BR122022004090B1 - PHARMACEUTICAL COMPOSITION, AND, USE OF THE PHARMACEUTICAL COMPOSITION - Google Patents

Abstract

Provided herein are antibodies that bind Fagales allergens, related Fagales allergens, birch pollen, Bet v 1, or compositions comprising the antibodies, nucleic acids encoding the antibodies, and methods of using the antibodies. According to certain embodiments, the antibodies are fully human monoclonal antibodies that bind to Bet v 1. The antibodies are useful for binding Bet v 1 in vivo, thereby preventing binding of the allergen to preformed IgE on the surface of mast cells or basophils. . In doing so, the antibodies act to prevent the release of histamine and other inflammatory mediators from mast cells and/or basophils, ameliorating the undesirable response to Fagales allergens in sensitized individuals.

Description

SPLIT OF BR112019025150-2, DEPOSITED ON 05/31/2018 FIELD OF INVENTION

[001] The present invention relates to human antibodies and fragments of human antibodies that bind to the antigen that bind to the birch pollen allergen, Bet v 1, therapeutic compositions comprising the antibodies and methods of using the antibodies.

SEQUENCE LISTING

[002] An official copy of the sequence listing is simultaneously submitted for specification electronically via EFS-Web as an ASCII-formatted sequence listing with a file name of 10301WO01_SEQ_LIST_ST25, a creation date of May 31, 2018, and a size of 137 kilobytes. The sequence listing contained in this ASCII-formatted document is part of the specification and is incorporated herein by reference in its entirety.

BASICS OF THE INVENTION

[003] Birch is the predominant trigger in 23% of US and 14% of European allergy patients (Datamonitor Allergic Rhinitis report, July 2010) and the leading cause of type 1 spring allergies in Europe, North America, Russia and Australia (Breiteneder ET AL., EMBO J. 1989, 8 (7): 1935-8). The Bet v 1 protein is one of the main birch allergens identified in the pollen of Betula verrucosa (European white birch, also synonymous with Betula pendula) and is responsible for binding IgE in more than 95% of patients allergic to birch pollen (Breiteneder , above). Bet v 1 is a small 7-stranded anti-parallel β-sheet 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): 3084-90). WO 94/10194 refers to peptides derived from trees of the order of Fagales.

[004] Sixty percent of patients allergic to birch pollen react 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 91 meters (100 yards) from the tree. Symptoms of birch pollen allergy can range from mild rhinitis and conjunctivitis to life-threatening asthmatic responses.

[005] Immunoglobulin E (IgE) is responsible for type 1 hypersensitivity, which manifests itself in allergic rhinitis, allergic conjunctivitis, hay fever, allergic asthma, bee venom allergy and food allergies. IgE circulates in the blood and binds to high-affinity FcεR1α 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 the high-affinity receptor on the surfaces of mast cells and basophils, resulting in the cross-linking of various IgE molecules and triggering the release of histamine and other inflammatory mediators, causing the various allergic symptoms.

[006] Treatment for allergies includes steroids to suppress immune activity and bronchial dilators to relieve asthma symptoms. Desensitization therapy is also used for severely allergic patients. Peptide vaccine combinations have been tested to desensitize individuals to specific allergens, e.g., Bet v 1 (see US 9,017,689). Antibodies have been proposed as a treatment for allergies because they can block the entry of allergenic molecules into mucosal tissues or can bind the allergen before it has the opportunity to bind to the IgE bound to the high-affinity receptor on the mast. cells or basophils, thus preventing the release of histamine and other inflammatory mediators from these cells.

[007] Patent no. US 5,670,626 describes the use of monoclonal antibodies for the treatment of IgE-mediated allergic diseases, such as allergic rhinitis, allergic asthma and allergic conjunctivitis, by blocking the binding of allergens to mucosal tissue. US patent 6,849,259 describes the use of allergen-specific antibodies to inhibit allergic inflammation in an in vivo mouse model of allergy. Milk and egg-based antibody systems have been described. For example, document US2003/0003133A1 discloses the use of milk as an allergen carrier to induce oral tolerance to birch pollen and other allergens. Compositions and methods for reducing an allergic response in an animal to an allergen in the environment through the use of a molecule that inhibits the ability of the allergen to bind to mast cells have been described in WO1994/024164A2. Other antibodies to Bet v 1 were mentioned in US 2010/0034812.

[008] The present invention is directed to overcoming one or more of the problems discussed above.

BRIEF SUMMARY OF THE INVENTION

[009] Provided herein are fully human monoclonal antibodies and antigen-binding fragments thereof that bind to birch pollen, e.g., natural Bet v 1, Betula pendula birch pollen extract (BPE), Betula nigra BPE or BPE from Betula populifolia. Antibodies may be useful for binding the Bet v 1 allergen in vivo following exposure of a sensitized patient to birch allergen and as such may act to promote the release of natural Bet v 1, Betula pendula birch pollen extract ( BPE), Betula nigra BPE or Betula populifolia BPE or to block allergen binding to preformed IgE on the surface of mast cells or basophils. In doing so, the antibodies described here may prevent the release of histamine or other inflammatory mediators from mast cells or basophils, preventing or lessening the undesirable effects seen 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 birch-related allergen, such as sneezing, congestion, nasal blockage, coughing, wheezing, bronchoconstriction, rhinitis, or conjunctivitis. In some embodiments, antibodies may be able to prevent even more serious in vivo complications associated with exposure to birch pollen allergen in sensitized individuals, such as asthmatic responses, anaphylaxis, or even death.

[0010] The antibodies provided herein may be complete, for example, an IgG1 or IgG4 antibody, or may comprise only an antigen-binding portion, for example, a Fab, F(ab')2 or scFv fragment, and may be modified to affect functionality, for example, to eliminate residual effector functions (Reddy et al., 2000, J. Immunol. 164: 1925 to 1933).

[0011] A first aspect of the invention provides an isolated human monoclonal antibody or antigen-binding fragment that binds to natural Bet v 1, birch pollen extract (BPE) from Betula pendula, BPE from Betula nigra and/or BPE of Betula populifolia.

[0012] In one embodiment, the isolated human monoclonal antibody or antigen-binding fragment thereof inhibits the binding of natural Bet v 1, Betula pendula BPE, Betula nigra BPE or Betula populifolia BPE that binds to specific IgE of allergens.

[0013] In one embodiment, the 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 binding fragment the antigen thereof binds to Bet v 1 with a KD ranging from about 10-8 to about 10-11 M. In one embodiment, the 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, the isolated human antibody or antigen-binding fragment thereof binds to Bet v 1, with an equal KD ranging from about 0.66 nM to about 27.9 nM.

[0014] In one embodiment, the isolated human antibody is a fully human monoclonal antibody.

[0015] 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 may 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 Lei c 8 and Lei d 8 (kiwi), Ara h 8 (peanut), Pru ar 1 (apricot), Pru av 1 (cherry), Pru p 1 (peach), Pyr c 1 (pear), Gly m 4 (soy), Vig r 1 (mung 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.

[0016] 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.

[0017] In one embodiment, the isolated human antibody or antigen-binding fragment thereof comprises the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained in 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 the three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained in 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 in the HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs in the specified HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Broadly speaking, Kabat's definition is based on sequence variability, Chothia's definition is based on the location of structural loop regions, and AbM's definition is a compromise between Kabat's and Chothia's approaches. See, for example, Kabat, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Maryland. 1991); Al-Lazikani et al., (1997), J. Mol. Biol. 273: 927 to 948; and Martin et al., (1989), Proc. Natl. Academic. Sci. USA 86: 9268 to 9272. Public databases are also available to identify CDR sequences within an antibody.

[0018] In one embodiment, the isolated human antibody or antigen-binding fragment thereof comprises the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained in any of the heavy chain variable region (HCVR) sequences selected from the group consisting of SEQ ID NO: 114, 146, 98 and 290; and the three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained in any of the light chain variable region (LCVR) sequences selected from the group consisting of SEQ ID NO: 122, 154, 106 and 298.

[0019] 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.

[0020] 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: 114, 146, 98 and 290.

[0021] 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.

[0022] 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: 122, 154, 106 and 298.

[0023] 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) 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.

[0024] 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: 114, 146, 98 and 290; and (b) an LCVR with an amino acid sequence selected from the group consisting of SEQ ID NO: 122, 154, 106 and 298.

[0025] In one embodiment, the isolated human antibody or antigen-binding fragment thereof comprises: (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.

[0026] In one embodiment, the isolated human antibody or antigen-binding fragment thereof comprises a pair of HCVR/LCVR amino acid 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.

[0027] In one embodiment, the isolated human antibody or antigen-binding fragment thereof comprises a pair of HCVR/LCVR amino acid sequences selected from the group consisting of SEQ ID NO: 114/122, 146/154, 98/106 and 290/298.

[0028] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds to Bet v 1 comprises a pair of HCVR/LCVR amino acid sequences selected from the group consisting of SEQ ID NO: 146/154 and 290/298.

[0029] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds to Bet v 1 interacts with at least one amino acid sequence selected from the group consisting of amino acid residues ranging from about position 23 at about position 44 of SEQ ID NO: 306; amino acid residues ranging from about position 44 to about 70 of SEQ ID NO: 306; amino acid residues ranging from about 2 to about 19 of SEQ ID NO: 306; amino acid residues ranging from about 57 to about 70 of SEQ ID NO: 306; and amino acid residues ranging from about 81 to about 96 of SEQ ID NO: 306.

[0030] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds to Bet v 1 interacts with amino acid residues ranging from about position 23 to about position 44 of SEQ ID NO: 306.

[0031] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds to Bet v 1 interacts with amino acid residues ranging from about position 23 to about position 43 of SEQ ID NO: 306.

[0032] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds to Bet v 1 interacts with amino acid residues ranging from about position 44 to about position 70 of SEQ ID NO: 306.

[0033] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds to Bet v 1 interacts with amino acid residues ranging from about position 44 to about position 56 of SEQ ID NO: 306.

[0034] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds to Bet v 1 interacts with amino acid residues ranging from about position 2 to about position 19 of SEQ ID NO: 306.

[0035] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds to Bet v 1 interacts with amino acid residues ranging from about position 57 to about position 70 of SEQ ID NO: 306.

[0036] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds to Bet v 1 interacts with amino acid residues ranging from about position 57 to about position 66 of SEQ ID NO: 306.

[0037] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds to Bet v 1 interacts with amino acid residues ranging from about position 81 to about position 96 of SEQ ID NO: 306.

[0038] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds to Bet v 1 interacts with amino acid residues ranging from about position 81 to about position 89 of SEQ ID NO: 306.

[0039] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds to Bet v 1 interacts with at least one amino acid sequence selected from the group consisting of SEQ ID NO: 307, 308, 309, 310 and 311. The epitopes comprising SEQ ID NO: 307, 308, 309, 310 or 311 may be extended by 1 to 5 amino acids or 5 to 10 amino acids, at the C-terminal end or at the N-terminal end. For example, the epitope of SEQ ID NO: 311 when extended by 5 to 10 amino acids encompasses the epitope of SEQ ID NO: 115. In other words, an epitope comprising SEQ ID NO: 311, for example, includes the epitope of SEQ ID NO: 315 .

[0040] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds to Bet v 1 interacts with SEQ ID NO: 307.

[0041] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds to Bet v 1 interacts with SEQ ID NO: 308.

[0042] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds to Bet v 1 interacts with SEQ ID NO: 309.

[0043] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds to Bet v 1 interacts with SEQ ID NO: 310.

[0044] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds to Bet v 1 interacts with SEQ ID NO: 311.

[0045] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds to Bet v 1 interacts with SEQ ID NO: 315.

[0046] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds to Bet v 1 interacts with at least one amino acid sequence selected from the group consisting of SEQ ID NO: 307, 308, 309, 310, 311 and 315 and comprises a pair of HCVR/LCVR sequences selected from the group consisting of SEQ ID NO: 114/122, 146/154, 98/106 and 290/298.

[0047] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that interacts with SEQ ID NO: 307 comprises the three HCDRs contained in the heavy chain variable region of SEQ ID NO: 146 and the three LCDRs contained in the variable region of the light chain of SEQ ID NO: 154.

[0048] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that interacts with SEQ ID NO: 310 comprises the three HCDRs contained in the heavy chain variable region of SEQ ID NO: 290 and the three LCDRs contained in the variable region of the light chain of SEQ ID NO: 298.

[0049] In one embodiment, the human antibody or antigen-binding fragment thereof that binds to Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 148, 150 and 152, respectively, and sequences of amino acids LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 156, 158 and 160, respectively.

[0050] In one embodiment, the human antibody or antigen-binding fragment thereof that binds to Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 292, 294 and 296, respectively and sequences of amino acids LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 300, 302 and 304, respectively.

[0051] In one embodiment, the human antibody or antigen-binding fragment thereof that binds to Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 4, 6 and 8, respectively, and the LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 12, 14 and 16, respectively.

[0052] In one embodiment, the human antibody or antigen-binding fragment thereof that binds to Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 20, 22 and 24, respectively, and the LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 28, 30 and 32, respectively.

[0053] In one embodiment, the human antibody or antigen-binding fragment thereof that binds to Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 36, 38 and 40, respectively, and the LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 44, 46 and 48, respectively.

[0054] In one embodiment, the human antibody or antigen-binding fragment thereof that binds to Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 52, 54 and 56, respectively and sequences of amino acids LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 60, 62 and 64, respectively.

[0055] In one embodiment, the human antibody or antigen-binding fragment thereof that binds to Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 68, 70 and 72, respectively, and sequences of amino acids LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 76, 78 and 80, respectively.

[0056] In one embodiment, the human antibody or antigen-binding fragment thereof that binds to Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 84, 86 and 88, respectively, and sequences amino acids LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 92, 94 and 96, respectively.

[0057] In one embodiment, the human antibody or antigen-binding fragment thereof that binds to Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 100, 102 and 104, respectively, and the LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 108, 110 and 112, respectively.

