(s) of the invention can then be isolated from the growth medium, cellular lysates, or cellular membrane fractions. The isolation and purification of the, e.g., recombinantly expressed antibodies or immunoglobulin chains of the invention may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibodies directed, e.g., against the constant region of the antibody of the invention. It will be apparent to those skilled in the art that the antibodies of the invention can be further coupled to other moieties for, e.g., drug targeting and imaging applications. Such coupling may be conducted chemically after expression of the antibody or antigen to site of attachment or the coupling product may be engineered into the antibody or antigen of the invention at the DNA level. The DNAs are then expressed in a suitable host system, and the expressed proteins are collected and renatured, if necessary.

Thus, one aspect of the invention relates to a nucleic acid molecule encoding a human monoclonal antibody or fragment thereof according to the invention.

One embodiment refers to an expression vector comprising the nucleic acid molecule encoding a human monoclonal antibody or fragment thereof as described herein.

Another embodiment refers to a host cell containing the expression vector as described herein. A further aspect refers to a method for preparing an anti-allergen antibody or allergenbinding fragment thereof, consisting of culturing the cell comprising a polynucleotide encoding the antibody or binding fragment thereof as described herein and isolating the antibody or allergen binding fragment thereof from the cell or culture medium of the cell.

EXAMPLE 1

Generation of Human antibodies

Aim

The aim of this present study was to generate fully human Bet v 1 specific antibodies for preventing effector cell bound patient IgE to bind to Bet v 1 and elicit an immediate reaction.

Material and Methods

Biotinylation of nBet v 1, nOue a 1 and OVA: To allow for Bet v 1 and Que a 1 specific memory B-cell isolation by FACS, Bet v 1 and Que a 1 were biotinylated and complexed with streptavidin PE (SA PE). nBet v 1, nQue a 1, were purified from pollen extract, with protein concentrations >1 mg/mL. EZ-Link™ Sulfo-NHS-LC-LC-biotin (Thermo Scientific) were diluted in water and immediately added to nBet v 1, nQue a 1, and OVA in biotimallergen ratios 2: 1, 1 :2, and 1 :2, respectively, and incubated under agitation at room temperature for 30 min with no light exposure.

Generation of fluorescent nBet v 1, nQue a 1, OVA (decoy) multimers: To generate biotinylated nBet v 1 and Que a 1 SA PE multimers, SA-PE (BioLegend) was added at a 1 :2 and 1:4 ratio, respectively. To generate biotinylated OVA SA PE multimers, SA-PE (BioLegend) was added at a 1 :4 ratio (1 parts biotinylated OVA to 4 parts SA APC).

Isolating and freezing PBMCs from blood donors: Peripheral blood samples were drawn from birch pollen allergic individuals. PBMCs were separated from the plasma and red blood cells using leucosep™. Cells were diluted to approximately 5xl0⁷ cells/mL in RPMI 1640 + GlutaMAX™ (Gibco) and counted and checked for viability using NucleoCounter NC-200 (ChemoMetec) with Vial Cassettes (ChemoMetec). Sinqle cell sortinq Bet v 1 and

B cells: Approximately 5-10' 10⁷ mononuclear cells (isolated as described above) were used. OVA decoy multimer was added to the cells at a final concentration of 5 nM, The respective antigen, nBet v 1 or nQue a 1, fluorescent multimer was added to a final concentration of 1 nM. Cells were enriched according to the protocol for anti-PE MicroBeads lyophilized (Miltenyi Biotec) the control sample were then stained with IgD FITC (Biolegend, IA6-2), IgM FITC (Biolegend, MHM-88), CD19 BV650 (Biolegend, SJ25C1), CD3 BV480 (Biolegend, UCHT1), CD14 BV480 (Biolegend, M5E2), live/dead™ fixable aqua dead cell kit (Thermo Scientific), CD38 BV421 (Biolegend, HIT2), and CD27 APC-H7 (Biolegend, M-T271) in Brilliant Stain Buffer (BD Horizon).

