EA045437B1 - HUMAN ANTIBODIES TO BET V 1 AND METHODS OF THEIR APPLICATION - Google Patents

Description

Field of technology to which the present invention relates

The present invention relates to human antibodies and antigen-binding fragments of human antibodies that bind to the birch pollen allergen, Bet v 1, therapeutic compositions containing antibodies, and methods of using antibodies.

List of sequences

An official copy of the sequence listing is filed simultaneously with the specification electronically via EFS-Web as an ASCII (American Standard Information Interchange Code) formatted sequence listing with file name 10301WO01_SEQ_LIST_ST25, creation date March 31, 2018, and size 137 KB . The sequence listing contained in this ASCII formatted document is part of the present specification and is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Birch is the predominant cause of allergy in 23% of allergy patients in the United States and 14% of allergy patients in Europe (Datamonitor report on Allergic Rhinitis, July 2010) and the leading cause of type 1 allergies in the spring in Europe, North America, Russia and Australia (Breiteneder et al., EMBO J. 1989, 8(7): 1935-8). Bet v 1 protein is the major birch allergen identified in the pollen of Betula verrucosa (European white birch tree, also synonymous with Betula pendula) and is responsible for binding IgE in more than 95% of patients with birch pollen allergy (Breiteneder, supra). Bet v 1 is a small 7-stranded antiparallel βsheet with three α-helices and a known crystal structure (Kofler et al., 2012, 422(1): 109-23; Markovic-Housley et al., J Mol Biol. 2003, 325(1): 123-33; Spangfort et al., J. Immunol. 2003, 171(6): 308490). International patent publication WO 94/10194 relates to peptides obtained from trees of the order Fagales.

Sixty percent of patients with birch pollen allergy respond exclusively to Bet v 1 (Jarolim et al., Int Arch Allergy Appl Immunol. 1989, 88(1-2): 180-2). A single birch tree can produce up to five million pollen grains that travel through the air up to 100 yards from the tree. Symptoms of a birch pollen allergy can range from mild rhinitis and conjunctivitis to life-threatening asthmatic reactions.

Immunoglobulin E (IgE) is responsible for type 1 hypersensitivity, which manifests itself in allergic rhinitis, allergic conjunctivitis, hay fever, allergic bronchial asthma, bee venom allergy and food allergies. IgE circulates in the blood and binds to the high-affinity IgE receptors FcεR1α on basophils and mast cells. In most allergic responses, allergens enter the body through inhalation, ingestion, or through the skin. The allergen then binds to preformed IgE, already bound to a high-affinity receptor on the surfaces of mast cells and basophils, resulting in cross-linking of several IgE molecules and triggering the release of histamine and other inflammatory mediators, causing various allergic symptoms.

Treatment for allergies includes steroids to suppress immune activity and bronchodilators to relieve asthma symptoms. Desensitization therapy is also used for patients with severe allergies. Combinations of peptide vaccines have been tested to desensitize people to certain allergens, such as Bet v 1 (see US Patent No. 9017689). Antibodies have been proposed for the treatment of allergies because they can block the penetration of allergen molecules into mucosal tissues or can bind the allergen before it has a chance to bind to IgE bound to the high affinity receptor on mast cells or basophils, thereby preventing the release of histamine and other inflammatory mediators from these cells.

US Pat. No. 5,670,626 describes the use of monoclonal antibodies to treat IgE-mediated allergic diseases such as allergic rhinitis, allergic bronchial asthma and allergic conjunctivitis by blocking the binding of allergens to mucosal tissue. US Pat. No. 6,849,259 describes the use of allergen-specific antibodies to inhibit allergic inflammation in an in vivo mouse model of allergy. Antibody systems based on milk and eggs have been described. For example, US Patent Application No. US 2003/0003133 A1 discloses the use of milk as an allergen carrier for inducing oral tolerance to birch pollen and other allergens. Compositions and methods of reducing the allergic response in an animal to an allergen in the environment by using a molecule that inhibits the ability of the allergen to bind to mast cells have been described in international patent publication WO 1994/024164 A2. Other antibodies to Bet v 1 were mentioned in US patent document No. 2010/0034812.

The present invention is directed to overcome one or more of the problems discussed above.

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Brief Disclosure of the Present Invention

Provided herein are fully human monoclonal antibodies and antigen binding fragments thereof that bind birch pollen, such as natural Bet v 1, Betula pendula birch pollen extract (BPE), Betula nigra BPE or Betula populifolia BPE. The antibodies may be useful for binding Bet v 1 allergen in vivo following exposure of a sensitized patient to birch allergen and, as such, may act to promote the removal of either natural Bet v 1, Betula pendula birch pollen extract (BPE), Betula nigra BPE or Betula BPE populifolia, or to block the binding of the allergen to pre-formed IgE on the surface of mast cells or basophils. Thus, the antibodies described herein may prevent the release of histamine or other inflammatory mediators from mast cells or basophils, thereby preventing or reducing the adverse effects observed in patients sensitized to birch allergen. In certain embodiments, the antibodies may be capable of reducing, minimizing, or preventing at least one symptom in a patient sensitive to a birch allergen or a birch allergen-related allergen, such as sneezing, congestion, nasal congestion, cough, shortness of breath, bronchospasm, rhinitis, or conjunctivitis. . In some embodiments, the antibodies may be capable of preventing even more serious in vivo complications associated with birch pollen allergen exposure in sensitized individuals, such as asthmatic reactions, anaphylaxis, or even death.

Antibodies provided herein may be full length, such as an IgG1 and/or IgG4 antibody, or may contain only an antigen binding portion, such as a Fab, F(ab')2, or scFv fragment, and may be modified to affect functionality, e.g. , to eliminate residual effector functions (Reddy et al., 2000, J. Immunol. 164: 1925-1933).

The first aspect of the present invention relates to an isolated human monoclonal antibody or antigen binding fragment thereof that binds to natural Bet v 1, Betula pendula birch pollen extract (BPE), Betula nigra BPE and/or Betula populifolia BPE.

In one embodiment, the isolated human monoclonal antibody or antigen-binding fragment thereof inhibits the binding of naturally occurring Bet v 1, Betula pendula BPE, Betula nigra BPE, or Betula populifolia BPE to allergen-specific IgE.

In one embodiment, an isolated human antibody or antigen-binding fragment thereof binds to Bet v 1 with a _KD equal to or less than 10 ^-8 M. In one embodiment, the human antibody or antigen-binding fragment thereof binds to Bet v 1 with a KD located at range from about 10 ^-8 to about 10 ^-11 M. In one embodiment, an isolated human antibody or antigen binding fragment thereof binds to Bet v 1 with a KD equal to or less than 27.9 nM. In one embodiment, an isolated human antibody or antigen binding fragment thereof binds to Bet v 1 with a K _D ranging from about 0.66 nM to about 27.9 nM.

In one embodiment, the isolated human antibody is a fully human monoclonal antibody.

In one embodiment, the isolated human monoclonal antibody or antigen-binding fragment thereof cross-reacts with one or more allergens selected from the group consisting of Aln g1, Cor a1, Car b1, Que a1, Api g2, Api g1, Dau c1, Mal d1, Ost c1, Fag s1 and Cas s1. Such allergens can also be called PR-10 proteins, or so-called pathogenesis-related (PR) proteins. Additional PR-10 proteins (members of the Bet v 1 family) also include Act c 8 and Act d 8 (kiwi), Ara h 8 (peanut), Pru ar 1 (apricot), Pru av 1 (cherry), Pru p 1 (peach), Pyr s 1 (pear), Gly m 4 (soybean), Vig r 1 (golden bean), Sola I 4 (tomato), Cuc m 3 (melon), Rub i 1 (raspberry), and Fra a 1 (strawberry). These allergens can also be considered Fagales-related allergens.

In one embodiment, the antibody or antigen binding fragment thereof cross-reacts with one or more allergens selected from the group consisting of Aln g1, Mal d1, Api g1, Car b1 and Cor a1.

In one embodiment, the isolated human antibody or antigen binding fragment thereof comprises three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within any of the heavy chain variable region (HCVR) sequences selected from the group consisting of SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 282 and 290; and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within any of the light chain variable region (LCVR) sequences selected from the group consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106 , 122, 138, 154, 170, 186, 202, 218, 234, 250, 266 and 298. Methods and techniques for identifying CDRs within the HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified amino acid sequences of HCVR and/or LCVR disclosed herein. Illustrative conventions that may be used to identify the boundaries of the CDR include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a combination of the Kabat and Chothia approaches. See, for example, Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., (1997), J. Mol. Biol. 273:927-948; and Martin et al., (1989), Proc. Natl. Acad. Sci. USA 56:9268–9272. Public databases are also available to identify CDR sequences within an antibody.

In one embodiment, the isolated human antibody or antigen binding fragment thereof comprises three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within any of the heavy chain variable region (HCVR) sequences selected from the group consisting of SEQ ID NO: 114, 146, 98 and 290; and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within any of the light chain variable region (LCVR) sequences selected from the group consisting of SEQ ID NOs: 122, 154, 106 and 298.

In one embodiment, the isolated human antibody or antigen binding fragment thereof comprises an HCVR with an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178 , 194, 210, 226, 242, 258, 274, 282 and 290.

In one embodiment, the isolated human antibody or antigen binding fragment thereof comprises an HCVR with an amino acid sequence selected from the group consisting of SEQ ID NOs: 114, 146, 98 and 290.

In one embodiment, the isolated human antibody or antigen binding fragment thereof comprises an LCVR with an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186 , 202, 218, 234, 250, 266 and 298.

In one embodiment, the isolated human antibody or antigen binding fragment thereof comprises an LCVR with an amino acid sequence selected from the group consisting of SEQ ID NOs: 122, 154, 106, and 298.

In one embodiment, the isolated human antibody or antigen binding fragment thereof comprises: (a) an HCVR with an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146 , 162, 178, 194, 210, 226, 242, 258, 274, 282 and 290; and (b) LCVR with an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250 , 266 and 298.

In one embodiment, an isolated human antibody or antigen binding fragment thereof comprises: (a) an HCVR with an amino acid sequence selected from the group consisting of SEQ ID NOs: 114, 146, 98 and 290; and (b) LCVR with an amino acid sequence selected from the group consisting of SEQ ID NOs: 122, 154, 106 and 298.

In one embodiment, the isolated human antibody or antigen binding fragment thereof comprises the following:

(a) an HCDR1 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244 , 260, 276, 284 and 292;

(b) an HCDR2 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246 , 262, 278, 286 and 294;

(c) an HCDR3 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248 , 264, 280, 288 and 296;

(d) an LCDR1 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252 , 268 and 300;

(e) an LCDR2 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 254 , 270 and 302; and (f) an LCDR3 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272 and 304.

In one embodiment, the isolated human antibody or antigen binding fragment thereof comprises a pair of amino acid sequences HCVR/LCVR selected from the group consisting of SEQ ID NO: 2/10, 18/26, 34/42, 50/58, 66/74, 82 /90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/266 , 282/266 and 290/298.

In one embodiment, the isolated human antibody or antigen binding fragment thereof comprises the amino acid sequence pair HCVR/LCVR selected from the group consisting of SEQ ID NOs: 114/122, 146/154, 98/106 and 290/298.

In one embodiment, an isolated human antibody or antigen binding fragment thereof that binds Bet v 1 comprises an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 146/154 and 290/298.

In one embodiment, an isolated human antibody or antigen binding fragment thereof that binds Bet v 1 reacts with at least one amino acid sequence selected from the group consisting of amino acid residues ranging from about position 23 to about position 44 according to SEQ ID NO: 306; amino acid residues ranging from approximately position 44 to - 3 045437

NO: 306; amino acid residues ranging from about position 44 to about 70 according to SEQ ID NO: 306; amino acid residues ranging from about 2 to about 19 according to SEQ ID NO: 306; amino acid residues ranging from about 57 to about 70 according to SEQ ID NO: 306; and amino acid residues ranging from about 81 to about 96 according to SEQ ID NO: 306.

In one embodiment, an isolated human antibody or antigen binding fragment thereof that binds Bet v 1 reacts with amino acid residues ranging from about position 23 to about position 44 according to SEQ ID NO: 306.

In one embodiment, an isolated human antibody or antigen binding fragment thereof that binds Bet v 1 reacts with amino acid residues ranging from about position 23 to about position 43 according to SEQ ID NO: 306.

In one embodiment, an isolated human antibody or antigen binding fragment thereof that binds Bet v 1 reacts with amino acid residues ranging from about position 44 to about position 70 according to SEQ ID NO: 306.

In one embodiment, an isolated human antibody or antigen binding fragment thereof that binds Bet v 1 reacts with amino acid residues ranging from about position 44 to about position 56 according to SEQ ID NO: 306.

In one embodiment, an isolated human antibody or antigen binding fragment thereof that binds Bet v 1 reacts with amino acid residues ranging from about position 2 to about position 19 according to SEQ ID NO: 306.

In one embodiment, an isolated human antibody or antigen binding fragment thereof that binds Bet v 1 reacts with amino acid residues ranging from about position 57 to about position 70 according to SEQ ID NO: 306.

In one embodiment, an isolated human antibody or antigen binding fragment thereof that binds Bet v 1 reacts with amino acid residues ranging from about position 57 to about position 66 according to SEQ ID NO: 306.

In one embodiment, an isolated human antibody or antigen binding fragment thereof that binds Bet v 1 reacts with amino acid residues ranging from about position 81 to about position 96 according to SEQ ID NO: 306.

In one embodiment, an isolated human antibody or antigen binding fragment thereof that binds Bet v 1 reacts with amino acid residues ranging from about position 81 to about position 89 according to SEQ ID NO: 306.

In one embodiment, an isolated human antibody or antigen binding fragment thereof that binds Bet v 1 reacts with at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 307, 308, 309, 310 and 311. Epitopes, containing SEQ ID NO: 307, 308, 309, 310 or 311, can be extended by 1-5 amino acids or 5-10 amino acids, either at the C-terminus or at the N-terminus. For example, the epitope of SEQ ID NO: 311, when extended by 5-10 amino acids, encompasses the epitope of SEQ ID NO: 115. In other words, the epitope containing SEQ ID NO: 311, for example, includes the epitope of SEQ ID NO: 315.

In one embodiment, an isolated human antibody or antigen binding fragment thereof that binds Bet v 1 is reacted with SEQ ID NO: 307.

In one embodiment, an isolated human antibody or antigen binding fragment thereof that binds Bet v 1 is reacted with SEQ ID NO: 308.

In one embodiment, an isolated human antibody or antigen binding fragment thereof that binds Bet v 1 is reacted with SEQ ID NO: 309.

In one embodiment, an isolated human antibody or antigen binding fragment thereof that binds Bet v 1 is reacted with SEQ ID NO: 310.

In one embodiment, an isolated human antibody or antigen binding fragment thereof that binds Bet v 1 is reacted with SEQ ID NO: 311.

In one embodiment, an isolated human antibody or antigen binding fragment thereof that binds Bet v 1 is reacted with SEQ ID NO: 315.

In one embodiment, an isolated human antibody or antigen-binding fragment thereof that binds Bet v 1 reacts with at least one amino acid sequence selected from the group consisting of SEQ ID NO: 307, 308, 309, 310, 311 and 315, and contain an HCVR/LCVR sequence pair selected from the group consisting of SEQ ID

NO: 114/122, 146/154, 98/106 and 290/298.

In one embodiment, an isolated human antibody or antigen binding fragment thereof that reacts with SEQ ID NO: 307 comprises three HCDRs contained in the heavy chain variable region of SEQ ID NO: 146 and three LCDRs contained in the light chain variable region of SEQ ID NO: 154.

In one embodiment, an isolated human antibody or antigen binding fragment thereof that reacts with SEQ ID NO: 310 comprises three HCDRs contained in the heavy chain variable region of SEQ ID NO: 290 and three LCDRs contained in the light chain variable region of SEQ ID NO: 298.

In one embodiment, a human antibody or antigen binding fragment thereof that binds Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 148, 150 and 152, respectively, and the amino acid sequences LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 156, 158 and 160 respectively.

In one embodiment, a human antibody or antigen binding fragment thereof that binds Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 292, 294 and 296, respectively, and the amino acid sequences LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 300, 302 and 304 respectively.

In one embodiment, a human antibody or antigen binding fragment thereof that binds Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 4, 6 and 8, respectively, and the amino acid sequences LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 12, 14 and 16 respectively.

In one embodiment, the human antibody or antigen binding fragment thereof that binds Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 20, 22 and 24, respectively, and the amino acid sequences LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 28, 30 and 32 respectively.

In one embodiment, a human antibody or antigen binding fragment thereof that binds Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 36, 38 and 40, respectively, and the amino acid sequences LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 44, 46 and 48 respectively.

In one embodiment, a human antibody or antigen binding fragment thereof that binds Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 52, 54 and 56, respectively, and the amino acid sequences LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 60, 62 and 64 respectively.

In one embodiment, the human antibody or antigen binding fragment thereof that binds Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 68, 70 and 72, respectively, and the amino acid sequences LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 76, 78 and 80 respectively.

In one embodiment, a human antibody or antigen binding fragment thereof that binds Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 84, 86 and 88, respectively, and the amino acid sequences LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 92, 94 and 96 respectively.

In one embodiment, a human antibody or antigen binding fragment thereof that binds Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 100, 102 and 104, respectively, and the amino acid sequences LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 108, 110 and 112 respectively.

In one embodiment, a human antibody or antigen binding fragment thereof that binds Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 116, 118 and 120, respectively, and the amino acid sequences LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 124, 126 and 128 respectively.

In one embodiment, the human antibody or antigen binding fragment thereof that binds Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 132, 134 and 136, respectively, and the amino acid sequences LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 140, 142 and 144 respectively.

In one embodiment, a human antibody or antigen binding fragment thereof that binds Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 164, 166 and 168, respectively, and the amino acid sequences LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 172, 174 and 176 respectively.

In one embodiment, a human antibody or antigen binding fragment thereof that binds Bet v 1 comprises the amino acid sequences HCDR1, HCDR2, and

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HCDR3 according to SEQ ID NO: 180, 182 and 184 respectively and amino acid sequences

LCDR1, LCDR2 and LCDR3 according to SEQ ID NO: 188, 190 and 192, respectively.

In one embodiment, a human antibody or antigen binding fragment thereof that binds Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 196, 198 and 200, respectively, and the amino acid sequences LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 204, 206 and 208 respectively.

In one embodiment, a human antibody or antigen binding fragment thereof that binds Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 212, 214 and 216, respectively, and the amino acid sequences LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 220, 222 and 224 respectively.

In one embodiment, a human antibody or antigen binding fragment thereof that binds Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 228, 230 and 232, respectively, and the amino acid sequences LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 236, 238 and 234 respectively.

In one embodiment, a human antibody or antigen binding fragment thereof that binds Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 244, 246 and 248, respectively, and the amino acid sequences LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 252, 254 and 256 respectively.

In one embodiment, a human antibody or antigen binding fragment thereof that binds Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 260, 262 and 264, respectively, and the amino acid sequences LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 268, 270 and 272 respectively.

In one embodiment, a human antibody or antigen binding fragment thereof that binds Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 276, 278 and 280, respectively, and the amino acid sequences LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 268, 270 and 272 respectively.

In one embodiment, a human antibody or antigen binding fragment thereof that binds Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 284, 286 and 288, respectively, and the amino acid sequences LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 268, 270 and 272 respectively.

In one embodiment, the present invention provides a fully human monoclonal antibody or antigen binding fragment thereof that binds to native Bet v 1, Betula pendula BPE, Betula nigra BPE or Betula populifolia BPE, wherein the antibody or fragment thereof exhibits one or more of the following characteristics: (i) contains HCVR with an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242 , 258, 274, 282 and 290, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (ii) contains an LCVR with an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250 , 266 and 298, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (iii) contains an HCDR3 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264, 280, 288 and 296, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and an LCDR3 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272 and 304, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (iv) contains an HCDR1 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260, 276, 284 and 292, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; HCDR2 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246, 262, 278, 286 and 294, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; LCDR1 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268 and 300, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and an LCDR2 domain with an amino acid sequence selected from

- 6 045437 group consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270 and 302, or substantially a similar sequence that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (v) binds to Bet v 1 with a K _D equal to or less than 10 ^-8 and ranging from about 10 ^-8 to about 10'¹¹; (vi) demonstrates efficacy in at least one animal model of anaphylaxis or inflammation; or (vii) competes with the reference antibody for binding to natural Bet v 1, Betula pendula birch BPE, Betula nigra BPE or Betula populifolia BPE.

In one embodiment, the reference antibody may include, for example, antibodies with a combination of heavy chain and light chain amino acid sequence pairs selected from the group consisting of 2/10, 18/26, 34/42, 50/58, 66/74 , 82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274 /266, 282/266 and 290/298.

The present invention relates to antibodies with a modified glycosylation profile. In some applications, modification to remove undesired glycosylation sites, or for example, removal of the fucose moiety to enhance antibody-dependent cellular cytotoxicity (ADCC) function may be useful (see Shield et al., 2002, JBC 277: 26733). In other applications, modification of galactosylation can be carried out to modify complement dependent cytotoxicity (CDC).

In a second aspect, an isolated antibody or antigen-binding fragment thereof is provided that competes for specific binding to native Bet v 1, Betula pendula birch BPE, Betula nigra BPE or Betula populifolia BPE with the antibody or antigen-binding fragment containing complementarity determining regions (CDRs) of the severe variable region chain (HCVR), wherein HCVR is characterized by an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226 , 242, 258, 274, 282 and 290; and a light chain variable region (LCVR) CDR, wherein the LCVR is characterized by an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266 and 298.

In one embodiment, an isolated antibody or antigen-binding fragment thereof is provided that competes for specific binding to native Bet v 1, Betula pendula BPE, Betula nigra BPE, or Betula populifolia BPE with the antibody or antigen-binding fragment containing complementarity determining regions (CDRs) of the variable region heavy chain (HCVR), wherein HCVR is characterized by an amino acid sequence selected from the group consisting of SEQ ID NOs: 114, 146, 98 and 290; and a light chain variable region (LCVR) CDR, wherein the LCVR is characterized by an amino acid sequence selected from the group consisting of SEQ ID NOs: 122, 154, 106 and 298.

In a related embodiment, the present invention provides an isolated antibody or antigen binding fragment thereof that competes for specific binding to native Bet v 1, Betula pendula birch BPE, Betula nigra BPE or Betula populifolia BPE with an antibody or antigen binding fragment containing heavy and light chain CDRs contained within pairs of heavy and light chain sequences selected from the group consisting of SEQ ID NO: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114 /122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/266, 282/266 and 290/298 .

According to a third aspect, there is provided an isolated antibody or antigen binding fragment thereof that binds to the same epitope on Bet v 1 as the antibody or antigen binding fragment comprising heavy chain variable region (HCVR) complementarity determining regions (CDRs), wherein the HCVR is characterized by an amino acid sequence, selected from the group consisting of SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 282 and 290; and a light chain variable region (LCVR) CDR, wherein the LCVR is characterized by an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266 and 298.

In one embodiment, an isolated antibody or antigen binding fragment thereof is provided that binds to the same epitope on Bet v 1 as the antibody or antigen binding fragment comprising heavy chain variable region (HCVR) complementarity determining regions (CDRs), wherein the HCVR is characterized by an amino acid sequence , selected from the group consisting of SEQ ID NO: 114, 146, 98 and 290; and a light chain variable region (LCVR) CDR, wherein the LCVR is characterized by an amino acid sequence selected from the group consisting of SEQ ID NOs: 122, 154, 106 and 298.

In a related embodiment, provided herein is an isolated antibody or antigen binding fragment thereof that binds to the same epitope on Bet v 1 as the antibody or antigen binding fragment comprising heavy and light chain CDRs contained within pairs of heavy and light chain sequences, selected from the group consisting of SEQ ID NO: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186,

- 7 045437

194/202, 210/218, 226/234, 242/250, 258/266, 274/266, 282/266 and 290/298.

According to a fourth aspect, the present invention relates to nucleic acid molecules encoding antibodies to Bet v 1 or fragments thereof. Recombinant expression vectors carrying such nucleic acids and host cells into which such vectors have been introduced are also provided herein, as are methods for producing antibodies by culturing host cells under conditions that allow antibody production and isolating the resulting antibodies.

In one embodiment, provided herein are nucleic acid molecules encoding a human monoclonal antibody or fragment thereof that binds native Bet v 1, Betula pendula BPE, Betula nigra BPE, or Betula populifolia BPE.

In one embodiment, provided herein is an antibody or fragment thereof comprising an HCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, 17, 33, 49, 65, 81, 97, 113, 129, 145 , 161, 177, 193, 209, 225, 241, 257, 273, 281, and 289, or a substantially identical sequence having at least 90, at least 95, at least 98, or at least 99% homology in attitude towards them.

In one embodiment, HCVR is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 113, 145, 257, and 289.

In one embodiment, the antibody or fragment thereof further comprises an LCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, 25, 41, 57, 73, 89, 105, 121, 137, 153, 169, 185 , 201, 217, 233, 249, 265 and 297, or a substantially identical sequence having at least 90, at least 95, at least 98 or at least 99% homology thereto.

In one embodiment, the LCVR is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 121, 153, 265, and 297.

In one embodiment, provided herein is an antibody or antigen binding fragment of an antibody comprising an HCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 7, 23, 39, 55, 71, 87, 103, 119, 135, 151, 167, 183, 199, 215, 231, 247, 263, 279, 287 and 295, or a substantially similar sequence thereof which is characterized by at least 90, at least 95, at least 98 or at least 99% sequence identity; and an LCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 15, 31, 47, 63, 79, 95, 111, 127, 143, 159, 175, 191, 207, 223, 239, 255, 271 and 303, or a substantially similar sequence thereto that has at least 90, at least 95, at least 98, or at least 99% sequence identity.