[0058] In one embodiment, the human antibody or antigen-binding fragment thereof that binds to Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 116, 118 and 120, respectively and sequences of amino acids LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 124, 126 and 128, respectively.

[0059] In one embodiment, the human antibody or antigen-binding fragment thereof that binds to Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 132, 134 and 136, respectively and sequences of amino acids LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 140, 142 and 144, respectively.

[0060] In one embodiment, the human antibody or antigen-binding fragment thereof that binds to Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 164, 166 and 168, respectively and sequences of amino acids LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 172, 174 and 176, respectively.

[0061] In one embodiment, the human antibody or antigen-binding fragment thereof that binds to Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 180, 182 and 184, respectively, and sequences of amino acids LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 188, 190 and 192, respectively.

[0062] In one embodiment, the human antibody or antigen-binding fragment thereof that binds to Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 196, 198 and 200, respectively and sequences of amino acids LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 204, 206 and 208, respectively.

[0063] In one embodiment, the human antibody or antigen-binding fragment thereof that binds to Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 212, 214 and 216, respectively, and sequences amino acids LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 220, 222 and 224, respectively.

[0064] In one embodiment, the human antibody or antigen-binding fragment thereof that binds to Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 228, 230 and 232, respectively, and the LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 236, 238 and 234, respectively.

[0065] In one embodiment, the human antibody or antigen-binding fragment thereof that binds to Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 244, 246 and 248, respectively and sequences of amino acids LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 252, 254 and 256, respectively.

[0066] In one embodiment, the human antibody or antigen-binding fragment thereof that binds to Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 260, 262 and 264, respectively and sequences of amino acids LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 268, 270 and 272, respectively.

[0067] In one embodiment, the human antibody or antigen-binding fragment thereof that binds to Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 276, 278 and 280, respectively, and sequences amino acids LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 268, 270 and 272, respectively.

[0068] In one embodiment, the human antibody or antigen-binding fragment thereof that binds to Bet v 1 comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 284, 286 and 288, respectively and sequences of amino acids LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 268, 270 and 272, respectively.

[0069] In one embodiment, the invention provides a fully human monoclonal antibody or antigen-binding fragment thereof that binds to natural Bet v 1, birch BPE from Betula pendula, BPE from Betula nigra or BPE from Betula populifolia, in that the antibody or fragment thereof exhibits one or more of the following characteristics: (i) 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, or a substantially similar sequence with at least 90%, at least 95%, at least 98% or at least 99 % sequence identity; (ii) 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, or a substantially similar sequence that has at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (iii) comprises 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 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 that has at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (iv) comprises 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 with at least 90%, at least 95%, at least 98% or at least 99% sequence identity; 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, or a substantially similar sequence that has at least 90%, at least 95%, at least 98% or at least 99% sequence identity; 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, 188, 204, 220, 236, 252, 268 and 300, or a substantially similar sequence 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 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 KD equal to or less than 10 -8 and in a range 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 natural Bet v 1, birch BPE from Betula pendula, BPE from Betula nigra or BPE from Betula populifolia.

[0070] In one embodiment, a “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.

[0071] The invention encompasses antibodies that have a modified glycosylation pattern. In some applications, modification to remove undesirable glycosylation sites may be useful, or for example, removal of a fucose moiety to enhance antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al., 2002, JBC 277 : 26733). In other applications, modification of galactosylation can be done to modify complement-dependent cytotoxicity (CDC).

[0072] A second aspect provides an isolated antibody or antigen-binding fragment thereof that competes for specific binding to natural Bet v 1, birch BPE from Betula pendula, BPE from Betula nigra or BPE from Betula populifolia with an antibody or fragment antigen binding component comprising the complementarity determining regions (CDRs) of a heavy chain variable region (HCVR), wherein the HCVR has 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 the CDRs of a light chain variable region (LCVR), wherein the LCVR has 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.

[0073] One embodiment provides an isolated antibody or antigen-binding fragment thereof that competes for specific binding to natural Bet v 1, birch BPE from Betula pendula, BPE from Betula nigra or BPE from Betula populifolia with an antibody or fragment of antigen binding comprising the complementarity determining regions (CDRs) of a heavy chain variable region (HCVR), wherein the HCVR has an amino acid sequence selected from the group consisting of SEQ ID NO: 114, 146, 98 and 290 ; and the CDRs of a light chain variable region (LCVR), wherein the LCVR has an amino acid sequence selected from the group consisting of SEQ ID NO: 122, 154, 106 and 298.

[0074] In a related embodiment, the invention provides an isolated antibody or antigen-binding fragment thereof that competes for specific binding to natural Bet v 1, birch BPE from Betula pendula, BPE from Betula nigra or BPE from Betula populifolia with an antibody or antigen-binding fragment comprising the heavy and light chain CDRs contained in 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.

[0075] A third aspect provides an isolated antibody or antigen-binding fragment thereof that binds the same epitope on Bet v 1 as an antibody or antigen-binding fragment comprising the complementarity determining regions (CDRs) of a variable region of the heavy chain (HCVR), wherein the HCVR has 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, 178, 194, 210, 226, 242, 258, 274, 282 and 290; and the CDRs of a light chain variable region (LCVR), wherein the LCVR has 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.

[0076] One embodiment provides an isolated antibody or antigen-binding fragment thereof that binds the same epitope on Bet v 1 as an antibody or antigen-binding fragment comprising the complementarity determining regions (CDRs) of a variable region of heavy chain (HCVR), wherein the HCVR has an amino acid sequence selected from the group consisting of SEQ ID NO: 114, 146, 98 and 290; and the CDRs of a light chain variable region (LCVR), wherein the LCVR has an amino acid sequence selected from the group consisting of SEQ ID NO: 122, 154, 106 and 298.

[0077] In a related embodiment, provided herein is an isolated antibody or antigen-binding fragment thereof that binds the same epitope on Bet v 1 as an antibody or antigen-binding fragment comprising the heavy and light chain CDRs contained therein. on 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.

[0078] In a fourth aspect, the invention provides nucleic acid molecules that encode Bet v 1 antibodies or fragments thereof. Recombinant expression vectors carrying these nucleic acids and the host cells into which these vectors have been introduced are also contemplated herein, as are methods for producing the antibodies by culturing the host cells under conditions that permit the production of the antibodies and the recovery of the antibodies. antibodies produced.

[0079] In one embodiment, herein provided are nucleic acid molecules that encode a human monoclonal antibody or a fragment thereof that binds to natural Bet v 1, BPE from Betula pendula, BPE from Betula nigra or BPE from Betula populifolia.

[0080] In one embodiment, there is provided herein an antibody or fragment thereof, which comprises 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 that is at least 90%, at least 95%, at least 98% or at least 99% homology with it.

[0081] In one embodiment, HCVR is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 113, 145, 257 and 289.

[0082] 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 that has at least 90%, at least 95%, at least 98% or at least 99% homology thereto. .

[0083] In one embodiment, the LCVR is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 121, 153, 265 and 297.

[0084] 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 with 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 that has at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0085] In one embodiment, provided herein is an antibody or fragment thereof that further comprises 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 with at least 90%, at least 95%, at least 98% or at least minus 99% sequence identity; an 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 that has at least 90%, at least 95%, at least 98% or at least 99% sequence identity; an 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 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 that has at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0086] A fifth aspect provides a pharmaceutical composition comprising a therapeutically effective amount of one or more isolated human antibodies or antigen-binding fragments that bind to natural Bet v 1, birch BPE from Betula pendula, BPE from Betula nigra or BPE from Betula populifolia, together with one or more pharmaceutically acceptable excipients.

[0087] In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of two or more isolated human antibodies or antigen-binding fragments thereof that bind Bet v 1 together with one or more pharmaceutically acceptable excipients.

[0088] In one embodiment, the pharmaceutical composition comprises: a) a first isolated human monoclonal antibody or antigen-binding fragment thereof that binds to Bet v 1, which comprises an HCVR with an amino acid sequence as set forth in SEQ ID NO: 146; and an LCVR with an 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 to Bet v 1, which comprises an HCVR with an amino acid sequence selected from the group consisting of SEQ ID NO: 114, 98 and 290; and an 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.

[0089] In one embodiment, the pharmaceutical composition comprises: a) a first isolated human monoclonal antibody or antigen-binding fragment thereof that binds to Bet v 1, which comprises an HCVR with an amino acid sequence as set forth in SEQ ID NO: 290; and an LCVR with an 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 to Bet v 1, which comprises an HCVR with an amino acid sequence selected from the group consisting of SEQ ID NO: 114, 146 and 98; and an 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.

[0090] In one embodiment, the pharmaceutical composition comprises: a) a first isolated human monoclonal antibody or antigen-binding fragment thereof that binds to Bet v 1, which comprises an HCVR with an amino acid sequence as set forth in SEQ ID NO: 146; and an LCVR with an 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 to Bet v 1, which comprises an HCVR with an amino acid sequence as set forth in SEQ ID NO: 290; and an LCVR with an amino acid sequence as set forth in SEQ ID NO: 298; and c) one or more pharmaceutically acceptable excipients.

[0091] In one embodiment, the pharmaceutical composition comprises: a) an isolated first human monoclonal antibody or antigen-binding fragment thereof, which comprises an HCVR with an amino acid sequence of SEQ ID NO: 146 and an LCVR with a sequence amino acid sequence of SEQ ID NO: 154; b) a second isolated human monoclonal antibody or antigen-binding fragment thereof, which comprises an HCVR with an amino acid sequence of SEQ ID NO: 290 and an LCVR with an amino acid sequence of SEQ ID NO: 298; c) one or more isolated human monoclonal antibodies or antigen-binding fragments comprising an HCVR with an amino acid sequence selected from the group consisting of SEQ ID NO: 114 and 98 and an LCVR with a sequence of amino acids selected from the group consisting of SEQ ID NO: 122 and 106; and d) one or more pharmaceutically acceptable excipients.

[0092] In one embodiment, the pharmaceutical composition comprises: a first isolated human monoclonal antibody or antigen-binding fragment thereof that binds to Bet v 1, which comprises a pair of HCVR/LCVR amino acid sequences consisting of SEQ ID NO: 146/154; a second isolated human monoclonal antibody or antigen-binding fragment thereof that binds to Bet v 1, comprising a pair of HCVR/LCVR amino acid sequences selected from the group consisting of SEQ ID NO: 114/122, 98/106 and 290/298; and one or more pharmaceutically acceptable excipients.

[0093] In one embodiment, the pharmaceutical composition comprises: a first isolated human monoclonal antibody or antigen-binding fragment thereof that binds to Bet v 1, which comprises a pair of HCVR/LCVR amino acid sequences consisting of SEQ ID NO: 146/154; a second isolated human monoclonal antibody or antigen-binding fragment thereof that binds to Bet v 1, comprising a pair of HCVR/LCVR amino acid sequences consisting of SEQ ID NO: 290/298; and one or more pharmaceutically acceptable excipients.

[0094] 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, which comprise 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.

[0095] 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, which comprise 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.

[0096] 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 the pairs of HCVR/LCVR amino acid sequence of SEQ ID NO: 114/122, 146/154, 98/106 and 290/298; and one or more pharmaceutically acceptable excipients.

[0097] In one embodiment, the pharmaceutical composition comprises the antibody designated H4H16992P with the HCVR/LCVR amino acid sequence pair of SEQ ID NO: 146/154; the antibody designated H4H17082P2 which has the HCVR/LCVR amino acid sequence pair of SEQ ID NO: 290/298; and the antibody designated H4H17038P2 which has the HCVR/LCVR amino acid sequence pair of SEQ ID NO: 98/106.

[0098] In one embodiment, the pharmaceutical composition comprises the antibody designated H4H16992P that has the HCVR/LCVR amino acid sequence pair of SEQ ID NO: 146/154; the antibody designated H4H17082P2 which has the HCVR/LCVR amino acid sequence pair of SEQ ID NO: 290/298; the antibody designated H4H17038P2 which has the HCVR/LCVR amino acid sequence pair of SEQ ID NO: 98/106; and the antibody designated H4H16987P which has the HCVR/LCVR amino acid sequence pair of SEQ ID NO: 114/122.

[0099] In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of an isolated first human monoclonal antibody or an antigen-binding fragment that binds to Bet v 1, wherein the first antibody or fragment thereof interacts with residues of amino acids ranging from about position 23 to about position 44 of SEQ ID NO: 306, and an isolated second human monoclonal antibody or antigen-binding fragment thereof that binds to Bet v 1, wherein the second antibody or fragment thereof interacts with amino acid residues ranging from about position 44 to about position 70 of SEQ ID NO: 306, together with one or more pharmaceutically acceptable excipients.

[00100] In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of an isolated first human monoclonal antibody or an antigen-binding fragment that binds to Bet v 1, together with one or more pharmaceutically acceptable excipients, wherein the first antibody or fragment thereof interacts with amino acid residues ranging from about position 23 to about position 43 of SEQ ID NO: 306, and an isolated second human monoclonal antibody or antigen-binding fragment thereof that binds to Bet v 1, wherein the second antibody or fragment thereof interacts with amino acid residues ranging from about position 44 to about position 56 of SEQ ID NO: 306.

[00101] In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of an isolated first human monoclonal antibody or an antigen-binding fragment that binds to Bet v 1, together with one or more pharmaceutically acceptable excipients, wherein the first antibody or fragment thereof interacts with the amino acid sequence of SEQ ID NO: 307 and a second isolated human monoclonal antibody or antigen-binding fragment thereof that binds to Bet v 1, wherein the second antibody or fragment thereof interacts with the amino acid sequence of SEQ ID NO: 308, 309, 310, 311 or 315.

[00102] In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of a first isolated human monoclonal antibody or an antigen-binding fragment thereof that binds to Bet v 1, and one or more isolated human monoclonal antibodies or fragments thereof. antigen binding thereof that bind to Bet v 1, together with one or more pharmaceutically acceptable excipients, wherein the first antibody or fragment thereof interacts with at least one amino acid sequence selected from the group consisting of amino acid residues that range from about position 23 to about position 43 of SEQ ID NO: 306; amino acid residues ranging from about position 44 to about 56 of SEQ ID NO: 306; amino acid residues ranging from about 2 to about 19 of SEQ ID NO: 306; amino acid residues ranging from about 57 to about position 70 of SEQ ID NO: 306; and amino acid residues ranging from about 81 to 89 or about 81 to about 96 of SEQ ID NO: 306.