150 single antigen-specific B cells (CD3-, CD14-, CD19⁺, IgD+IgM-, allergen-PE⁺, and decoy- AF647 ) were single-cell sorted 96-well plates.

cDNA and libraries were generated from single cells (obtained as described above) according to SMART-Seq® Single Cell Kit User Manual (version 101619). cDNA was then amplified and purified before quantification with Qubit 4 (Invitrogen) and quality assessment with TapeStation 4150 (Agilent). Library preparation for Illumina sequencing was completed with NovaSeq 6000 Reagent Kits (Illumina; 20028400). cDNA was fragmented, index labelled, amplified, and pooled prior to sequencing. Sequencing was then performed on Illumina NovaSeq 6000. The sequencing results from the tetramer sorted memory B cells were analyzed using the RNA-sequence aligner STAR (v. 2.7.10a). B cell receptor sequences were reconstructed using a modified version of the BraCeR pipeline (docker pull nielsphk/bracer: 1.3), originally created by Lindeman et al. 2018 (Lindeman et al., 2018).

137 heavy- and light chain paired B-cell receptor sequences were generated from the single cell sorted B-cells.

Plasmid design Human VH and VL sequences derived from selected B-cells were cloned into mammalian expression vectors.

Heavy chain expression vector: A synthetic gene consisting of a signal peptide followed by VH + human IgHG4 (with the so called S228P mutation) was cloned as a Hindlll/BamHI fragment into expression vector pcDNA3. 1(+).

Light chain expression vector: A synthetic gene consisting of a signal peptide followed by VL + human IgK or IgL was cloned as a Hindlll/BamHI fragment into expression vector pcDNA3. !(+)■ Sequences were codon optimized for mammalian expression. Transfection grade plasmid preparations was obtained from Genscript (NJ, USA)

Expression of mAbs: Antibodies were expressed using a 1 : 1 ratio of heavy: light chain encoding plasmid following the "Expi293™ Expression System User Guide" (ThermoFisher, publication number: MAN0019402, revision number: B.O. The supernatants were harvested 6 days after transfection by centrifugation at 2700g for 1 hour at 4°C and filtered using a 0,45 pm filter.

Results

150 B-cells were single-cell sorted and underwent scRNAseq, leading to 76 paired heavy- and light chain sequences, where 70 antibodies were expressed, yielding 36 Bet v 1 specific, antibodies.

EXAMPLE 2

Affinity determination of antibodies binding tree pollen allergens

Aim

The aim of the present example is to determine the affinity of the obtained antibodies to major tree pollen allergens Bet v 1, Ain g 1, Cor a 1, and Que a 1.

Material and method

Equilibrium dissociation constants (KD) for different related allergens binding to monoclonal antibodies were determined utilizing biolayer interferometry (BLI) on an Octet RED96e instrument. All binding studies were performed in 8mM NaH2PO4, 150mM NaCI, 3mM KCI, 2mM KH2PO4, 0.1% BSA, and 0.05% v/v surfactant Tween-20, pH 7.4 kinetic buffer at 25°C and 1000 rpm shake speed. For the antibodies expressed as IgG4, Octet® ProA Biosensors were loaded with the monoclonal antibodies before binding the allergen. For the antibodies expressed as IgE, Octet® SAX Biosensors were loaded with biotinylated anti-IgE VHH followed by capture of the monoclonal antibodies before binding the allergen. Binding studies were performed on the following allergens: Birch (nBet v 1 and rBet v 1.0112), Alder (Ain g 1), Hazel (Cor a 1) and White Oak (Que a 1). Immobilized monoclonal antibody were dipped in 2-fold serially diluted allergens for 5 minutes to allow for association while the dissociation of monoclonal antibody bound allergens was monitored for 30 minutes in kinetic buffer. Double reference subtraction was performed. Maximum allergen capture level, global association rate (k_a), dissociation (kd) rate and dissociation equilibrium constants (KD) were determined by fitting the real-time binding sensorgrams to a 1 : 1 fitting model using Data Analysis HT 11.1 software (Sartorius). The antibodies REGN5713, REGN5714 and REGN5715 from Regeneron are added for comparison.