In one embodiment, provided herein is an antibody or fragment thereof further comprising an HCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, 19, 35, 51, 67, 83, 99, 115, 131, 147, 163, 179, 195, 211, 227, 243, 259, 275, 283 and 291, or a substantially similar sequence thereof that is characterized by at least 90, at least 95, at least 98, or at least 99% sequence identity; HCDR2 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 5, 21, 37, 53, 69, 85, 101, 117, 133, 149, 165, 181, 197, 213, 229, 245, 261 , 277, 285 and 293, or a substantially similar sequence thereto that has at least 90, at least 95, at least 98, or at least 99% sequence identity; LCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 11, 27, 43, 59, 75, 91, 107, 123, 139, 155, 171, 187, 203, 219, 235, 251, 267 and 299, or a substantially similar sequence thereto that has at least 90, at least 95, at least 98, or at least 99% sequence identity; and an LCDR2 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 13, 29, 45, 61, 77, 93, 109, 125, 141, 157, 173, 189, 205, 221, 237, 253, 269 and 301, or a substantially similar sequence thereto that has at least 90, at least 95, at least 98, or at least 99% sequence identity.

According to a fifth aspect, there is provided a pharmaceutical composition comprising a therapeutically effective amount of one or more isolated human antibodies or antigen-binding fragments thereof that bind to natural Bet v 1, Betula pendula birch BPE, Betula nigra BPE or Betula populifolia BPE, together with one or more pharmaceutically acceptable excipients.

In one embodiment, the pharmaceutical composition contains a therapeutically effective amount of two or more isolated human antibodies or antigen binding fragments thereof that bind to Bet v 1, together with one or more pharmaceutically acceptable excipients.

According to one embodiment, the pharmaceutical composition contains the following:

- 8 045437

a) a first isolated human monoclonal antibody or antigen binding fragment thereof that binds Bet v 1, which contains HCVR with the amino acid sequence as set forth in SEQ ID NO: 146; and LCVR with amino acid sequence as presented in

SEQ ID NO: 154;

b) a second isolated human monoclonal antibody or antigen binding fragment thereof that binds Bet v 1, which contains an HCVR with an amino acid sequence selected from the group consisting of SEQ ID NOs: 114, 98 and 290; and LCVR with an amino acid sequence selected from the group consisting of SEQ ID NO: 122, 106 and 298; And

c) one or more pharmaceutically acceptable excipients.

According to one embodiment, the pharmaceutical composition contains the following:

a) a first isolated human monoclonal antibody or antigen binding fragment thereof that binds Bet v 1, which contains an HCVR with the amino acid sequence as set forth in SEQ ID NO: 290; and LCVR with the amino acid sequence as set forth in SEQ ID NO: 298;

b) a second isolated human monoclonal antibody or antigen binding fragment thereof that binds Bet v 1, which contains an HCVR with an amino acid sequence selected from the group consisting of SEQ ID NOs: 114, 146 and 98; and LCVR with an amino acid sequence selected from the group consisting of SEQ ID NO: 122, 154 and 106; And

c) one or more pharmaceutically acceptable excipients.

According to one embodiment, the pharmaceutical composition contains the following:

a) a first isolated human monoclonal antibody or antigen binding fragment thereof that binds Bet v 1, which contains HCVR with the amino acid sequence as set forth in SEQ ID NO: 146; and LCVR with the amino acid sequence as set forth in SEQ ID NO: 154;

b) a second isolated human monoclonal antibody or antigen binding fragment thereof that binds Bet v 1, which contains an HCVR with the amino acid sequence as set forth in SEQ ID NO: 290; and LCVR with the amino acid sequence as set forth in SEQ ID NO: 298; And

c) one or more pharmaceutically acceptable excipients.

According to one embodiment, the pharmaceutical composition contains the following:

a) a first isolated human monoclonal antibody or antigen-binding fragment thereof, comprising an HCVR with the amino acid sequence according to SEQ ID NO: 146, and an LCVR with the amino acid sequence according to SEQ ID NO: 154;

b) a second isolated human monoclonal antibody or an antigen binding fragment thereof, comprising an HCVR having the amino acid sequence of SEQ ID NO: 290, and an LCVR having the amino acid sequence of SEQ ID NO: 298;

c) one or more additional isolated human monoclonal antibodies or antigen binding fragments comprising an HCVR with an amino acid sequence selected from the group consisting of SEQ ID NOs: 114 and 98, and an LCVR with an amino acid sequence selected from the group consisting of SEQ ID NOs: 122 and 106; And

d) one or more pharmaceutically acceptable excipients.

According to one embodiment, the pharmaceutical composition contains the following:

a first isolated human monoclonal antibody or antigen binding fragment thereof that binds Bet v 1, comprising the amino acid sequence pair HCVR/LCVR consisting of SEQ ID NO: 146/154;

a second isolated human monoclonal antibody or antigen binding fragment thereof that binds Bet v 1, comprising a pair of amino acid sequences HCVR/LCVR selected from the group consisting of SEQ ID NOs: 114/122, 98/106 and 290/298; and one or more pharmaceutically acceptable excipients.

According to one embodiment, the pharmaceutical composition contains the following:

a first isolated human monoclonal antibody or antigen binding fragment thereof that binds Bet v 1, comprising the amino acid sequence pair HCVR/LCVR consisting of SEQ ID NO: 146/154;

a second isolated human monoclonal antibody or antigen binding fragment thereof that binds Bet v 1, comprising the amino acid sequence pair HCVR/LCVR consisting of SEQ ID NO: 290/298; and one or more pharmaceutically acceptable excipients.

In one embodiment, the pharmaceutical composition comprises two or more isolated human monoclonal antibodies or antigen binding fragments thereof that bind to Bet v 1 containing HCVR/LCVR amino acid sequence pairs selected from the group consisting of SEQ ID NO: 2/10, 18/ 26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/266, 282/266 and 290/298; and one

- 9 045437 or several pharmaceutically acceptable excipients.

In one embodiment, the pharmaceutical composition comprises three or more isolated human monoclonal antibodies or antigen binding fragments thereof that bind to Bet v 1 containing HCVR/LCVR amino acid sequence pairs selected from the group consisting of SEQ ID NO: 2/10, 18/ 26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/266, 282/266 and 290/298; and one or more pharmaceutically acceptable excipients.

In one embodiment, the pharmaceutical composition comprises four isolated human monoclonal antibodies that bind to Bet v 1, or antigen binding fragments thereof, wherein the human antibodies or antigen binding fragments thereof comprise HCVR/LCVR amino acid sequence pairs according to SEQ ID NO: 114/122, 146/ 154, 98/106 and 290/298; and one or more pharmaceutically acceptable excipients.

According to one embodiment, the pharmaceutical composition contains an antibody designated H4H16992P, characterized by the amino acid sequence pair HCVR/LCVR according to SEQ ID NO: 146/154; an antibody designated H4H17082P2, characterized by the amino acid sequence pair HCVR/LCVR according to SEQ ID NO: 290/298; and an antibody designated H4H17038P2, characterized by the amino acid sequence pair HCVR/LCVR according to SEQ ID NO: 98/106.

According to one embodiment, the pharmaceutical composition contains an antibody designated H4H16992P, characterized by the amino acid sequence pair HCVR/LCVR according to SEQ ID NO: 146/154; an antibody designated H4H17082P2, characterized by the amino acid sequence pair HCVR/LCVR according to SEQ ID NO: 290/298; an antibody designated H4H17038P2, characterized by the amino acid sequence pair HCVR/LCVR according to SEQ ID NO: 98/106; and an antibody designated H4H16987P, characterized by the amino acid sequence pair HCVR/LCVR according to SEQ ID NO: 114/122.

In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of a first isolated human monoclonal antibody or antigen binding fragment thereof that binds Bet v 1, wherein the first antibody or fragment thereof reacts with amino acid residues ranging from about position 23 to about position 44 according to SEQ ID NO: 306, and a second isolated human monoclonal antibody or antigen binding fragment thereof that binds Bet v 1, wherein the second antibody or fragment thereof reacts with amino acid residues ranging from about position 44 to about position 70 according to SEQ ID NO: : 306, together with one or more pharmaceutically acceptable excipients.

In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of a first isolated human monoclonal antibody or antigen-binding fragment thereof that binds Bet v 1, together with one or more pharmaceutically acceptable excipients, wherein the first antibody or fragment thereof reacts with amino acid residues located in range from about position 23 to about position 43 according to SEQ ID NO: 306, and a second isolated human monoclonal antibody or antigen binding fragment thereof that binds Bet v 1, wherein the second antibody or fragment thereof reacts with amino acid residues ranging from about position 44 to approximately position 56 according to SEQ ID NO: 306.

In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of a first isolated human monoclonal antibody or antigen binding fragment thereof that binds Bet v 1, together with one or more pharmaceutically acceptable excipients, wherein the first antibody or fragment thereof reacts with an amino acid sequence according to SEQ ID NO: 307, and a second isolated human monoclonal antibody or antigen binding fragment thereof that binds Bet v 1, wherein the second antibody or fragment thereof reacts with the amino acid sequence of SEQ ID NO: 308, 309, 310, 311 or 315.

In one embodiment, the pharmaceutical composition contains a therapeutically effective amount of a first isolated human monoclonal antibody or antigen binding fragment thereof that binds Bet v 1, and one or more additional isolated human monoclonal antibodies or antigen binding fragments thereof that bind Bet v 1, together with one or more pharmaceutically acceptable excipients, wherein the first antibody or fragment thereof reacts with at least one amino acid sequence selected from the group consisting of amino acid residues ranging from about position 23 to about position 43 according to SEQ ID NO: 306; amino acid residues ranging from about position 44 to about 56 according to SEQ ID NO: 306; amino acid residues ranging from about 2 to about 19 according to SEQ ID NO: 306; amino acid residues ranging from about 57 to about 70 according to SEQ ID NO: 306; and amino acid residues ranging from about 81 to about 89 or about 81 to about 96 according to SEQ ID NO: 306.

In one embodiment, one or more additional isolated human monoclonal antibodies or fragments thereof react with at least one amino acid sequence selected from the group consisting of amino acid residues ranging from about position 23 to about position 43 according to SEQ ID NO: 306 ; amino acid residues ranging from about position 44 to about 56 according to SEQ ID NO: 306; amino acid residues ranging from about 2 to about 19 according to SEQ ID NO: 306; amino acid residues ranging from about 57 to about 70 according to SEQ ID NO: 306; and amino acid residues ranging from about 81 to about 89 or about 81 to about 96 according to SEQ ID NO: 306, wherein at least one of the one or more additional isolated human monoclonal antibodies reacts with a different amino acid sequence than the first isolated monoclonal antibody person.

In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of a first isolated human monoclonal antibody or antigen binding fragment thereof that binds Bet v 1, and one or more additional isolated human monoclonal antibodies or antigen binding fragments thereof that bind Bet v 1, together with one or more pharmaceutically acceptable excipients, wherein the first antibody or fragment thereof is reacted with an amino acid sequence according to SEQ ID NO: 307, and wherein one or more additional antibodies or fragments thereof are reacted with an amino acid sequence selected from the group consisting of SEQ ID NO: 308, 309, 310, 311 and 315.

In one embodiment, the pharmaceutical composition contains a therapeutically effective amount of at least two isolated human monoclonal antibodies or antigen-binding fragments thereof that bind to naturally occurring Bet v 1 or BPE, together with one or more pharmaceutically acceptable excipients, wherein the at least two antibodies do not compete for binding with natural Bet v 1 or BPE. In some aspects, the antibodies or antigen binding fragments thereof are antibodies to Bet v 1, H4H16992P and H4H17082P2 comprising the HCVR/LCVR amino acid sequence pairs of SEQ ID NO: 146/154 and 290/298, respectively.

In one embodiment, the pharmaceutical composition contains a therapeutically effective amount of at least three isolated human monoclonal antibodies or antigen-binding fragments thereof that bind to naturally occurring Bet v 1 or BPE, together with one or more pharmaceutically acceptable excipients, wherein the at least three antibodies are not compete for binding with natural Bet v 1 or BPE. In some aspects, the antibodies or antigen binding fragments thereof are antibodies to Bet v 1, H4H16992P, H4H17082P2 and H4H17038P2 comprising the HCVR/LCVR amino acid sequence pairs of SEQ ID NO: 146/154, 290/298 and 98/106, respectively.

In one embodiment, the pharmaceutical composition contains a therapeutically effective amount of at least four isolated human monoclonal antibodies or antigen-binding fragments thereof that bind to naturally occurring Bet v 1 or BPE, together with one or more pharmaceutically acceptable excipients, wherein the at least four antibodies are not compete for binding with natural Bet v 1 or BPE. In some aspects, the antibodies or antigen binding fragments thereof are antibodies to Bet v 1, H4H16992P, H4H17082P2, H4H17038P2, and H4H16987P, comprising the HCVR/LCVR amino acid sequence pairs of SEQ ID NOs: 146/154, 290/298, 98/106, and 114 /122 respectively.

In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of an isolated human monoclonal antibody or antigen-binding fragment thereof that binds Bet v 1, together with one or more pharmaceutically acceptable excipients, wherein the antibody or antigen-binding fragment thereof cross-reacts with one or more allergens selected from the group consisting of Aln g1, Cor a1, Car b1, Que a1, Api g2, Api g1, Dau c1, Mal d1, Ost c1, Fag s1 and Cas s1. In some embodiments, the antibody or antigen-binding fragment thereof cross-reacts with one or more allergens selected from the group consisting of Aln g1, Mal d1, Api g1, Car b1, and Cor a1.

In one embodiment, the present invention provides a composition that is a combination of a therapeutically effective amount of one or more anti-Bet v 1 antibodies or antigen binding fragments thereof according to the present invention and a therapeutically effective amount of a second therapeutic agent together with one or more pharmaceutically acceptable excipients.

- 11 045437

The second therapeutic agent may be a small molecule drug, a protein/polypeptide, an antibody, a nucleic acid molecule such as an antisense molecule, or a siRNA. The second therapeutic agent may be synthetic or derived from a natural source.

The second therapeutic agent may be any agent that is effectively combined with an antibody or fragment thereof of the present invention, for example, a second antibody other than those described herein that is capable of blocking the binding of Bet v 1 to IgE present on mast cells or basophils. The second therapeutic agent may also be any agent that is used as a standard of care for the treatment of an allergic response to any allergen. Such second therapeutic agent may be an antihistamine, epinephrine, decongestant, corticosteroid, or peptide vaccine.

In certain embodiments, the second therapeutic agent may be an agent that helps neutralize or reduce any potential side effect(s) associated with the antibody or antigen binding fragment of an antibody of the present invention, if any. side effect(s) should occur.

It should also be understood that the antibodies and pharmaceutically acceptable compositions of the present invention can be used in combination therapies, i.e. The antibodies and pharmaceutically acceptable compositions can be administered simultaneously, before or after one or more other desired therapeutic agents or medical procedures, including, for example, in combination with an allergen-specific immunotherapy (SIT) regimen where the antibodies are administered before or during SIT. The specific combination of treatments (therapeutic agents or procedures) to be used in a combination regimen will take into account the compatibility of the required therapeutic agents and/or procedures and the desired therapeutic effect to be achieved. It should also be understood that the therapies used may achieve the desired effect for the same disorder (e.g., the antibody may be administered concomitantly with another agent used to treat the same disorder), or they may achieve different effects (e.g., control of any adverse effects). . As used herein, additional therapeutic agents that are typically administered to treat or prevent a specific disease or condition are appropriate for the disease or condition being treated.

When multiple therapeutic agents are coadministered, dosages can be adjusted accordingly, as established in the art.

According to a sixth aspect, there is provided a method of treating a patient who exhibits a sensitivity or allergic response to Fagales protein, a Fagales-related allergen, birch pollen or an extract thereof, or Bet v 1 protein, or treating at least one symptom or complication associated with the sensitivity or allergic response. a response to Fagales protein, Fagales-related allergen, birch pollen or an extract thereof, or Bet v 1 protein, involving the administration of an effective amount of one or more isolated human monoclonal antibodies or antigen-binding fragments thereof that bind to natural Bet v 1, BPE Betula pendula, BPE Betula nigra or BPE Betula populifolia, or a pharmaceutical composition containing an effective amount of one or more isolated human monoclonal antibodies or fragments thereof that bind to natural Bet v 1, BPE Betula pendula, BPE Betula nigra or BPE Betula populifolia, to a patient in need thereof wherein sensitivity or allergic response to Fagales protein, Fagales-related allergen, birch pollen or an extract thereof, or Bet v 1 protein is either prevented or reduced in severity and/or duration, or at least one symptom or complication associated with sensitivity or allergic response to Fagales protein, Fagales-related allergen, birch pollen or extract thereof, or Bet v 1 protein is prevented or reduced in intensity, or the frequency and/or duration or severity of sensitivity or allergic response to Fagales protein, related Fagales, birch pollen or its extract or protein Bet v 1 is reduced after administration of one or more isolated human monoclonal antibodies or fragments thereof that bind to natural Bet v 1, BPE Betula pendula, BPE Betula nigra or BPE Betula populifolia, or after administration a composition containing any one or more of the above antibodies.

In some embodiments, the birch pollen extract is selected from the group consisting of natural Bet v 1, BPE Betula pendula, BPE Betula nigra, and BPE Betula populifolia.

In some embodiments, the treatment results in a reduction in allergic rhinitis, allergic conjunctivitis, allergic bronchial asthma, or anaphylactic reaction following exposure of the patient to Fagales protein, a Fagales-related allergen, birch pollen or an extract thereof, or Bet v 1 protein.

In some embodiments, the method further comprises administering an effective amount of a second therapeutic agent useful for attenuating the allergic response to Fagales protein, Fagales-related allergen, birch pollen or extract thereof, or Bet v 1 protein. The second therapeutic agent may be selected from the group consisting of a corticosteroid,

- 12 045437 bronchodilator, antihistamine, epinephrine, decongestant, other different Bet v 1 antibody and peptide vaccine.

In some embodiments, the method further comprises treating the patient with an allergen-specific immunotherapy (SIT) regimen immediately following or concurrently with the antibodies or fragments thereof or a pharmaceutical composition containing the antibodies.

In one embodiment, the present invention provides a method of treating a patient with a Fagales allergy who exhibits a sensitivity or allergic response to one or more Fagales allergens or related Fagales allergens, or treating at least one symptom or complication associated with the sensitivity or allergic response. a response to one or more Fagales allergens or related Fagales allergens, comprising the administration of an effective amount of one or more isolated human monoclonal antibodies or antigen binding fragments thereof that bind to Bet v 1, or a pharmaceutical composition containing an effective amount of one or more isolated human monoclonal antibodies human or fragments thereof that bind to Bet v 1, to a patient in need thereof, wherein sensitivity or allergic response to the Fagales or Fagales-related allergen is either prevented or reduced in severity and/or duration, or at least one symptom or a complication associated with a sensitivity or allergic response to the Fagales or Fagales-related allergen is prevented or reduced in intensity, or the frequency and/or duration or severity of the sensitivity or allergic response to the Fagales or Fagales-related allergen is reduced after administration one or more isolated human monoclonal antibodies or fragments thereof that bind to Bet v 1, or after administration of a composition containing any one or more of the above antibodies.

In some embodiments, the one or more Fagales allergens are selected from the group consisting of Bet v 1, Aln g1, Cor a1, Car b1, and Que a1.

In one embodiment, the present invention provides a pharmaceutical composition comprising one or more antibodies described herein that bind to natural Bet v 1, BPE Betula pendula, BPE Betula nigra and BPE Betula populifolia, for use in treating a patient who exhibits sensitivity or allergic response to Fagales protein, birch pollen or extract thereof, or Bet v 1 protein, or for the treatment of at least one symptom or complication associated with sensitivity or allergic response to Fagales protein, birch pollen or extract thereof, or Bet v 1 protein, wherein sensitivity or allergic response to Fagales protein, birch pollen or an extract thereof, or Bet v 1 protein is either prevented or reduced in severity and/or duration, or at least one symptom or complication associated with sensitivity or allergic response to Fagales protein, birch pollen or extract thereof, or Bet v 1 protein, is prevented or reduced in intensity, or the frequency and/or duration or severity of sensitivity or allergic response to Fagales protein, birch pollen or its extract or protein Bet v 1 decreases.

In one embodiment, the present invention relates to the use of a pharmaceutical composition containing one or more antibodies of the present invention that bind to Bet v 1 in the preparation of a medicament for use in treating a patient who exhibits a sensitivity or allergic response to birch pollen or an extract thereof or to the Bet v 1 protein, or for the treatment of at least one symptom or complication associated with a sensitivity or allergic response to birch pollen or an extract thereof, or to the Bet v 1 protein, wherein the sensitivity or allergic response to birch pollen or extract thereof or protein Bet v 1 is either prevented or reduced in severity and/or duration, or at least one symptom or complication associated with sensitivity or allergic response to birch pollen or extract thereof or protein Bet v 1 are prevented or reduced in intensity, or the frequency and/or duration or severity of sensitivity or allergic response to birch pollen or extract thereof or to Bet v 1 protein is reduced.

In one embodiment, the present invention relates to the use of a pharmaceutical composition as described above, wherein the composition is administered in combination with a second therapeutic agent useful for reducing an allergic response to Fagales protein, birch pollen or extract thereof, or Bet v 1 protein. In one embodiment In an embodiment, the present invention relates to the use of a pharmaceutical composition as described above, wherein the second therapeutic agent is selected from a corticosteroid, a bronchodilator, an antihistamine, epinephrine, a decongestant, another different anti-Bet v 1 antibody, and a peptide vaccine.

In certain embodiments, antibodies of the present invention that bind to Bet v 1 may be capable of reducing, minimizing, or preventing

- 13 045437 at least one symptom in a patient sensitive to birch pollen or Bet v 1, such as sneezing, congestion, nasal congestion, cough, shortness of breath, bronchospasm, rhinitis or conjunctivitis.

In one embodiment, antibodies of the present invention that bind to Bet v 1, or a composition containing one or more antibodies of the present invention, can be used to prevent more serious in vivo complications associated with Bet v 1 allergy, including asthmatic reactions, anaphylactic shock, or even death due to anaphylaxis.

In one embodiment, the pharmaceutical composition is administered to a patient in combination with a second therapeutic agent.

In another embodiment, the second therapeutic agent is selected from the group consisting of an antihistamine, epinephrine, a decongestant, a corticosteroid, another different anti-Bet v 1 antibody, a peptide vaccine, and any other palliative therapy useful to reduce the severity of the allergic response or to reduce the intensity at least one symptom associated with an allergic response.

In another embodiment, the pharmaceutical composition is administered simultaneously with, before, or after one or more other desired therapeutic agents or medical procedures, including, for example, administration before or simultaneously with an allergen-specific immunotherapy (SIT) regimen. In some aspects, use of a SIT regimen in conjunction with antibodies provided herein provides a synergistic effect.

In one embodiment, there is provided a method of enhancing the effectiveness and/or safety of an allergen-specific immunotherapy (SIT) regimen, the method comprising administering an effective amount of one or more isolated human monoclonal antibodies or antigen-binding fragments thereof, as provided herein, or a pharmaceutical composition comprising an effective amount of one or more isolated human monoclonal antibodies or fragments thereof, to a patient in need thereof, immediately prior to or concurrently with the SIT regimen, wherein the severity of the allergic response to the SIT regimen is reduced. In some embodiments, the SIT regimen comprises an escalating dose phase followed by a maintenance phase. In some embodiments, the SIT scheme is a rush SIT scheme.

In a further aspect, there is provided a method of preventing or reducing mast cell degranulation associated with Fagales protein, a Fagales-related allergen, birch pollen or birch pollen extract, or sensitization with Bet v 1, the method comprising administering a pharmaceutical composition described herein to a person in need thereof. to the patient.

In some embodiments, the BPE is selected from the group consisting of natural Bet v 1, Betula pendula BPE, Betula nigra BPE, and Betula populifolia BPE.

Other embodiments will become apparent from a review of the following detailed description.

Brief description of the figures

In fig. Figure 1 shows epitope mapping based on H-D/MS exchange for the interaction of H4H16992P with Bet v 1.

In fig. Figure 2 shows epitope mapping based on H-D/MS exchange for the interaction of H4H17082P with Bet v 1.

In fig. Figure 3 shows epitope mapping based on H-D/MS exchange for the interaction of H4H17038P2 with Bet v 1.

In fig. Figure 4 shows epitope mapping based on H-D/MS exchange for the interaction of H4H16987P with Bet v 1.

In fig. Figure 5 shows epitope mapping based on H-D/MS exchange for the interaction of H4H16992P, H4H17082P, H4H17038P2 and H4H16987P with Bet v 1.

In fig. 6 is a diagram of the protocol used to determine the effectiveness of antibody combinations in blocking Bet v 1-induced mast cell degranulation in a humanized mouse model of PCA.

In fig. 7 shows the ability of a combination of anti-Bet v 1 antibodies to block mast cell degranulation in a humanized mouse model of RSA using IgE-containing sera obtained from three Bet v 1-sensitive donors.

In fig. 8 shows two graphs, the first showing representative data showing combinations of antibodies to Bet v 1 that block basophil activation in PBMCs obtained from a single birch pollen allergic donor. The histogram presents data showing the percentage of blocking of basophil activation in PBMCs obtained from several donors using combinations of antibodies to Bet v 1, in relation to each antibody separately.