[00103] In one embodiment, the one or more isolated human monoclonal antibodies or fragments thereof 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 43 of SEQ ID NO: 306; amino acid residues ranging from about position 44 to about 56 of SEQ ID NO: 306; amino acid residues ranging from about 2 to about 19 of SEQ ID NO: 306; amino acid residues ranging from about 57 to about 70 of SEQ ID NO: 306; and amino acid residues ranging from about 81 to 89 or about 81 to about 96 of SEQ ID NO: 306, wherein at least one of the one or more isolated human monoclonal antibodies interacts with an amino acid sequence different from the first antibody isolated human monoclonal.

[00104] In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of a first isolated human monoclonal antibody or an antigen-binding fragment thereof that binds to Bet v 1, and one or more isolated human monoclonal antibodies or fragments thereof. antigen binding thereof that bind to Bet v 1, together with one or more pharmaceutically acceptable excipients, wherein the first antibody or fragment thereof interacts with the amino acid sequence of SEQ ID NO: 307 and wherein the one or more Additional antibodies or fragments interact with an amino acid sequence selected from the group consisting of SEQ ID NO: 308, 309, 310, 311 and 315.

[00105] In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of at least two isolated human monoclonal antibodies or antigen-binding fragments thereof that bind to natural Bet v 1 or BPE, together with one or more pharmaceutical excipients acceptable, in which the at least two antibodies do not compete for binding to natural Bet v 1 or BPE. In some aspects, the antibodies or antigen-binding fragments thereof are the Bet v 1 antibodies H4H16992P and H4H17082P2 comprising the HCVR/LCVR amino acid sequence pairs of SEQ ID NO: 146/154 and 290/298, respectively.

[00106] In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of at least three isolated human monoclonal antibodies or antigen-binding fragments thereof that bind to natural Bet v 1 or BPE, together with one or more pharmaceutical excipients acceptable, in which the at least three antibodies do not compete for binding to natural Bet v 1 or BPE. In some aspects, the antibodies or antigen-binding fragments thereof are the Bet v 1 antibodies H4H16992P, H4H17082P2 and H4H17038P2 comprising the HCVR/LCVR amino acid sequence pairs of SEQ ID NO: 146/154, 290/298 and 98/ 106, respectively.

[00107] In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of at least four isolated human monoclonal antibodies or antigen-binding fragments thereof that bind to natural Bet v 1 or BPE, together with one or more pharmaceutical excipients acceptable, in which the at least four antibodies do not compete for binding to natural Bet v 1 or BPE. In some aspects, the antibodies or antigen-binding fragments thereof are the Bet v 1 antibodies H4H16992P, H4H17082P2, H4H17038P2 and H4H16987P comprising the HCVR/LCVR amino acid sequence pairs of SEQ ID NO: 146/154, 290/298 , 98/106 and 114/122, respectively.

[00108] In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of an isolated human monoclonal antibody or antigen-binding fragment thereof that binds to 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.

[00109] In one embodiment, the invention features a composition, which is a combination of a therapeutically effective amount of one or more anti-Bet v 1 antibodies or antigen-binding fragments thereof of the invention and a therapeutically effective amount of a second therapeutic agent, together with one or more pharmaceutically acceptable excipients.

[00110] The second therapeutic agent can be a small molecule drug, a protein/polypeptide, an antibody, a nucleic acid molecule, such as an antisense molecule, or an siRNA. The second therapeutic agent may be synthetic or naturally derived.

[00111] The second therapeutic agent can be any agent that is advantageously combined with an antibody or fragment thereof of the 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 in mast cells or basophils. A second therapeutic agent can also be any agent used as standard of care in treating an allergic response to any allergen. This second therapeutic agent may be an antihistamine, epinephrine, decongestant, corticosteroid, or peptide vaccine.

[00112] In certain embodiments, the second therapeutic agent may be an agent that helps neutralize or reduce any possible side effect associated with the antibody or antigen-binding fragment of an antibody of the invention, if such side effect occurs.

[00113] It will also be appreciated that the antibodies and pharmaceutically acceptable compositions of the present invention can be employed in combination therapies, that is, the antibodies and pharmaceutically acceptable compositions can be administered concomitantly with, before or after, one or more other therapeutic products or desired medical procedures, including, for example, in combination with an allergen-specific immunotherapy (SIT) regimen, in which antibodies are administered before or during the SIT. The particular combination of therapies (therapeutic products or procedures) to be employed in a combination regimen will take into account the compatibility of the desired therapeutic products and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (e.g., an antibody may be administered concomitantly with another agent used to treat the same disorder) or may achieve different effects (e.g., control of adverse effects). . As used herein, additional therapeutic agents that are normally administered to treat or prevent a specific disease or condition are appropriate for the disease or condition being treated.

[00114] When multiple therapeutic products are co-administered, dosages can be adjusted accordingly, as is recognized in the relevant art.

[00115] A sixth aspect provides a method for treating a patient who demonstrates sensitivity or allergic reaction to a Fagales protein, a Fagales-related allergen, birch pollen or an extract thereof or Bet v 1 protein or to treat at least one symptom or complication associated with a sensitivity or allergic reaction to a Fagales protein, Fagales-related allergen, birch pollen or an extract thereof or Bet v 1 protein, comprising administering an effective amount of one or more isolated human monoclonal antibodies or antigens - binding fragments thereof that bind to natural Bet v 1, BPE from Betula pendula, BPE from Betula nigra or BPE from Betula populifolia, or a pharmaceutical composition comprising an effective amount of one or more isolated human monoclonal antibodies or fragments thereof that bind to Bet v natural 1, BPE from Betula pendula, BPE from Betula nigra or BPE from Betula populifolia, to a patient in need thereof, in whom sensitivity or an allergic reaction against a Fagales Protein, an allergen related to Fagales , birch pollen or an extract thereof, or the Bet v 1 protein is prevented or decreased in severity and/or duration, or at least one symptom or complication associated with sensitivity to, or allergic reaction against, a Fagales protein, a related allergen a Fagales, birch pollen or an extract thereof, or Bet v 1 protein is prevented or ameliorated, or that the frequency and/or duration of, or the severity of sensitivity to or allergic reaction against, a Fagales protein, a protein related to Fagales, birch pollen or an extract thereof, or Bet v 1 protein is reduced following administration of one or more isolated human monoclonal antibodies or fragments thereof that bind to natural Bet v 1, Betula pendula BPE, Betula BPE nigra, or BPE from Betula populifolia, or after administration of a composition comprising any one or more of the foregoing antibodies.

[00116] In some embodiments, the birch pollen extract is selected from the group consisting of natural Bet v 1, Betula pendula BPE, Betula nigra BPE and Betula populifolia BPE.

[00117] In some embodiments, treatment results in a reduction in allergic rhinitis, allergic conjunctivitis, allergic asthma, or an anaphylactic response following exposure of the patient to a Fagales protein, Fagales-related allergen, birch pollen or extract thereof or Protein Bet v 1.

[00118] In some embodiments, the method further comprises administering an effective amount of a second therapeutic agent useful for decreasing an allergic reaction to a Fagales protein, a Fagales-related allergen, birch pollen or an extract thereof or Bet v 1 protein The second therapeutic agent may be selected from the group consisting of a corticosteroid, a bronchial dilator, an antihistamine, epinephrine, a decongestant, another antibody other than Bet v 1, and a peptide vaccine.

[00119] In some embodiments, the method further comprises treating the patient with an allergen-specific immunotherapy (SIT) regimen shortly after or simultaneously with the antibodies or fragments thereof or with the pharmaceutical composition comprising the antibodies.

[00120] In one embodiment, the invention provides a method for treating a Fagales-allergic patient who demonstrates sensitivity or allergic reaction to one or more Fagales allergens or Fagales-related allergens, or for treating at least one associated symptom or complication to a sensitivity or allergic reaction to one or more Fagales allergens, or Fagales-related allergens, comprising administering 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 comprising an amount of one or more isolated human monoclonal antibodies or fragments thereof that bind to Bet v 1, to a patient in need, in whom sensitivity to or an allergic reaction against a Fagales allergen or related allergen Fagales is prevented, or less in severity and/or duration, or at least one symptom or complication associated with sensitivity or allergic response action against, a Fagales allergen or Fagales-related allergen, is prevented or improved, or that the frequency and/or or duration of, or the severity of sensitivity to or allergic reaction against, a Fagales allergen or Fagales-related allergen, is reduced following administration of one or more of the isolated human monoclonal antibodies or fragments thereof that bind to Bet v 1, or following administration of a composition comprising any one or more of the foregoing antibodies.

[00121] 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.

[00122] In one embodiment, the invention provides a pharmaceutical composition comprising one or more of the antibodies described herein that bind to natural Bet v 1, BPE from Betula pendula, BPE from Betula nigra and BPE from Betula populifolia for use in the treatment of a patient who demonstrates sensitivity to, or an allergic reaction to, a Fagales protein, birch pollen or an extract thereof, or Bet v 1 protein, or to treat at least one symptom or complication associated with a sensitivity or allergic reaction to a protein Fagales, birch pollen or an extract thereof, or Bet v 1 protein, wherein sensitivity or an allergic reaction to a Fagales protein, birch pollen or an extract thereof, or the Bet v 1 protein is prevented or diminished in severity and/or duration, or at least one symptom or complication associated with sensitivity or allergic reaction to a Fagales protein, birch pollen or an extract thereof, or the Bet v 1 protein is prevented or ameliorated or that the frequency and /or duration or severity of sensitivity or allergic reaction against a Fagales protein, birch pollen or an extract thereof or Bet v 1 protein is reduced.

[00123] In one embodiment, the invention provides the use of a pharmaceutical composition comprising one or more of the antibodies of the invention that bind to Bet v 1 in the manufacture of a medicament for use in treating a patient who demonstrates sensitivity or allergy. reaction against, birch pollen or an extract thereof, or to Bet v 1 protein, or to treat at least one symptom or complication associated with a sensitivity or allergic reaction to birch pollen or an extract thereof, or to Bet v protein 1, wherein sensitivity to, or an allergic reaction against, birch pollen or an extract thereof, or to the Bet v 1 protein is prevented or lessened in severity and/or duration, or at least one symptom or complication associated with the sensitivity the, or allergic reaction against, birch pollen or an extract thereof, or to the Bet v 1 protein is prevented or ameliorated, or that the frequency and/or duration or severity of the sensitivity or allergic reaction to birch pollen either an extract thereof or the Bet v 1 protein is reduced.

[00124] In one embodiment, the invention provides the use of a pharmaceutical composition as described above, wherein the composition is administered in combination with a second therapeutic agent useful for decreasing an allergic reaction to a Fagales protein, birch pollen or a extract thereof, or Bet Protein v 1. In one embodiment, the invention provides the use of the pharmaceutical composition as described above, wherein the second therapeutic agent is selected from a corticosteroid, a bronchial dilator, an antihistamine, epinephrine , a decongestant, another different antibody to Bet v 1 and a vaccine peptide.

[00125] In certain embodiments, antibodies of the invention that bind to Bet v 1 may be able to reduce, minimize or prevent at least one symptom in a patient sensitive to birch pollen or Bet v 1, such as sneezing, congestion, blockage nasal pain, cough, wheezing, bronchoconstriction, rhinitis or conjunctivitis.

[00126] In one embodiment, antibodies of the invention that bind to Bet v 1, or a composition comprising one or more antibodies of the invention can be used to prevent more serious in vivo complications associated with an allergy to Bet v 1, including responses asthma, anaphylactic shock or even death resulting from anaphylaxis.

[00127] In one embodiment, the pharmaceutical composition is administered to the patient in combination with a second therapeutic agent.

[00128] In another embodiment, the second therapeutic agent is selected from the group consisting of an antihistamine, epinephrine, a decongestant, a corticosteroid, another antibody other than Bet v 1, a peptide vaccine, and any other palliative therapy useful for reducing the severity of the allergy. reaction or to improve at least one symptom associated with the allergic reaction.

[00129] In yet another embodiment, the pharmaceutical composition is administered concomitantly with, before or after one or more other desired therapeutic or medical procedures, including, for example, administered before or concomitantly with an allergen-specific immunotherapy (SIT) regimen. In some aspects, the use of an SIT regimen in conjunction with the antibodies provided herein provides a synergistic effect.

[00130] In one embodiment, a method is provided for improving the efficacy and/or safety of an allergen-specific immunotherapy (SIT) regimen, wherein the method comprises administering an effective amount of one or more isolated human monoclonal antibodies or fragments of antigen-binding antibodies 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 before or at the same time as the SIT regimen. , in which the severity of an allergic reaction to the SIT regimen is mitigated. In some embodiments, the SIT regimen comprises a higher dosage phase followed by a maintenance phase. In some embodiments, the SIT regime is an urgent SIT regime.

[00131] In a further aspect, there is provided a method for preventing or reducing mast cell degranulation associated with Fagales protein, Fagales-related allergen, birch pollen or birch pollen extract or Bet v 1 sensitization, wherein the method comprises administering a pharmaceutical composition described herein to a patient in need thereof.

[00132] 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.

[00133] Other embodiments will be apparent from a review of the detailed description below.

BRIEF DESCRIPTION OF FIGURES

[00134] Figure 1 provides the H/D exchange/MS epitope mapping for H4H16992P interaction with Bet v 1.

[00135] Figure 2 provides H/D exchange/MS epitope mapping for H4H17082P interaction with Bet v 1.

[00136] Figure 3 provides the H/D exchange/MS epitope mapping for the interaction of H4H17038P2 with Bet v 1.

[00137] Figure 4 provides H/D exchange/MS epitope mapping for H4H16987P interaction with Bet v 1.