Results

Binding kinetics parameters for natural and recombinant Bet v 1, natural Ain g 1, natural Cor a 1, and natural Que a 1 to different monoclonal antibodies of the invention at 25 °C are shown in Table 3 through Table 8.

The affinity (Kd) for the antibodies of the invention is below the control antibodies. The antibodies 2E02, 2C10, 2B04, 2E07, A07, BIO and 2_10 all has an affinity to the Bet v 1, Ain g 1, Cor a 1, and Que a 1 allergen higher than the REGN5713, REGN5714 and REGN5715. Table 3 - affinity (Kd) of rBet v 1, nAln g 1, nCor a 1 and nQue a 1, binding to the monoclonal antibodies of the invention at 25°C

As shown in table 3, 7 of the 36 tested antibodies can bind all 4 tested allergens. Further, only one (REGN5715) of the three control antibodies (REGN5713, REGN5714 and REGN5715) were able to bind all four allergens, indicating that binding affinity to Bet v 1, does not always give cross reactivity to the three other allergens.

Table 4 - Binding kinetics parameters of recombinant Bet v 1 binding to monoclonal antibodies at 25°C

As shown in table 4, all the 36 monoclonal antibodies of the invention demonstrated measurable binding to recombinant Bet v 1 with KD values ranging from 249 pM to 468 nM. Further, the antibodies 2B04, 2C10, 2e02 and A07, all demonstrated a stronger affinity compared to REGN5713, REGN5714 and REGN5715.

Table 5 - Binding kinetics parameters of natural Bet v 1 binding to monoclonal antibodies at 25°C

"ND" indicates that the affinity was not determined.

As shown in Table , all the 34 of 36 monoclonal antibodies of the invention demonstrated measurable binding to natural Bet v 1 with KD values ranging from 219 pM to 506 nM.

Further, the antibodies 2E02, 2B04, A07 and BIO all demonstrated greater affinity to natural Bet V 1 compared to the control antibodies REGN5713, REGN5714 and REGN5715. Table 6 - Binding kinetics parameters of natural Ain g 1 binding to monoclonal antibodies at

25°C

"NB" indicates that no binding was observed under the current experimental conditions (data now shown) As shown in Table , 15 of the monoclonal antibodies of the invention demonstrated measurable binding to natural Ain g 1 with KD values ranging from 626 pM to 4.34 uM. The other 21 antibodies did not demonstrate any measurable binding to Ain g 1 under the tested conditions. Further 2_9, 2A03, 2A09, 2B04, 2C10, 2E02, 2E07, A07 and BIO all demonstrated a greater affinity compared to the control antibodies REGN5713, REGN5714, and REGN5715.

Table 7 - Binding kinetics parameters of natural Cor a 1 binding to monoclonal antibodies at

25°C

NB indicates that no binding was observed under the current experimental conditions (data now shown)

As shown in Table , 14 of the monoclonal antibodies of the invention demonstrated measurable binding to natural Cor a 1 with KD values ranging from 2.69 nM to 2.18 uM. The other 22 antibodies did not demonstrate any measurable binding to Cor a 1 under the tested conditions. 2_20, 2B04, 2C10, 2E02, 2E07, A07, and BIO all demonstrated greater affinity to Cor a 1 compared to REGN5713, REGN5714 and REGN5715

Table 8 - Binding kinetics parameters of natural Que a 1 binding to monoclonal antibodies at 25°C

As shown in Table , seven of the monoclonal antibodies of the invention demonstrated measurable binding to natural Que a 1 with KD values ranging from 660 pM to 158 nM. The other 29 antibodies did not demonstrate any measurable binding to Que a 1 under the tested conditions. Further, the antibodies 2_10, 2E02, 2E07, 2C10, 2B04, and A07 all demonstrated greater affinity to Que a 1 compared to REGN5713, REGN5714 and REGN5715

Conclusion

Here we show that the antibodies 2_10, 2B04, 2C10, 2E02, 2E07, and A07 binds all four tree pollen allergens tested (Bet v 1, Ain g 1, Cor a 1, and Que a 1) and therefore exhibit greater cross-reactivity compared to the control antibodies REGN5713 , REGN5714, and REGN5715. EXAMPLE 3