Detailed Disclosure of the Present Invention

Before the present compositions and methods are described, it should be understood that the present

- 14 045437 the invention is not limited to the specific compositions and methods described, as well as experimental conditions, since such methods, compositions and conditions may vary. It should also be understood that the terminology used herein is intended to describe specific embodiments only and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention relates. As used herein, the term approximate, when used in relation to a specific numerical value given, means that the value may differ from the given value by no more than 1%. For example, as used herein, the expression approximately 100 includes 99 and 101 and all values in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, preferred methods and materials are described below.

Definitions

As used herein, the term Bet v 1 refers to at least one Bet v 1 protein, either in natural/native form or produced recombinantly. The Bet v 1 protein contains the amino acid sequence of SEQ ID NO: 306 and the nucleic acid sequence of SEQ ID NO: 305. The native Bet v 1 protein is approximately 17 kDa in size and exists as a 7-stranded antiparallel β-sheet (β1 -β7), two short α-helices (α1 and α2) connecting β1 and β2, a long C-terminal α-helix (α3), and a glycine-rich loop motif between β2 and β3 (Kofler et al. (2012) J. Mol Biol 422(1): 109-123). The recombinantly produced mutant Bet v 1, SEQ ID NO: 312, contains G2-N160 according to Uniprot P15494 with an S85A substitution and contains a Myc-Myc-hexahistidine tag. Amino acid sequence of Bet v 1 from Uniprot: P15494, i.e. SEQ ID NO: 314, may also refer to Bet v 1.

Bet v 1 is a polypeptide containing, or alternatively consisting of, the amino acid sequence of SEQ ID NO: 306 or SEQ ID NO: 314 or a sequence homologous thereto. As used herein, the phrase sequence homologous to SEQ ID NO: 306 or SEQ ID NO: 314 refers to a polypeptide that has an identity to SEQ ID NO: 306 or SEQ ID NO: 314 that is greater than 70, preferably greater than 80, more preferably greater than 90 and even more preferably greater than 95%.

As used herein, the term Bet v 1 fragment refers to a polypeptide containing, or alternatively consisting of, at least one Bet v 1 antigenic site. In one embodiment, the term Bet v 1 fragment, as used herein, refers to a polypeptide containing or, alternatively, consisting of at least two Bet v 1 antigenic sites. In one embodiment, the antigenic sites are linked covalently. In one embodiment, the antigenic sites are linked by at least one peptide bond. In one embodiment, two antigenic sites are linked by at least one peptide bond and a spacer between the antigenic sites. In one embodiment, the at least two antigenic sites comprise amino acid sequences 23-44 and 44-56 according to Uniprot P15494. In one embodiment, at least two antigenic sites comprise an amino acid sequence within any of SEQ ID NOs: 306, 307, 308, 309, 310, 311, and 315. In one embodiment, any of the Bet v 1 fragments is capable of inducing antibody production in vivo that specifically bind to naturally occurring Bet v 1 or recombinantly produced Bet v 1.

As used herein, the term antibody means any antigen-binding molecule or molecular complex containing at least one complementarity determining region (CDR) that specifically binds or interacts with a particular antigen (eg, Bet v 1). The term antibody, as used herein, is intended to include immunoglobulin molecules consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains linked together by disulfide bonds (i.e., complete antibody molecules), and, as well as their multimers (eg, IgM) or their antigen-binding fragments. Each heavy chain consists of a heavy chain variable region (HCVR or VH) and a heavy chain constant region (consisting of CH1, _CH2 and CH3 domains). Each light chain consists 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 hypervariable regions called complementarity determining regions (CDRs), which alternate with regions that are more conserved, which are called framework regions (FR).Each VH and _VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1,

- 15 045437

CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present invention, the FR antibody (or antigen binding fragment thereof) may be identical to human germline sequences or may be naturally or synthetically modified. The amino acid consensus sequence can be determined based on parallel analysis of two or more CDRs.

In addition, it is possible to replace one or more CDR residues or omit one or more CDRs. The scientific literature describes antibodies that can dispense with one or two CDRs to bind. Padlan et al. (1995 FASEB J. 9: 133-139) analyzed the contact regions between antibodies and their antigens based on published crystal structures and concluded that only approximately one-fifth to one-third of the CDR residues actually contact the antigen. Padlan has also found many antibodies in which one or two CDRs do not have antigen-contacting amino acids (see also Vajdos et al. 2002 J Mol Biol 320:415-428).

Non-antigen contact CDR residues can be identified based on previous studies (eg, residues H60-H65 in CDRH2 are often not required) from Kabat CDR regions outside the Chothia CDR, by molecular modeling and/or empirically. If a CDR or residue(s) thereof is omitted, it is typically replaced with an amino acid occupying the corresponding position in another human antibody sequence or consensus of such sequences. The positions to be replaced within the CDR and the amino acid to be replaced can also be selected empirically. Empirical substitutions may be conservative or non-conservative substitutions.

Fully human monoclonal antibodies that specifically bind to Bet v 1, as disclosed herein, may contain one or more amino acid substitutions, insertions and/or deletions in the framework regions and/or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from publicly available antibody sequence databases. The present invention provides antibodies and antigen binding fragments thereof that are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework regions and/or CDR regions are mutated to the corresponding residue(s) the germline from which the antibody is 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 collectively referred to herein as germline mutations). Starting with the heavy and light chain variable region sequences disclosed herein, one of ordinary skill in the art can readily produce various antibodies and antigen binding fragments that contain one or more individual germline mutations or combinations thereof. In certain embodiments, all framework and/or CDR region residues within the V _H and/or V _L domains are backmutated to residues found in the original germline sequence from which the antibody is derived. In other embodiments, only certain residues are backmutated to the original germline sequence, e.g., only mutated residues occurring within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only mutated residues occurring within CDR1, CDR2, or CDR3 . In other embodiments, one or more residues of the framework region and/or CDR region are mutated to the corresponding residue(s) of another germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody initially).

Moreover, antibodies of the present invention may contain any combination of two or more germline mutations within framework regions and/or CDR regions, for example, wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence, and other certain residues that differ from the original germline sequence and are retained or mutated to the corresponding residue of the divergent germline sequence. Once produced, antibodies and antigen binding fragments that contain one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (depending on the specific situations), reduced immunogenicity, etc. Antibodies and antigen binding fragments produced by this general method are provided by the present invention.

The present invention also provides fully human monoclonal antibodies containing variants of any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the present invention provides antibodies with amino acid sequences HCVR, LCVR and/or CDR, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc. conservative amino acid substitutions with respect to any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein.

As used herein, the term human antibody includes non-naturally occurring human antibodies. The term includes antibodies that are recombinantly produced in the body of a non-human mammal or in the cells of a non-human mammal. The term is intended to include antibodies isolated from or produced in the body of a human subject.

In some embodiments, the antibodies of the present invention may be recombinant and/or non-naturally occurring human antibodies. As used herein, the term recombinant human antibody is intended to include all human antibodies that are produced, expressed, generated, or isolated by recombinant methods, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further). below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (for example, a mouse) that is transgenic for human immunoglobulin genes (see, for example, Taylor et al. ( 1992) Nucl. Acids Res. 20:6287-6295) or antibodies produced, expressed, created or isolated by any other means that involve splicing human immunoglobulin gene sequences with other DNA sequences. In certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, if an animal that is transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V _H and V _L regions of the recombinant antibodies are sequences which, while related to human germline _VH and _VL sequences, may not naturally exist within the human germline antibody repertoire in vivo.

Human antibodies can exist in two forms, which are related to the heterogeneity of the hinge. In one form, the immunoglobulin molecule contains a stable four-chain construct of approximately 150-160 kDa in which the dimers are held together by a disulfide bond between the heavy chains. In the second form, the dimers are not linked through interchain disulfide bonds, and a molecule of approximately 75-80 kDa is formed, consisting of covalently linked light and heavy chains (half-antibodies). These forms are extremely difficult to separate, even after affinity purification.

The frequency of occurrence of the second form in different intact IgG isotypes is due, without limitation, to structural differences associated with the isotype of the antibody hinge region. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the occurrence of the second form (Angal et al., 1993, Molecular Immunology 30:105) to levels typically observed using the human IgG1 hinge. The present invention covers antibodies characterized by one or more mutations in the hinge, CH2 or CH3 region, which may be desirable, for example, in production, to improve the yield of the desired form of the antibody.

As used herein, the expression antigen binding molecule means a protein, polypeptide or molecular complex containing or consisting of at least one complementarity determining region (CDR), which alone or in combination with one or more additional CDRs and/or framework regions (FR), binds specifically to a specific antigen. In certain embodiments, the antigen binding molecule is an antibody or antibody fragment, as those terms are defined elsewhere herein.

The phrase specifically binds or specifically binds to or the like means that the antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding may have an equilibrium dissociation constant of at least about 1x10'6 M or less (eg, a lower KD value indicates tighter binding). Methods for determining whether two molecules are specifically binding are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, antibodies that specifically bind to Bet v 1 have been identified using surface plasmon resonance, such as BIACORE™.

The phrase high-affinity antibody refers to those monoclonal antibodies that have a binding affinity for Bet v 1, expressed as a KD, of at least ^10'8

- 17 045437

M; preferably 10 ^-9 M; more preferably 10 ^-10 M, even more preferably 10 ^-11 M, even more preferably 10 ^-12 M, as measured by surface plasmon resonance, e.g.

BIACORE™ or solution affinity ELISA.

By the term slow dissociation rate, Koff or kd is meant an antibody that dissociates from Bet v 1 with a rate constant of 1x10' ³ -c'1 or less, preferably 1x10' ⁴ -c'1 or less, as determined by surface plasmon resonance eg BIACORE™.

As used herein, the terms antigen binding portion of an antibody, antigen binding fragment of an antibody, and the like include any naturally occurring, enzymatically produced, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. As used herein, the terms antigen binding antibody fragment or antibody fragment refer to one or more antibody fragments that retain the ability to bind Bet v 1.

In specific embodiments, the antibody or antibody fragments of the present invention may be conjugated to a therapeutic moiety (immunoconjugate), such as a corticosteroid, a second anti-Bet v 1 antibody, or epinephrine, a vaccine, or any other therapeutic moiety useful for treating an allergic response to Bet v 1 .

The antibodies of the present invention may be isolated antibodies. As used herein, the term isolated antibody means an antibody that has been identified and separated and/or recovered from at least one component of its natural environment. For example, an antibody that is separated or removed from at least one component of the body or from a tissue or cell in which the antibody naturally exists or is produced in nature is an isolated antibody for purposes of the present invention. The isolated antibody also includes the antibody in situ in the recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. In certain embodiments, the isolated antibody may be substantially free of other cellular material and/or chemical compounds.

In certain embodiments, the isolated antibody may be substantially free of other antibodies (Abs) having different antigen specificities (e.g., an isolated antibody that specifically binds to Bet v 1 or a fragment thereof is substantially free of antibodies that specifically bind to antigens different from Fagales antigens or, in some aspects, different from Bet v 1.

As used herein, the term blocking antibody or neutralizing antibody (or antibody that neutralizes the activity of Bet v 1) is intended to mean an antibody or antigen binding portion thereof whose binding to Bet v 1 results in inhibition of at least one biological activity of Bet v 1. For example, an antibody of the present invention may help prevent a primary allergic response to Bet v 1. Alternatively, an antibody of the present invention may demonstrate the ability to prevent a secondary allergic response to Bet v 1 or at least one symptom of an allergic response to Bet v 1, including sneezing, coughing, asthmatic condition or anaphylactic reaction 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 the biological activity of Bet v 1 using one or more 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 (eg, other animal models to evaluate protection from challenge with Bet v 1 following administration of one or more antibodies described herein document).

As used herein, the term surface plasmon resonance refers to an optical phenomenon that allows the analysis of biomolecular interactions in real time by detecting changes in protein concentrations within a biosensor matrix, for example, using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, New Jersey).

The term K _D as used herein is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

The term epitope refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of the antibody molecule, known as the paratope. A single antigen may contain more than one epitope. Thus, different antibodies may bind to different regions on the antigen and may have different biological effects. The term epitope also refers to the site on an antigen to which B and/or T cells respond. This also refers to the region of the antigen that is bound by the antibody. Epitopes can be either linear or conformational. A linear epitope is formed by adjacent amino acid residues in a polypeptide chain. The conformational epitope is formed by spatially adjacent

- 18 045437 amino acids from various segments of the unbranched polypeptide chain. In certain embodiments, epitopes may include determinants that are reactive surface groups of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structure characteristics and/or specific charge characteristics. Epitopes can be defined as structural or functional. Functional epitopes are generally a subclass of structural epitopes and contain those residues that directly contribute to the affinity of the interaction. Epitopes formed from adjacent amino acids are typically retained when exposed to denaturing solvents, whereas epitopes formed by folding into a tertiary structure are typically lost when exposed to denaturing solvents. An epitope typically includes at least 3, and more typically at least 5 or 8-10 amino acids in a unique spatial conformation.

The term substantial identity or substantially identical, when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with corresponding nucleotide insertions or deletions with another nucleic acid (or a strand complementary thereto), there is at least nucleotide sequence identity about 90%, and more preferably at least about 95, 96, 97, 98, or 99% of the nucleotide bases, as measured by any known sequence identity measurement algorithm, such as FASTA, BLAST, or GAP, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

As applied to polypeptides, the term substantially similar or substantially similar means that the two peptide sequences, when optimally aligned, for example using the GAP or BESTFIT programs using default gap penalties, have at least 90% sequence identity, even more preferably at least 95, 98 or 99% sequence identity. Preferably, positions of residues that are not identical are distinguished by conservative amino acid substitutions. A conservative amino acid substitution is one in which an amino acid residue is replaced by another amino acid residue containing a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). Typically, a conservative amino acid substitution will not substantially change the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage sequence identity or percentage similarity may be adjusted upward to correct for the conservative nature of the substitution. The means for making this adjustment are well known to those skilled in the art. (See, for example, Pearson, 1994, Methods Mol. Biol. 24: 307-331). Examples of groups of amino acids that contain side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine and tryptophan; (5) main side chains: lysine, arginine and histidine; (6) acid side chains: aspartate and glutamate, and (7) sulfur side chains: cysteine and methionine. Preferred groups of conservative amino acid substitutions are as follows: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate and asparagyne glutamine. Alternatively, a conservative replacement is any change characterized by a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992, Science 256: 1443-1445). A moderately conservative substitution is any change characterized by a non-negative value in the PAM250 log-likelihood matrix.

Sequence similarity for polypeptides is typically measured using sequence analysis software.

Protein analysis software matches similar sequences using similarity scores assigned to various substitutions, deletions and other modifications, including conserved amino acid substitutions. For example, GCG software contains programs such as GAP and BESTFIT that can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and its mutein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percentage sequence identities of regions of the highest

- 19 045437 better overlap between the query sequence and the search sequences (Pearson, 2000, earlier). Another preferred algorithm when comparing a sequence of the present invention to a database containing a large number of sequences from various organisms is the BLAST computer program, especially BLASTP or TBLASTN, using default parameters. (See, for example, Altschul et al., 1990, J. Mol. Biol. 215: 403-410 and 1997 Nucleic Acids Res. 25:3389-3402)

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

Antibodies of the present invention can be used to desensitize an individual allergic to Fagales. The term desensitize is defined herein as reducing the allergic reactivity of an individual with a Fagales allergy to exposure to a Fagales allergen, such as birch pollen, for example, Bet v 1, Aln g1, Cor a1, Car b1, Que a1, Api g2, Api g1, Dau c1, Mal d1, Ost c1, Fag s1 and/or Cas s1 (to a level less than what an individual allergic to Fagales would otherwise experience), or a Fagales-related allergen. The term desensitize is further defined herein as reducing an individual's allergic reactivity to PR-10 proteins, including Act c 8 and Act d 8 (kiwi), Ara h 8 (peanut), Pru ar 1 (apricot), Pru av 1 (cherry), Pru p 1 (peach), Pyr s 1 (pear), Gly m 4 (soybean), Vig r 1 (golden bean), Sola I 4 (tomato), Cuc m 3 (melon), Rub i 1 (raspberry), and Fra a 1 (strawberry).

General description.

Trees belonging to the order Fagales are the source of spring allergy symptoms, and the major birch allergen, Bet v 1, is responsible for IgE binding in more than 95% of patients with birch pollen allergy (Breiteneder, EMBO J. 1989, 8(7): 1935-8). Birch is the predominant cause of allergy in 23% of allergy patients in the United States and 14% of allergy patients in Europe. (DataMonitor report on Allergic Rhinitis, July 2010). The severity of symptoms in people who demonstrate sensitivity to birch pollen ranges from relatively mild rhinitis and conjunctivitis to a potentially life-threatening asthmatic condition. It has been shown that more than 60% of patients suffering from birch pollen allergy have IgE antibodies to this Bet v 1 (Jarolim et al., Int Arch Allergy Appl Immunol. 1989, 88(1-2): 180-2) .

Fagales trees show a clear geographical distribution, with birch and alder being endemic to the northern parts of Europe and North America, while hazel, hornbeam and oak prefer warmer climates, thus inhabiting more southern parts of these continents. Joint populations of all five species often occur in temperate zones (Spieksma FTM. Regional European pollen calendars. D'Amato G, Spieksma FTM, Bonini S, eds. Allergenic pollen and pollinosis in Europe. Hoboken, NJ: Wiley-Blackwell; 1991. pp. 49-65). Several Betulaceae trees, including alder, hazel, and hornbeam, are capable of initiating sensitization to Bet v 1-like allergens in susceptible individuals, resulting in the production of highly cross-reactive IgE antibodies. (Hauser, et al., 2011, Clin Exp Allergy. 41: 1804-14).

Allergens of the order Fagales or Fagales allergens as defined herein include birch pollen (Bet v 1), alder pollen (Aln g1 and Aln g4), hazel pollen (Cor a1, Cor a2, Cor a8, Cor a9, Cor a10, Cor a11, Cor a12, Cor a13 and Cor a14), hornbeam pollen (Car b1), hop hornbeam pollen (Ost c1), chestnut pollen (Cas s1, Cas s5, Cas s8 and Cas s9), beech pollen (Fag s1i ) and white oak pollen (Que a1 and Que a2). Of the allergens that are not birch pollen allergens, Aln g1, Cor a1, Car b1, Ost c1, Cas s1, Fag s1 and Que a1 are similar or related to Bet v 1, i.e. are related Fagales allergens. These allergens are also expressed in the nuts of Fagales trees and in the fruits of unrelated trees belonging to the order Rosales.

Cross-reactivity of these allergens can cause food allergy symptoms in patients with pollen allergies. Typical cross-reactive forms of food allergies include allergies to apples, cherries, apricots, pears, alfalfa, peas, soybeans, tomatoes, celery, carrots, and asparagus. (Allergens and Allergen Immunotherapy: Subcutaneous, Sublingual, and Oral, 5th ed. Richard F. Lockey, Dennis K. Ledford, editors. CRC Press, Taylor & Francis Group, London, NY, 2014. pp. 114, 118119) .

As used herein, the phrase Fagales allergen includes Fagales-related allergens. In some embodiments, a Fagales related allergen is defined as a protein having at least about 35% overall sequence homology with Bet v 1, or sequence homology with Bet v 1 epitope 1 of at least about 50%, or sequence homology with epitope 2 of Bet v 1 of at least about 40%, or sequence homology to epitope 3 of Bet v 1 of at least about 25%, or sequence homology of epitope 4 of Bet v 1 of at least about 15%. In some embodiments, a Fagales related allergen is defined as a protein that cross-reacts with

- 20 045437 antibodies to Bet v 1 are generated, including allergens from the Rosales order.

Immunoglobulin E (IgE) is responsible for type 1 hypersensitivity, which manifests itself in allergic rhinitis, allergic conjunctivitis, hay fever, allergic bronchial asthma, bee venom allergy and food allergies. IgE circulates in the blood and binds to high-affinity Fc receptors for IgE on basophils and mast cells. In most allergic responses, allergens enter the body through inhalation, ingestion, or through the skin. The allergen then binds to preformed IgE, already bound to a high-affinity receptor on the surfaces of mast cells and basophils, resulting in cross-linking of several IgE molecules and triggering the release of histamine and other inflammatory mediators, causing various allergic symptoms.

Treatment of birch pollen allergy involves desensitization therapy, which involves repeated injections with increasing dosages of either crude birch pollen extract or short peptides derived from Bet v 1. Disadvantages of allergen-specific immunotherapy include the long duration of treatment, which leads to to problems with patient compliance with the treatment regimen and frequent allergic responses (up to 30%) to the administered protein. Desensitization therapy may take several years before treatment is considered effective. Successful treatment depends on the composition and quality of the extract, and treatment is contraindicated in patients with severe asthma/food allergies due to the risk of severe IgE-mediated adverse effects. Accordingly, there is a need in the field of birch pollen allergy treatment for alternative treatment strategies for patients sensitive to Fagales allergens, in particular Bet v 1.

Antibodies have been proposed as a general strategy for the treatment of allergies because they can block the penetration of allergen molecules into mucosal tissues or can bind the allergen before it can bind to 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. US Pat. No. 5,670,626 describes the use of monoclonal antibodies to treat IgE-mediated allergic diseases such as allergic rhinitis, allergic bronchial asthma and allergic conjunctivitis by blocking the binding of allergens to mucosal tissue. US Pat. No. 6,849,259 describes the use of allergen-specific antibodies to inhibit allergic inflammation in an in vivo model of allergy in mice. Antibody systems based on milk and eggs have been described. For example, US 20030003133 A1 discloses the use of milk as an allergen carrier for inducing oral tolerance to birch pollen and other allergens. Compositions and methods of reducing the allergic response in an animal to an allergen in the environment by using a molecule that inhibits the ability of the allergen to bind to mast cells have been described in international patent publication WO 1994/024164 A2. Other antibodies to Bet v 1 were mentioned in US patent document No. 2010/0034812.

The fully human antibodies described herein exhibit specific binding to Bet v 1 and may be useful for the treatment of patients suffering from birch pollen allergy, in particular in patients who demonstrate sensitivity to the Bet v 1 allergen. Use of such antibodies may be effective treatment for patients allergic to Fagales tree pollen, or can be used to prevent an increased response to Bet v 1 upon secondary exposure or associated allergy-related symptoms, or can be used to reduce the severity and/or duration of an allergic response associated with primary exposure to birch pollen allergen or with relapse of symptoms upon secondary exposure. They can be used alone or as an adjuvant therapy with other therapeutic components or methods known in the art for the treatment of such allergies, such as, but not limited to, treatment with corticosteroids or epinephrine. They can be used in combination with a second or third different antibody specific for Bet v 1. They can be used with allergen-specific immunotherapy (SIT). In some embodiments, the combination with SIT results in a synergistically effective combination.

In contrast to desensitization therapy, treatment with the antibodies described herein can provide effective relief within approximately 2 weeks of initiation of treatment or within approximately 10 days or approximately 8 days of initiation of treatment. When combined with exposure to Bet v 1 protein or peptides or one or more additional Fagales allergens, treatment with the fully human antibodies described herein may not only block the allergic response, but may more effectively or synergistically desensitize patients allergic to pollen from Fagales trees.

In certain embodiments, the antibodies of the present invention are obtained from mice immunized with a primary immunogen, such as natural Bet v 1, which can be purchased commercially (for example, from Indoor Biotechnologies, VA, Cat. No. NA-BV1-1) or can obtained recombinantly. The full length amino acid sequence of Bet v 1 is shown as SEQ ID NO: 306. The full length amino acid sequence of Bet v 1 can also be found in SEQ ID NO: 314 from Uniprot: P15494.

The immunogen may be a biologically active and/or immunogenic fragment of natural, native or recombinantly produced Bet v 1 or DNA encoding its active fragment. The fragment may be derived from either the N-terminus or the C-terminus of Bet v 1 or from any site in the amino acid sequence of Bet v 1.

Antigen-Binding Antibody Fragments Unless specifically stated otherwise, the term antibody as used herein should be understood to include antibody molecules containing two immunoglobulin heavy chains and two immunoglobulin light chains (ie, complete antibody molecules), as well as antigen-binding fragments thereof. As used herein, the terms antigen binding portion of an antibody, antigen binding fragment of an antibody, and the like include any naturally occurring, enzymatically produced, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the terms antigen binding portion of an antibody or antibody fragment refer to one or more antibody fragments that retain the ability to specifically bind to Bet v 1. An antibody fragment may include a Fab fragment, an F(ab')2 fragment, an Fv fragment, a dAb, fragment containing CDR, or isolated CDR. Antigen-binding antibody fragments can be prepared, for example, from complete antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving DNA manipulation and expression of DNA encoding antibody variable and (optionally) constant domains. Such DNA is known and/or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example to arrange one or more variable and/or constant domains into a suitable configuration or to introduce codons, create cysteine residues, modify, add or remove amino acids, etc. .

Non-limiting examples of antigen binding fragments include the following: (i) Fab fragments; (ii) fragments F(ab') ₂ ; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv molecules (scFv); (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (eg, a dedicated complementarity determining region (CDR) such as the CDR3 peptide) or a conformationally restricted peptide FR3-CDR3-FR4. Other engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, tri-bodies, tetrabodies, mini-antibodies, nanoantibodies (e.g. monovalent nanoantibodies, bivalent nanoantibodies, etc.), immunopharmaceuticals small modular protein (SMIP) and shark IgNAR variable domains are also included within the scope of the expression antigen binding fragment as used herein.

An antigen binding fragment of an antibody will typically contain at least one variable domain. The variable domain can be of any size or amino acid composition and will typically contain at least one CDR that is adjacent or in the same reading frame as one or more framework sequences. In antigen binding fragments containing a V _H domain associated with a V _L domain, the V _H and V _L domains may be located relative to each other in any suitable arrangement. For example, the variable region may be dimeric and may contain VH-VH, VH-VL, or VLVL dimers. Alternatively, the antigen binding fragment of the antibody may comprise a monomeric VH or VL domain.