[00138] Figure 5 provides H/D exchange/MS epitope mapping for interaction of H4H16992P, H4H17082P, H4H17038P2 and H4H16987P with Bet v 1.

[00139] Figure 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 PDA model.

[00140] Figure 7 shows the ability of anti-Bet v 1 antibody combinations to block mast cell degranulation in a humanized mouse PDA model using IgE-containing sera obtained from three Bet v 1-sensitive donors.

[00141] Figure 8 shows two graphs, the first providing representative data demonstrating combinations of anti-Bet v 1 antibodies that block basophil activation in PBMCs obtained from a birch allergic donor. The bar graph provides data representing the percentage of blockade of basophil activation in PBMCs obtained from various donors by combinations of anti-Bet v 1 antibodies relative to each antibody alone.

DESCRIPTION

[00142] Before the present compositions and methods are described, it should be understood that this invention is not limited to particular compositions and methods, and experimental conditions described, as such methods, compositions and conditions may vary. It should also be understood that the terminology used herein is only to describe particular embodiments and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[00143] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is commonly understood by one skilled in the art to which this invention belongs. As used herein, the term “about,” when used in reference to a specific recited numerical value, means that the value may vary from the recited value by no more than 1%. For example, as used herein, the expression “about 100” includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[00144] 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 now described.

DEFINITIONS

[00145] The term “Bet v 1”, as used here, refers to at least one Bet v 1 protein, in natural/native form or produced recombinantly. The Bet v 1 protein comprises the amino acid sequence of SEQ ID NO: 306 and the nucleic acid sequence of SEQ ID NO: 305. The natural Bet v 1 protein is approximately 17 kD and exists as an anti-parallel β-sheet of 7 strands (β1 - β7), two short α-helices (α1 and α2) connecting β1 and β2, a long terminal C-helix (α3) and the glycine-rich loop motif between β2 and β3 (Kofler et al. (2012 ) J. Mol. Biol. 422 (1): 109-123). A recombinantly produced Bet v 1 mutant, SEQ ID NO: 312, comprises G2-N160 of Uniprot P15494 with an S85A substitution and contains a Myc-Myc-hexahistidine tag. Uniprot's Bet v 1 amino acid sequence: P15494, i.e. SEQ ID NO: 314, may also refer to Bet v 1.

[00146] “Bet v 1” is a polypeptide comprising, or alternatively, consisting of, an amino acid sequence of SEQ ID NO: 306 or SEQ ID NO: 314, or a homologous sequence thereof. The phrase “sequence homologous to SEQ ID NO: 306 or SEQ ID NO: 314”, as used herein, refers to a polypeptide that has an identity with 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%.

[00147] The term “Bet v 1 fragment”, as used herein, refers to a polypeptide that comprises or alternatively consists of at least one Bet v 1 antigenic site. In one embodiment, the term “Bet v 1 fragment v 1", as used herein, refers to a polypeptide comprising or consisting of at least two Bet v 1 antigenic sites. In one embodiment, the antigenic sites are covalently linked. In one embodiment, the antigenic sites are linked by at least one peptide bond. In one embodiment, the 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 of Uniprot P15494. In one embodiment, the at least two antigenic sites comprise an amino acid sequence within any of SEQ ID NO: 306, 307, 308, 309, 310, 311 and 315. In one embodiment, any of the Bet v 1 fragments have the ability to induce the production of antibodies in vivo that specifically bind to naturally occurring Bet v 1 or recombinantly produced Bet v 1.

[00148] The term “antibody,” as used herein, means any antigen-binding molecule or molecular complex that comprises at least one complementarity-determining region (CDR) that specifically binds to or interacts with a specific antigen (e.g., Bet v 1). The term “antibody,” as used herein, is intended to refer to immunoglobulin molecules composed of four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds (i.e., “complete antibody molecules”). ), as well as multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain is composed of a heavy chain variable region (“HCVR” or “VH”) and a heavy chain constant region (composed of the CH1, CH2 and CH3 domains). Each light chain is composed of a light chain variable region (“LCVR or” VL”) and a light chain constant region (CL). The VH and VL regions can further be subdivided into regions of hypervariability, called complementarity determining regions (CDR), interspersed with more conserved regions, called structural regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the invention, the FRs of the antibody (or antigen-binding fragment thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence can be defined based on a side-by-side analysis of two or more CDRs.

[00149] Replacing one or more CDR residues or omitting one or more CDRs is also possible. Antibodies have been described in the scientific literature in which one or two CDRs can be dispensed for binding. 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 one-fifth to one-third of the CDR residues come into contact with the antigen . Padlan also found many antibodies in which one or two CDRs were missing amino acids in contact with an antigen (see also, Vajdos et al., 2002, J Mol Biol 320: 415428).

[00150] CDR residues that do not come into contact with the antigen can be identified based on previous studies (for example, residues H60-H65 in CDRH2 are generally not required), from regions of Kabat CDRs located outside the CDRs of Chothia, by molecular modeling and/or empirically. If a CDR or residue (or residues) is omitted, it is generally replaced by an amino acid that occupies the corresponding position in another human antibody sequence or a consensus of such sequences. Positions for substitution within CDRs and amino acids to replace can also be selected empirically. Empirical substitutions can be conservative or non-conservative substitutions.

[00151] Fully human monoclonal antibodies that specifically bind to Bet v 1, as disclosed herein, may comprise one or more amino acid substitutions, insertions and/or deletions in the structure and/or CDR regions of the heavy chain variable domains and mild compared to corresponding germline sequences. Such mutations can be readily determined by comparing the amino acid sequences disclosed herein with germline sequences available in, for example, public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework regions and/or CDR are mutated to the corresponding residue (or residues) of the germline sequence from which the antibody was derived, or the corresponding residue (or residues) of another human germline sequence, or a conservative amino acid substitution of the germline residue (or residues) (or germlines) as “germline mutations”). One skilled in the art, starting with the heavy and light chain variable region sequences disclosed herein, can readily produce numerous antibodies and antigen-binding fragments comprising one or more individual germline mutations or combinations thereof. In certain embodiments, all framework and/or CDR residues within the VH and/or VL domains are mutated back to residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, only the mutated residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues found in CDR1, CDR2 or CDR3. In other embodiments, one or more of the dungeon (or dungeons) and/or CDR residue (or residues) are mutated into the corresponding residue (or residues) of a different germline sequence (i.e., a germline sequence). germline sequence that is different from the germline sequence from which the antibody was originally derived).

[00152] Furthermore, the antibodies of the present invention may contain any combination of two or more germline mutations within the frame and/or CDR regions, for example, wherein certain individual residues are mutated to the corresponding residue of a specific sequence of the germline, while other other residues differing from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antibodies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, enhanced antagonistic or agonistic biological properties. or enhanced (as applicable) be reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are included in the present invention.

[00153] The present invention also includes fully human monoclonal antibodies comprising variants of any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes antibodies with HCVR, LCVR and/or CDR amino acid sequences of, 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.

[00154] The term “human antibody”, as used herein, is intended to include non-naturally occurring human antibodies. The term includes antibodies that are produced recombinantly in a non-human mammal or in cells of a non-human mammal. The term is not intended to include antibodies isolated or generated in a human subject.

[00155] The antibodies of the invention may, in some embodiments, be recombinant and/or non-naturally occurring human antibodies. The term “recombinant human antibody” as used herein is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, 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 (e.g., a mouse) that is transgenic for human immunoglobulin genes (see, e.g., Taylor et al. , 1992, Nucl. Acids Res. 20: 6287 to 6295) or antibodies prepared, expressed, created or isolated by any other means that involve joining human immunoglobulin gene sequences to other DNA sequences. In certain embodiments, these recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, although related to human germline VH and VL sequences, may not naturally exist in the germline repertoire of human antibodies in vivo.

[00156] Human antibodies can exist in two forms associated with hinge heterogeneity. In one form, an immunoglobulin molecule comprises a stable four-chain construct of approximately 150-160 kDa, in which the dimers are held together by an interchain heavy chain disulfide bond. In a second form, the dimers are not linked via interchain disulfide bonds and a molecule of about 75-80 kDa composed of a covalently coupled light and heavy chain (half-antibody) is formed. These forms have been extremely difficult to separate, even after affinity purification.

[00157] The frequency of appearance of the second form in various intact IgG isotypes is due to, without limitation, structural differences associated with the isotype of the hinge region of the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the appearance of the second form (Angal et al., 1993, Molecular Immunology 30:105) to levels normally observed using a human IgG1 hinge. The present invention encompasses antibodies that have one or more mutations in the hinge region, CH2 or CH3, which may be desirable, for example, in production, to improve the yield of the desired antibody form.

[00158] As used herein, the term “antigen-binding molecule” means a protein, polypeptide or molecular complex that comprises or consists of at least one complementarity determining region (CDR) that alone, or in combination with one or more CDRs and/or additional framework regions (FRs), specifically binds to a specific antigen. In certain embodiments, an antigen-binding molecule is an antibody or a fragment of an antibody, as those terms are defined elsewhere herein.

[00159] The phrase “specifically binds” or “specifically binds to” or similar means that an antibody or antigen-binding fragment likewise forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding can be distinguished by an equilibrium dissociation constant of at least about 1x10-6 M or less (e.g., a lower KD indicates tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, antibodies were identified by surface plasmon resonance, for example, BIACORE™, which specifically binds to Bet v 1.

[00160] The phrase “high affinity” antibody refers to monoclonal antibodies that have a binding affinity to Bet v 1, expressed as KD, of at least 10-8 M; preferably 10-9 M; more preferably M-10, even more preferably M-11, even more preferably M-12, as measured by surface plasmon resonance, e.g., BIACORE™ or solution affinity ELISA.

[00161] By the term “slowing rate”, “Koff” or “kd” means an antibody that dissociates from Bet v 1, with a constant rate of 1 x 10-3s- 1 or less, preferably 1 x 10-4s -1 or less as determined by surface plasmon resonance, e.g. BIACORE™.

[00162] The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtained, synthesized, or genetically modified polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The terms “antigen-binding portion” of an antibody, or “antibody fragment”, as used herein, refers to one or more fragments of an antibody that retain the ability to bind Bet v 1.

[00163] The specific embodiments, antibody or antibody fragments of the invention can 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 to treat an allergic response to Bet v 1.

[00164] The antibodies of the invention can be isolated antibodies. An “isolated antibody”, as used herein, 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 has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally exists or is produced naturally, is an “isolated antibody” for purposes of the present invention. An isolated antibody also includes an antibody in situ within a recombinant cell. Isolated antibodies are antibodies that have undergone at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and/or chemicals.

[00165] According to certain embodiments, an isolated antibody may be substantially free of other antibodies (Abs) with different antigenic specificities (for example, an isolated antibody that specifically binds to Bet v 1, or a fragment thereof, is substantially free of Abs that specifically bind to other antigens Fagales antigens, or in some respects, except Bet v 1.

[00166] A “blocking antibody” or “neutralizing antibody”, as used herein (or an “antibody that neutralizes the activity of Bet v 1”), is intended to refer to an antibody, or an antigen-binding moiety, whose binding Bet v 1 results in the inhibition of at least one biological activity of Bet v 1. For example, an antibody of the invention may assist in preventing the primary allergic response to Bet v 1. Alternatively, an antibody of the 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, an asthmatic condition, or an anaphylactic response caused by Bet v 1. This inhibition of the biological activity of Bet v 1 can be assessed by measuring one or more indicators of the biological activity of Bet v 1 by one or more of several standard in vitro or in vivo assays (such as a passive cutaneous anaphylaxis assay, as described herein) or other in vivo assays known in the art ( for example, other animal models to examine protection against Bet v 1 challenge following administration of one or more of the antibodies described herein).

[00167] The term “surface plasmon resonance”, as used herein, refers to an optical phenomenon that allows analysis of biomolecular interactions in real time by detecting changes in protein concentrations within a biosensor array, e.g. using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

[00168] The term “KD”, as used herein, is intended to refer to the dissociation equilibrium constant of a given antibody-antigen interaction.

[00169] 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 can have more than one epitope. Thus, different antibodies can bind to different areas on an antigen and can have different biological effects. The term “epitope” also refers to a site on an antigen to which B and/or T cells respond. Also refers to a region of an antigen that is bound by an antibody. Epitopes can be linear or conformational. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural features and/or specific charge characteristics. . Epitopes can also be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and contain those residues that directly contribute to the affinity of the interaction. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically includes at least 3 and, more usually, at least 5 or 8 to 10 amino acids in a unique spatial conformation.

[00170] The term “substantial identity” or “substantially identical”, when referring to a nucleic acid or fragment thereof, indicates that, when perfectly aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand ), there is nucleotide sequence identity in at least about 90% and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known sequence identity algorithm. sequence, such as FASTA, BLAST, or GAP, as discussed below. A nucleic acid molecule having substantial identity with a reference nucleic acid molecule may, in certain cases, encode a polypeptide with the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[00171] As applied to polypeptides, the term “substantial similarity” or “substantially similar” means that two peptide sequences, when perfectly aligned, as in the GAP or BESTFIT programs using standard range weights, share at least 90% identity of sequence, even more preferably at least 95%, 98% or 99% sequence identity. Preferably, the positions of the residues, which are not identical, differ by conserved amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is replaced by another amino acid residue with a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution does not substantially alter the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of 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 to 331). Examples of groups of amino acids that have 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) basic side chains: lysine, arginine and histidine; 6) acidic side chains: aspartate and glutamate and 7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate and asparagine-glutamine. Alternatively, a conservative replacement is any change that has a positive value in the PAM250 likelihood matrix revealed in Gonnet et al. (1992 Science 256: 1443-45). A “moderately conservative” replacement is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[00172] Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and a mutein of the same. See, for example, GCG Version 6. 1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters; a program in GCG Version 6. 1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percentage sequence identity of the regions with best overlap between query and search sequences (Pearson, 2000 supra). Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different 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 to 3402).