Epitope binning on Bet v 1

Aim

The aim of this present study was to epitope bin a subset of mAbs on Bet v 1

Materials and methods

Epitope binning : Pair-wise epitope binning was performed by BLI on the Octet RED96e platform at 1000 rpm agitation and 30°C. Kinetic buffer (8 mM NaH2PO4, 3 mM KCI, 2 mM KH2PO4, 0.1% BSA, and 0.02% Tween-20) was used for all experiments. Specifically, each mAb, position 1, were immobilized to a sensor tip and dipped into nBet v 1 at 100 nM for 120 seconds. The tips were then dipped into all mAbs, position 2, individually at 9 ug/mL for 300 seconds to assess if the other mAbs could bind to an epitope not occupied by the immobilized mAb. Overlapping and non-overlapping clones were determined by comparing mAbs with the same mAb at position 1 and 2 as a negative control. Fejl! Henvisningskilde ikke fundet. shows the epitope binning matrix.

Results

As seen from figure 3, the different antibodies can be divided into groups depending on their epitope specificity. Each ring represents one of the antibodies in table 4. Overlapping rings illustrate that the antibodies are binding to overlapping epitopes on Bet v 1, whereas nonoverlapping rings shows antibodies binding to distinct non-overlapping epitopes.

Based on the binding to each individual epitope, the antibodies can be put into groups, able to bind simultaneous to Bet v 1.

Examples of such groups are:

• A07, 2B04 and 2E02 or

• A07, 2B04 and 2_10 or

• A07, 2B04 and 2C10 or

• BIO, 2B04 and 2E02 or BIO, 2B04 and 2_10 or

BIO, 2B04 and 2C10.

Conclusion

In conclusion, we were able to divide the individual antibodies into groups depending on the epitope specificity to Bet v 1. Further, this grouping allows for defining groups of antibodies that bind simultaneous to Bet v 1.

EXAMPLE 4

Determination of Simultaneous Binding of Three Anti-Bet v 1 Monoclonal Antibodies to Bet v 1

Aim

The aim of this experiment was to use the prediction from example 3 and show that the Bet v 1 epitopes bound by three selected Bet v 1 monoclonal antibodies were unique and that, irrespective of the order of monoclonal antibody binding, no steric hindrance was exhibited upon simultaneous binding of these three antibodies. Order dependent binding of the three Bet v 1 monoclonal antibodies was also assessed.

Material and methods

Simultaneous binding of three anti-Bet v 1 monoclonal antibodies to Bet v 1 was determined using real time, label-free biolayer interferometry (BLI) based Octet RED96e (Sartorius). The experiment was performed at 25°C in kinetic buffer (8 mM NaH2PO4, 3 mM KCI, 2 mM KH2PO4, 0.1% BSA, and 0.02% Tween-20).

In three separate experiments, each of the three IgG4 mAbs analysed in this example (A07, 2B04 and 2E02) that were previously shown to bind Bet v 1 with high affinity and being highly cross reactive to PR-10 allergens from related tree species (example 2) as well as being able to bind pair-wise simultaneously to Bet v 1 (example 3) were initially captured via Protein A sensor tips to a level of 2.5-3.3 nm (table 5). Remaining Protein A binding sites were subsequently blocked with high concentrations (> 10-fold higher concentration) of an irrelevant non-Bet v 1 IgG4 mAb. The sensor tips were then dipped into wells containing 4.25 |jg/ml Bet v 1 (for 5 min) that was then captured by the Protein A bound anti-Bet v 1 antibody (table 5) to a level of 0.3-0.5 nm. This was followed by sequential dipping the sensor tips into wells containing either the same anti-Bet v 1 mAb as directly captured on the Protein A tips (negative control) or the two additional mAb clones (for 5 min in each step) (mAb-1-3, tabl 5). See example sensorgram figure 4.

Baseline steps i.e. dipping of the sensor tips in kinetic buffer was included in between all of the above mentioned steps.