In certain embodiments, an antigen binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen binding fragment of an antibody of the present invention include the following: (i) V _H -C _H 1; (ii) V _H -C _H 2; (iii) V _H -C _H 3; (iv) V _H Ch1-Ch2; (v) Vh -Ch1-Ch2-Ch3; (vi) Vh -Ch2-Ch3; (vii) Vh -Cl; (viii) Vl -Ch1; (ix) Vl -Ch2; (x) Vl -Ch3; (xi) V _L -C _h 1-C _h 2; (xii) V _L -C _h 1-C _h 2-C _h 3; (xiii) V _L -C _h 2-C _h 3; and (xiv) V _L -C _L . In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can either be directly linked to each other or can be linked through a full or partial hinge or linker region. The hinge region may consist of at least 2 (eg, 5, 10, 15, 20, 40, 60 or more) amino acids, resulting in a flexible or semi-flexible connection between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, the antigen binding fragment of an antibody of the present invention may comprise a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with each other and/or with one or more monomeric VH domains or V _L (for example, via disulfide bond(s))

Obtaining human antibodies.

Methods for producing human antibodies in transgenic mice are known in the art. Any such known methods can be used in the context of the present invention to obtain human antibodies that specifically bind to Bet v 1.

When using VELOCIMMUNE™ technology (see, for example, US Patent No. 6596541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for creating monoclonal antibodies, first, high-affinity chimeric antibodies to Bet v 1 with a human variable region and a mouse constant region are isolated. VELOCIMMUNE® technology involves the creation of a transgenic mouse with a genome containing human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces an antibody containing the human variable region and the mouse constant region in response to antigen stimulation . DNA encoding the variable regions of the heavy and light chains of the antibody is isolated and operably linked to DNA encoding the constant regions of the human heavy and light chains. The DNA is then expressed in a cell capable of expressing a fully human antibody.

Typically, a VELOCIMMUNE® mouse is sensitized with an antigen of interest, and lymphatic cells (such as B cells) are recovered from the mice that express the antibodies. Lymphatic cells can be fused with a myeloma cell line to produce immortalized hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific for the antigen of interest. DNA encoding the heavy chain and light chain variable regions can be isolated and linked to the desired heavy chain and light chain isotype constant regions. Such an antibody protein may be produced in a cell, such as a CHO cell. Alternatively, DNA encoding antigen-specific chimeric antibodies or light and heavy chain variable domains can be isolated directly from antigen-specific lymphocytes.

First, high-affinity chimeric antibodies with a human variable region and a mouse constant region are isolated. As in the experiments section below, antibody characteristics are determined and antibodies are selected for desired characteristics including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with the desired human constant region, for example, wild-type IgG1 or IgG4 or modified IgG1 or IgG4 to create a fully human antibody of the present invention. While the selected constant region may vary depending on the specific application, the characteristics of high antigen binding affinity and target specificity are found in the variable region.

In general, the antibodies of the present invention have very high affinities, typically with a K _D of from about 10 ^-12 to about 10 ^-9 M when measured by binding to antigen, either immobilized on a solid phase or in a liquid phase. Mouse constant regions are replaced with the desired human constant regions to create fully human antibodies according to the present invention. While the selected constant region may vary depending on the particular application, the high affinity antigen binding characteristics and target specificity characteristics are in the variable region.

Bioequivalents.

The anti-Bet v 1 antibodies and antibody fragments of the present invention cover proteins with amino acid sequences that differ from the amino acid sequences of the described antibodies, but which retain the ability to bind to Bet v 1. Such variant antibodies and antibody fragments contain one or more insertions, deletions or substitutions amino acids compared to the original sequence, but exhibit biological activity that is substantially equivalent to the activity of the described antibodies. Likewise, antibody-encoding DNA sequences of the present invention include sequences that contain one or more insertions, deletions, or nucleotide substitutions relative to the disclosed sequence, but that encode an antibody or antibody fragment that is substantially bioequivalent to the antibody or antibody fragment of the present invention.

Two antigen-binding proteins or antibodies are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show significant differences when administered at the same molar dose under similar experimental conditions or as a single dose , or in the form of several doses. Some antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in their extent of absorption, but not in their rate of absorption, and may still be considered bioequivalent because such differences in rate of absorption are intentional and reflected on the label, not are essential to achieving effective concentrations of the drug in the body, for example, with chronic use, and are considered medically insignificant for the particular drug product being studied.

In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity, or potency.

In one embodiment, two antigen binding proteins are bioequivalent if the patient can switch one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity or reduced efficacy, compared to continuing therapy without such switching .

In one embodiment, two antigen binding proteins are bioequivalent if they both act through a common mechanism or mechanisms of action for the condition or conditions of use, to the extent such mechanisms are known.

Bioequivalence can be demonstrated using in vivo and in vitro methods. Bioequivalence measurements include, for example, (a) an in vivo test in humans or other mammals in which the concentration of an antibody or its metabolites is measured in blood, plasma, serum or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of in vivo bioavailability data in humans; (c) an in vivo test in humans or other mammals in which the relevant acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) a well-controlled clinical trial that establishes the safety, efficacy or bioavailability or bioequivalence of the antibody.

Bioequivalent variants of the antibodies of the present invention can be constructed, for example, by making various substitutions of residues or sequences or removing terminal or internal residues or sequences that are not essential for biological activity. For example, cysteine residues that are not essential for biological activity can be removed or replaced with other amino acids to prevent the formation of unnecessary or irregular intramolecular disulfide bridges upon renaturation. In other contexts, bioequivalent antibodies may include antibody variants containing amino acid substitutions that modify the glycosylation characteristics of the antibodies, such as mutations that eliminate or remove glycosylation.

Biological characteristics of antibodies.

In general, antibodies of the present invention may function by binding to Bet v 1, a fragment of Bet v 1, or Bet v 1 and one or more Fagales allergens or Fagales-related allergens.

In certain embodiments, antibodies of the present invention may bind to an epitope or fragment located within the Bet v 1 protein, for example, an epitope or fragment spanning amino acid residues ranging from about position 23 to about position 43 according to SEQ ID NO: 306 ; an epitope or fragment comprising amino acid residues ranging from about position 44 to about 56 according to SEQ ID NO: 306; an epitope or fragment comprising amino acid residues ranging from about 2 to about 19 according to SEQ ID NO: 306; an epitope or fragment comprising amino acid residues ranging from about 57 to about 70 according to SEQ ID NO: 306; or an epitope or fragment comprising amino acid residues ranging from about 81 to about 89 or about 81 to about 96 according to SEQ ID NO: 306. In certain embodiments, the antibodies of the present invention can bind to at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 307, 308, 309, 310, 311 and 315, wherein the epitope sequence can be extended by 1-5 amino acids or about 5 to about 10 amino acids at either the C-terminus or the N-terminus.

In certain embodiments, the antibodies of the present invention may function by blocking or inhibiting the binding of IgE to mast cells or basophils in a patient sensitive to the Bet v 1 allergen. In certain embodiments, the antibodies provided herein inhibit or block the activation of basophils, for example, at least at least approximately 70% compared to the isotype control antibody. In certain embodiments, the antibodies inhibit or block mast cell degranulation, for example, by at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, compared to the isotype control antibody.

In one embodiment, the present invention provides a fully human monoclonal antibody or antigen binding fragment thereof that binds Bet v 1, wherein the antibody or fragment thereof exhibits one or more of the following characteristics: (i) contains an HCVR with an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 282 and 290, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (ii) contains an LCVR with an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250 , 266 and 298, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (iii) contains an HCDR3 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264, 280, 288 and 296, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and an LCDR3 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272 and 304, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (iv) contains an HCDR1 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260, 276, 284 and 292, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; HCDR2 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246, 262, 278, 286 and 294, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; LCDR1 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268 and 300, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and an LCDR2 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270 and 302, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (v) associated with a Bet v 1c KD equal to or less than 10 ^-8 or ranging from about 10 ^-8 to about 10 ^-11 ; (vi) demonstrates efficacy in at least one animal model of anaphylaxis or inflammation; or (vii) competes with a reference antibody for binding to Bet v 1.

In one embodiment, the present invention relates to the use of a combination of two or more fully human antibodies of the present invention or fragments thereof to produce a composition, wherein the antibodies bind to Bet v 1, and wherein each antibody or fragment thereof contained in the composition exhibits one or more the following characteristics: (i) contains HCVR with an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 282 and 290, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (ii) contains an LCVR with an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250 , 266 and 298, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (iii) contains an HCDR3 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264, 280, 288 and 296, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and an LCDR3 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 24o, 256, 272 and 304, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (iv) contains an HCDR1 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260, 276, 284 and 292, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; HCDR2 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 22, 38, 54, 70, 86, 102, 118,

- 25 045437

134, 150, 166, 182, 198, 214, 230, 246, 262, 278, 286 and 294, or a substantially similar sequence thereto that is characterized by at least 90%, at least 95%, at least 98 % or at least 99% sequence identity; LCDR1 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268 and 300, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and an LCDR2 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270 and 302, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (v) binds to Bet v 1 with a K _D equal to or less than 10 ^-8 or ranging from about 10 ^-8 to about 10 ^-11 ; (vi) demonstrates efficacy in at least one animal model of anaphylaxis or inflammation; or (vii) competes with a reference antibody for binding to Bet v 1.

Certain anti-Bet v 1 antibodies of the present invention, when used alone or in combination, are capable of binding to and neutralizing at least one biological effect of Bet v 1, as determined by in vitro or in vivo assays. The ability of the antibodies of the present invention to bind and neutralize Bet v 1 activity can be measured using any standard method known to those skilled in the art, including binding assays or neutralizing activity assays (eg, protection against anaphylaxis), as described herein.

Non-limiting exemplary in vitro assays for measuring binding activity are illustrated in Example 3 herein. In Example 3, the binding affinities and kinetic constants of human antibodies to Bet v 1 were determined using surface plasmon resonance.

Bet v 1 proteins or peptides can be modified to include the insertion or substitution of certain residues for labeling or for conjugation with carrier molecules such as KLH. For example, a cysteine can be added at either the N-terminus or the C-terminus of the peptide, or a linker sequence can be added to produce a peptide for conjugation, for example, with KLH for immunization. Antibodies specific for Bet v 1 may not contain additional tags or fragments, or they may contain an N-terminal or C-terminal tag or fragment. In one embodiment, the tag or moiety is biotin. In a binding assay, the location of the label (if present) can determine the orientation of the peptide relative to the surface to which the peptide is bound. For example, if the surface is coated with avidin, the peptide containing N-terminal biotin will be oriented such that the C-terminal part of the peptide is distal to the surface.

Epitope mapping and related technologies.

As used herein, 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 contain more than one epitope. Thus, different antibodies may bind to different regions on the antigen and may have different biological effects. Epitopes can be either conformational or linear. A conformational epitope is formed by spatially adjacent amino acids from different segments of an unbranched polypeptide chain. A linear epitope is formed by adjacent amino acid residues in a polypeptide chain. In certain cases, the epitope may include saccharide moieties, phosphorylated groups, or sulfonyl groups on the antigen.

Provided herein are anti-Bet v 1 antibodies that react with one or more amino acids found within the Bet v 1 molecule, including, for example, any Bet v 1 fragment shown in SEQ ID NO: 306, or within comparable regions of the recombinantly produced Bet v 1 protein. The epitope to which antibodies bind may consist of one contiguous sequence of 3 or more (for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20 or more) amino acids located within the Bet v 1 molecule. Exemplary contiguous sequences include amino acid residues ranging from about position 23 to about position 44 according to SEQ ID NO: 306; amino acid residues ranging from about position 23 to about position 43 according to SEQ ID NO: 306; amino acid residues ranging from about position 23 to about position 38 according to SEQ ID NO: 306; amino acid residues ranging from about position 23 to about position 41 according to SEQ ID NO: 306; amino acid residues ranging from about position 26 to about position 43 according to SEQ ID NO: 306; amino acid residues ranging from about position 29 to about position 43 according to SEQ ID NO: 306; amino acid residues ranging from about position 44 to about position 70 according to SEQ ID NO: 306; amino acid residues in the range

- 26 045437 not from about position 44 to about position 56 according to SEQ ID NO: 306; amino acid residues ranging from about position 45 to about position 56 according to SEQ ID NO: 306; amino acid residues ranging from about position 2 to about position 19 according to SEQ ID NO: 306; amino acid residues ranging from about position 5 to about position 10 according to SEQ ID NO: 306; amino acid residues ranging from about position 5 to about position 19 according to SEQ ID NO: 306; amino acid residues ranging from about position 8 to about position 19 according to SEQ ID NO: 306; amino acid residues ranging from about position 11 to about position 19 according to SEQ ID NO: 306; amino acid residues ranging from about position 57 to about position 70 according to SEQ ID NO: 306; amino acid residues ranging from about position 57 to about position 66 according to SEQ ID NO: 306; amino acid residues ranging from about position 81 to about position 96 according to SEQ ID NO: 306; amino acid residues ranging from about position 84 to about position 96 according to SEQ ID NO: 306; amino acid residues ranging from about position 85 to about position 96 according to SEQ ID NO: 306; and amino acid residues ranging from about position 81 to about position 89 according to SEQ ID NO: 306. Additional exemplary contiguous sequences include at least one amino acid sequence selected from the group consisting of SEQ ID NO: 307, 308, 309, 310, 311 and 315, which sequence may be extended by about 1 to about 5 amino acids or about 5 to about 10 amino acids, either at the C-terminus or the N-terminus. Alternatively, the epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) located within the Bet v 1 molecule (eg, a conformational epitope).

Various techniques known to those of ordinary skill in the art can be used to determine whether an antibody reacts with one or more amino acids within a polypeptide or protein. Exemplary techniques include, for example, routine cross-blocking assays such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY). Other methods include alanine scanning mutagenesis analysis, peptide blotting (Reineke (2004) Methods Mol Biol 248:443-63), peptide digestion assay, crystallographic studies and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer (2000) Protein Science 9:487-496). Another method that can be used to identify amino acids in a polypeptide with which an antibody reacts is hydrogen-deuterium exchange, detected by mass spectrometry. In general terms, the hydrogen-deuterium exchange method involves labeling a protein of interest with deuterium, followed by binding of the antibody to the deuterium-labeled protein. The protein-antibody complex is then transferred to water and the exchangeable protons in the amino acids that are protected by the antibody complex undergo back exchange of deuterium for hydrogen at a slower rate than the exchangeable protons in the amino acids that are not part of the interface. As a result, amino acids that form part of the protein/antibody interface may retain deuterium and therefore exhibit relatively greater mass compared to amino acids not included in the interface. Once the antibody is dissociated, the target protein is subjected to protease digestion and mass spectrometric analysis, thereby identifying deuterium-labeled residues that correspond to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A. X-ray diffraction analysis of the antibody-antigen complex can also be used for epitope mapping purposes.

Modification-Assisted Profiling (MAP), also known as Antigen Structure-based Antibody Profiling (ASAP), is a method of distributing categories of large numbers of monoclonal antibodies directed against the same antigen, according to the similarities in the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (US 2004/0101920). Each category may reflect a unique epitope, either clearly different from or partially overlapping with the epitope represented by another category. This technology allows genetically identical antibodies to be quickly filtered out so that characterization can focus on genetically distinct antibodies. When used for hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce monoclonal antibodies with the desired characteristics. MAP can be used to sort the antibodies of the present invention into groups of antibodies that bind to different epitopes.

- 27 045437

In certain embodiments, anti-Bet v 1 antibodies or antigen-binding fragments thereof bind to an epitope within Bet v 1, in natural or native form, as illustrated in SEQ ID NO: 306 or SEQ ID NO: 314 (amino acid sequence of Bet v 1 from Uniprot : P15494), or a recombinantly obtained protein or a fragment thereof. According to certain embodiments of the antibodies of the present invention, as shown in table. 1, interact with at least one amino acid sequence selected from the group consisting of amino acid residues ranging from about position 23 to about position 44 according to SEQ ID NO: 306; amino acid residues ranging from about position 23 to about position 43 according to SEQ ID NO: 306; amino acid residues ranging from about position 23 to about position 38 according to SEQ ID NO: 306; amino acid residues ranging from about position 23 to about position 41 according to SEQ ID NO: 306; amino acid residues ranging from about position 26 to about position 43 according to SEQ ID NO: 306; amino acid residues ranging from about position 29 to about position 43 according to SEQ ID NO: 306; amino acid residues ranging from about position 44 to about position 70 according to SEQ ID NO: 306; amino acid residues ranging from about position 44 to about position 56 according to SEQ ID NO: 306; amino acid residues ranging from about position 45 to about position 56 according to SEQ ID NO: 306; amino acid residues ranging from about position 2 to about position 19 according to SEQ ID NO: 306; amino acid residues ranging from about position 5 to about position 10 according to SEQ ID NO: 306; amino acid residues ranging from about position 5 to about position 19 according to SEQ ID NO: 306; amino acid residues ranging from about position 8 to about position 19 according to SEQ ID NO: 306; amino acid residues ranging from about position 11 to about position 19 according to SEQ ID NO: 306; amino acid residues ranging from about position 57 to about position 70 according to SEQ ID NO: 306; amino acid residues ranging from about position 57 to about position 66 according to SEQ ID NO: 306; amino acid residues ranging from about position 81 to about position 96 according to SEQ ID NO: 306; amino acid residues ranging from about position 84 to about position 96 according to SEQ ID NO: 306; amino acid residues ranging from about position 85 to about position 96 according to SEQ ID NO: 306; and amino acid residues ranging from about position 81 to about position 89 according to SEQ ID NO: 306. These regions are further illustrated in SEQ ID NO: 307, 308, 309, 310, 311 and 315.

The present invention also provides anti-Bet v 1 antibodies that bind to the same epitope or portion of an epitope as any of the specific exemplary antibodies described herein in Table. 1, or an antibody with the CDR sequences of any of the exemplary antibodies described in table. 1. Likewise, the present invention also provides antibodies to Bet v 1 that compete for binding to Bet v 1 or a fragment of Bet v 1 with any of the specific exemplary antibodies described herein in Table 1, or an antibody with the CDR sequences of any of the exemplary antibodies described in table. 1.

Whether an antibody binds to the same epitope as or competes with the reference anti-Bet v 1 antibody can be readily determined using routine methods known in the art. For example, to determine whether a test antibody binds to the same epitope as a reference anti-Bet v 1 antibody of the present invention, the reference antibody is allowed to bind to the Bet v 1 protein or peptide under saturating conditions. The ability of the test antibody to bind to the Bet v 1 molecule is then assessed. If the test antibody is able to bind to Bet v 1 after saturating binding to the reference Bet v 1 antibody, it can be concluded that the test antibody binds to a different epitope compared to the reference Bet antibody v 1. On the other hand, if the test antibody is unable to bind to the Bet v 1 molecule after saturating binding to the reference Bet v 1 antibody, then the test antibody may bind to the same epitope as the epitope bound by the reference Bet v 1 antibody antibody according to the present invention.

To determine whether an antibody competes for binding to a reference Bet v 1 antibody, the binding methodology described above is performed in two orientations: in the first orientation, the reference antibody is allowed to bind to the Bet v 1 molecule under saturating conditions, followed by assessment of binding of the test antibody to the Bet molecule v 1. In the second orientation, the test antibody is allowed to bind to the Bet v 1 molecule under saturating conditions, followed by an assessment of the binding of the reference antibody to the Bet v 1 molecule. If in both orientations only the first (saturating) antibody is able to bind to the Bet v 1 molecule, then do conclusion that the test antibody and the reference antibody compete for binding to Bet v 1. One skilled in the art will appreciate that an antibody that competes for binding to the reference antibody may not necessarily bind to the same epitope as the reference antibody, but can sterically block binding of the reference antibody by binding to an overlapping or adjacent epitope.

Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) the other's binding to the antigen. That is, a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, but preferably by 75%, 90%, or even 99%, as measured in a competitive binding assay (see ., for example, Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies are characterized by the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other. Two antibodies are characterized by overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other.

Additional routine experiments (e.g., peptide mutations and binding assays) can then be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference antibody, or whether steric blocking (or other phenomenon) is reason for the lack of observed binding. Experiments of this nature can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

Immunoconjugates.

The present invention includes a human monoclonal antibody to Bet v 1 conjugated to a therapeutic moiety (immunoconjugate), such as an agent capable of reducing the severity of the allergic response to the Bet v 1 allergen, or in an environment in which Fagales trees are present, or reducing the intensity at least one symptom associated with exposure to birch pollen or Bet v 1 allergen, including rhinitis, conjunctivitis or difficulty breathing or the severity thereof. Such an agent may be a corticosteroid, a second different Bet v 1 antibody, or a vaccine. The type of therapeutic moiety that can be conjugated to the anti-Bet v 1 antibody will take into account the condition being treated and the desired therapeutic effect to be achieved. Alternatively, if the desired therapeutic effect is to treat complications or symptoms associated with exposure to the Bet v 1 allergen, or any other condition resulting from such exposure, such as, without limitation, rhinitis or conjunctivitis, it may be advantageous to conjugate an agent suitable for treating the consequences or symptoms of a condition or to reduce any side effects of antibodies according to the present invention. Examples of suitable means for forming immunoconjugates are known in the art, see, for example, international patent publication WO 05/103081.

Therapeutic administration and formulations.

The present invention relates to therapeutic compositions containing antibodies to Bet v 1 or antigen binding fragments thereof according to the present invention. Administration of therapeutic compositions in accordance with the present invention will be carried out by suitable routes, including, without limitation, intravenously, subcutaneously, intramuscularly, intranasally, with suitable carriers, excipients and other means that are included in the formulations to provide improved transport, delivery, tolerability and the like. similar. Many acceptable formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid containing (cationic or anionic) vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorbent pastes, oil-in-emulsions. water and water-in-oil, carbowax emulsions (polyethylene glycols with different molecular weights), semi-solid gels and semi-solid mixtures containing carbowax. See also Powell et al. Compendium of excipients for parenteral formulations PDA (1998) J Pharm Sci Technol 52:238–311.

The dosage of the antibody may vary depending on the age and size of the patient being dosed, the target disease, conditions, route of administration, and the like. When an antibody of the present invention is used to treat rhinitis or conjunctivitis associated with exposure to Fagales tree pollen, or birch pollen, or Bet v 1, in an individual with sensitivity to Bet v 1, or to prevent an anaphylactic reaction to a Fagales allergen or to reduce the severity allergic response, it is advisable to administer intravenously the antibody of the present invention, typically in a single dose of about 0.01 to about 30 mg/kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 - approximately 3 mg/kg body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. In certain embodiments, an antibody or antigen-binding fragment thereof according to the present

- 29 045437 the invention can be administered as an initial dose of at least about 0.1 mg - about 800 mg, about 1 - about 500 mg, about 5 - about 300 mg, or about 10 - about 200 mg, up to about 100 mg or up to approximately 50 mg. In certain embodiments, the initial dose may be followed by a second or multiple subsequent doses of an antibody or antigen-binding fragment thereof in an amount that may be approximately the same as or less than the amount of the initial dose, with the interval between subsequent doses being at least 1 day to 3 days. day; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

Various delivery systems are known that can be used to administer the pharmaceutical composition of the present invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al. (1987) J Biol Chem 262:4429–4432). Routes of administration include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucous membranes (for example, through the oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with other biological active means. Administration may be systemic or local.

The pharmaceutical composition can be delivered in a vesicle, in particular a liposome (see, for example, Langer, 1990, Science 249: 1527-1533).

In certain situations, the pharmaceutical composition can be delivered in a controlled release system. According to one embodiment, a pump may be used. According to another embodiment, polymeric materials can be used. In yet another embodiment, the controlled release system can be placed in close proximity to the target of the composition, thus requiring only a fraction of the systemic dose.

Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, etc. These injectable preparations can be prepared by generally known methods. For example, injectable preparations can be prepared, for example, by dissolving, suspending or emulsifying the above-described antibody or a salt thereof in a sterile aqueous or oily medium commonly used for injection. As the aqueous injection medium, there are, for example, physiological saline solution, isotonic solution containing glucose and other auxiliaries, etc., which can be used in combination with a suitable solubilizing agent such as alcohol (eg ethanol), polyhydric alcohol (eg propylene glycol, polyethylene glycol), nonionic surfactant [eg polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oil medium, for example, sesame oil, soybean oil, etc. are used, which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus obtained is preferably filled into a suitable ampoule.

The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. Moreover, with respect to subcutaneous delivery, the pen delivery device easily finds use in delivering the pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device typically uses a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. The disposable pen delivery device does not have a replaceable cartridge. In contrast, a disposable pen delivery device comes pre-filled with a pharmaceutical composition contained in a reservoir within the device. Once the reservoir is empty of the pharmaceutical composition, the entire device is discarded.

Numerous reusable pen and auto-injector delivery devices are used in the subcutaneous delivery of the pharmaceutical composition of the present invention. Examples include, without limitation, the following: AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75/25™ pen, pen HUMALOG™, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, Inn.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo

- 30 045437

Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™ and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany), to name just a few . Examples of disposable pen delivery devices useful in the subcutaneous delivery of a pharmaceutical composition of the present invention include, but are not limited to, the following: SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk) and KWIKPEN™ (Eli Lilly ), SURECLICK™ autoinjector (Amgen, Thousand Oaks, Calif.), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and HUMIRA™ pen (Abbott Labs, Abbott Park, 111.), to name only several of them.