[00173] The expression “therapeutically effective amount” means an amount that produces the desired effect for which it is administered. The exact amount will depend on the purpose of the treatment and will be verified by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[00174] The antibodies of the invention can be used to “desensitize” an individual allergic to Fagales. The term “desensitize” is defined here to decrease the allergic reactivity of a Fagales-allergic individual to exposure to a Fagales allergen such as birch pollen, e.g., 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 (at a level lower than what the Fagales allergic individual would experience) or to a Fagales-related allergen. The term “desensitize” is herein further defined to decrease an individual's allergic reactivity to PR-10 proteins, including Lei c 8 and Lei d 8 (kiwi), Ara h 8 (peanut), Pru ar 1 (apricot) , Pru av 1 (cherry), Pru p 1 (peach), Pyr c 1 (pear), Gly m 4 (soy), Vig r 1 (mung bean), Sola I 4 (tomato), Cuc m 3 (melon) , Rub i 1 (raspberry) and Fra a 1 (strawberry).

GENERAL DESCRIPTION

[00175] Trees belonging to the order Fagales are the source of spring allergy symptoms and the main birch allergen, Bet v 1, is responsible for IgE binding in more than 95% of patients allergic to birch pollen (Breiteneder, EMBO J. 1989, 8(7):1935-8). Birch is the predominant trigger in 23% of US and 14% of European allergy patients. (DataMonitor Allergic Rhinitis Report, July 2010). The severity of symptoms in individuals demonstrating sensitivity to birch pollen ranges from a relatively mild rhinitis and conjunctivitis to a potentially fatal asthmatic condition. It has been shown that more than 60% of patients allergic to birch pollen have IgE antibodies to this Bet v 1 (Jarolim et al., Int Arch Allergy Appl Immunol. 1989, 88 (1-2):180-2).

[00176] Fagales trees show a distinct geographic distribution, where birch and alder are endemic to the northern parts of Europe and North America, while hazel, hornbeam and oak prefer a warmer climate, populating more parts south of these continents. Co-populations of all five species are often found in temperate climate zones 5. (Spieksma FTM. Regional European pollen calendars. D'Amato G, Spieksma FTM, Bonini S, editors. Allergenic pollen and pollinosis in Europe. Hoboken, NJ : Wiley-Blackwell; 1991. pages 49 to 65). Several Betulaceae trees, including alder, hazel and ray, have the potential to initiate sensitization to Bet v 1 allergens in susceptible individuals, resulting in the production of highly reactive IgE antibodies. (Hauser et al., 2011, Clin Exp Allergy. 41: 1804-14)

[00177] Fagales order allergens, or Fagales allergens, as defined herein include birch pollen (Bet v 1), alder pollen (Aln g1 and Aln g4), hazelnut pollen (Cor a1, Cor a2, Cor a8, Color a9, Color a9, Color a10, Color a11, Color a12, Color a13 and Color a14), horn ray pollen (Car b1), hop ray pollen (Ost c1), chestnut pollen (Cas s1 , Cas s5, Cas s8 and Cas s9), beech pollen (Fag s1) and white oak pollen (Que a1 and Que a2). Of the non-birch pollens, 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., Fagales-related allergens. These allergens are also expressed in the nuts of Fagales trees and the fruits of unrelated trees belonging to the order Rosales. Cross-reactivity of these allergens can lead to food allergy symptoms in pollen-allergic patients. Examples of cross-reactive food allergies include apple, cherry, apricot, pear, medicago, pea, soy, tomato, celery, carrot, and asparagus. (Allergens and Allergen Immunotherapy: Subcutaneous, Sublingual, and Oral, 5th Edition. Richard F. Lockey, Dennis K. Ledford, editors. CRC Press, Taylor & Francis Group, London, NY, 2014. pages 114, 118-119).

[00178] As used herein, the phrase “Fagales allergen” includes allergens related to Fagales. In some embodiments, the "Fagales-related allergen" is defined as a protein with an overall sequence homology to Bet v 1 of at least about 35%, or a sequence homology to Bet v 1 epitope 1 of at least about 35%. of 50%, or a sequence homology to Bet v 1 epitope 2 of at least about 40%, or a sequence homology to Bet v 1 epitope 3 of at least about 25%, or a sequence homology with Bet v 1 epitope 4 of at least about 15%. In some embodiments, the “Fagales-related allergen” is defined as a protein to which anti-Bet v 1 antibodies cross-react, including allergens of the order of Rosales.

[00179] Immunoglobulin E (IgE) is responsible for type 1 hypersensitivity, which manifests itself in allergic rhinitis, allergic conjunctivitis, hay fever, allergic 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 the high-affinity receptor on the surfaces of mast cells and basophils, resulting in the cross-linking of various IgE molecules and triggering the release of histamine and other inflammatory mediators, causing the various allergic symptoms.

[00180] Treatment for birch pollen allergies includes desensitization therapy, which involves repeated injections with increasing doses of a crude extract of birch pollen or short peptides derived from Bet v 1. The shortcomings of allergen-specific immunotherapy include the long duration of treatment, resulting in problems with patient compliance and frequent allergic reactions (up to 30%) to the injected protein. Desensitization therapy can take several years before the 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 serious IgE-mediated adverse events. Therefore, in the field of birch pollen allergy treatment, alternative strategies for the treatment of patients sensitive to Fagales allergens, in particular Bet v 1, are needed.

[00181] Antibodies have been proposed as a general treatment strategy for allergies, as they can block the entry of allergenic molecules into mucosal tissues or can bind the allergen before it has the opportunity to bind to IgE bound to the receptor. high affinity. in mast cells or basophils, preventing the release of histamine and other inflammatory mediators from these cells. Patent No. US 5,670,626 describes the use of monoclonal antibodies for the treatment of IgE-mediated allergic diseases such as allergic rhinitis, allergic asthma and allergic conjunctivitis by blocking the binding of allergens to mucosal tissue. Patent No. 6,849,259 describes the use of allergen-specific antibodies to inhibit allergic inflammation in an in vivo mouse model of allergy. Milk and egg-based antibody systems have been described. For example, US20030003133A1 discloses the use of milk as an allergen carrier to induce oral tolerance to birch pollen and other allergens. Compositions and methods for reducing an allergic response in an animal to an allergen in the environment through the use of a molecule that inhibits the allergen's ability to bind to mast cells have been described in WO1994/024164A2. Other antibodies to Bet v 1 were mentioned in US 2010/0034812.

[00182] The fully human antibodies described here demonstrate specific binding to Bet v 1 and may be useful in the treatment of patients suffering from birch pollen allergies, in particular in patients who demonstrate sensitivity to the Bet v 1 allergen. antibodies may be an effective means of treating patients suffering from allergies to Fagales tree pollen, or may be used to prevent an increased response to Bet v 1 following secondary exposure or the symptoms associated with the allergy, or may be used to decrease the severity and/or duration of the allergic response associated with primary exposure to birch pollen allergen or recurrence of symptoms following secondary exposure. They can be used alone or as adjuvant therapy with other therapeutic moieties or modalities known in the art for treating such allergies, such as, without limitation, treatment with corticosteroids or epinephrine. They can be used in conjunction with a different second or third antibody specific for Bet v 1. They can be used with allergen-specific immunotherapy (SIT). In some embodiments, the combination with SIT results is synergistically effective.

[00183] Unlike desensitization therapy, treatment with the antibodies described herein can provide effective relief within about 2 weeks after starting treatment, or within about 10 days or about 8 days after starting treatment. . In combination with exposure to the Bet v 1 proteins or peptides, or one or more additional Fagales allergens, the fully human antibody treatment described here may not only block an allergic reaction, but may also more effectively or synergistically desensitize the patients suffering from allergies to Fagales tree pollen.

[00184] In certain embodiments, the antibodies of the invention are obtained from mice immunized with a primary immunogen, such as natural Bet v 1, which can be purchased commercially (e.g., from Indoor Biotechnologies, VA, in NA-BV1-1) or it can be produced recombinantly. The full-length amino acid sequence of Bet v 1 is shown as SEQ ID NO: 306. The complete Bet v 1 amino acid sequence can also be found in SEQ ID NO: 314, from Uniprot: P15494.

[00185] The immunogen can 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 the N-terminus or C-terminus of Bet v 1, or from any site within the Bet v 1 amino acid sequence.

ANTIBODY ANTIGEN BINDING FRAGMENTS

[00186] Unless otherwise indicated, the term “antibody”, as used herein, should be understood to encompass antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., “complete antibody molecules”), as well as fragments of their binding to antigen. The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtained, synthesized, or genetically modified polypeptide or glycoprotein that specifically binds to an antibody. antigen to form a complex. The terms “antigen-binding portion” of an antibody, or “antibody fragment” as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to Bet v 1. A fragment of Antibody may include a Fab fragment, an F(ab')2 fragment, an Fv fragment, a dAb fragment, a fragment containing a CDR, or an isolated CDR. Antigen-binding fragments of an antibody can be derived, for example, from complete antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques, involving the manipulation and expression of the DNA encoding the variable domain of the antibody. antibody and (optionally) constant domains. Such DNA is known and/or readily available in, for example, 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 organize one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids. etc.

[00187] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab’)2 fragments; (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 (e.g., an isolated complementarity determining region (CDR), such as a CDR3 peptide) or a restricted FR3-CDR3-FR4 peptide. Other engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabetics, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.) , small modular immunopharmaceuticals (SMIPs) and shark variable IgNAR domains are also included in the term “antigen-binding fragment” as used herein.

[00188] An antigen-binding fragment of an antibody typically comprises at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR, which is adjacent to or in-frame with one or more framework sequences. In antigen-binding fragments that have a VH domain associated with a VL domain, the VH and VL domains can be located relative to each other in any suitable arrangement. For example, the variable region may be dimeric and contain VH - VH, VH - VL or VL - VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.

[00189] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variables and constant domains that may be found within an antigen-binding fragment thereof of an antibody of the present invention include: (i) VH-CH1; (ii) VH-CH2;(iii) VH-CH3; (iv) VH-CH1-C2H; (v) VH-CH1-CH2-CH3; (VI) VH-CH2-CH3; (vii) VH-CG; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-C2H; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge region or hinge connection. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in a flexible or semi-flexible link between adjacent variable and/or constant domains in a single polypeptide molecule. Furthermore, an 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 noncovalent association with each other and/or with one or more plus monomeric VH or VL domains (e.g., by disulfide bond (or bonds).

PREPARATION OF HUMAN ANTIBODIES

[00190] Methods for generating human antibodies in transgenic mice are known in the art. Any known methods can be used in the context of the present invention to produce human antibodies that specifically bind to Bet v 1.

[00191] Using VELOCIMMUNE™ technology (see, for example, US 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for generating monoclonal antibodies, high-affinity chimeric antibodies to Bet v 1 are initially isolated with a human variable region and a mouse constant region. VELOCIMMUNE® technology involves the generation of a transgenic mouse with a genome comprising human heavy and light chain variable regions operatively linked to endogenous mouse constant region sites, such that the mouse produces an antibody comprising a human variable region and a constant of the mouse in response to antigen stimulation. The DNA encoding the variable regions of the antibody heavy and light chains is isolated and operably linked to the DNA encoding the constant regions of the human heavy and light chains. The DNA is then expressed in a cell capable of expressing the fully human antibody.

[00192] Generally, a VELOCIMMUNE® mouse is challenged with the antigen of interest and lymphatic cells (such as B cells) are recovered from the antibody-expressing mice. Lymphatic cells can be fused with a myeloma cell line to prepare immortal hybridoma cell lines, and these hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the heavy chain and light chain variable regions can be isolated and ligated to desirable isotypic constant regions of the heavy chain and light chain. This antibody protein can 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.

[00193] Initially, high-affinity chimeric antibodies are isolated having a human variable region and a mouse constant region. As in the experimental section below, antibodies are distinguished and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate the fully human antibody of the invention, for example wild-type or modified IgG1 or IgG4. Although the constant region selected may vary depending on the specific use, the characteristics of high-affinity antigen binding and target specificity reside in the variable region.

[00194] In general, the antibodies of the present invention have very high affinities, typically having KD of about 10 -12 through about 10 -9 M, when measured by binding to the antigen, whether immobilized on the solid phase or in the solid phase. solution. The mouse constant regions are replaced with the desired human constant regions to generate the fully human antibodies of the invention. Although the constant region selected may vary depending on the specific use, the characteristics of high-affinity antigen binding and target specificity reside in the variable region.

BIOEQUIVALENTS

[00195] The anti-Bet v 1 antibodies and antibody fragments of the present invention encompass proteins that have amino acid sequences that vary from those of the described antibodies, but which retain the ability to bind Bet v 1. Such variant antibodies and fragments of Antibodies comprise one or more amino acid additions, deletions or substitutions when compared to the parental sequence, but exhibit biological activity that is essentially equivalent to that of the described antibodies. Likewise, the antibody-encoding DNA sequences of the present invention encompass sequences that comprise one or more nucleotide additions, deletions, or substitutions as compared to the disclosed sequence, but that encode an antibody or antibody fragment that is essentially bioequivalent to an antibody. or antibody fragment of the invention.

[00196] 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 show no significant difference when administered at the same molar dose under similar experimental conditions, single dose or multiple dose. Some antibodies will be considered equivalent or pharmaceutical alternatives if they are equivalent in the extent of their absorption, but not in their rate of absorption, and can still be considered bioequivalent because these differences in absorption rate are intentional and reflected in the labeling. are not essential for obtaining effective drug concentrations in the body, for example, in chronic use, and are considered clinically insignificant for the drug in question studied.

[00197] In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity and potency.

[00198] In one embodiment, two antigen-binding proteins are bioequivalent if a patient can be switched 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 decreased efficacy, compared to continued therapy without this switch.

[00199] In one embodiment, two antigen-binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.

[00200] Bioequivalence can be demonstrated by in vivo and/or in vitro methods. Bioequivalence measures include, for example, (a) an in vivo test in humans or other mammals, in which the concentration of the 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 human bioavailability data; (c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in a well-controlled clinical trial that establishes the safety, efficacy or bioavailability or bioequivalence of an antibody.