Results

The level of binding (nm) was measured using Data Analysis HT 11.1.3.50 (Molecular Devices) and results are shown in table 9. A signal of less than 0.05 nm indicates no binding, whereas signals greater than 0.1 nm represents binding i.e. no competition of the mAbs for binding to Bet v 1. All three Bet v 1 monoclonal antibodies included in this example were able to simultaneously bind to Bet v 1 and binding responses were not affected by the order in which the antibodies were added. Table 9:

NB=No binding (<0.05 nm)

Conclusion

This shows that a group of 3 antibodies with different epitope specificity, were able to bind

Bet v 1 simultaneously. Regardless of the order by which each of the three mAbs binds to Bet v 1, there was no competition impeding the simultaneous binding of all three antibodies, implying that mAb clone A07, 2B04, and 2E02 bind to non-overlapping, distinct epitopes without steric hindrance as predicted in example 4.

EXAMPLE 5

Inhibition of allergen-specific IgE binding tree pollen allergens

Aim

The aim of this present study was to determine if the mAbs of the invention were able to block binding of IgE to nBet v 1, nQue a 1, nAln g 1, and nCor a 1 and inhibit activation of basophils from tree pollen allergic individuals, when challenged with either nBet v 1, nQue a 1, nAln g 1, or nCor a 1.

Materials and methods

The mAbs of the invention were tested for blocking of IgE epitopes and inhibition of basophil activation. Blocking of IgE epitopes and inhibition of basophil activation are key in vitro assays to assess the potency of an antibody cocktail in inhibiting the allergic reaction, as previously suggested (Orengo et al., 2018; Atanasio et al., 2022).

PBMC were isolated from blood freshly drawn from tree pollen allergic donors. The cells were preincubated with IL-3 before mixing with single tree pollen allergens preincubated for 1 h with various mAbs (either a mix or single mAbs). Activation of basophils were done at 37°C for 1 h. Cells were then washed and stained with antibodies for specific surface cell proteins (CD123, CD203c and CD63) for analysis by flow cytometry. Basophil activation was measured via CD63 upregulation on Basophils identified within PBMC based on CD123 and CD203c.

Results

Different mixes of mAbs were tested (table 10) and compared to the effect by the individual mAbs. Table 10:

The experiments were done with basophils isolated from 6 tree pollen allergic donors. Basophils were inhibited with mAb mixes comprising 4nM of each mAb in the mix (figure 5- 8). Allergen stimulation was done using the allergnes; nBet v 1 (figure 5A-F), nAln g 1 (figure 6A-F), nCor a 1 (figure 7A-F), and nQue a 1 (figure 8A-F).

As seen in figure 5A-F, activation was strongly inhibited by the antibody mixes, compared to the buffer control in all 6 donors.

When basophils were stimulated with nAln g 1, nCor a 1, and nQue a 1, only ALK-1 and ALK- 2 mix where able to inhibit the activation, whereas the control-1 showed no-, or very limited effect on the activation in all 6 donors (figure 6-8A-F).

Next, basophil activation was inhibited with increasing concentrations of each mAb (from 5xl0e-4 nM to 200 nM) (figure 9-10 and table 11-18). The concentration of allergen (nBet v 1, nAln g 1, nCor a 1 and nQue a 1) used for stimulation of the basophils were selected individually for each donors A-F (due to variation in sensitivity for each donor). Figure 9-10A- D shows two representative individual donors stimulated (one by one) with all 4 allergens (Donor D and F from table 11-18).

As seen in figure 9A and 10A, ALK-1, ALK-2 and control-1 were able to inhibit the nBet v 1 induced activation to a greater degree than the individual antibodies.

When the basophils were stimulated with anyone of, nAln g 1, nCor a 1 and nQue a 1, ALK-1 and ALK-2 did show the strongest inhibition among all tested donors compared to both control-1, and the individual antibodies (figure 9B-D, 10B-D and table 11-14). Table 11: Percent inhibition of basophil activation in 6 individual donors, using different combination of mAbs, after activation with nBet v 1.