The pharmaceutical compositions for oral or parenteral use described above are advantageously prepared in unit dose dosage forms that correspond to the dose of the active ingredients. Such unit dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the above-mentioned antibody contained therein is generally from about 5 to about 500 mg per unit dose dosage form; especially in injection form, it is preferred that the antibody be contained in an amount of from about 5 to about 100 mg and from about 10 to about 250 mg for other dosage forms.

Therapeutic applications of antibodies.

Due to their interaction with Bet v 1, the antibodies of the present invention are useful for treating the primary response following human exposure to Fagales allergen, birch pollen, or an environment containing the Bet v 1 protein, or at least one symptom associated with an allergic response, such as itchy eyes, conjunctivitis, rhinitis, shortness of breath, difficulty breathing, or to prevent a secondary response to the Bet v 1 allergen, including a more severe anaphylactic reaction, or to reduce the severity, duration and/or frequency of symptoms after re-exposure to the Fagales allergen. Accordingly, it is contemplated that the antibodies of the present invention may be used prophylactically or therapeutically.

According to a further embodiment of the present invention, the antibodies of the present invention are used to produce a pharmaceutical composition for the treatment of patients suffering from sensitivity to birch pollen or its extract and/or Bet v 1 protein. In yet another embodiment of the present invention, the antibodies of the present invention are used to obtain a pharmaceutical compositions for reducing the severity of initial exposure to Bet v 1 or to reduce the severity, duration and/or number of allergic responses to Bet v 1. In a further embodiment of the present invention, the antibodies of the present invention are used as an adjuvant therapy with any other agent useful for treating Fagales allergens, including corticosteroids, vaccines, allergen-specific immunotherapy (SIT), or any other palliative therapy known to those skilled in the art.

Types of combination therapy.

Combination therapies may include an anti-Bet v 1 antibody of the present invention and any additional therapeutic agent that can be effectively combined with an antibody of the present invention or a biologically active fragment of an antibody of the present invention.

For example, a second therapeutic agent may be used to help reduce allergic symptoms following exposure to Fagales allergen, birch pollen or extract thereof, or Bet v 1, or exposure to an environment in which Fagales trees are present and flowering, such as a corticosteroid. The antibodies may also be used in combination with other therapies, such as a Bet v 1 allergen-specific vaccine. Additional therapeutically active component(s) may be administered before, simultaneously with, or after administration of the Bet v 1 antibody herein. invention. For purposes of the present disclosure, such administration regimens are considered to be administration of an anti-Bet v 1 antibody in combination with a second therapeutically active component.

Allergen-specific immunotherapy (SIT).

As used herein, the expressions allergen-specific immunotherapy, specific immunotherapy, SIT, SIT regimen, and the like refer to the repeated administration of an allergen to a patient over time as a means to treat or prevent allergies and allergic responses or to reduce or eliminate allergic responses. In a typical SIT regimen, small amounts of allergen are initially administered to the allergic patient, followed by administration of increasing amounts of allergen. In certain cases, the SIT regimen includes at least two sequential phases: (1) an escalating dose phase and (2) a maintenance phase. In the escalating dose phase, increasing doses of the allergen are administered until an effective and safe dose is reached. The dose that is established at the end of the escalating phase is then administered to the patient during the maintenance phase. The duration of the increasing dose phase may be several weeks or several months.

- 31 045437 tsev. However, in certain embodiments, the escalating dose phase is characterized by a substantially shorter duration (eg, less than one week, less than 6 days, less than 5 days, less than 4 days, less than 3 days, or less than 2 days). SIT regimens that include an escalating dose phase of less than 5 days are sometimes called fulminant immunotherapy or fulminant SIT. The maintenance phase of the SIT regimen may last several weeks, several months, several years, or indefinitely.

Administration schemes.

In accordance with certain embodiments of the present invention, multiple doses of one or more anti-Bet v 1 antibodies (antibody combinations) may be administered to a patient over a period of time. Methods according to this aspect of the present invention involve sequentially administering multiple doses of an antibody, a combination of antibodies, to a patient. As used herein, the term sequential administration means that each dose of the antibody or combination of antibodies is administered to the patient at different points in time, for example, on different days separated by a predetermined interval (for example, hours, days, weeks or months). The present invention provides methods that involve sequentially administering to a patient one initial dose of an antibody or combination of antibodies, followed by one or more secondary doses of the antibody, and optionally followed by one or more tertiary doses of the antibody.

The terms initial dose, secondary doses and tertiary doses refer to the time sequence of administration of the antibody or combination of antibodies according to the present invention. Thus, the starting dose is the dose that is administered at the beginning of the treatment regimen (also called the initial dose); secondary doses are doses that are administered after the initial dose; and tertiary doses are doses that are administered after secondary doses. The initial, secondary and tertiary doses may contain the same amount of antibody or combination of antibodies, but generally may differ from each other in terms of frequency of administration. However, in certain embodiments, the amount of antibody or combination of antibodies contained in the initial, secondary and/or tertiary doses varies relative to each other (eg, it is adjusted higher or lower depending on the situation) during the course of treatment. In certain embodiments, two or more (eg, 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as loading doses, followed by subsequent doses that are administered less frequently (eg, maintenance doses).

According to one exemplary embodiment of the present invention, each secondary and/or tertiary dose is administered at 1-26 intervals (e.g., 1.1%, 2.2%, 3.3%, 4.4%, 5.5%, 6.6 %, 7, 7%, 8, 8%, 9, 9%, 10, 10%, 11, 11%, 12, 12%, 13, 13%, 14, 14%, 15, 15%, 16, 16 %, 17, 17%, 18, 18%, 19, 19%, 20, 20%, 21, 21%, 22, 22%, 23, 23%, 24, 24%, 25, 25%, 26, 26 % or more) weeks after the immediately preceding dose. As used herein, the phrase immediately preceding dose in a sequence of administrations means a dose of an antibody or combination of antibodies that is administered to a patient before the administration of the immediately next dose in a sequence, without any intervening doses.

Methods according to this aspect of the present invention may involve administering to a patient any number of secondary and/or tertiary doses of an antibody or combination of antibodies. For example, in certain embodiments, only one secondary dose is administered to the patient. In other embodiments, the patient is administered two or more (eg, 2, 3, 4, 5, 6, 7, 8, or more) secondary doses. Likewise, in certain embodiments, only one tertiary dose is administered to the patient. In other embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses are administered to the patient.

In embodiments that include multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose can be administered to the patient 1-2 weeks after the immediately preceding dose. Likewise, in embodiments involving multiple tertiary doses, each tertiary dose can be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2-4 weeks after the immediately preceding dose. Alternatively, the frequency with which secondary and/or tertiary doses are administered to the patient may vary during the course of treatment. The frequency of administration may also be adjusted during the course of treatment by the physician depending on the needs of the individual patient after clinical examination.

Diagnostic applications of antibodies.

Antibodies to Bet v 1 according to the present invention can also be used to detect and/or measure Bet v 1 in a sample, for example, for diagnostic purposes. It is contemplated that confirmation of an allergic response suspected of being caused by Bet v 1 can be accomplished by measuring the presence of any Bet v 1 using any one or more antibodies of the present invention. Illustrative diagnostic tests regarding Bet v 1 may

- 32 045437 include, for example, contacting a sample obtained from a patient with an antibody to Bet v 1 according to the present invention, wherein the antibody to Bet v 1 is labeled with a detectable label or reporter molecule or used as a capture ligand for selective isolation of the protein Bet v 1 from patient samples. Alternatively, an unlabeled anti-Bet v 1 antibody can be used for diagnostic purposes in combination with a secondary antibody that is itself labeled with a detectable label. The detectable label or reporter molecule may be a radioactive isotope such as ³ H, ¹⁴ C, ³² P, ³⁵ S or ¹²⁵ I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase or luciferase. Specific exemplary assays that can be used to detect or measure Bet v 1 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

Samples that can be used in Bet v 1 diagnostic assays in accordance with the present invention include any tissue or fluid sample obtained from a patient that contains detectable amounts of Bet v 1 protein or fragments thereof under normal or pathological conditions. Typically, the Bet v 1 content of a particular sample obtained from a healthy/non-allergic patient (eg, a patient not affected by sensitivities associated with the presence of Bet v 1) will be measured to initially establish a baseline, or standard Bet v 1 content. this initial Bet v 1 content can be compared with Bet v 1 contents measured in samples obtained from individuals suspected of having sensitivity to Bet v 1 in birch pollen or symptoms associated with such a condition.

While the present invention has been specifically shown and described with reference to a number of embodiments, those skilled in the art will appreciate that changes in form and detail may be made to the various embodiments disclosed herein without departing from the spirit and the scope of the present invention and that the various embodiments disclosed herein are not intended to limit the scope of the claims of the present invention.

Examples

The following examples are provided so that those of ordinary skill in the art will have full disclosure and description of how to make and apply the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors of the present invention believe to be their invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. quantities, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise noted, parts are by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

Example 1. Generation of human antibodies to Bet v 1.

An immunogen containing any of the following can be used to generate antibodies to Bet v 1. In certain embodiments, the antibodies of the present invention are obtained from mice immunized with a primary immunogen, such as full-length natural Bet v 1 (nBet v 1), which is commercially available commercially (eg, Stallergenes Greer, Lenoir, NC, cat. no. XP527D3A25) or isolated from birch pollen (see, for example, Buters, et al. (2012), Atomospheric Environment 55:496-505) or which can be obtain recombinantly (see GenBank accession number P 15494 for the full-length amino acid sequence of Bet v 1) or fragments of the Bet v 1 protein, followed by immunization with a secondary immunogen or an immunogenically active fragment of the native protein. Various constructs can be prepared using portions of the Bet v 1 protein known to those skilled in the art. These constructs can be used alone or in various combinations to induce antibody responses in vivo. For example, recombinant Bet v 1 constructs, such as the examples set forth in SEQ ID NOs: 307, 308, 309, 310, 311 and 315, or fragments thereof, can be used as immunogens.

In certain embodiments, the antibodies of the present invention are obtained from mice immunized with a primary immunogen, such as a biologically active and/or immunogenic fragment of native Bet v 1 or DNA encoding an active fragment thereof. The fragment may be derived from the N-terminal or C-terminal portion of Bet v 1.

In certain embodiments, the recombinant Bet v 1 protein constructs used in the studies described herein may also include a C-terminal tag (myc-myc-hexahistidine tag) as described below. In other embodiments, the recombinant Bet v 1 protein construct includes amino acids G2-N160 according to Uniprot P15494. In some embodiments, the construct comprises an S85A replacement. The proteins were expressed in Chinese hamster ovary (CHO) cells. The exogenous signal sequence used to stimulate expression in CHO cells is not included in the sequence listings.

- 33 045437

In certain embodiments, the immunogen may be a Bet v 1 fragment or fusion protein that contains any one or more of the following: i) Bet v 1 amino acid residues 23-43 (see Uniprot P15494 as well as SEQ ID NO: 307); ii) amino acid residues 4456 Bet v 1 (see Uniprot P15494 and also SEQ ID NO: 308); iii) amino acid residues 2-19 Bet v 1 (see Uniprot P15494 and also SEQ ID NO: 309); iv) amino acid residues 57-70 Bet v 1 (see Uniprot P15494 and also SEQ ID NO: 310); v) amino acid residues 81-89 Bet v 1 (see Uniprot P15494 and SEQ ID NO: 311) and vi) amino acid residues 81-96 Bet v 1 (see Uniprot P15494 and SEQ ID NO: 315).

In certain embodiments, antibodies that bind specifically to Bet v 1 can be prepared using fragments of the above regions or peptides that extend beyond the designated regions by about 5 to about 20 amino acid residues from one or both of the N- or C- end of the areas described in this document. In certain embodiments, any combination of the above regions or fragments thereof can be used in the production of Bet v 1 specific antibodies. In certain embodiments, any one or more of the above Bet v 1 regions or fragments thereof can be used to produce monospecific, bispecific, or multispecific antibodies.

The full-length proteins or fragments thereof, which were used as immunogens as described above, were administered directly, with an adjuvant to stimulate an immune response, to a VELOCIMMUNE® mouse containing DNA encoding the variable regions of the human immunoglobulin heavy and kappa light chains.

Antibodies to Bet v 1 are isolated directly from antigen-positive B cells without fusion with myeloma cells, as described in US patent No. 7582298. Using this method, several fully human antibodies to Bet v 1 (ie antibodies having variable human domains and human constant domains); Exemplary antibodies thus formed are designated as follows: H4H16943P, H4H16946P, H4H16950P, H4H16960P, H4H16967P, H4H16971P, H4H16979P, H4H16987P, H4H16991P, H4H16992P, H4H1 7001R, N4N17015R, N4N17027R, N4N17028R, N4N17031R, N4N17033R, N4N17038R2, N4N17045R2, N4N17067R2 and N4N17082R2 .

The biological properties of exemplary antibodies generated in accordance with the methods of this example are described in detail in the examples presented below.

Example 2 Amino acid sequences of the heavy and light chains.

In table 1a shows identifiers of amino acid sequences of the variable regions of the heavy and light chains and CDRs of selected antibodies to Bet v 1. Table. 1b shows nucleic acid sequence identifiers of the heavy and light chain variable regions and CDRs of selected antibodies to Bet v 1.

Table 1a ______Aino acid sequence identifiers______

SEQ ID NO: Antibody designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 N4N16943R 2 4 6 8 10 12 14 16 N4N16946R 18 20 22 24 26 28 thirty 32 N4N16950R 34 36 38 40 42 44 46 48 N4N16960R 50 52 54 56 58 60 62 64 N4N16967R 66 68 70 72 74 76 78 80 N4N16971R 82 84 86 88 90 92 94 96 N4N17038R2 98 100 102 104 106 108 110 112 N4N16987R 114 116 118 120 122 124 126 128 Н4Н16991Р 130 132 134 136 138 140 142 144 N4N16992R 146 148 150 152 154 156 158 160 N4N17001R 162 164 166 168 170 172 174 176 N4N17015R 178 180 182 184 186 188 190 192 N4N17027R 194 196 198 200 202 204 206 208 N4N17028R 210 212 214 216 218 220 222 224 N4N17031R 226 228 230 232 234 236 238 240 N4N17033R 242 244 246 248 250 252 254 256 N4N16979R 258 260 262 264 266 268 270 272

- 34 045437

N4N17045R2 274 276 278 280 266 268 270 272 N4N17067R2 282 284 286 288 266 268 270 272 N4N17082R2 290 292 294 296 298 300 302 304

lb table

Nucleic acid sequence identifiers

SEQ ID NO: Antibody designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 N4N16943R 1 3 5 7 9 eleven 13 15 N4N16946R 17 19 21 23 25 27 29 31 N4N16950R 33 35 37 39 41 43 45 47 N4N16960R 49 51 53 55 57 59 61 63 N4N16967R 65 67 69 71 73 75 77 79 N4N16971R 81 83 85 87 89 91 93 95 N4N17038R2 97 99 101 103 105 107 109 111 N4N16987R FROM 115 117 119 121 123 125 127 Н4Н16991Р 129 131 133 135 137 139 141 143 N4N16992R 145 147 149 151 153 155 157 159 N4N17001R 161 163 165 167 169 171 173 175 N4N17015R 177 179 181 183 185 187 189 191 N4N17027R 193 195 197 199 201 203 205 207 N4N17028R 209 211 213 215 217 219 221 223 N4N17031R 225 227 229 231 233 235 237 239 N4N17033R 241 243 245 247 249 251 253 255 N4N16979R 257 259 261 263 265 267 269 271 N4N17045R2 273 275 277 279 265 267 269 271 N4N17067R2 281 283 285 287 265 267 269 271 N4N17082R2 289 291 293 295 297 299 301 303

Antibodies are generally referred to herein according to the following nomenclature: the prefix Fc (e.g., Ή4Η, Ή2Μ, etc.) followed by a numerical identifier (e.g., 16943 17001, etc.), followed by the suffix “P.” or P2. The prefixes H4H and H2M in the designations of antibodies used herein indicate a particular isotype of the Fe region of the antibody. Thus, according to this nomenclature, the antibody herein may be referred to as, for example, Ή4Η16943Ρ, etc. since Ή4Η denotes an antibody that contains a human IgG4 Fc (all variable regions are fully human, as indicated by the first Ή in the antibody designation). One of ordinary skill in the art will appreciate that an antibody with a particular Fc isotype can be converted into an antibody with a different Fc isotype (e.g., a mouse IgGl Fc antibody can be converted into a human IgG4 antibody, etc.), but in any case the variable domains (including CDRs), which are indicated using the numeric identifiers shown in table. 1 will remain the same and the antigen binding properties are expected to be identical or substantially similar regardless of the nature of the Fc domain.

In table Figure 1c shows the amino acid sequence identifiers of the full-length heavy and light chains of selected antibodies to Bet ν 1.

Table 1c

Heavy and light chain amino acid sequence identifiers

SEQ ID NO: Antibody designation Heavy chain Light chain H4H17038P2 316 317 H4H16987P 318 319 H4H16992P 320 321 H4H17082P2 322 323

Example 3 Antibody Binding to Bet ν 1 as Determined by Surface Plasmon

-35045437 resonance.

Dissociation equilibrium constants (Ko) for the binding of native Bet v1 to purified monoclonal antibodies to Bet v 1 were determined using a real-time surface plasmon resonance biosensor (SPR-Biacore) using a Biacore 4000 instrument. All binding studies were performed in a running buffer containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% v/v. surfactant Tween-20, pH 7.4 (HBS-ET) at 25°C and 37°C. The Biacore sensor surface was first derivatized by amine coupling with a mouse monoclonal anti-human Fc antibody (GE, Cat. No. BR-1008-39) to capture the monoclonal antibodies to Bet v 1. Various concentrations of natural Bet v 1 (Indoor, Cat. No. BR-1008-39) NA-BV1-1) or a reagent produced by CHO recombinant Bet v 1 containing the S85A mutation with a C-terminal myc-myc hexahistidine tag (mutant Bet v 1-MMH; SEQ ID NO: 312) was first prepared in HBS-ET running buffer ( 100 nM - 1.23 nM; serially diluted 3 times) and were injected onto a surface containing anti-Bet v 1 monoclonal antibodies captured by anti-human Fc antibodies for 4 minutes at a flow rate of 30 μl/min, while dissociation of Bet v 1 monoclonal antibody reagent bound was monitored for 10 min in HBS-ET running buffer. Kinetic rate constants for association (k _a ) and dissociation (k d ) were determined by fitting real-time binding sensorgrams to a 1:1 mass transfer-limited binding model using Scrubber 2.0c curve fitting software. Equilibrium dissociation constants for binding (KD) and dissociative half-lives (t½) were calculated from the kinetic rate constants as follows:

kd 1п(2)

K _D = 5—, and th = ~gg ka kd

The binding kinetics parameters for natural Bet v 1 and mutant Bet v 1-MMH with various monoclonal antibodies to Bet v 1 according to the present invention at 25°C and 37°C are shown in table. 2-5.

At 25°C, all monoclonal antibodies to Bet v 1 according to the present invention bound to natural Bet v 1 with KD values ranging from 0.66 nM to 13.5 nM, as shown in table. 2. At 37°C, all monoclonal antibodies to Bet v 1 according to the present invention bound to natural Bet v 1 with KD values ranging from 1.59 nM to 27.9 nM, as shown in table. 3. At both 25°C and 37°C, the isotype control antibody did not demonstrate any measurable binding to native Bet v 1.

At both 25°C and 37°C, 3 of 20 monoclonal antibodies to Bet v 1 according to the present invention did not bind to mutant Bet v 1-MMH. At 25°C, 17 of 20 monoclonal antibodies to Bet v 1 bound to mutant Bet v 1-MMH with Kd values ranging from 348 pM to 43.8 nM, as shown in table. 4. At 37°C, 17 of the 20 monoclonal antibodies to Bet v 1 according to the present invention bound to the mutant Bet v 1-MMH with K _D values ranging from 655 pM to 106 nM, as shown in table. 5. At both 25°C and 37°C, the isotype control antibody did not show any measurable binding to the Betv mutant 1-MMH.

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table 2

Parameters of the binding kinetics of natural Bet v 1 binding to __________Monoclonal antibodies to Bet v 1 at 25°C_______

Antibody mAb capture level (PE, resonance unit) 100 nM bound natural Bet ν 1 (PE) k _a (1/Ms) k _d (1/s) Kd(M) tH (min) N4N16943R 346 ± 1.5 71 1.64E±05 2.30E-04 1.40E-09 50 N4N16946R 355 ± 1.8 74 2.05E±05 2.01E-04 9.82E-10 58 N4N16950R 357 ±2.3 58 9.61E±04 5.03E-04 5.23E-09 23 N4N16960R 342 ± 2.2 57 4.22E±05 6.90E-04 1.64E-09 17 N4N16967R 332 ±0.9 27 3.76E±04 1.49E-04 3.98E-09 77 N4N16971R 343 ± 0.5 57 2.40E±05 7.81E-04 3.26E-09 15 N4N16979R 295 ± 0.8 41 2.30E±05 2.78E-04 1.21E-09 42 N4N16987R 334 ±0.5 61 4.10E±05 1.14E-03 2.77E-09 10 Н4Н16991Р 313 ±0.4 57 2.95E±05 3.12E-04 1.06E-09 37 N4N16992R 351 ±0.7 64 3.73E±05 2.48E-04 6.65E-10 47 N4N17001R 352 ±0.6 71 3.29E±05 3.29E-04 1.00E-09 35 N4N17015R 370 ± 0.7 68 1.84E±05 2.46E-04 1.34E-09 47 N4N17027R 340 ± 0.6 48 6.24E±04 2.70E-04 4.32E-09 43 N4N17028R 350 ±0.7 63 2.81E±05 1.07E-03 3.79E-09 AND N4N17031R 335 ±0.7 54 1.24E±05 1.68E-03 1.35E-08 6.8 N4N17033R 336 ±0.4 61 3.94E±05 4.69E-04 1.19E-09 25 N4N17038R2 324 ± 0.9 64 2.77E±05 3.64E-04 1.32E-09 32 N4N17045R2 368 ± 0.9 69 1.22E±05 1.49E-04 1.22E-09 78 N4N17067R2 344 ± 0.5 58 1.13E±05 4.02E-04 3.56E-09 29 N4N17082R2 366 ± 0.6 72 5.45E±05 5.97E-04 1.09E-09 19 Isotypic control 345 ±0.5 1 NB* NB* NB* NB*

*NB indicates that binding was not observed under the current experimental conditions.

- 37 045437

Table 3

Binding kinetics parameters of natural Bet v 1 binding to monoclonal antibodies to Bet v 1 at 37°C

Antibody Capture level mAL (PE) 100 nM bound natural Bet ν 1 (PE) k _a (1/M-s) k _d (1/s) Kd(M) tH (min) N4N16943R 451 ±2.7 88 2.60E±05 1.27E-03 4.89E-09 9.1 N4N16946R 469 ± 2.6 93 2.89E±05 6.01E-04 2.08E-09 19 N4N16950R 471 ±2.1 76 1.41E±05 1.21E-03 8.57E-09 10 N4N16960R 422 ± 0.9 65 6.62E±05 1.68E-03 2.54E-09 6.8 N4N16967R 412 ± 1.9 45 5.04E±04 7.19E-04 1.43E-08 16 N4N16971R 433 ± 1.3 66 3.52E±05 2.44E-03 6.93E-09 4.7 N4N16979R 352 ± 1.3 52 3.42E±05 6.16E-04 1.80E-09 19 N4N16987R 436 ± 1.7 74 5.20E±05 4.77E-03 9.16E-09 2.4 Н4Н16991Р 379 ± 1.0 66 5.00E±05 1.08E-03 2.16E-09 AND N4N16992R 436 ± 1.1 73 5.38E±05 8.53E-04 1.59E-09 14 N4N17001R 457 ±0.8 87 4.85E±05 1.22E-03 2.52E-09 9.5 N4N17015R 464 ± 1.4 84 2.93E±05 7.92E-04 2.70E-09 15 N4N17027R 427 ± 0.9 67 9.81E±04 9, ZOE-04 9.48E-09 12 N4N17028R 435 ± 1.3 71 3.73E±05 2.90E-03 7.77E-09 3.9 N4N17031R 419 ± 1.8 63 4.85E±05 5.44E-03 2.79E-08 2.1 N4N17033R 417 ± 1.4 74 6.27E±05 1D9E-03 1.90E-09 10 N4N17038R2 406 ± 1.4 74 3.73E±05 1.62E-03 4,ZZE-09 7.1 N4N17045R2 465 ± 1.4 90 2.37E±05 4.41E-04 1.86E-09 26 N4N17067R2 440 ± 1.2 80 2.68E±05 1.47E-03 5.48E-09 7.9 N4N17082R2 453 ± 1.1 83 7.48E±05 1.82E-03 2.43E-09 6.3 Isotypic control 426 ± 1 1 NB* NB* NB* NB*

*NB indicates that binding was not observed under the current experimental conditions.