[00201] Bioequivalent variants of the antibodies of the invention can be constructed, for example, by making various substitutions of residues or sequences or deleting terminal or internal residues or sequences, not necessary for biological activity. For example, cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bonds after renaturation. In other contexts, bioequivalent antibodies may include antibody variants comprising amino acid changes, which modify the glycosylation characteristics of the antibodies, for example, mutations that eliminate or remove glycosylation.

BIOLOGICAL CHARACTERISTICS OF ANTIBODIES

[00202] In general, the antibodies of the present invention can function by binding Bet v 1, a fragment of Bet v 1, or Bet v 1 and one or more Fagales allergens or Fagales-related allergens.

[00203] In certain embodiments, the antibodies of the present invention can bind to an epitope or fragment located within the Bet v 1 protein, for example, an epitope or fragment encompassing amino acid residues ranging from about position 23 to about position 43 of SEQ ID NO: 306; an epitope or fragment encompassing amino acid residues ranging from about position 44 to about 56 of SEQ ID NO: 306; an epitope or fragment encompassing amino acid residues ranging from about 2 to about 19 of SEQ ID NO: 306; an epitope or fragment encompassing amino acid residues ranging from about 57 to about 70 of SEQ ID NO: 306; or an epitope or fragment encompassing amino acid residues ranging from about 81 to about 89 or about 81 to about 96 of SEQ ID NO: 306. In certain embodiments, the antibodies of the present invention can bind to at least an amino acid sequence selected from the group consisting of SEQ ID NO: 307, 308, 309, 310, 311 and 315, wherein an epitope sequence may be extended by 1 to 5 amino acids or about 5 to about 10 amino acids in C-terminal end or N-terminal end.

[00204] 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 by, for example, at least about 70% when compared to an isotype control antibody. In certain embodiments, the antibodies inhibit or block mast cell degranulation by, for example, 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%, when compared to an isotype control antibody.

[00205] In one embodiment, the invention provides a fully human monoclonal antibody or antigen-binding fragment thereof that binds to Bet v 1, wherein the antibody or fragment thereof exhibits one or more of the following characteristics: (i) 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, or a substantially similar sequence thereof, with at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (ii) 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, or a substantially similar sequence that has at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (iii) comprises 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 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 that has at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (iv) comprises 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 with at least 90%, at least 95%, at least 98% or at least 99% sequence identity; 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, or a substantially similar sequence that has at least 90%, at least 95%, at least 98% or at least 99% sequence identity; 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, 188, 204, 220, 236, 252, 268 and 300, or a substantially similar sequence 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 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 KD equal to or less than 10-8 or in a range between 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.

[00206] In one embodiment, the invention provides the use of a combination of two or more fully human antibodies of the invention, or fragments thereof, for the preparation of a composition, in which the antibodies bind to Bet v 1 and in which each antibody or fragment thereof contained in the composition exhibits one or more of the following characteristics: (i) 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, or a substantially similar sequence with at least 90%, at least 95%, at least 98% or at least minus 99% sequence identity; (ii) 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, or a substantially similar sequence that has at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (iii) comprises 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 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 that has at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (iv) comprises 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 with at least 90%, at least 95%, at least 98% or at least 99% sequence identity; 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, or a substantially similar sequence that has at least 90%, at least 95%, at least 98% or at least 99% sequence identity; 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, 188, 204, 220, 236, 252, 268 and 300, or a substantially similar sequence 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 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 KD equal to or less than 10-8 or in a range between 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.

[00207] Certain Bet v 1 antibodies of the present invention, when used alone or in combination, are capable of binding 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 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 activity neutralization assays (e.g., protection against anaphylaxis), such as described here.

[00208] Exemplary non-limiting in vitro assays for measuring binding activity are illustrated in Example 3, herein. In Example 3, the binding affinities and kinetic constants of human anti-Bet v 1 antibodies were determined by surface plasmon resonance.

[00209] Bet v 1 proteins or peptides can be modified to include addition or substitution of certain residues for labeling or for the purpose of conjugation with carrier molecules, such as KLH. For example, a cysteine can be added at the N-terminus or the C-terminus of a peptide, or a linker sequence can be added to prepare the peptide for conjugation with, for example, KLH for immunization. Antibodies specific for Bet v 1 may contain no additional labels or portions, or may contain an N-terminal or C-terminal label or portion. In one embodiment, the marker or moiety is biotin. In a binding assay, the location of a tag (if any) can determine the orientation of the peptide relative to the surface to which the peptide is bound. For example, if a surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented so that the C-terminal portion of the peptide is distal to the surface.

EPITOPES MAPPING AND RELATED TECHNOLOGIES

[00210] The term “epitope”, as used herein, 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 can have more than one epitope. Thus, different antibodies can bind to different areas on an antigen and can have different biological effects. Epitopes can be conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, an epitope may include saccharide moieties, phosphoryl groups, or sulfonyl groups on the antigen.

[00211] Provided herein are anti-Bet v 1 antibodies that interact with one or more amino acids found in the Bet v 1 molecule, including, for example, any fragment of Bet v 1 shown in SEQ ID NO: 306, or in comparable regions of a recombinant product produced Bet v 1 protein. The epitope to which antibodies bind may consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located in the Bet v 1 molecule. Exemplary contiguous sequences include amino acid residues ranging from about position 23 to about position 44 of SEQ ID NO: 306; amino acid residues ranging from about position 23 to about position 43 of SEQ ID NO: 306; amino acid residues ranging from about position 23 to about position 38 of SEQ ID NO: 306; amino acid residues ranging from about position 23 to about position 41 of SEQ ID NO: 306; amino acid residues ranging from about position 26 to about position 43 of SEQ ID NO: 306; amino acid residues ranging from about position 29 to about position 43 of SEQ ID NO: 306; amino acid residues ranging from about position 44 to about position 70 of SEQ ID NO: 306; amino acid residues ranging from about position 44 to about position 56 of SEQ ID NO: 306; amino acid residues ranging from about position 45 to about position 56 of SEQ ID NO: 306; amino acid residues ranging from about position 2 to about position 19 of SEQ ID NO: 306; amino acid residues ranging from about position 5 to about position 10 of SEQ ID NO: 306; amino acid residues ranging from about position 5 to about position 19 of SEQ ID NO: 306; amino acid residues ranging from about position 8 to about position 19 of SEQ ID NO: 306; amino acid residues ranging from about position 11 to about position 19 of SEQ ID NO: 306; amino acid residues ranging from about position 57 to about position 70 of SEQ ID NO: 306; amino acid residues ranging from about position 57 to about position 66 of SEQ ID NO: 306; amino acid residues ranging from about position 81 to about position 96 of SEQ ID NO: 306; amino acid residues ranging from about position 84 to about position 96 of SEQ ID NO: 306; amino acid residues ranging from about position 85 to about position 96 of SEQ ID NO: 306; and amino acid residues ranging from about position 81 to about position 89 of 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, wherein this sequence may be extended by about 1 to about 5 amino acids, or about 5 to about 10 amino acids, at the C-terminal end or at the N-terminal end. Alternatively, the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) located within the Bet v 1 molecule (for example, a conformational epitope).

[00212] Various techniques known to those skilled in the art can be used to determine whether an antibody “interacts 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 mutational analysis, peptide transfer analysis (Reineke (2004) Methods Mol Biol 248: 443-63), peptide cleavage analysis crystallographic studies, and NMR analysis. Furthermore, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer (2000) Protein Science 9: 487 to 496). Another method that can be used to identify the amino acids within a polypeptide that an antibody interacts with is the hydrogen/deuterium exchange detected by mass spectrometry. Broadly speaking, the hydrogen/deuterium exchange method involves labeling the protein of interest in deuterium, followed by binding of the antibody to the deuterium-labeled protein. Next, the protein/antibody complex is transferred to water and the exchangeable protons within the amino acids that are protected by the antibody complex undergo reverse exchange from deuterium to hydrogen at a slower rate than the exchangeable protons in the amino acids that are not part of it. of the interface. As a result, amino acids that are part of the protein/antibody interface can retain deuterium and therefore exhibit relatively greater mass compared to amino acids not included in the interface. After antibody dissociation, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing 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 to 259; Engen and Smith (2001) Anal. Chem. 73:256A-265A. X-ray crystallography of the antigen/antibody complex can also be used for epitope mapping purposes.

[00213] Modification Assisted Profiling (MAP), also known as Antigen Structure-Based Antibody Profiling (ASAP) is a method that categorizes a large number of monoclonal antibodies (monoclonal antibodies) directed against the same antigen, according to the similarities of the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (document US 2004/0101920). Each category may reflect a unique epitope that is distinctly different from or partially overlapping with the epitope represented by another category. This technology allows for rapid filtering of genetically identical antibodies so that characterization can be focused on genetically distinct antibodies. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce monoclonal antibodies with desired characteristics. MAP can be used to classify antibodies of the invention into groups of antibodies that bind to different epitopes.

[00214] In certain embodiments, anti-Bet v 1 antibodies or antigen-binding fragments thereof bind to an epitope on Bet v 1, in natural or native form, as exemplified in SEQ ID NO: 306 or in SEQ ID NO: 314 (Bet v 1 amino acid sequence from Uniprot: P15494), or produced recombinantly, or a fragment thereof. In certain embodiments, the antibodies of the 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 of SEQ ID NO: 306 ; amino acid residues ranging from about position 23 to about position 43 of SEQ ID NO: 306; amino acid residues ranging from about position 23 to about position 38 of SEQ ID NO: 306; amino acid residues ranging from about position 23 to about position 41 of SEQ ID NO: 306; amino acid residues ranging from about position 26 to about position 43 of SEQ ID NO: 306; amino acid residues ranging from about position 29 to about position 43 of SEQ ID NO: 306; amino acid residues ranging from about position 44 to about position 70 of SEQ ID NO: 306; amino acid residues ranging from about position 44 to about position 56 of SEQ ID NO: 306; amino acid residues ranging from about position 45 to about position 56 of SEQ ID NO: 306; amino acid residues ranging from about position 2 to about position 19 of SEQ ID NO: 306; amino acid residues ranging from about position 5 to about position 10 of SEQ ID NO: 306; amino acid residues ranging from about position 5 to about position 19 of SEQ ID NO: 306; amino acid residues ranging from about position 8 to about position 19 of SEQ ID NO: 306; amino acid residues ranging from about position 11 to about position 19 of SEQ ID NO: 306; amino acid residues ranging from about position 57 to about position 70 of SEQ ID NO: 306; amino acid residues ranging from about position 57 to about position 66 of SEQ ID NO: 306; amino acid residues ranging from about position 81 to about position 96 of SEQ ID NO: 306; amino acid residues ranging from about position 84 to about position 96 of SEQ ID NO: 306; amino acid residues ranging from about position 85 to about position 96 of SEQ ID NO: 306; and amino acid residues ranging from about position 81 to about position 89 of SEQ ID NO: 306. These regions are further exemplified in SEQ ID NO: 307, 308, 309, 310, 311 and 315.

[00215] The present invention also includes anti-Bet v 1 antibodies that bind to the same epitope, or a portion of the epitope, as any of the specific exemplary antibodies described herein in Table 1, or an antibody that has the CDR sequences of any of the exemplary antibodies described in Table 1. Likewise, the present invention also includes anti-Bet v 1 antibodies 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.

[00216] One can easily determine whether an antibody binds to the same epitope or competes for binding with a reference anti-Bet v 1 antibody using routine methods known in the art. For example, to determine whether a test antibody binds to the same epitope as an anti-Bet v 1 reference antibody of the invention, the reference antibody is allowed to bind to a Bet v 1 protein or peptide under saturating conditions. Next, the ability of a test antibody to bind to the Bet v 1 molecule is assessed. Whether the test antibody has the ability to bind to Bet v 1 after saturation binding with the anti-Bet v 1 reference antibody , it can be concluded that the test antibody binds to a different epitope than the anti-Bet v 1 reference antibody. On the other hand, if the test antibody does not have the ability to bind to the Bet v 1 molecule after binding saturation with the reference anti-Bet v 1 antibody, the test antibody may bind to the same epitope as the epitope bound by the reference anti-Bet v 1 antibody of the invention.

[00217] To determine whether an antibody competes for binding with an anti-Bet v 1 reference antibody, the binding methodology described above is performed in two orientations: In a first orientation, the reference antibody can bind to a molecule of Bet v 1 under saturation conditions followed by evaluation of the binding of the test antibody to the Bet v 1 molecule. In a second orientation, the test antibody is allowed to bind to a Bet v 1 molecule under saturation conditions, followed by evaluation of the binding of the reference antibody to the Bet v 1 molecule. If, in both orientations, only the first antibody (saturated) had the ability to bind to the Bet v 1 molecule, it is concluded that the test antibody and the reference antibody reference antibody compete for binding to Bet v 1. As will be appreciated by one skilled in the art, an antibody that competes for binding with a reference antibody may not necessarily bind to the identical epitope as the reference antibody, but may sterically block binding of the antibody. reference linking an overlapping or adjacent epitope.

[00218] Two antibodies bind to the same epitope or overlap if each one competitively inhibits (blocks) the binding of the other to the antigen. That is, a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits binding of the other by at least 50%, but preferably 75%, 90%, or even 99%, as measured in a binding assay. competitive (see, e.g., Junghans et al., Cancer Res. 1990 50: 1495 to 1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[00219] Additional routine experiments (e.g., peptide mutation and binding analyses) can then be performed to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or whether the Steric blocking (or other phenomenon) is responsible for the observed lack of binding. Experiments of this type 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

[00220] The invention encompasses an anti-Bet v 1 human monoclonal antibody conjugated to a therapeutic moiety (“immunoconjugate”), as an agent that has the ability to reduce the severity of an allergic response to the Bet v 1 allergen or in an environment in which Fagales trees are present or to improve at least one symptom associated with exposure to birch pollen or Bet v 1 allergen, including rhinitis, conjunctivitis or breathing difficulties, or the severity thereof. This agent may be a corticosteroid, a second antibody other than Bet v 1, or a vaccine. The type of therapeutic moiety that can be conjugated to the Bet v 1 antibody will take into account the condition to be treated and the desired therapeutic effect to be achieved. Alternatively, if the desired therapeutic effect is to treat the sequelae or symptoms associated with exposure to the Bet v 1 allergen or any other condition resulting from such exposure, such as, but not limited to, rhinitis or conjunctivitis, it may be advantageous to combine an appropriate agent to treat the sequelae. or symptoms of the condition or to alleviate any side effects of the antibodies of the invention. Examples of suitable agents for forming immunoconjugates are known in the art, see, for example, WO 05/103081.