Table 12: Percent inhibition of basophil activation in 6 individual donors, using different combination of mAbs, after activation with nAln g 1.

Table 13: Percent inhibition of basophil activation in 6 individual donors, using different combination of mAbs, after activation with nCor a 1.

Table 14: Percent inhibition of basophil activation in 6 individual donors, using different combination of mAbs, after activation with nQue a 1.

ALK-3 and ALK-4 was tested for the ability to inhibit basophil activation after stimulation with nAln g 1, nCor a 1 and nQue (table 15-18). Both ALK-3 and ALK-4 showed a stronger inhibition of basophil activation compared to the individual antibodies, in all 6 donors.

Table 15: Percent inhibition of basophil activation in 6 individual donors, using different combination of mAbs, after activation with nBet vl.

Table 16: Percent inhibition of basophil activation in 6 individual donors, using different combination of mAbs, after activation with nAln g 1.

Table 17: Percent inhibition of basophil activation in 6 individual donors, using different combination of mAbs, after activation with nCor a 1.

Table 18: Percent inhibition of basophil activation in 6 individual donors, using different combination of mAbs, after activation with nQue a 1.

Conclusion

Here it is shown that the ALK-1 and ALK-2, are able block the binding of IgE to the allergens (nBet v 1, nAln g 1, nCor a 1 and nCor a 1) leading to inhibition of basophil activation. Inhibition by ALK-1 and ALK-2, of nBet v 1 induced basophil activation, was comparable to the reference mix, "control-1" (figure 5A-F, 9A and 10A). In contrast ALK-1 and ALK-2 show more inhibition of basophil activation by nAln g 1, nCor a 1 and nQue a 1 compared to "control-1" (figure 6-8B-F, 9B-D and 10B-D).

Further, It was shown that a mix of two antibodies were able to inhibit basophil activation by all four allergens to a greater extent than the individual ABs. REFERENCES

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Claims

1. An antibody or antigen-binding fragment thereof, which comprises three heavy chains CDRs (HCDR1, HCDR2 and HCDR3) and three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein;

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence contained within SEQ ID NO: 21, a HCDR2 amino acid sequence contained within SEQ ID NO: 21, a HCDR3 amino acid sequence contained within SEQ ID NO: 21, a LCDR1 amino acid sequence contained within SEQ ID NO: 22, a LCDR2 amino acid sequence contained within SEQ ID NO: 22 and a LCDR3 amino acid sequence contained within SEQ ID NO: 22; or

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence contained within SEQ ID NO: 41, a HCDR2 amino acid sequence contained within SEQ ID NO: 41, a HCDR3 amino acid sequence contained within SEQ ID NO: 41, a LCDR1 amino acid sequence contained within SEQ ID NO: 42, a LCDR2 amino acid sequence contained within SEQ ID NO: 42 and a LCDR3 amino acid sequence contained within SEQ ID NO: 42; or

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence contained within SEQ ID NO: 121, a HCDR2 amino acid sequence contained within SEQ ID NO: 121, a HCDR3 amino acid sequence contained within SEQ ID NO: 121, a LCDR1 amino acid sequence contained within SEQ ID NO: 122, a LCDR2 amino acid sequence contained within SEQ ID NO: 122 and a LCDR3 amino acid sequence contained within SEQ ID NO: 122, wherein each of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 may be determined by either IMGT, Kabat or Chothia, and optionally wherein each of the CDR regions determined by either IMGT, Kabat or Chothia may be subject to 1, 2, or 3 amino acid substitutions, such that each HCDR1 may contain 1, 2, or 3 amino acid substitutions, each HCDR2 may contain 1, 2, or 3 amino acid substitutions, each HCDR3 may contain 1, 2, or 3 amino acid substitutions, each LCDR1 may contain 1, 2, or 3 amino acid substitutions, each LCDR2 may contain 1, 2, or 3 amino acid substitutions, and/or each LCDR3 may contain 1, 2, or 3 amino acid substitutions.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the CDR region is determined by IMGT method.