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Table 4

Binding kinetics parameters of mutant Bet v 1-MMH binding to monoclonal antibodies to Bet v 1 at 25°C

Antibody Capture level mAL (PE) 100 nM bound mutant Bet ν 1MMN (PE) ka (1/Ms) kd (1/s) Kd(M) t'/2 (min) N4N16943R 344 ± 0.8 0 NB* NB* NB* NB* N4N16946R 354 ±0.5 83 4.05E±05 1.41E-04 3.48E-10 82 N4N16950R 357 ±0.6 70 1.51E±05 3.35E-04 2.21E-09 35 N4N16960R 343 ± 0.8 64 5.62E±05 4.43E-04 7.89E-10 26 N4N16967R 331 ±0.3 0 N.B. N.B. N.B. N.B. N4N16971R 343 ± 0.5 58 4.07E±05 3.86E-03 9.48E-09 3.0 N4N16979R 293 ± 1.0 46 2.86E±05 2, ZOE-04 8.04E-10 50 N4N16987R 334 ±0.4 46 4.90E±05 2.15E-02 4.38E-08 0.5 Н4Н16991Р 312 ±0.5 61 3.90E±05 2.87E-04 7.35E-10 40 N4N16992R 350 ±0.4 70 4.59E±05 2.76E-04 6.01E-10 42 N4N17001R 351 ±0.7 1 NB* NB* NB* NB* N4N17015R 368 ±0.8 76 2.72E±05 2.81E-04 1.03E-09 41 N4N17027R 340 ± 0.4 63 1.10E±05 3.19E-04 2.90E-09 36 N4N17028R 349 ± 0.5 71 8.11E+05 6.93E-04 8.54E-10 17 N4N17031R 333 ±0.5 63 1.87E±05 1.05E-03 5.61E-09 AND N4N17033R 336 ±0.5 67 5.40E±05 4.15E-04 7.68E-10 28 N4N17038R2 324 ± 1 63 6.42E±05 6.41E-03 9.98E-09 1.8 N4N17045R2 368 ± 1.1 60 8.00E±04 4.98E-04 6.23E-09 23 N4N17067R2 344 ± 0.3 55 1.00E±05 1.17E-03 1.17E-08 9.9 N4N17082R2 366 ±0.5 78 5.30E±05 6.12E-04 1.16E-09 19 Isotypic control 344 ±0.8 1 NB* NB* NB* NB*

*NB indicates that binding was not observed under the current experimental conditions.

- 39 045437

Table 5

Binding kinetics parameters of mutant Bet v 1-MMH binding to monoclonal antibodies to Bet v 1 at 37°C

Antibody Capture level mAL (PE) 100 nM bound mutant Bet ν 1MMN (PE) ka (1/Ms) kd (1/s) Kd(M) 1½ (min) N4N16943R 441 ± 1.3 0 NB* NB* NB* NB* N4N16946R 459 ± 1.8 97 6.03E±05 3.95E-04 6.55E-10 29 N4N16950R 461 ± 1.2 83 2.15E±05 1.25E-03 5.81E-09 9.2 N4N16960R 417 ± 1.4 72 8.79E±05 1.77E-03 2.01E-09 6.5 N4N16967R 405 ± 0.8 -1 NB* NB* NB* NB* N4N16971R 427 ± 1.2 60 6.48E±05 1.20E-02 1.86E-08 1.0 N4N16979R 348 ± 0.7 56 4.62E±05 7, ZOE-04 1.58E-09 16 N4N16987R 429 ± 1.1 36 7.95E±05 8.47E-02 1.06E-07 0.14 Н4Н16991Р 376 ± 0.7 67 7.17E±05 1.06E-03 1.48E-09 AND N4N16992R 432 ± 0.6 78 7.84E±05 8.85E-04 1.1 ZE-09 13 N4N17001R 451 ±0.8 2 NB* NB* NB* NB* N4N17015R 458 ± 1.1 89 4.45E±05 8.99E-04 2.02E-09 13 N4N17027R 421 ± 0.8 78 1.71E±05 1.16E-03 6.80E-09 9.9 N4N17028R 430 ±0.8 77 1.01E+06 2.35E-03 2.32E-09 4.9 N4N17031R 413 ± 1.4 71 2.78E±05 4.06E-03 1.46E-08 2.8 N4N17033R 414 ±0.8 79 8.83E±05 1.18E-03 1.34E-09 9.8 N4N17038R2 399 ± 0.9 56 8,ЗЗЭ±05 2.96E-02 3.56E-08 0.39 N4N17045R2 458 ± 1.2 84 1.62E±05 2.32E-03 1.43E-08 5.0 N4N17067R2 435 ± 0.7 76 2.53E±05 4.28E-03 1.69E-08 2.7 N4N17082R2 448 ± 1 89 8.21E±05 1.90E-03 2.31E-09 6.1 Isotypic control 421 ± 0.7 -2 NB* NB* NB* NB*

*NB indicates that binding was not observed under the current experimental conditions.

Example 4 Antibody binding to related allergens as determined by surface plasmon resonance.

Dissociation equilibrium constants (KD) for the binding of various related allergens to purified anti-Bet v 1 monoclonal antibodies were determined using a real-time surface plasmon resonance biosensor (SPR-Biacore) using a Biacore 4000 instrument. All binding studies were performed in a running buffer containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% v/v. surfactant Tween-20, pH 7.4 (HBS-ET) at 25°C. The Biacore sensor surface was first derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (GE, cat. no. BR-1008-39) to capture the monoclonal antibodies to Bet v 1. Binding studies were performed on the following related allergens: alder (Aln g 1 , MyBiosource, cat. no. MBS1041484), apple (Mal d 1, MyBiosource, cat. no. MBS1224919), carrot (Dau with 1.2, MyBiosource, cat. no. MBS1212920), celery (Api g 1, MyBiosource, cat. no. cat. MBS1171376), celery (Api g 2, MyBiosource, cat. no. MBS1047880), common hornbeam (Car b 1 isoform 1A and 1B, MyBiosource, cat. no. MBS1200018), common hornbeam (Car b 1 isoform 2, MyBiosource , cat. no. MBS1043940), hazel (Cor A 1, MyBiosource, cat. no. MBS5304600) and white oak (Que a 1, MyBiosource, cat. no. MBS1258822). Various concentrations of related allergens were prepared in HBS-ET working buffer (100 nM - 6.25 nM; serially diluted 4-fold) and then applied to a surface containing monoclonal antibodies to Bet v 1 captured by antibodies to human Fc for 3 min at a flow rate of 30 μl/min, while the dissociation of monoclonal antibody-bound allergens was monitored for 8 min in HBSET running buffer. Kinetic rate constants for association ( _ka ) and dissociation (kd) were determined by fitting real-time binding sensorgrams to a 1:1 mass transfer-limited binding model using Scrubber 2.0c curve fitting software. Dissociation equilibrium constants for binding ( _KD ) and dissociative half-lives (t½) were calculated from the kinetic rate constants as follows:

- 40 045437 ln(2) kd kd

Kd = t— , and 1½ = ka

The binding kinetics parameters for related allergens with various monoclonal antibodies to Bet v 1 according to the present invention at 25°C are shown in table. 6-11.

As shown in table. 6 at 25°C 9 of 20 anti-Bet v 1 antibodies according to the present invention showed measurable binding to Aln g 1 with KD values ranging from 1.03 nM - 175 nM. The other 11 antibodies did not demonstrate any measurable binding to Aln g 1 under the conditions tested.

As shown in table. 7 at 25°C, two of the 20 anti-Bet v 1 antibodies of the present invention showed measurable binding to Mal d 1 with K _D values of 29.8 nM and 494 nM. The other 18 antibodies did not demonstrate any measurable binding to Mal d 1 under the conditions tested.

As shown in table. 8 at 25°C, one of the anti-Bet v 1 antibodies of the present invention showed measurable binding to Api g 1 with a KD value of 167 nM. The other 19 antibodies did not demonstrate any measurable binding to Api g 1 under the conditions tested.

As shown in table. 9 at 25°C 8 of the 20 anti-Bet v 1 antibodies of the present invention demonstrated measurable binding to Car b 1 isoform 1A and 1B with KD values ranging from 1.2 nM to 380 nM. The other 12 antibodies did not demonstrate any measurable binding to Car b 1 isoforms 1A and 1B under the conditions tested.

As shown in table. 10 at 25°C, 14 of 20 anti-Bet v 1 antibodies according to the present invention showed measurable binding to Car b 1 isoform 2 with K _D values ranging from 335 pM to 564 nM. The other 6 antibodies did not demonstrate any measurable binding to Car b 1 isoform 2 under the conditions tested.

As shown in table. 11 at 25°C, one of the anti-Bet v 1 antibodies of the present invention showed measurable binding to Cor A 1 with a KD value of 396 nM. The other 19 antibodies did not demonstrate any measurable binding to Cor A 1 under the conditions tested.

None of the antibodies of the present invention demonstrated measurable binding to Dau c 1.2, Api g 2 or Que a 1 under the conditions tested (data not shown). The isotype control antibody did not demonstrate any measurable binding to any allergen tested.

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Table 6

Binding kinetics parameters of alder allergen (Aln g1) binding to monoclonal antibodies to Bet v 1 at 25°C

Antibody Capture level mAL (PE) 100 nM bound analyte (PE) k _a (1/Ms) ka (1/s) Kd(M) 1½ (min) N4N16943R 675 ± 0.9 1 NB* NB* NB* NB* N4N16946R 668 ± 1.2 2 NB* NB* NB* NB* N4N16950R 634 ± 1.7 15 9.12E±04 1.60E-02 1.75E-07 0.7 N4N16960R 681 ±2.1 29 2.26E±05 3.32E-02 1.47E-07 o, s N4N16967R 666 ±3.1 0 NB* NB* NB* NB* N4N16971R 668 ±2.1 41 9.30E±04 1.18E-02 1.26E-07 1.0 N4N16979R 545 ± 0.4 0 NB* NB* NB* NB* N4N16987R 667 ± 1.6 139 2.21E±05 8.70E-04 3.94E-09 13 Н4Н16991Р 608 ± 0.4 3 NB* NB* NB* NB* N4N16992R 653 ± 1.3 5 NB* NB* NB* NB* N4N17001R 630 ± 1.6 49 2.64E±05 2.78E-02 1.05E-07 0.4 N4N17015R 745 ±3 8 NB* NB* NB* NB* N4N17027R 772 ± 0.4 9 9.29E±04 6.43E-03 6.92E-08 1.8 N4N17028R 708 ± 1.5 16 5.17E±04 1.90E-03 3.67E-08 6.1 N4N17031R 727 ± 1.3 59 5.29E±04 2.38E-03 4.50E-08 4.8 N4N17033R 649 ± 0.2 3 NB* NB* NB* NB* N4N17038R2 660 ± 1.3 2 NB* NB* NB* NB* N4N17045R2 746 ± 1.2 0 NB* NB* NB* NB* N4N17067R2 617 ±2.2 2 NB* NB* NB* NB* N4N17082R2 662 ± 3.6 132 3.12E±05 3.23E-04 1.03E-09 36 Isotypic control 686 ± 1.3 0 NB* NB* NB* NB*

*NB indicates that binding was not observed under the current experimental conditions.

- 42 045437

Table 7

Binding kinetics parameters of apple allergen (Mal d 1) binding to monoclonal antibodies to Bet v 1 at 25°C

Antibody Capture level mAL (PE) 100 nM bound analyte (PE) ka (1/Ms) kd (1/s) Kd(M) 1½ (min) N4N16943R 676 ± 0.7 -1 NB* NB* NB* NB* N4N16946R 668 ± 2.2 0 NB* NB* NB* NB* N4N16950R 635 ± 1.9 -1 NB* NB* NB* NB* N4N16960R 690 ± 8.2 1 NB* NB* NB* NB* N4N16967R 671 ±3.2 -2 NB* NB* NB* NB* N4N16971R 667 ± 3.4 25 1.61E±05 7.95E-02 4.94E-07 0.2 N4N16979R 545 ± 0.5 1 NB* NB* NB* NB* N4N16987R 668 ± 0.8 2 NB* NB* NB* NB* Н4Н16991Р 608 ± 1.4 0 NB* NB* NB* NB* N4N16992R 652 ± 0.8 3 NB* NB* NB* NB* N4N17001R 626 ± 0.8 0 NB* NB* NB* NB* N4N17015R 741 ±2.6 0 NB* NB* NB* NB* N4N17027R 767 ± 1.4 0 NB* NB* NB* NB* N4N17028R 704 ± 2.3 1 NB* NB* NB* NB* N4N17031R 727 ± 1.6 0 NB* NB* NB* NB* N4N17033R 650 ± 1.6 1 NB* NB* NB* NB* N4N17038R2 658 ± 1.5 -1 NB* NB* NB* NB* N4N17045R2 747 ± 1.3 -1 NB* NB* NB* NB* N4N17067R2 616±2 -1 NB* NB* NB* NB* N4N17082R2 662 ± 1.9 112 1.25E±06 3.74E-02 2.98E-08 0.3 Isotypic control 686 ± 1.1 0 NB* NB* NB* NB*

*NB indicates that binding was not observed under the current experimental conditions.

- 43 045437

Table 8

Parameters of the binding kinetics of celery allergen (Api g 1) binding to _____________monoclonal antibodies to Bet v 1 at 25°C_____________

Antibody mAb capture level (PE) 100 nM bound analyte (PE) k _a (1/M-s) k _d (1/s) Kd(M) 1½ (min) N4N16943R 674 ± 0.7 0 NB* NB* NB* NB* N4N16946R 666 ± 0.4 1 NB* NB* NB* NB* N4N16950R 632 ±3 0 NB* NB* NB* NB* N4N16960R 684 ±3.6 0 NB* NB* NB* NB* N4N16967R 668 ± 4.2 -1 NB* NB* NB* NB* N4N16971R 666 ± 0.7 0 NB* NB* NB* NB* N4N16979R 546 ± 1.1 0 NB* NB* NB* NB* N4N16987R 668 ± 0.8 1 NB* NB* NB* NB* Н4Н16991Р 607 ± 1.3 0 NB* NB* NB* NB* N4N16992R 651 ±2.9 1 NB* NB* NB* NB* N4N17001R 625 ± 1.3 -1 NB* NB* NB* NB* N4N17015R 740 ± 1.5 0 NB* NB* NB* NB* N4N17027R 765 ± 1.1 0 NB* NB* NB* NB* N4N17028R 705 ± 2.2 1 NB* NB* NB* NB* N4N17031R 727 ± 1.3 0 NB* NB* NB* NB* N4N17033R 648 ± 0.3 39 4.75E±05 7.91E-02 1.67E-07 0.2 N4N17038R2 659 ± 0.6 -1 NB* NB* NB* NB* N4N17045R2 747 ± 0.5 0 NB* NB* NB* NB* N4N17067R2 613 ±0.6 0 NB* NB* NB* NB* N4N17082R2 660 ±3.1 0 NB* NB* NB* NB* Isotypic control 684 ±3.1 -1 NB* NB* NB* NB*

*NB indicates that binding was not observed under the current experimental conditions.

- 44 045437

Table 9

Parameters of the binding kinetics of the common hornbeam allergen (isoforms 1A and 1B Car b 1), ____binding with monoclonal antibodies to Bet v 1 at 25°C____

Antibody mAb capture level (PE) 100 nM bound analyte (PE) ka (1/M-s) k _d (1/s) Kd(M) 1½ (min) N4N16943R 674 ± 1 26 1.52E+05 5.78E-02 3.80E-07 0.2 N4N16946R 667 ± 1.6 63 1.43E+05 2.40E-02 1.68E-07 0.5 N4N16950R 633 ± 1.6 -1 NB* NB* NB* NB* N4N16960R 687 ± 0.8 4 NB* NB* NB* NB* N4N16967R 666 ± 3.3 -1 NB* NB* NB* NB* N4N16971R 669 ± 4.6 1 NB* NB* NB* NB* N4N16979R 545 ± 0.4 1 NB* NB* NB* NB* N4N16987R 668 ± 0.3 0 NB* NB* NB* NB* Н4Н16991Р 608 ± 1.8 1 NB* NB* NB* NB* N4N16992R 653 ± 0.6 161 2.81E±05 3.37E-04 1.20E-09 34 N4N17001R 626 ± 1 0 NB* NB* NB* NB* N4N17015R 742 ± 1.8 0 NB* NB* NB* NB* N4N17027R 767 ± 0.3 41 3.14E±04 6.73E-03 2.14E-07 1.7 N4N17028R 705 ± 0.7 159 2.10E±05 5.11E-04 2.43E-09 23 N4N17031R 726 ± 3.2 126 8.04E±04 Ι,ΙΙΕ-03 1.38E-08 10 N4N17033R 650 ± 1.3 0 NB* NB* NB* NB* N4N17038R2 659 ± 0.9 105 1.51E±05 6.23E-03 4.14E-08 1.9 N4N17045R2 748 ± 1 5 NB* NB* NB* NB* N4N17067R2 616± 1 0 NB* NB* NB* NB* N4N17082R2 664 ± 1.6 69 3.90E±05 3.53E-02 9.05E-08 0.3 Isotypic control 686 ±0.7 0 NB* NB* NB* NB*

*NB indicates that binding was not observed under the current experimental conditions.

- 45 045437

Table 10

Binding kinetics parameters of the common hornbeam allergen (car b1 isoform 2) binding to monoclonal antibodies to Bet v 1 at 25°C

Antibody Capture level mAL (PE) 100 nM bound analyte (PE) ka (1/M-s) k _d (1/s) Kd(M) 11*2 (min) N4N16943R 675 ± 1.6 183 1.11E±06 3.71E-04 3.35E-10 31 N4N16946R 666 ± 0.7 63 7.69E±04 1.38E-02 1.79E-07 0.8 N4N16950R 633 ± 3 0 NB* NB* NB* NB* N4N16960R 682 ± 4.5 1 NB* NB* NB* NB* N4N16967R 663 ± 5.4 0 NB* NB* NB* NB* N4N16971R 668 ±4.1 23 2.59E±05 1.46E-01 5.64E-07 0 N4N16979R 547 ± 1.1 114 9.71E±05 2.41E-03 2.48E-09 4.8 N4N16987R 667 ± 1.3 153 1.01E±06 5.61E-03 5.56E-09 2.1 Н4Н16991Р 607 ± 3.4 93 1.07E±06 4.04E-02 3.78E-08 o, s N4N16992R 652 ± 0.6 156 1.40E±06 8.61E-04 6.17E-10 13 N4N17001R 625 ± 1.1 157 2.03E±06 3.20E-03 1.58E-09 3.6 N4N17015R 741 ± 1.6 2 NB* NB* NB* NB* N4N17027R 765 ± 1.8 42 5.84E±04 5.77E-03 9.89E-08 2.0 N4N17028R 703 ±4.1 95 9.71E±04 4.37E-03 4.50E-08 2.6 N4N17031R 727 ± 1.2 160 3.95E±05 5.17E-03 1.31E-08 2.2 N4N17033R 650 ± 1.2 111 1.34E±06 4.47E-02 3.34E-08 o, s N4N17038R2 660 ± 1.6 73 1.28E±05 6.48E-03 5.05E-08 1.8 N4N17045R2 747 ± 1.2 5 NB* NB* NB* NB* N4N17067R2 616 ± 1 1 NB* NB* NB* NB* N4N17082R2 661 ±2 166 2.18E±06 1.24E-03 5.67E-10 9.3 Isotypic control 685 ± 1.2 0 NB* NB* NB* NB*

*NB indicates that binding was not observed under the current experimental conditions.

- 46 045437

Table 11

Parameters of the binding kinetics of hazel allergen (Cor A 1) binding to monoclonal antibodies to Bet v 1 at 25°C

Antibody Capture level mAL (PE) 100 nM bound analyte (PE) k _a (1/M-s) k _d (1/s) Kd(M) tH (min) N4N16943R 673 ± 0.4 0 NB* NB* NB* NB* N4N16946R 665 ± 0.6 -1 NB* NB* NB* NB* N4N16950R 635 ±3.3 0 NB* NB* NB* NB* N4N16960R 687 ±7 0 NB* NB* NB* NB* N4N16967R 668 ± 1.7 0 NB* NB* NB* NB* N4N16971R 666 ± 5.8 0 NB* NB* NB* NB* N4N16979R 546 ± 1.1 0 NB* NB* NB* NB* N4N16987R 668 ± 0.7 0 NB* NB* NB* NB* Н4Н16991Р 607 ± 1.3 0 NB* NB* NB* NB* N4N16992R 652 ± 1.6 0 NB* NB* NB* NB* N4N17001R 626 ± 0.4 0 NB* NB* NB* NB* N4N17015R 740 ± 0.7 1 NB* NB* NB* NB* N4N17027R 766 ± 2.8 0 NB* NB* NB* NB* N4N17028R 704 ± 1.3 0 NB* NB* NB* NB* N4N17031R 727 ± 1.2 0 NB* NB* NB* NB* N4N17033R 649 ± 1.5 0 NB* NB* NB* NB* N4N17038R2 659 ±0.1 0 NB* NB* NB* NB* N4N17045R2 748 ± 0.8 0 NB* NB* NB* NB* N4N17067R2 614 ±0.7 -1 NB* NB* NB* NB* N4N17082R2 661 ±0.8 20 1.06E±05 4.18E-02 3.96E-07 0.3 Isotypic control 685 ±0.5 1 NB* NB* NB* NB*

*NB indicates that binding was not observed under the current experimental conditions.

Example 5. Blocking the binding of Bet v 1 to allergen-specific IgE with IgG antibodies to Bet v 1.

The ability of individual anti-Bet v 1 antibodies of the present invention or combinations of anti-Bet v 1 antibodies of the present invention to block the binding of Bet v 1 to plate-captured IgE from the plasma/serum of an allergic human donor was determined using ELISA. Antibodies were tested either alone or in polyclonal mixtures. For the assay, microtiter plates were coated overnight at 4°C with human FcεR1α (high affinity receptor for IgE) extracellular domain protein, which was prepared with a C-terminal mouse Fc tag (hFc8R1a-mFc; SEQ ID NO: 313). The plates were then blocked with 0.5% BSA (w/v) for 1 h at room temperature. Plasma from allergic donors was diluted and then total IgE was captured on the receptor-coated surface. A constant amount of 0.1 nM biotin-labeled native Bet v 1 (Indoor Biotechnologies, cat. no. NA-BV1-1) was premixed with antibodies to Bet v 1, at a single concentration of 1 μg/ml or in serial dilutions starting from 10 µg/ml or 1 µg/ml of each antibody and incubated for 1 hour at room temperature to allow the interaction of Bet v 1 with the antibody to reach equilibrium. The antibody-Bet v 1 mixture was then added to the IgE-coated plate for 1 hour. The plates were then washed and the amount of native Bet v 1 bound to the plate was detected using horseradish peroxidase-conjugated streptavidin (Thermo Scientific, cat. no. N200/ QJ223091), diluted 1:10000 and incubated for 1 hour at room temperature. The plates were then washed with PBS-T between each step of the ELISA protocol described above. To develop a colorimetric reaction, the TMB/H2O2 substrate (BD Pharmingen, reagent A, cat. no. 51-2602KC + reagent B, cat. no. 51-2607KC) was added to the plates and incubated for 20 min at room temperature. The reaction was stopped using 2N. sulfuric acid (H ₂ SO ₄ ; VWR, cat. no. BDH3500-1). Subsequently, the absorbance was measured using a spectrophotometer (Victor, Perkin Elmer) at 450 nm. Percent blocking was calculated using the highest antibody concentration used in each assay as described below.

- 47 045437

Blocking, % = (A450 without antibody) - (A450 at the highest antibody concentration) x 100 (A450 without antibody) antibodies to Bet v 1 were tested as individual antibodies for their ability to block the binding of Bet v 1 to plate-captured plasma IgE donor - a person with allergies using the described ELISA test. Individual monoclonal antibodies were able to partially block IgE binding to Bet v 1 by 8.5-64%, highlighting the polyclonality of IgE. 7 antibodies showed blocking ranging from 36-64%, at the highest antibody concentration tested, 10 μg/ml, as shown in table. 12.

Four antibodies to Bet v 1, H4H17082P2, H4H17038P2, H4H16987P and H4H16992P, were tested in a single point blocking assay. The combination of H4H17082P2 and H4H16992P demonstrated greater than 90% blocking in 7 of 10 IgE donors. Combinations of three and four monoclonal antibodies showed similar results and did not appear to add any additional blocking effect compared to the combination of two antibodies, H4H17082P2 and H4H16992P, as shown in Table 1. 13.

Four monoclonal antibodies, H4H17082P2, H4H17038P2, H4H16987P and H4H16992P, were subsequently tested as single antibodies and combinations of 2, 3 and 4 monoclonal antibodies in a dose response blocking assay with samples from 3 IgE donors. The results showed that the combination of two monoclonal antibodies to Bet v 1, H4H17082P2 and H4H16992P, blocked the binding of Bet v 1 to allergen-specific IgE by more than 90% and close to baseline in 3 donors, as shown in Table 1. 14. The studied combinations of 3- and 4-antibodies showed a similar breadth and intensity of blocking activity. As a positive control, purified mouse polyclonal IgG to Bet v 1 demonstrated >90% blocking.

Table 12

Antibodies to Bet v 1, blocking the binding of Bet v 1 to allergen-specific IgE

Donor ID number 23397-RV 24606-AV Antibodies % blocking of Bet vic binding by captured IgE N4N17082R2 MAV2 56.6 60.3 N4N16971R 35.4 12.8 N4N17027R 23.0 28.4 N4N17028R 19.7 16.6 N4N16946R 27.6 22.8 N4N17038R2 MAVZ 49.0 55.3 N4N16950R 32.3 20.7 N4N16987R MAV4 15.2 12.8 N4N17045R2 27.4 27.2 N4N17067R2 25.7 22.2 N4N16967R 21.8 23.5 N4N17015R 41.8 63.4 N4N16979R 33.6 12.9 Н4Н16991Р 30.0 32.6 N4N17033R 51.8 49.0 N4N16992 ΜΑΒΙ 36.7 64.4 N4N16960R 8.5 17.6 N4N17031 16.7 20.5 N4N17001R 47.2 52.7 N4N16943R 47.7 52.5

For these tests, plasma from an allergic donor was diluted 1:50.