THERAPEUTIC ADMINISTRATION AND FORMULATIONS

[00221] The invention provides therapeutic compositions comprising anti-Bet v 1 antibodies or antigen-binding fragments thereof of the present invention. Administration of therapeutic compositions according to the invention will be administered by a suitable route, including, without limitation, intravenously, subcutaneously, intramuscularly, intranasally, with suitable carriers, excipients and other agents, which are incorporated into the formulations to provide improved transfer. , delivery, tolerance and the like. A variety of suitable formulations can be found in the form known to all pharmacists: Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. Such formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing vesicles (cationic or anionic) (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water, and water emulsions. in oil, carbowax emulsions (polyethylene glycols of various 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 to 311.

[00222] The dose of the antibody may vary depending on the age and size of a patient to be administered, target disease, conditions, route of administration and the like. When the antibody of the present invention is used to treat rhinitis or conjunctivitis associated with exposure to pollen from a Fagales tree, or birch pollen or Bet v 1, in an individual with sensitivity to Bet v 1, or to prevent an anaphylactic disease response to Fagales allergen, or to lessen the severity of the allergic response, it is advantageous to intravenously administer the antibody of the present invention typically in a single dose of about 0.01 to about 30 mg/kg of body weight, with more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg of body weight. Depending on the severity of the condition, the frequency and duration of treatment may be adjusted. In certain embodiments, the antibody or antigen-binding fragment thereof of the invention may be administered as an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 500 mg, about 5 to about 300 mg or about 10 to about 200 mg, to about 100 mg or to about 50 mg. In certain embodiments, the initial dose may be followed by administration of a second or several subsequent doses of the antibody or antigen-binding fragment thereof in an amount that may be approximately the same or less than that of the initial dose, wherein the as subsequent doses are separated by at least 1 day to 3 days; at least a 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.

[00223] Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells with the ability to express mutant viruses, receptor-mediated endocytosis (see, e.g. example, Wu et al., 1987, J. Biol. Chem. 262: 4429 to 4432). Methods of introduction include, without limitation, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The composition may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered in conjunction with other biologically active agents. Administration can be systemic or local.

[00224] The pharmaceutical composition can also be delivered in a vesicle, in particular a liposome (see, for example, Langer, 1990, Science 249: 1527 to 1533).

[00225] In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, polymeric materials may be used. In yet another embodiment, a controlled release system can be placed near the target of the composition, thus requiring only a fraction of the systemic dose.

[00226] Injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations can be prepared by publicly known methods. For example, injectable preparations can be prepared, for example, by dissolving, suspending or emulsifying the antibody or salt thereof described above in a sterile aqueous medium or in an oily medium conventionally used for injections. As an aqueous medium for injections, there are, for example, physiological saline solution, an isotonic solution containing glucose and other auxiliary agents, etc., which can be used in combination with a suitable solubilizing agent such as an alcohol (e.g. ethanol), a polyalcohol (e.g. propylene glycol, polyethylene glycol), a non-ionic surfactant [e.g. polysorbate 80, HCO-50 (polyoxyethylene adduct (50 mol) of hydrogenated castor oil)], etc. As an oily medium, for example, sesame oil, soybean oil, etc. are employed, which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled into a suitable ampoule.

[00227] A pharmaceutical composition of the present invention can be administered subcutaneously or intravenously with a standard needle and syringe. Furthermore, with respect to subcutaneous administration, a pen administration device has ready applications in administering a pharmaceutical composition of the present invention. A delivery device for such a pen may be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can be readily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can be reused. In a disposable pen delivery device, there is no replaceable cartridge. Instead, the disposable pen delivery device comes preloaded with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[00228] Numerous reusable pen and auto-injector administration devices have applications in the subcutaneous administration of a pharmaceutical composition of the present invention. Examples include, but are certainly not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, United Kingdom), DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, pen HUMALIN 70/30™ (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen ( Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™ and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to name just a few. Examples of disposable pen delivery devices with applications in the subcutaneous delivery of a pharmaceutical composition of the present invention include, but are certainly not limited to, the SOLOSTAR™ pen (sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™ autoinjector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA™ pen (Abbott Labs, Abbott Park, IL), to name just a few .

[00229] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in dosage forms in a unit dose suitable for adjusting a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforementioned antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in injection form, it is preferred that the aforementioned antibody is contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms.

THERAPEUTIC USES OF ANTIBODIES

[00230] Due to the interaction with Bet v 1, the present antibodies are useful for treating the primary response following exposure of an individual to Fagales allergen, birch pollen or an environment containing the Bet v 1 protein, or at least a symptom associated with the allergic response, such as itchy eyes, conjunctivitis, rhinitis, wheezing, breathing difficulties, or to prevent a secondary response to the Bet v 1 allergen, including a more severe anaphylactic response, or to decrease the severity, duration and/or frequency of symptoms following exposure to Fagales allergen. Therefore, it is anticipated that the antibodies of the present invention may be used prophylactically or therapeutically.

[00231] In yet another embodiment of the invention, the present antibodies are used for the preparation of a pharmaceutical composition for the treatment of patients suffering from sensitivity to birch pollen or an extract thereof and/or the Bet v 1 protein. In yet another embodiment of the invention, the present antibodies are used for the preparation of a pharmaceutical composition to reduce the severity of primary exposure to Bet v 1, or to reduce the severity, duration and/or number of allergic responses to Bet v 1 In As a further embodiment of the invention, the present antibodies are used as adjuvant therapy with any other agent useful in the treatment of Fagales allergens, including corticosteroids, vaccines, allergen-specific immunotherapy (SIT) or any other palliative therapy known to those skilled in the art.

COMBINED THERAPIES

[00232] Combination therapies may include an anti-Bet v 1 antibody of the invention and any additional therapeutic agent that can be advantageously combined with an antibody of the invention or with a biologically active fragment of an antibody of the invention.

[00233] For example, a second therapeutic agent may be employed to help reduce allergic symptoms following exposure to a Fagales allergen, birch pollen or an extract thereof, or Bet v 1, or exposure to an environment in which the Fagales trees are present and flourishing, like a corticosteroid. The antibodies can also be used in conjunction with other therapies, such as a vaccine specific for the Bet v 1 allergen. Additional therapeutically active components can be administered before, simultaneously with, or after administration of the anti-Bet v 1 antibody of the present invention. For purposes of the present disclosure, such administration regimens are considered to be the administration of an anti-Bet v 1 antibody “in combination with” a second therapeutically active component.

ALLERGEN-SPECIFIC IMMUNOTHERAPY (SIT)

[00234] As used herein, the terms “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 form of treating or preventing allergies and allergic reactions, or to reduce or eliminate allergic responses. In a typical SIT regimen, small amounts of allergen are initially administered to an allergic patient, followed by the administration of larger amounts of allergen. In certain cases, the SIT regimen comprises at least two consecutive phases: (1) a dosing phase and (2) a maintenance phase. In the dosing phase, increasing doses of allergen are administered until an effective and safe dose is reached. The dose established at the end of the dosing phase is then administered to the patient throughout the course of the maintenance phase. The duration of the dosing phase may be several weeks or several months. In certain embodiments, however, the dosing phase is of substantially shorter duration (e.g., less than a 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 comprise a dosing phase of less than 5 days are sometimes referred to as “Rush” immunotherapy or “Rush SIT.” The maintenance phase of an SIT regimen may last several weeks, several months, several years, or indefinitely.

ADMINISTRATION REGIMES

[00235] In accordance with certain embodiments of the present invention, multiple doses of one or more anti-Bet v 1 antibodies (a combination of antibodies) can be administered to a patient over a defined period of time. Methods according to this aspect of the invention comprise sequentially administering to a patient multiple doses of an antibody, antibody combination. As used herein, “sequential administration” means that each dose of an antibody or combination of antibodies is administered to the patient at a different time, e.g., on different days, separated by a predetermined interval (e.g., hours, days, weeks, or months). The present invention includes methods, which comprise sequentially administering to the patient a single initial dose of an antibody or antibody combination, followed by one or more secondary doses of the antibody, and optionally followed by one or more tertiary doses of the antibody.

[00236] The terms “initial dose”, “secondary doses” and “tertiary doses” refer to the temporal sequence of administration of an antibody or combination of antibodies provided herein. Thus, the “initial dose” is the dose administered at the beginning of the treatment regimen (also called “base dose”); “secondary doses” are the doses administered after the initial dose; and “tertiary doses” are the doses administered after the secondary doses. The initial, secondary, and tertiary doses may all contain the same amount of an antibody or combination of antibodies, but may generally differ from each other in terms of frequency of administration. In certain embodiments, however, the amount of an antibody or combination of antibodies contained in the initial, secondary, and/or tertiary doses varies from one another (e.g., adjusted up or down, as appropriate) during the course of treatment. In certain embodiments, two or more doses (e.g., 2, 3, 4, or 5) are administered at the beginning of the treatment regimen as “loading doses” followed by subsequent doses that are administered less frequently (e.g., “loading doses” maintenance “).

[00237] In an exemplary embodiment of the present invention, each secondary and/or tertiary dose is administered 1 to 26 (e.g., 1, 11/?, 2, 2^, 3, 3^, 4, 4^2, 5 , 5^2, 6, 6^2, 7, 7^, 8, 8^, 9, 9^, 10, 10^, 11, 11^, 12, 12^, 13, 13^2, 14, 14 ^2, 15, 15 ^2, 16, 16^, 17, 17^, 18, 18^, 19, 19, 20, 20^, 21, 21^, 22, 22^2, 23, 23^, 24 , 24^, 25, 25^, 26, 26^ or more) weeks after the immediately preceding dose. The phrase "the immediately preceding dose", as used herein, means, in a sequence of several administrations, the dose of an antibody or combination of antibodies, which is administered to a patient prior to administration of the next dose in the sequence with no intervening doses. .

[00238] Methods according to this aspect of the invention may comprise 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 a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) secondary doses are administered to the patient. Likewise, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses are administered to the patient.

[00239] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the immediately preceding dose. Likewise, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the immediately preceding dose. Alternatively, the frequency with which secondary and/or tertiary doses are administered to a patient may vary throughout the treatment regimen. The frequency of administration can also be adjusted during the course of treatment by a doctor depending on the needs of each patient after clinical examination.

DIAGNOSTIC USES OF ANTIBODIES

[00240] The anti-Bet v 1 antibodies of 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 anticipated that confirmation of an allergic response believed to be caused by Bet v 1 can be made by measuring the presence of Bet v 1 through the use of any one or more of the antibodies of the invention. Exemplary diagnostic assays for Bet v 1 may comprise, for example, contacting a sample obtained from a patient with an anti-Bet v 1 antibody of the invention, wherein the anti-Bet v 1 antibody is labeled with a reporter molecule. or detectable marker or used as a capture ligand to selectively isolate the Bet v 1 protein from patient samples. Alternatively, an unlabeled anti-Bet v1 antibody can be used in diagnostic applications in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule may be a radioisotope, such as 3H, 14C, 32P, 35S or 125I; a fluorescent or chemiluminescent moiety, such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, p-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).

[00241] Samples that can be used in the Bet v 1 diagnostic assays according to the present invention include any tissue or fluid sample obtained from a patient, which contains detectable amounts of Bet v 1 protein, or fragments thereof, in normal or pathological conditions. Generally, Bet v 1 levels in a specific sample obtained from a healthy/non-allergic patient (e.g., a patient not affected by a sensitivity associated with the presence of Bet v 1) will be measured to initially establish a baseline or standard. , level of Bet v 1. This baseline level of Bet v 1 can then be compared with levels of Bet v 1 measured in samples obtained from individuals suspected of having sensitivity to Bet v 1 in birch pollen or symptoms associated with this condition.

[00242] Although the invention has been particularly shown and described with reference to various embodiments, it would be understood by those skilled in the art that changes in form and details may be made in the various embodiments disclosed herein without departing from the spirit and scope of the invention and that the various embodiments disclosed herein are not intended to act as limitations on the scope of the claims.

EXAMPLES

[00243] The following examples are presented in order to provide those skilled in the art with a complete description and description of how to make and use the methods and compositions of the invention and are not intended to limit the scope of what the inventors consider to be their invention. Efforts have been made to ensure accuracy regarding the numbers used (e.g. quantities, temperature, etc.), but some errors and experimental deviations must be considered. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

EXAMPLE 1: GENERATION OF HUMAN ANTIBODIES TO BET V 1

[00244] An immunogen comprising any of the following can be used to generate antibodies to Bet v 1. In certain embodiments, the antibodies of the invention are obtained from mice immunized with a primary immunogen, such as full-length natural Bet v 1 (nBet v 1), which can be purchased commercially (e.g., from Stallergenes Greer, Lenoir, NC, at XP527D3A25) or isolated from birch pollen (see, e.g., Buters, et al. (2012), Atomospheric Environment 55:496 a 505), or which can be produced recombinantly (see GenBank accession number P 15494 for the complete amino acid sequence of Bet v 1) or fragments of the Bet v 1 protein, followed by immunization with a secondary immunogen or with a fragment immunogenically active natural 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 elicit antibody responses in vivo. For example, recombinant Bet v 1 constructs, such as those exemplified in SEQ ID NO: 307, 308, 309, 310, 311 and 315, or fragments thereof, can be used as immunogens.

[00245] In certain embodiments, the antibodies of the invention are obtained from mice immunized with a primary immunogen, such as a biologically active and/or immunogenic fragment of natural Bet v 1, or DNA encoding its active fragment. The fragment can be derived from the N-terminal or C-terminal portion of Bet v 1.