3. An antibody or antigen-binding fragment thereof, wherein;

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence of SEQ ID NO: 3, a HCDR2 amino acid sequence of SEQ ID NO: 4, a HCDR3 amino acid sequence of SEQ ID NO: 5, a LCDR1 amino acid sequence of SEQ ID NO: 6, a LCDR2 amino acid sequence of SEQ ID NO: 7 and a LCDR3 amino acid sequence of SEQ ID NO: 8; or • the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence of SEQ ID NO: 23, a HCDR2 amino acid sequence of SEQ ID NO: 24, a HCDR3 amino acid sequence of SEQ ID NO: 25, a LCDR1 amino acid sequence of SEQ ID NO: 26, a LCDR2 amino acid sequence of SEQ ID NO: 27 and a LCDR3 amino acid sequence of SEQ ID NO: 28; or

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence of SEQ ID NO: 83, a HCDR2 amino acid sequence of SEQ ID NO: 84, a HCDR3 amino acid sequence of SEQ ID NO: 85, a LCDR1 amino acid sequence of SEQ ID NO: 86, a LCDR2 amino acid sequence of SEQ ID NO: 87 and a LCDR3 amino acid sequence of SEQ ID NO: 88; or

• the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence of SEQ ID NO: 103, a HCDR2 amino acid sequence of SEQ ID NO: 104, a HCDR3 amino acid sequence of SEQ ID NO: 105, a LCDR1 amino acid sequence of SEQ ID NO: 106, a LCDR2 amino acid sequence of SEQ ID NO: 107 and a LCDR3 amino acid sequence of SEQ ID NO: 108; or

4. An antibody or antigen-binding fragment thereof, wherein

• the antibody or antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 41 and a LCVR having the amino acid sequence of SEQ ID NO: 42; or

• the antibody or antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 61 and a LCVR having the amino acid sequence of SEQ ID NO: 62; or

• the antibody or antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 81 and a LCVR having the amino acid sequence of SEQ ID NO: 82; or

• the antibody or antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 121 and a LCVR having the amino acid sequence of SEQ ID NO: 122; or wherein each HCVR may contain 1, 2, or 3 amino acid substitutions, and/or each LCVR may contain 1, 2, or 3 amino acid substitutions.

5. The antibody or antigen-binding fragment thereof according to anyone of the preceding claims, wherein the antibody or antigen-binding fragment thereof, is capable of reducing or inhibiting the binding of an IgE antibody to one, two, three or four of the allergens selected from the group consisting of Bet v 1, Cor a 1, Ain g 1, and Que a 1.

6. The antibody or antigen-binding fragment thereof according to anyone of the preceding claims, wherein the antibody comprises a Fc region of human origin.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein the Fc region is IgGl-Fc or IgG4-Fc.

8. The antibody or antigen-binding fragment thereof according to claim 6, wherein the Fc region is IgG4-Fc.

9. The antibody or antigen-binding fragment thereof according to claim 8, wherein the IgG4-Fc comprises a S228P mutation.

10. The antibody or antigen-binding fragment thereof according to anyone of the preceding claims, wherein the antibody is a human antibody.

11. A multi-specific antibody comprising two or more antigen-binding fragments selected among antigen-binding fragments according to any one of claims 1-2 and claims 5-10 as dependent on claims 1-2; or selected among antibodies or antigen-binding fragments thereof according to claim 3 and claims 5-10 as dependent on claim 3; or selected among antibodies or antigen-binding fragments thereof according to claim 4 and claims 5-10 as dependent on claim 4.

12. A composition comprising two antibodies or antigen-binding fragments thereof selected among antibodies or antigen-binding fragments thereof according to any one of claims 1-2 and claims 5-10 as dependent on claims 1-2; or selected among antibodies or antigenbinding fragments thereof according to claim 3 and claims 5-10 as dependent on claim 3; or selected among antibodies or antigen-binding fragments thereof according to claim 4 and claims 5-10 as dependent on claim 4.