- 48 045437

Table 13

Single antibodies and combinations of antibodies that block binding

Bet v 1 with allergen-specific IgE

Donor ID: 23658MD 23939mn 23035- B.L. 25414CW 25340- R.R. 25299- R.J. 25609- MS 26532- SS 29718MW 22627- ΜΝ Antibodies % blocking (at a concentration of 1 μg/ml of each antibody) N4N17082R2 MAV2 62 79 52 38 81 66 51 74 49 61 N4N17038R2 MAVZ 24 17 36 23 28 24 17 23 14 23 N4N16987R MAB4 27 16 -46 -4 15 9 16 4 13 12 N4N16992R ΜΑΒΙ 44 53 43 53 67 70 89 80 82 62 N4N17082R2 + N4N17038R2 70 85 73 57 90 78 65 83 60 79 N4N17082R2 + N4N16987R 74 88 21 45 84 69 65 75 61 76 N4N17082R2 + N4N16992R 79 93 72 77 91 91 98 94 94 96 N4N17038R2 + N4N16987R 47 31 16 28 41 29 33 29 28 47 N4N17038R2 + N4N16992R 58 62 51 77 83 75 93 87 87 77 N4N16987R + N4N16992R 61 61 -6 63 75 69 94 81 89 64

- 49 045437

N4N17082R2 + N4N17038R2 + N4N16987R 78 88 39 63 91 79 72 83 68 85 N4N17082R2 + N4N17038R2 + N4N16992R 83 92 65 91 96 89 98 94 94 90 N4N17082R2 + N4N16987R + N4N16992R 83 91 16 85 91 79 96 90 95 78 N4N17038R2 + N4N16987R + N4N16992R 71 69 3 82 88 71 95 86 93 76 N4N17082R2 + N4N17038R2 + N4N16987R + N4N16992R 86 93 44 89 95 84 98 94 97 87 Biotinyl. Bet v 1 (without antibody) in conc. 0.1 nM 0 0 0 0 0 0 0 0 0 0 Isotypic control -3 -2 -5 -4 -1 -4 -2 -4 -2 -3

Plasma from allergic donors was normalized to a titer of either 1:20 (6 donors), 1:10 (3 donors), or 1:9 (1 donor) Bet v 1-specific IgE and then diluted 1:50 for this assay.

Table 14

Combinations of antibodies that block the binding of Bet v 1 to allergen-specific IgE

Donor ID: 23939-MN 25340-RR 25609-MS Antibody % blocking (at a concentration of 1 μg/ml of each antibody) N4N17082R2 82.9 82.7 56.4 N4N17038R2 20.2 23.2 22.8 N4N16987R 19.6 11.1 21.6

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N4N16992R 63.3 70.2 86.9 N4N17082R2 + N4N16992R 94.8 93.8 96.5 N4N17082R2 + N4N17038R2 + N4N16992R 93.4 96.4 95.2 N4N17082R2 + N4N16987R + N4N16992R 91.5 93.1 94.2 N4N17082R2 + N4N17038R2 + N4N16987R + N4N16992R 92.6 95.4 95.7 Isotypic control 16.8 10.6 8.8 Purified mouse IgG to Bet ν 1 in conc. 333.3 nM 92.7 93.9 94.6

Example 6 Epitope mapping of anti-Bet v 1 antibodies binding to Bet v 1 using hydrogen-deuterium (H/D) exchange.

To determine the amino acid residues of Bet v 1 [(amino acids M1-N160 according to Uniprot P15494], with which H4H16992P2, H4H17082P2, H4H17038P2 and H4H16987P interact, epitope mapping was performed using H/D exchange with mass spectrometry. General description of the H/ exchange method D is set out, for example, in Ehring (1999) Analytical Biochemistry 267(2):252-259 and Engen and Smith (2001) Anal Chem 73:256A-265A.

HDX-MS experiments were performed on an integrated Waters HDX/MS platform consisting of a Leaptec HDX PAL system for deuterium labeling, a Waters Acquity M-Class (solvent management auxiliary) for sample processing and loading, a Waters Acquity M-Class (μBinary solvent management device) ) for analytical gradient column and Synapt G2-Si mass spectrometer to measure peptide mass.

The labeling solution was prepared in 10 mM PBS buffer in D2O at pD 7.0 (equivalent to pH 6.6). For deuterium labeling, 3.8 µl of natural Bet v 1 (Indoor Biotech, Cat. No. NA-BV1-1, 28 pmol/µl), Bet v 1 premixed with each antibody, or a mixture of all 4 Bet v 1 antibodies at a molar ratio of 1:1 incubated with 56.2 μl of D ₂ O-labeling solution at various time points (eg, non-deuterated control = 0 s, labeled for 1 min, 5 min, 10 min and 20 min). Deuteration was quenched by transferring 50 μL of sample to 50 μL of pre-cooled 0.2 M TCEP, 6 M guanidine chloride in 100 mM phosphate buffer, pH 2.5 (quenching buffer), and the mixed sample was incubated at 1.0°C for 2 min. . The quenched sample was then introduced into the Waters HDX Manager for online pepsin/protease XIII digestion. Digested peptides were captured on an ACQUITY UPLC VEN C18 1.7 μm, 2.1 x 5 mm VanGuard precolumn at 0°C and eluted on an ACQUITY UPLC VEN C18 1.7 μm, 1.0 x 50 mm analytical column over a 9-minute 5-minute gradient separation. 40% B (mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile). The mass spectrometer was set to a cone voltage of 37 V, a scan time of 0.5 s, and a mass/charge range of 50–1700 Th.

To identify peptides from Bet v 1, LC-MS ^E data from the non-deuterated sample were processed and a database search including Bet v 1 and its randomized sequence was performed using Waters ProteinLynx Global Server (PLGS) software. Identified peptides were imported into DynamX software and filtered using two criteria: 1) minimum number of products per amino acid: 0.3 and 2) replication file threshold: 3. DynamX software then automatically determined the deuterium uptake of each peptide based on retention time and high accuracy mass (<10 ppm) at several time points with 3 repetitions at each time point.

Using an online pepsin/protease XIII column coupled with MS ^E data collection, a total of 36 peptides from Bet v 1 were reproducibly identified in the absence or presence of antibody, representing 91.2% coverage sequences. Peptides with significantly reduced deuteration uptake when coupled to antibody combinations H4H16992P2, H4H17082P2, H4H17038P2, H4H16987P are illustrated in FIG. 1-4 and 5 respectively. The recorded peptide mass corresponds to the average value of the centroid mass of MH+ from three parallels of Bet v 1 in complex with the antibody or antibodies to Bet v 1.

As shown in FIG. 1, peptides corresponding to amino acids 23-43 (FILDGDNLFPKVAPQAISSVE, SEQ ID NO: 307) were characterized by a slower rate of deuteration in the presence of H4H16992P.

As shown in FIG. 2, peptides corresponding to amino acids 44-56 (NIEGNGGPGTIKK, SEQ ID NO: 308) were characterized by a slower rate of deuteration in the presence of H4H17082P.

As shown in FIG. 3, peptides corresponding to amino acids 2-19 (GVFNYETETTSVIPAARL, SEQ ID NO: 309) were characterized by a slower rate of deuteration in the presence of H4H17038P2.

As shown in FIG. 4, peptides corresponding to amino acids 57-70 (ISFPEGFPFKYVKD, SEQ ID NO: 310) and 81-96 (KYNYSVIEGGPIGDTL, SEQ ID NO: 315) were characterized by a slower rate of deuteration in the presence of H4H16987P.

As shown in FIG. 5, peptides corresponding to amino acids 23-43 (FILDGDNLFPKVAPQAISSVE, SEQ ID NO: 307), amino acids 44-56 (NIEGNGGPGTIKK, SEQ ID NO: 308), 2-19 (GVFNYETETTSVIPAARL, SEQ ID NO: 309), amino acids 57-70 (ISFPEGFPFKYVKD, SEQ ID NO: 310) and 81-96 (KYNYSVIEGGPIGDTL, SEQ ID NO: 315), were characterized by slower deuteration rates in the presence of a combination of 4 antibodies to Bet v 1 (H4H16992P, H4H17082P, H4H17038P2 and H4H16987P).

In addition, a low level of protection was observed for peptide 23-43 in the presence of H4H17082P, H4H17038P2 and H4H16987P, indicating that this region could represent a secondary epitope for these monoclonal antibodies.

Example 7: Effect of anti-Bet v 1 antibodies on an in vivo model of passive cutaneous anaphylaxis (PCA).

To determine the effectiveness of the anti-Bet v 1 antibodies of the present invention in blocking allergen-induced mast cell degranulation, an in vivo passive cutaneous anaphylaxis (PCA) model was used. This model involves intradermal injection of an allergen-specific immune serum into a local area on the skin, followed by intravenous injection of antigen along with dye. The allergic response causes dilation of capillaries and increased vascular permeability at the site of sensitization, which leads to the preferential accumulation of dye in this place. The dye can be extracted from the tissue and quantified spectrophotometrically. Extravasation of dye into tissue sensitized with the immune serum of interest can then be compared with extravasation into tissue sensitized with the irrelevant immune serum.

Immune sera were prepared for use in the assay by immunizing Balb/c mice with 5 μg of native Bet v 1 protein (Indoor Biotechnologies, cat. no. NA-BV1-1) in a solution of 1 mg/ml alum in 1x phosphate buffered saline (PBS). on day 0. A week later (day 7), sensitized mice were given a booster dose of 5 μg of natural Bet v 1 protein in a solution of 1 mg/ml alum in 1x PBS. Two weeks after the boost dose, mice were given intranasal airway challenge with 0.5 μg of native Bet v 1 protein in 20 μl of PBS on days 21, 24, and 28. Mice were then sacrificed on day 31 and serum was collected. Total IgE concentration in isolated immune serum was determined using the OptEIA™ ELISA kit (BD Biosciences, Cat. No. 555248) according to the manufacturer's instructions. The final concentration of Bet v 1 immune serum was diluted to 2600 ng/ml IgE in PBS.

Immune sera used as a negative control in the assay were prepared by immunizing Balb/c mice with 5 μg of natural Fel d 1 protein purified from cat hair extract (Indoor Biotechnologies, Cat. No. LTN-FD1-1) in solution 1 mg/ml alum in PBS on day 0. Mice were given a booster dose of 5 μg of Fel d 1 protein with a solution of 1 mg/ml alum in PBS on days 14 and 21. One week after the last booster dose (day 28), mice were sacrificed and serum was collected. Total IgE concentration in isolated immune serum was determined using the OptEIA™ ELISA kit (BD Biosciences, Cat. No. 555248) according to the manufacturer's instructions. The final concentration of immune sera was diluted to 2500 ng/ml IgE in PBS.

For PCA assays, groups of Balb/c mice (n>5 per experiment) were first subcutaneously injected with either an isotype control antibody, an anti-Bet v 1 antibody, or a combination of anti-Bet v 1 antibodies at a dose of 1 mg/kg (total antibody dose). . Three days after antibody administration, 10 μl of either 1.5 ng Bet v 1 immune serum or 3 ng negative control immune serum were injected into the right and left ears of mice in each group, respectively. Twenty-four hours after local administration of allergen-specific immune sera, mice were stimulated by intravenous injection (100 μl per mouse) of a solution of 1 μg/ml natural Bet v 1 dissolved in PBS containing 0.5% (wt. /vol.) Evans blue dye (Sigma Aldrich, cat. No. E2129).

One hour after antigen stimulation, mice were sacrificed, their ears were cut off, placed in 1 ml of formamide and then incubated for 3 days at 50-56°C to extract Evans blue dye.

The ear tissue was then removed from the formamide, blotted to remove excess liquid, and weighed. Two hundred microliter aliquots of each formamide extract were transferred into 96-well plates in duplicate and then their absorbance was measured at 620 nm. The measured absorbance was converted to Evans blue concentration using a standard curve and reported as ng Evans blue per mg tissue. Means ± standard deviations are shown in Table 15 for each group. Mean differences compared to isotype controls were calculated using Bonferroni's multiple comparisons test in GraphPad Prism.

As shown in table. 15, in the first study, a single anti-Bet v 1 antibody, H4H17082P2, did not demonstrate a significant reduction in dye extravasation compared with the isotype control. In contrast, in Study 2, the combination of two anti-Bet v 1 antibodies according to the present invention (H4H16992P and H4H17082P2) and the combination of four anti-Bet v 1 antibodies according to the present invention (H4H16992P, H4H17082P2, H4H17038P2 and H4H16987P) showed a significant reduction in dye extravasation compared with treatment with isotype control, with a decrease of 35.26 and 36.49 ng/mg, respectively. Similarly, in Study 3, the combination of two anti-Bet v 1 antibodies of the present invention (H4H16992P and H4H17082P2) again demonstrated a significant reduction in dye extravasation compared to isotype control treatment with a reduction of 41.09 ng/mg. As previously demonstrated in Study 1, in Study 3 a single anti-Bet v 1 antibody, H4H17082P2, did not demonstrate a significant reduction in dye extravasation compared with the isotype control. However, another single antibody, H4H16992P, showed a significant reduction in dye extravasation compared to the isotype control with a reduction of 25.86 ng/mg. From the studies conducted, a single antibody H4H16992P, a combination of two antibodies H4H17082P2 + H4H16992P, and a combination of four antibodies H4H17082P2 + H4H16992P + H4H17038P2 + H4H16987P were able to block mast cell degranulation, as evidenced by a significant decrease in dye extravasation compared to the isotype control in the passive model cutaneous anaphylaxis in vivo as determined by two-way ANOVA with Bonferroni post hoc test. Antibody H4H17082P2 alone failed to block mast cell degranulation, as indicated by increased dye extravasation compared with isotype in two studies performed. The number of mice used per group (n) is indicated in parentheses in the table.

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Table 15

Effect of antibodies to Bet v 1 in an in vivo model of passive cutaneous anaphylaxis

Allergen Difference Difference negative average for Betv 1 (ng Evans average for Treatment group control (ng Evans compared to blue/mg compared to isotypic isotypic blue/mg tissue ±SD) control tissue ±SD) control Study 1 N4N17082R2 (n=8) 2.1±0.9 -0.31 46.6 ±17.3 7.5 Study 2 N4N17082R2 + 6.3±3.8 1,807 8.3±7.7 -35.26 (****) N4N16992R (n=10) N4N17082R2 + N4N16992R + 7.6±5.5 3,090 7.1±4.1 -36.49 (****) N4N17038R2 + N4N16987R (n=10) Study 3 N4N17082R2 (n=5) 6.5±1.4 -0.311 89.4±28.3 27.37 (*) Н4Н16992Р (n=5) 6.4±2.1 -0.324 36.2±14 -25.86 (*) N4N17082R2 + 3.6±.31 -3.118 21±9.6 -41.09 (***) Н4Н16992Р (n=5)

mg/kg is the total antibody concentration used for all antibody treatment groups.

*P<.05, ***P<.001, ***P<.0001 p=number of mice in each group.

Example 8: Effect of anti-Bet v 1 antibodies against three different birch pollen extracts in an in vivo model of passive cutaneous anaphylaxis (PCA).

Anti-Bet v 1 antibodies provided herein were tested for effectiveness in blocking allergen-induced mast cell degranulation in an in vivo model of passive cutaneous anaphylaxis (PCA). Allergen-specific immune serum was administered transdermally by injection into a local area on the skin, followed by intravenous injection of the antigen along with the dye. The allergic response causes dilation of capillaries and increased vascular permeability at the site of sensitization, which leads to the preferential accumulation of dye in this place. The dye can be extracted from the tissue and quantified spectrophotometrically. Extravasation of dye into tissue sensitized with the immune serum of interest can then be compared with extravasation into tissue sensitized with an irrelevant immune serum.

Immune sera to natural Bet v 1, birch pollen extract (BPE) Betula pendula (also known as Betula verrucosa, BPE Betula nigra and BPE Betula populifolia) were prepared for use in the assay by immunizing Balb/c mice with 5 μg of natural Bet v 1 protein (Indoor Biotechnologies, Cat. No. NA-BV1-1, Lot No. 36164) or 5 μg BPE Betula pendula (Stallargenes Greer, Cat. No. XP527D3A25, Lot No. 277329), Betula nigra (Stallargenes Greer Cat. No. XP79D3A25, Lot No. 285077) or Betula populifolia (Stallargenes Greer Cat No. XP80D3A2.5, Lot No. 273622) in a solution of 1 mg/ml alum in 1x phosphate buffered saline (PBS) on day 0. One week later (day 7) in sensitized mice a booster dose of 5 μg of natural protein Bet v 1 or 5 μg of the corresponding BPE (Betula pendula, nigra or populifolia) in a solution of 1 mg/ml alum in 1 x PBS was administered.Two weeks after the boost dose, mice were subjected to intranasal stimulation through the respiratory tract with 0.5 μg of native Bet v 1 protein or 0.5 μg of the corresponding birch pollen extract in 20 μl of 1x PBS on days 21, 24, and 28. Mice were then sacrificed on day 31 and serum was collected. Total IgE concentration in isolated immune serum batches was determined using the OptEIA™ ELISA kit (BD Biosciences, Cat. No. 555248) according to the manufacturer's instructions. The final concentration of Bet v 1 immune sera was diluted to 2500 ng/ml IgE in PBS and the final concentration of birch pollen extract immune sera batches was diluted to 3000 ng/ml for Betula pendula, 1900 ng/ml for Betula nigra and 3700 ng/ml for Betula populifolia .

Immune sera used as a negative control in the assay were obtained by immunizing Balb/c mice with 5 μg of natural Fel d 1 protein purified from cat hair extract (Indoor Biotechnologies, Cat. No. LTN-FD1-1, Lot No. 36099 ), in a solution of 1 mg/ml alum in 1x PBS. Mice were administered a booster dose of 5 μg of Fel d 1 protein with a solution of 1 mg/ml alum in 1x PBS on and 21 days. One week after the last booster dose (day 28), mice were sacrificed and serum was collected. Total IgE concentration in isolated immune serum was determined using the OptEIA™ ELISA kit (BD Biosciences, Cat. No. 555248) according to the manufacturer's instructions.

The final concentration of immune sera was diluted to 4800 ng/ml IgE in PBS.

For PCA assays, groups of Balb/c mice (n>4 per experiment, repeated three times) were first subcutaneously injected with either an isotype control antibody or a combination of two anti-Bet v 1 antibodies at a dose of 1 mg/kg (total antibody dose). Three days after antibody administration, 10 μl of either 1 ng Bet v 1 immune serum, 25 ng Betula pendula immune serum, 25 ng Betula nigra immune serum, or 25 ng Betula populifolia immune serum was injected into the right of mice in each respective group. The left ears were injected with 1 ng or 25 ng Fel d 1 (negative control) to match the immune serum concentration in the corresponding right ear. Twenty-four hours after local administration of allergen-specific immune sera, mice were challenged by intravenous injection (100 μl per mouse) of a 1 μg/ml solution of natural Bet v 1 (cat. no. NA-BV1-1, lot no. 36164) or 1 µg/ml of the corresponding BPE (Stallargenes Greer, Cat. No. XP527D3A25, Lot No. 277329, Cat. No. XP79D3A25, Lot No. 285077 and Cat. No. XP80D3A2.5, Lot No. 273622) dissolved in 1x PBS containing 0. 5% (w/v) Evans blue dye (Sigma Aldrich, cat. no. E2129). One hour after antigen stimulation, mice were sacrificed, their ears were cut off, placed in 1 ml of formamide and then incubated for 3 days at 50°C to extract Evans blue dye. The ear tissue was then removed from the formamide, blotted to remove excess liquid, and weighed. Two hundred microliter aliquots of each formamide extract were transferred into 96-well plates in duplicate. The optical density of the resulting supernatants was measured at 620 nm. The measured absorbance was converted to Evans blue concentration using a standard curve and reported as ng Evans blue per mg ear tissue. Mean values ± standard deviation are given in table. 16 for each group. Mean differences compared to isotype controls were calculated using Bonferroni's multiple comparisons test in GraphPad Prism.

In table 16 demonstrates the effectiveness of the combination of two antibodies to Bet v 1, H4H16992P and H4H17082P2, as indicated by a significant decrease in dye extravasation compared to the isotype control in all studied groups. As shown, a combination of two monoclonal antibodies to Bet v 1, H4H17082P2/H4H16992P, blocks mast cell degranulation in an in vivo model of passive cutaneous anaphylaxis in response to sensitization and subsequent stimulation with natural Bet v 1 compared to isotype control, demonstrating a significant reduction extravasation of dye, amounting to 88.34. Similarly, a decrease in dye extravasation was also observed in the H4H17082P2/H4H16992P treated group for all three birch pollen extracts compared to the corresponding isotype control groups with statistically significant reductions of 62.52 for Betula pendula, 71.19 for Betula nigra and 91.47 for Betula populifolia. The difference in means compared to the isotype control was calculated using two-way analysis of variance with Bonferroni's post hoc test. The number of mice used per group (n) is indicated in parentheses in the tables.

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Table 16

Effect of antibodies to Bet v 1 in a model of passive cutaneous anaphylaxis (PCA) in vivo

Sensitization and treatment Negative allergen control (ng Evans blue/mg tissue ±SD) Difference average compared to isotype control Betv 1 or BPE (ng Evans blue/mg tissue +SD) Difference average compared to isotype control 1 ng nBet ν 1 N4N17082R2+ N4N16992R (p>14) 7.59 + 3.03 0.7623 9.56 + 3.53 -88.34 (****) 25 ng Betula pendula Н4Н17082Р2+ Н4Н16992Р (n>14) 7.76 + 3.38 0.7337 15.54 + 8.21 -62.52 (****) 25 ng Betula nigra Н4Н17082Р2+ Н4Н16992Р (n>14) 8.16 + 4.44 1.05 23.94 ± 18.32 -71.19 (****) 25 ng Betula populifolia Н4Н17082Р2+ Н4Н16992Р (n>14) 7.11 + 2.37 0.5416 10.09 + 5.90 -91.47 (****)

mg/kg is the total antibody concentration used for all antibody treatment groups. *P<.05, ***P<.001, ***P<.0001 n=number of mice per group.

Example 9. Cross competition between monoclonal antibodies to Bet v 1.

Binding competition within a panel of different anti-Bet v 1 mAbs was determined using real-time label-free biolayer interferometry analysis on the Octet HTX biosensor platform (Pall ForteBio Corp.). The entire experiment was performed at 25°C in a buffer containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v/v. surfactant Tween-20, 1 mg/ml BSA, pH 7.4 (HBS-EBT) with shaking the plate at a speed of 1000 rpm. To assess whether two antibodies are able to compete with each other for binding to their respective epitopes on a recombinant mutant Bet v 1 expressed with a C-terminal myc-myc-hexahistidine tag (mutant Bet v 1-MMH; SEQ ID NO: 312) , approximately ∼0.21 nM mutant Bet v 1-MMH was first captured on Octet biosensor tips coated with anti-pentahistidine tag antibodies (Fortebio Inc, cat. no. 18-5122) by dipping the biosensor tips for 90 s into wells containing 5 µg/ml solution of mutant Bet v 1-MMN. The antigen-captured biosensor tips were then saturated with the first monoclonal antibody to Bet v 1 (then called mAb-1) by dipping into wells containing 50 μg/mL mAb-1 solution for 4 min. The biosensor tips were then sequentially immersed into wells containing a 50 μg/mL solution of a second monoclonal antibody to Bet v 1 (then called mAb-2) for 3 min. The biosensor tips were washed in HBS-EBT buffer between each experimental step. The binding response was monitored in real time throughout the experiment and the binding response was recorded at the end of each step. The binding response of mAb-2 to mutant Bet v 1-MMN, which had previously formed a complex with mAb-1, was compared and the competitive/non-competitive behavior of various monoclonal antibodies to Bet v 1 was determined, as shown in table. 17.

Three of 20 monoclonal antibodies to Bet v 1 did not bind to mutant Bet v 1-MMH and the cross-competition data were considered inconclusive.

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Table 17

Cross competition between monoclonal antibodies to Bet v 1 mAb-1 mAb-2, which competes with mAb-1

N4N17082R2 N4N16971R N4N16971R N4N17082R2 N4N17027R N4N17028R N4N16946R N4N17038R2 N4N17027R N4N16971R N4N17028R N4N16946R N4N17038R2 N4N16950R N4N17028R N4N16971R N4N17027R N4N16946R N4N17038R2 N4N16950R N4N16946R N4N16971R N4N17027R N4N17028R N4N17038R2 N4N16950R N4N17038R2 N4N16971R N4N17027R N4N17028R N4N16946R N4N16950R N4N16979R N4N16950R N4N17027R N4N17028R

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H4H16946P H4H17038P2 H4H17015P H4H16987P H4H17045P2 H4H17067P2 H4H16967P H4H17045P2 H4H16987P H4H17067P2 H4H16967P H4H17015P H4H17067P2 H4H16987P H4H17045P2 H4H16967P H4H17015P H4H16967P H4H16987P H4H17045P2 H4H17067P2 H4H17015P H4H16992P H4H17015P H4H16950P H4H17045P2 H4H17067P2 H4H16967P H4H16979P H4H16991P H4H17033P H4H16992P H4H16979P H4H17038P2 H4H17015P H4H16991P H4H17033P H4H16992P H4H16991P H4H17015P H4H16979P

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N4N17033R N4N16992R N4N17033R N4N17015R N4N16979R Н4Н16991Р N4N16992R N4N16992R N4N16967R N4N17015R N4N16979R Н4Н16991Р N4N17033R N4N16960R N4N17031R N4N16960R N4N16992R N4N17031R N4N17031R N4N16992R N4N16960R N4N16943R IC* N4N17001R IC*

*IC indicates that monoclonal antibodies to Bet v 1 did not bind to the Bet v 1MMH mutant and the cross-competition data were considered inconclusive.

Example 10. The ability of combinations of antibodies to Bet v 1 to block mast cell degranulation induced by Bet v 1 in a humanized mouse model of RSA.