13. The composition according to claim 12, wherein the composition at least comprises an antibody or antigen-binding fragment thereof which comprises three heavy chains CDRs (HCDR1, HCDR2 and HCDR3) and three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein each of HCDR1, HCDR2 and HCDR3 has an amino acid sequence contained within SEQ ID NO: 1 and wherein each of LCDR1, LCDR2 and LCDR3 has an amino acid sequence contained within SEQ ID NO: 2.

14. The antibody composition according to any one of claims 12 and 13, wherein the composition at least comprises an antibody or antigen-binding fragment thereof which comprises three heavy chains CDRs (HCDR1, HCDR2 and HCDR3) and three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein each of HCDR1, HCDR2 and HCDR3 has an amino acid sequence contained within SEQ ID NO: 81 and wherein each of LCDR1, LCDR2 and LCDR3 has an amino acid sequence contained within SEQ ID NO: 82.

15. The composition according to claim 12, wherein the composition comprises a first antibody and a second antibody each comprising three heavy chains CDRs (HCDR1, HCDR2 and HCDR3) and three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein

• the first antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 1 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 2, and the second antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 81 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 82; or

• the first antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 21 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 22, and the second antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 41 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 42; or

• the first antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 21 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 22, and the second antibody comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 81 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 82; or • the first antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 41 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 42, and the second antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 81 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 82.

16. The composition according to any one of claims 12-14, comprising three antibodies or antigen-binding fragments thereof selected among antibodies or antigen-binding fragments thereof according to any one of claims 1-2 and claims 5-10 as dependent on claims 1-2; or selected among antibodies or antigen-binding fragments thereof according to claim 3 and claims 5-10 as dependent on claim 3; or selected among antibodies or antigen-binding fragments thereof according to claim 4 and claims 5-10 as dependent on claim 4.

17. The composition according to claim 16, comprising a first antibody, a second antibody and a third antibody each comprising three heavy chains CDRs (HCDR1, HCDR2 and HCDR3) and three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein,

• the first antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 1 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 2, the second antibody comprises HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 41 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 42, and the third antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 81 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 82; or

• the first antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 1 and a LCDR1, LCDR2 AND LCDR3 with the amino acid sequence contained within SEQ ID NO: 2, the second antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 41 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 42 and the third antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 101 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 102; or • the first antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 21 and a LCDR1, LCDR2 and LCDR3 with the amino acid sequence contained within SEQ ID NO: 22, the second antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 41 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 42 and the third antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 81 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 82; or

• the first antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 21 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 22, the second antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 41 and comprises LCDR1, LCDR2 and LCDR3 with the amino acid sequence of SEQ ID NO: 42 and the third antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 101 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 102; or

• the first antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 41 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 42, the second antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 101 and a LCDR1, LCDR2 AND LCDR3 with the amino acid sequence contained within SEQ ID NO: 102 and the third antibody comprises each of HCDR1, HCDR2 and HCDR3 with an amino acid sequence contained within SEQ ID NO: 121 and comprises each of LCDR1, LCDR2 and LCDR3 with an amino acid sequence contained within SEQ ID NO: 122.

18. A pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to anyone of claims 1-10, the multispecific antibody according to claim 11, or the composition according to anyone of claims 12- 17, together with one or more pharmaceutically acceptable excipients.

19. A method of treating a tree pollen allergy, the method comprising administering a pharmaceutical composition according to claim 19, wherein the tree pollen allergy is associated with allergy towards one or more of the allergens Bet v 1, Cor a 1, Ain g 1 and Que a 1.

20. An antibody or antigen-binding fragment thereof according to anyone of claims 1-10, the multi-specific antibody according to claim 11, or the composition according to anyone of claims 12-17 for use in a method of treating a tree pollen allergy, wherein the tree pollen allergy is associated with allergy towards one or more of the allergens Bet v 1, Cor a 1, Ain g 1 and Que a 1.

21. A nucleic acid molecule encoding a human monoclonal antibody or fragment thereof according to any one of claims 1-10.

22. An expression vector comprising the nucleic acid molecule encoding a human monoclonal antibody or fragment thereof according to claim 21.

23. A host cell containing the expression vector of claim 22.