To study the polyclonality of the allergen-specific IgE response in humans with birch allergy, an FcεR1α humanized mouse model was used to facilitate binding of human IgE to FcεR1α on the surface of mouse mast cells. Since human IgE cannot bind to murine FcεR1α, a genetically modified mouse was created where the endogenous murine FcεR1α was replaced with the corresponding human FcεR1α sequence and designated FcεR1α ^hu/hu . FcεR1α ^hu/hu mice were validated for use in this model, demonstrating surface expression of human FccR1a and the ability to respond to allergen:IgE activation in a passive cutaneous anaphylaxis (PCA) model in a manner comparable to wild-type mice. This RSA model involves intradermal injection of allergen-specific human serums into a local area on the skin, followed by intravenous injection of the relevant allergen along with a dye. The allergic response causes dilation of capillaries and increased vascular permeability at the site of sensitization, which leads to the preferential accumulation of dye in this place. The dye can be extracted from the tissue and quantified spectrophotometrically. Dye extravasation in tissue sensitized with the test immune serum is compared with extravasation in tissue sensitized with non-allergen-specific human sera.

Ways.

To determine the effect of anti-Bet v 1 antibodies on mast cell degranulation in this model, FcεR1α humanized mice received a subcutaneous injection of an isotype control antibody or a combination of anti-Bet v 1 antibodies on day 1. Two independent experiments were performed for each human donor, n= 5 mice per group with pooled data. The groups consisted of the following: no monoclonal antibody negative control, negative isotype control, two Bet v 1 antibody treatment group REGN5713+REGN5715 and a three Bet v 1 antibody treatment group REGN5713+REGN5714+REGN5715. The total or combined antibody concentration was 1 mg/kg or the IgG4 isotype control antibody concentration (anti-IL6R2 antibody as a negative isotype control). Three days later, serum from patients with birch allergy or serum from patients without birch allergy (negative control) was injected intradermally (ID) into the right and left ears, respectively, allowing allergen-specific IgE to bind to FcεRI on mast cells. To ensure that the experiment used the same amount of allergen-specific IgE from each donor, each immune serum injection was normalized to the ImmunoCAP® Bet v 1-specific IgE concentration of 10 kU _a /L.

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[Twenty-four hours after topical administration of allergen-specific antibodies, mice were stimulated with an intravenous injection of 1 μg of Bet v 1 diluted in PBS containing 0.5% Evans blue dye. One hour after allergen stimulation, mice were sacrificed. Evans blue dye was extracted from ear tissue and quantified spectrophotometrically using a standard curve. (See Figure 6 for a diagram of the protocol used.) The reduction in Evans Blue extravasation was calculated on average by subtracting the Evans Blue concentration (normalized to ear tissue mass) for the birch allergen-specific serum-treated ear in the antibody-treated group, B(mAb, i), from the group treated with isotype control antibody, B (isotype, mean). This number was then divided by the difference between B(isotype, mean) and the concentration of ear dye injected with non-allergen-specific serum in the antibody-treated group [N(mAb, i)], and multiplied by 100 s obtaining an overall average percentage reduction in dye extravasation (% reduction). The equation is shown below:

.% reduction (average) = 100* [B(isotype, average) - B(mAb, 1)]/[B(isotype, average) N(mAb, i)]

The increase in % reduction in dye leakage in the anti-Bet v 1 antibody-treated group compared with the negative isotype control group is a measure of the effectiveness of the anti-Bet v 1 antibody or combinations of antibodies in blocking mast cell degranulation.

Results.

In this model, the combined use of antibodies to Bet v 1, designated H4H16992P (also designated REGN5713), H4H17038P2 (also designated REGN5714), and H4H17082P2 (also designated REGN5715), demonstrated maximal blocking of the IgE-mediated response when using sera containing IgE against 3/3 of donors are allergic to birch (see Fig. 7). The use of sera from a donor with an allergy to birch 25609, H4H16992R, H4H17038R2 and H4H17082R2 in combination showed a 95% blockade of mast cell degranulation compared to the isotype control (mean difference -42.04 +/-6.9 (p <0.0001)) and the combined use of H4H16992P and H4H17082P2 showed a 93% block of mast cell degranulation compared with isotype control (mean difference -41.74 +/-3.7 (p < 0.0001)). The use of sera from a donor with an allergy to birch 23658, H4H16992R, H4H17038R2 and H4H17082R2 in combination showed a 90% blockade of mast cell degranulation compared to the isotype control (mean difference -53.67 +/7.1 (p <0.0001)) and H4H16992P in combination with H4H17082P2 showed a 74% blockade of mast cell degranulation compared to isotype control (mean difference -44.58 +/-11.4 (p < 0.0001)). As a result, the use of sera from a donor with an allergy to birch 25414, H4H16992R, H4H17038R2 and H4H17082R2 in combination showed a 92% blockade of mast cell degranulation compared to the isotype control (mean difference -39.72 +/-7.5 (p < 0. 0001)) and H4H16992P in combination with H4H17082P2 showed an 80% blockade of mast cell degranulation compared to the isotype control (mean difference -34.27 +/-7.8 (p < 0.0001)).

Example 11: Ability of anti-Bet v 1 antibody combinations to block basophil activation in a phosphorescence flow cytometry assay using phospho-Erk.

The human IgE response was examined by testing the effect of various combinations of anti-Bet v 1 antibodies H4H16992P (also referred to as REGN5713), H4H17038P2 (also referred to as REGN5714) and H4H17082P2 (also referred to as REGN5715) on inhibition of basophil activation using samples from 8 individuals allergic to birch More specifically, to assess FcsR binding and activation, basophils were examined in a phosphorescence flow cytometry-based functional assay that measures phosphorylation of ERK kinase, a proxy for basophil activation and degranulation (Liu, Y. et al. (2007), J Exp Med 204 , 93-103.

Ways.

Blood was collected from patients with birch allergy (n=8) and PBMCs were isolated by density centrifugation on a Ficoll layer, washed, resuspended and seeded as individual spots in a 96-well format. A 2-fold stimulation plate was prepared in parallel, which included the dose effect of purified Bet v 1, as well as the effects of doses of anti-Bet v 1 antibodies and combinations of antibodies (2.56 pM-200 nM) mixed with a constant dose (final concentration 100 pM ) purified natural Bet v 1. Cells were stimulated and then stained with an antibody cocktail containing pErk-Alexa 488, CD123-BUV395 and HLA-DR-APC antibodies. After staining, data were acquired using the LSR-Fortessa instrument and analyzed by calculating the MFI of phosphorylated Erk staining within the basophil gate. The percentage of maximum inhibition was calculated as: 100-((100x maximum antibody response)/isotype response). The maximum antibody response was the average median fluorescence intensity (MFI) of phosphorylated Erk at the top three antibody doses on the dose response curve (plateau of the curve) minus the baseline MFI value (average of unstimulated samples in parallel), and the isotype response was the average of all MFI values in a dose response isotype control antibody produced by Regeneron (REGN1945 anti-Fel d 1 IgG4 ^P antibody)

- 60 045437 minus the original MFI value.

Results.

Basophils from all 8 individuals with birch pollen allergy responded to Bet v 1 stimulation with varying intensities. See fig. 8. H4H16992P, H4H17038P2 and H4H17082P2 inhibited at least 70% of basophil activation in 8/8 donors, while the combination of H4H16992P with H4H17082P2 achieved the same amount of inhibition in 6/8 donors. Notably, individual antibodies, when tested separately, showed a high degree of variability in their ability to influence allergen binding to IgE. H4H16992P achieved >70% blockade in 3/8 donors, and H4H17082P2 achieved 70% blockade of basophil activation in 4/8 donors. H4H17038P2 demonstrated 70% blocking in only 1/8 of the donors tested.

Example 12. Determination of simultaneous binding of three monoclonal antibodies to Bet v 1 with natural Bet v 1.

This experiment was performed to ensure that the binding epitopes of the three selected anti-Bet v 1 monoclonal antibodies were unique and that, regardless of the order of binding of the monoclonal antibodies, steric hindrance did not occur when the three antibodies were bound simultaneously. In addition, order-dependent competition between three anti-Bet v 1 monoclonal antibodies was assessed.

Simultaneous binding of three anti-Bet v 1 monoclonal antibodies to the same Bet v 1 was determined using the Biacore 3000 real-time label-free surface plasmon resonance biosensing platform (GE Healthcare). The entire experiment was performed at 25°C in a running buffer containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v/v. surfactant Tween-20, pH 7.4 (HBS-ET). Antibodies were immobilized on different surfaces of the CM5 sensor using EDC/NHS chemistry to achieve immobilization levels of 5000-13000 RU. REGN1945 (monoclonal antibody against Fel d 1) was also immobilized as a negative control. Natural Bet v 1 (nBet v 1), 10 nM or 20 nM, was injected on top of various sensor surfaces immobilized with monoclonal antibodies to Bet v 1 for 10-12 seconds, followed by sequential injection of different monoclonal antibodies to Bet v 1 for 6 min at a flow rate of 15 µl/min.

The binding of various anti-Bet v 1 monoclonal antibodies to nBet v 1 bound to the monoclonal antibody-immobilized sensor surface was measured using Scrubber 2.0c. The results are shown in table. 18. A binding signal of less than 1 RU (RU, resonance unit) indicates that no binding was observed upon administration of the anti-Bet v 1 monoclonal antibody, while a higher binding signal (greater than 2 RU) represents no competition. All three anti-Bet v 1 monoclonal antibodies included in this example were able to bind nBet v 1 simultaneously, and the binding reaction was not affected by the order in which the antibodies were added.

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Claims (41)

Table 18 Competition of simultaneous binding of antibodies to Bet v 1 Sequential binding of 3 monoclonal antibodies to Bet ν 1 Monoclonal antibody to Bet ν 1, immobilized on the surface Communication nBet ν 1 (PE) mAb-1 Communication mAb-1 (PE) mAb-2 Communication mAb-2 (PE) mAb-3 Communication mAb-3 (PE) REGN5713 10 REGN5713 0 REGN5714 59 REGN5715 44 eleven 0 REGN5715 48 REGN5714 53 10 REGN5714 59 REGN5713 -3 REGN5715 43 10 60 REGN5715 43 REGN5713 -6 10 REGN5715 46 REGN5713 -4 REGN5714 54 10 46 REGN5714 54 REGN5713 -5 REGN5714 14 REGN5713 67 14 63 13 REGN5714 1 13 1 12 REGN5715 42 12 40 REGN5714 -4 REGN5715 38 REGN5713 48 REGN5715 35 REGN5713 47 REGN5714 -2 REGN5715 40 REGN5714 -5 REGN5715 thirty REGN5713 42 REGN5714 -3 REGN5713 44 REGN5715 14 REGN5713 62 REGN5714 69 REGN5715 -5 14 60 REGN5715 -4 REGN5714 67 13 REGN5714 73 REGN5713 56 REGN5715 -5 13 73 REGN5715 -3 REGN5713 54 12 REGN5715 -1 REGN5713 51 REGN5714 65 12 0 REGN5714 69 REGN5713 47 Data represent the average of at least 3 independent injections of anti-Bet v 1 monoclonal antibody per nBet v 1 complex and immobilized anti-Bet v 1 monoclonal antibody. Summary. Regardless of the order of antibody binding to Bet v 1, there was no competition to prevent all three antibodies from binding simultaneously, suggesting that REGN5713, REGN5714, and REGN5715 bind to non-overlapping distinct epitopes. CLAIM 1. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to natural Bet v 1 or birch pollen extract (BPE), wherein the antibody or fragment thereof contains: heavy chain complementarity determining region (HCDR) 1 containing the amino acid sequence of SEQ ID NO: 292; HCDR2 containing the amino acid sequence SEQ ID NO: 294; HCDR3 containing the amino acid sequence SEQ ID NO: 296; a light chain complementarity determining region (LCDR)1 containing the amino acid sequence of SEQ ID NO: 300; LCDR2 containing the amino acid sequence SEQ ID NO: 302; and LCDR3 containing the amino acid sequence of SEQ ID NO: 304. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is a fully human monoclonal antibody. 3. The antibody or antigen binding fragment thereof according to claim 1, wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region (HCVR) amino acid sequence set forth in SEQ ID NO: 290 and a light chain variable region (LCVR) amino acid sequence set forth in SEQ ID NO: 298. 4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the BPE is a Betula pendula BPE, a Betula nigra BPE, or a Betula populifolia BPE. 5. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof cross-reacts with one or more allergens selected from the group consisting of Aln g1, Cor a1, Car b1, Que a1, Api g2, Api g1, Dau c1, Mal d1, Ost c1, Fag s1 and Cas s1. 6. The antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the antibody or antigen-binding fragment thereof binds Bet v 1 with a K _D equal to or less than 10-8 M, as measured by surface plasmon resonance. 7. An antibody or an antigen-binding fragment thereof according to claim 1, where the antibody or its antigen-binding fragment - 62 045437 this fragment binds to a fragment of the amino acid sequence Bet v 1 selected from the group consisting of SEQ ID NOs: 307, 308, 309, 310 and 311. 8. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof inhibits the binding of natural Bet v 1, BPE Betula pendula, BPE Betula nigra or BPE Betula populifolia with allergen-specific IgE. 9. A nucleic acid molecule encoding an isolated monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8. 10. An expression vector containing a nucleic acid molecule according to claim 9. 11. A host cell containing the expression vector according to claim 10. 12. A pharmaceutical composition containing a therapeutically effective amount of a first antibody or an antigen-binding fragment thereof according to claim 1 and a second isolated monoclonal antibody or an antigen-binding fragment thereof that binds to natural Bet v 1 or birch pollen extract (BPE), containing: (b) HCDR1 containing the amino acid sequence of SEQ ID NO: 148; HCDR2 containing the amino acid sequence SEQ ID NO: 150; HCDR3 containing the amino acid sequence SEQ ID NO: 152; a light chain complementarity determining region (LCDR)1 containing the amino acid sequence of SEQ ID NO: 156; LCDR2 containing the amino acid sequence SEQ ID NO: 158; and LCDR3 containing the amino acid sequence SEQ ID NO: 160. 13. A pharmaceutical composition containing a therapeutically effective amount of a first antibody or an antigen-binding fragment thereof according to claim 1 and a second isolated monoclonal antibody or an antigen-binding fragment thereof that binds to natural Bet v 1 or birch pollen extract (BPE), containing: (c) HCDR1 containing the amino acid sequence of SEQ ID NO: 100; HCDR2 containing the amino acid sequence SEQ ID NO: 102; HCDR3 containing the amino acid sequence SEQ ID NO: 104; LCDR1 containing the amino acid sequence SEQ ID NO: 108; LCDR2 containing the amino acid sequence SEQ ID NO: 110; and LCDR3 containing the amino acid sequence of SEQ ID NO: 112. 14. A pharmaceutical composition containing a therapeutically effective amount of a first antibody or an antigen-binding fragment thereof according to claim 1 and a second isolated monoclonal antibody or an antigen-binding fragment thereof that binds to natural Bet v 1 or birch pollen extract (BPE), containing: (d) HCDR1 containing the amino acid sequence of SEQ ID NO: 116; HCDR2 containing the amino acid sequence SEQ ID NO: 118; HCDR3 containing the amino acid sequence SEQ ID NO: 120; LCDR1 containing the amino acid sequence SEQ ID NO: 124; LCDR2 containing the amino acid sequence SEQ ID NO: 126; and LCDR3 containing the amino acid sequence SEQ ID NO: 128; and a pharmaceutically acceptable excipient. 15. The pharmaceutical composition according to claim 12, additionally containing a third isolated monoclonal antibody or an antigen-binding fragment thereof, which binds to natural Bet v 1 or BPE, containing: (c) HCDR1 containing the amino acid sequence of SEQ ID NO: 100; HCDR2 containing the amino acid sequence SEQ ID NO: 102; HCDR3 containing the amino acid sequence SEQ ID NO: 104; LCDR1 containing the amino acid sequence SEQ ID NO: 108; LCDR2 containing the amino acid sequence SEQ ID NO: 110; and LCDR3 containing the amino acid sequence of SEQ ID NO: 112. 16. The pharmaceutical composition according to claim 12, additionally containing a third isolated monoclonal antibody or an antigen-binding fragment thereof, which binds to natural Bet v 1 or BPE, containing: (d) HCDR1 containing the amino acid sequence of SEQ ID NO: 116; HCDR2 containing the amino acid sequence SEQ ID NO: 118; HCDR3 containing the amino acid sequence SEQ ID NO: 120; LCDR1 containing the amino acid sequence SEQ ID NO: 124; LCDR2 containing the amino acid sequence SEQ ID NO: 126; and LCDR3 containing the amino acid sequence of SEQ ID NO: 128. 17. The pharmaceutical composition according to claim 13, additionally containing a third isolated monoclonal antibody or an antigen-binding fragment thereof, which binds to natural Bet v 1 or BPE, containing: (d) HCDR1 containing the amino acid sequence of SEQ ID NO: 116; HCDR2 containing the amino acid sequence SEQ ID NO: 118; HCDR3 containing the amino acid sequence SEQ ID NO: 120; LCDR1 containing the amino acid sequence SEQ ID NO: 124; LCDR2 containing the amino acid sequence SEQ ID NO: 126; and LCDR3 containing the amino acid sequence of SEQ ID NO: 128. - 63 045437 18. The pharmaceutical composition according to claim 15, additionally containing a fourth isolated monoclonal antibody or an antigen-binding fragment thereof, which binds to natural Bet v 1 or BPE containing: (d) HCDR1 containing the amino acid sequence of SEQ ID NO: 116; HCDR2 containing the amino acid sequence SEQ ID NO: 118; HCDR3 containing the amino acid sequence SEQ ID NO: 120; LCDR1 containing the amino acid sequence SEQ ID NO: 124; LCDR2 containing the amino acid sequence SEQ ID NO: 126; and LCDR3 containing the amino acid sequence of SEQ ID NO: 128. 19. Pharmaceutical composition according to any one of claims 12-18, formulated for subcutaneous or intravenous administration. 20. A pharmaceutical composition containing a therapeutically effective amount of a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8 together with one or more pharmaceutically acceptable excipients. 21. Pharmaceutical composition according to claim 20, containing: (a) an isolated monoclonal antibody or antigen binding fragment thereof comprising HCVR having the amino acid sequence of SEQ ID NO: 290 and LCVR having the amino acid sequence of SEQ ID NO: 298; and (b) an isolated monoclonal antibody or antigen binding fragment thereof comprising an HCVR having the amino acid sequence of SEQ ID NO: 146 and an LCVR having the amino acid sequence of SEQ ID NO: 154. 22. Pharmaceutical composition according to claim 20, containing: (a) an isolated monoclonal antibody or antigen binding fragment thereof comprising HCVR having the amino acid sequence of SEQ ID NO: 290 and LCVR having the amino acid sequence of SEQ ID NO: 298; and (c) an isolated monoclonal antibody or antigen binding fragment thereof comprising HCVR having the amino acid sequence of SEQ ID NO: 98 and LCVR having the amino acid sequence of SEQ ID NO: 106. 23. Pharmaceutical composition according to claim 20, containing: (a) an isolated monoclonal antibody or antigen binding fragment thereof comprising HCVR having the amino acid sequence of SEQ ID NO: 290 and LCVR having the amino acid sequence of SEQ ID NO: 298; and (d) an isolated monoclonal antibody or antigen binding fragment thereof comprising HCVR having the amino acid sequence of SEQ ID NO: 114 and LCVR having the amino acid sequence of SEQ ID NO: 122. 24. Pharmaceutical composition according to claim 20, containing: (a) an isolated monoclonal antibody or antigen binding fragment thereof comprising HCVR having the amino acid sequence of SEQ ID NO: 290 and LCVR having the amino acid sequence of SEQ ID NO: 298; (b) an isolated monoclonal antibody or antigen binding fragment thereof comprising HCVR having the amino acid sequence of SEQ ID NO: 146 and LCVR having the amino acid sequence of SEQ ID NO: 154; and (c) an isolated monoclonal antibody or antigen binding fragment thereof comprising HCVR having the amino acid sequence of SEQ ID NO: 98 and LCVR having the amino acid sequence of SEQ ID NO: 106. 25. Pharmaceutical composition according to claim 20, containing: (a) an isolated monoclonal antibody or antigen binding fragment thereof comprising HCVR having the amino acid sequence of SEQ ID NO: 290 and LCVR having the amino acid sequence of SEQ ID NO: 298; (b) an isolated monoclonal antibody or antigen binding fragment thereof comprising HCVR having the amino acid sequence of SEQ ID NO: 146 and LCVR having the amino acid sequence of SEQ ID NO: 154; and (d) an isolated monoclonal antibody or antigen binding fragment thereof comprising HCVR having the amino acid sequence of SEQ ID NO: 114 and LCVR having the amino acid sequence of SEQ ID NO: 122. 26. Pharmaceutical composition according to claim 20, containing: (a) an isolated monoclonal antibody or antigen binding fragment thereof comprising HCVR having the amino acid sequence of SEQ ID NO: 290 and LCVR having the amino acid sequence of SEQ ID NO: 298; (c) an isolated monoclonal antibody or antigen binding fragment thereof comprising HCVR having the amino acid sequence of SEQ ID NO: 98 and LCVR having the amino acid sequence of SEQ ID NO: 106; and (d) an isolated monoclonal antibody or antigen-binding fragment thereof containing - 64 045437 HCVR having the amino acid sequence of SEQ ID NO: 114, and LCVR having the amino acid sequence of SEQ ID NO: 122. 27. Pharmaceutical composition according to claim 20, containing: (a) an isolated monoclonal antibody or antigen binding fragment thereof comprising HCVR having the amino acid sequence of SEQ ID NO: 290 and LCVR having the amino acid sequence of SEQ ID NO: 298; (b) an isolated monoclonal antibody or antigen binding fragment thereof comprising HCVR having the amino acid sequence of SEQ ID NO: 146 and LCVR having the amino acid sequence of SEQ ID NO: 154; (c) an isolated monoclonal antibody or antigen binding fragment thereof comprising HCVR having the amino acid sequence of SEQ ID NO: 98 and LCVR having the amino acid sequence of SEQ ID NO: 106; and (d) an isolated monoclonal antibody or antigen binding fragment thereof comprising HCVR having the amino acid sequence of SEQ ID NO: 114 and LCVR having the amino acid sequence of SEQ ID NO: 122. 28. The pharmaceutical composition according to any one of claims 20 to 27, which further comprises a therapeutically effective amount of a second therapeutic agent. 29. The pharmaceutical composition according to claim 28, wherein the second therapeutic agent is selected from the group consisting of a corticosteroid, a bronchodilator, an antihistamine, epinephrine, a decongestant, another anti-Bet v 1 antibody, and a peptide vaccine. 30. A method of preventing or reducing mast cell degranulation or blocking basophil activation associated with sensitization by natural Bet v 1, BPE Betula pendula, BPE Betula nigra or BPE Betula populifolia, wherein the method involves administering an antibody or an antigen-binding fragment thereof according to any one of claims 1- 8 or a pharmaceutical composition according to any one of claims 12-29 to a patient in need thereof. 31. Use of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8, or a pharmaceutical composition according to any of claims 12 to 29, in the preparation of a medicament for preventing or reducing mast cell degranulation or blocking basophil activation associated with sensitization by natural Bet v 1, BPE Betula pendula, BPE Betula nigra or BPE Betula populifolia. 32. A method of treating a patient who exhibits sensitivity or allergic response to Fagales protein, Fagales allergen, birch pollen or extract thereof, or Bet v 1 protein, or treating at least one symptom or complication associated with sensitivity or allergic response to to Fagales protein, Fagales allergen, birch pollen or an extract thereof or Bet v 1 protein, the method comprising administering an effective amount of one or more isolated monoclonal antibodies or antigen-binding fragments thereof according to any one of claims 1 to 8, or a pharmaceutical composition according to any one of claims .12-29 to the patient in need. 33. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 or a pharmaceutical composition according to any one of claims 12 to 29 in the preparation of a medicament for treating a patient who exhibits sensitivity or an allergic response to Fagales protein, Fagales allergen, birch pollen or an extract thereof or Bet v 1 protein, or for the treatment of at least one symptom or complication associated with a sensitivity or allergic response to Fagales protein, Fagales allergen, birch pollen or an extract thereof or Bet v 1 protein. 34. The method according to claim 32, wherein the birch pollen extract is selected from the group consisting of natural Bet v 1, BPE Betula pendula, BPE Betula nigra and BPE Betula populifolia. 35. Use according to claim 33, wherein the birch pollen extract is selected from the group consisting of natural Bet v 1, BPE Betula pendula, BPE Betula nigra and BPE Betula populifolia. 36. The method according to claim 32, wherein the Fagales allergen is selected from the group consisting of Bet v 1, Aln g1, Cor a1, Car b1 and Que a1. 37. Use according to claim 30, wherein the Fagales allergen is selected from the group consisting of Bet v 1, Aln g1, Cor a1, Car b1 and Que a1. 38. The method of claim 32, further comprising a second therapeutic agent useful for reducing the allergic response to Fagales protein, Fagales allergen, birch pollen or extract thereof, or Bet v 1 protein. 39. The use of claim 33, further comprising a second therapeutic agent useful for reducing the allergic response to Fagales protein, Fagales allergen, birch pollen or extract thereof, or Bet v 1 protein. 40. The method of claim 38, wherein the second therapeutic agent is selected from the group consisting of a corticosteroid, a bronchodilator, an antihistamine, epinephrine, a decongestant, another anti-Bet v 1 antibody, and a peptide vaccine. 41. Use according to claim 39, wherein the second therapeutic agent is selected from the group consisting of -