[00246] 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 indicated below. In other embodiments, the recombinant Bet v 1 protein construct includes amino acids G2 to N160 of Uniprot P15494. In some embodiments, the construction comprises a replacement of S85A. The proteins were expressed in Chinese hamster ovary (CHO) cells. An exogenous signal sequence used to promote expression in CHO cells is not included in the sequence listings.

[00247] In certain embodiments, the immunogen may be a Bet v 1 fragment or fusion protein comprising any one or more of the following: i) amino acid residues 23-43 of Bet v 1 (see Uniprot P15494 and also SEQ ID NO: 307); ii) amino acid residues 44-56 of Bet v 1 (see Uniprot P15494 and also SEQ ID NO: 308); iii) amino acid residues 2-19 of Bet v 1 (see Uniprot P15494 and also SEQ ID NO: 309); iv) amino acid residues 57-70 of Bet v 1 (see Uniprot P15494 and also SEQ ID NO: 310); v) amino acid residues 81-89 of Bet v 1 ((see Uniprot P15494 and also SEQ ID NO: 311) and vi) amino acid residues 81-96 of Bet v 1 ((see Uniprot P15494 and also SEQ ID NO: 315 )

[00248] In certain embodiments, antibodies that specifically bind to Bet v 1 can be prepared using fragments of the aforementioned regions or peptides that extend beyond the designated regions by about 5 to about 20 amino acid residues of one or more both the N or C-terminal ends of the regions described herein. In certain embodiments, any combination of the aforementioned regions or fragments thereof can be used in preparing Bet v 1 specific antibodies. In certain embodiments, any one or more of the aforementioned regions of Bet v 1, or fragments thereof, can be used. for the preparation of monospecific, bispecific or multispecific antibodies.

[00249] The complete proteins, or fragments thereof, that were used as immunogens, as noted above, were administered directly, with an adjuvant to stimulate the immune response, to a VELOCIMMUNE® mouse comprising DNA encoding light chain heavy variable regions kappa and human immunoglobulin psad.

[00250] Anti-Bet v 1 antibodies were isolated directly from antigen-positive B cells without fusion with myeloma cells, as described in US Patent 7,582,298. Using this method, several fully human anti-Bet v 1 antibodies (i.e., antibodies that have human variable domains and human constant domains) have been obtained; Examples of antibodies generated in this way were designated as follows: H4H16943P, H4H16946P, H4H16950P, H4H16960P, H4H16967P, H4H16971P, H4H16979P, H4H16987P, H4H16991P, H4H16992P, H4H1700 1P, H4H17015P, H4H17027P, H4H17028P, H4H17031P, H4H17033P, H4H17038P2, H4H17045P2, H4H17067P2 , and H4H17082P2.

[00251] The biological properties of exemplary antibodies generated according to the methods of this Example are described in detail in the Examples presented below.

EXAMPLE 2: HEAVY AND LIGHT CHAIN AMINO ACID SEQUENCES

[00252] Table 1a provides the amino acid sequence identifiers of the variable regions of the heavy and light chains and CDRs of selected anti-Bet v 1 antibodies. Table 1b provides the nucleic acid sequence identifiers of the heavy and light chain variable regions and CDRs of selected anti-Bet v1 antibodies. TABLE 1A: AMZNOACID SEQUENCE IDENTIFIERS TABLE 1B: NUCLEIC ACID SEQUENCE IDENTIFIERS

[00253] Antibodies are typically referred to herein according to the following nomenclature: Fc prefix (e.g., “H4H”, “H2M”, etc.), followed by a numeric identifier (e.g. “16943”, “17001” , etc., as shown in Table 1), followed by a “P” or “P2” suffix. The prefixes H4H and H2M in the antibody designations used herein indicate the particular isotype of the Fc region of the antibody. Thus, according to this nomenclature, an antibody may be referred to herein as, for example, “H4H16943P”, etc., as the “H4H” designates the antibody has a human IgG4 Fc (all variable regions are fully human, as indicated by the first 'H' in the antibody designation). As will be appreciated by one skilled in the art, an antibody with a specific Fc isotype can be converted into an antibody with a different Fc isotype (e.g., an antibody with a human IgG1 Fc can be converted into an antibody with a human IgG4, etc. .), but in any case the variable domains (including the CDRs) - indicated by the numerical identifiers shown in Table 1 - will remain the same, and the antigen-binding properties are expected to be identical or substantially similar regardless of nature of the Fc domain.

[00254] Table 1c provides the amino acid sequence identifiers of the full-length heavy and light chains of selected anti-Bet v 1 antibodies. TABLE 1C: AMZNOACID SEQUENCE IDENTIFIERS

EXAMPLE 3: ANTIBODY BINDING TO BET V 1 AS DETERMINED BY SURFACE PLASMONIC RESONANCE

[00255] The equilibrium dissociation constants (KD) for the binding of natural Bet v 1 to purified anti-Bet v 1 monoclonal antibodies were determined using a real-time surface plasmon resonance biosensor (SPR-Biacore) on an instrument Biacore 4000. All binding studies were performed in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v/v surfactant Tween-20, pH 7.4 (HBS-ET) running buffer at 25°C and 37°C. The surface of the Biacore CM5 sensor was first derivatized by amine coupling with a mouse anti-human Fc monoclonal antibody (GE, no BR-1008-39) to subsequently capture anti-Bet v 1 monoclonal antibodies. natural (Indoor, Cat no NA-BV1-1) or CHO produced recombinant Bet v 1 containing an S85A mutation with a C-terminal hexahistidine myc-myc tag (Bet v 1 mutant-MMH; SEQ ID NO: 312) the reagent was prepared for the first time in HBS-ET running buffer (100 nM - 1.23 nM; serially diluted three times) and were injected onto the surface of the anti-Bet v 1 monoclonal antibody captured by the anti-human Fc for 4 minutes at a flow rate of 30 μl/minute, while the dissociation of Bet v 1 reagent bound to the monoclonal antibody was monitored for 10 minutes in HBS-ET running buffer. Association (ka) and dissociation (kd) rate constants were determined by fitting real-time binding sensor diagrams to a 1:1 binding model with mass transport limitation using Scrubber 2.0c curve fitting software. . Bond dissociation equilibrium constants (KD) and dissociative half-lives (d/2) were calculated from the kinetic rate constants as:

[00256] The binding kinetics parameters for natural Bet v 1 and mutant Bet v 1-MMH to different anti-Bet v 1 monoclonal antibodies of the invention at 25°C and 37°C are shown in Tables 2 to 5.

[00257] At 25°C, all anti-Bet v 1 monoclonal antibodies of the invention bound to natural Bet v 1 with KD values ranging from 0.66 nM to 13.5 nM, as shown in Table 2. 37°C, all anti-Bet v 1 monoclonal antibodies of the 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 25°C and 37°C, the isotype control antibody demonstrated no measurable binding to natural Bet v 1.

[00258] Both at 25°C and 37°C, 3 of 20 anti-Bet v 1 monoclonal antibodies of the invention did not bind to Bet v 1 mutant-MMH. At 25°C, 17 of 20 anti-Bet v 1 monoclonal antibodies 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 anti-Bet v 1 monoclonal antibodies of the invention bound to Bet v 1 mutant-MMH with KD values ranging from 655 pM to 106 nM, as shown in Table 5. At 25°C and 37°C, the control antibody of the isotype demonstrated no measurable binding to the Bet v 1 mutant- MMH. TABLE 2: BINDING KINETICS PARAMETERS OF NATURAL BET V 1 BINDING TO BET V 1 MONOCLONAL ANTIBODIES AT 25°C. TABLE 3: BINDING KINETICS PARAMETERS OF NATURAL BET V 1 BINDING TO BET V 1 MONOCLONAL ANTIBODIES AT 37°C. TABLE 4: BINDING KINETICS PARAMETERS OF BET V 1 MUTANT-MMH THAT BIND TO BET V 1 MONOCLONAL ANTIBODIES AT 25°C. TABLE 5: BINDING KINETICS PARAMETERS OF BET V 1 MUTANT-MMH THAT BIND TO BET V 1 MONOCLONAL ANTIBODIES AT 37°C.

EXAMPLE 4: BINDING OF ANTIBODIES TO RELATED ALLERGENS AS DETERMINED BY SURFACE PLASM RESONANCE

[00259] Equilibrium dissociation constants (KD) for different related allergens that bind to purified anti-Bet v1 monoclonal antibodies were determined using a real-time surface plasmon resonance biosensor (SPR-Biacore) on a Biacore 4000 instrument. All binding studies were performed in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v/v Tween-20 surfactant, pH 7.4 (HBS-ET) buffer. run at 25°C. The Biacore CM5 sensor surface was first derivatized by amine coupling with a mouse anti-human Fc monoclonal antibody (GE, no BR-1008-39) to capture the anti-Bet v1 monoclonal antibodies. in the following related allergens: Alder (Aln g 1, MyBiosource, Cat no MBS1041484), Apple (Mal d 1, MyBiosource, Cat no MBS1224919), Carrot (Dau c 1. 2, MyBiosource, Cat no MBS1212920), Celery (Api g 1, MyBiosource, Cat no MBS1171376), Celery (Api g 2, MyBiosource, Cat no MBS1047880), European Hornbeam (Car b 1 isoform 1A and 1B, MyBiosource, cat # MBS1200018), European Hornbeam (Car b 1 isoform 2, MyBiosource , Cat no MBS1043940), Hazel (Color A 1, MyBiosource, Cat no MBS5304600) and White Oak (Que a 1, MyBiosource, Cat no MBS1258822). Different concentrations of the related allergens were prepared in HBS-ET running buffer (100 nM - 6.25 nM; 4-fold serial dilution) and then were injected onto the surface of the anti-Bet v 1 monoclonal antibody captured by the Human Fc for 3 minutes at a flow rate of 30 μl/minute, while dissociation of allergens bound to monoclonal antibodies was monitored for 8 minutes in HBS-ET running buffer. The rate constants of association (ka) and dissociation (Kd) were determined by fitting real-time binding sensorgrams to a 1:1 binding model with mass transport limitation via the Purifier 2.0c curve fitting software. . The dissociation equilibrium binding constants (KD) and dissociation half-life (t/) were calculated from the kinetic rate constants as:

[00260] Binding kinetic parameters for allergens related to different anti-Bet v 1 monoclonal antibodies of the invention at 25°C are shown in Tables 6 to 11.

[00261] As shown in Table 6, at 25°C, nine of the 20 anti-Bet v 1 antibodies of the invention demonstrated measurable Aln g 1 binding with KD values ranging from 1.03 nM to 175 nM. The other 11 antibodies did not demonstrate any measurable binding to Alng 1 under the conditions tested.

[00262] As shown in Table 7, at 25°C, two of the 20 anti-Bet v 1 antibodies of the invention demonstrated measurable binding to Mal d 1, with Kd values of 29.8 nM and 494 nM. The other 18 antibodies demonstrated no measurable binding to Mal d1 under the conditions tested.

[00263] As shown in Table 8, at 25°C, one of the anti-Bet v 1 antibodies of the invention demonstrated measurable binding to Api g 1 with a KD value of 167 nM. The other 19 antibodies demonstrated no measurable binding to Api g 1 under the conditions tested.

[00264] As shown in Table 9, at 25°C, eight of the 20 anti-Bet v 1 antibodies of the invention demonstrated measurable binding to a Car b 1 isoform 1A and 1B with KD values between 1.2 nM to 380nM. The other 12 antibodies demonstrated no measurable binding to the Car b 1 1A and 1B isoform under the conditions tested.

[00265] As shown in Table 10 at 25°C, 14 to 20 of one of the anti-Bet v 1 antibodies of the invention demonstrated measurable binding to a Car b 2 isoform with KD values ranging from 335 pM to 564 nM. The other 6 antibodies did not demonstrate any measurable binding to the Car b 1 2 isoform under the conditions tested.

[00266] As shown in Table 11 at 25°C, one of the anti-Bet v 1 antibodies of the invention demonstrated measurable binding to Co A 1 with a KD value of 396 nM. The other 19 antibodies demonstrated no measurable binding to Cor A 1 under the conditions tested.

[00267] None of the antibodies of the 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 demonstrated no measurable binding to any of the allergens tested.TABLE 6: BINDING KINETICS PARAMETERS OF ALDER (ALN G1) BINDING TO BET V 1 MONOCLONAL ANTIBODIES AT 25°C. TABLE 7: BINDING KINETICS PARAMETERS OF APPLE (MAL D1) BINDING TO BET V 1 A MONOCLONAL ANTIBODIES TABLE 8: BINDING KINETICS PARAMETERS OF CELERY (API G 1) BINDING TO BET V 1 MONOCLONAL ANTIBODIES AT 25°C. TABLE 9: BINDING KINETICS PARAMETERS OF EUROPEAN HORNBEAM (ISOFORM CAR B 1 1A & 1B) WHICH BINDS BET V 1 MONOCLONAL ANTIBODIES AT 25°C. TABLE 10: BINDING KINETICS PARAMETERS OF EUROPEAN HORNBEAM (CAR B1 2 ISOFORM) BINDING TO BET V 1 MONOCLONAL ANTIBODIES AT 25°C. TABLE 11: BINDING KINETICS PARAMETERS OF HAZEL HAZEL (COLOR A 1) BINDING TO ANTIBODIES

EXAMPLE 5: BLOCKADE OF BET V 1 BINDING TO ALLERGEN-SPECIFIC IgE BY ANTI-BET V 1 IgG ANTIBODIES

[00268] The ability of single anti-Bet v 1 antibodies of the invention or combinations of anti-Bet v 1 antibodies of the invention to block the binding of Bet v 1 to plate-captured IgE from allergic human donor plasma/serum has been determined using ELISA. Antibodies were tested alone or in polyclonal mixtures. For the assay, microtiter plates were coated overnight at 4°C with human extracellular domain protein of FcεR1α (the high-affinity receptor for IgE) that was produced with a C-terminal mouse Fc tag (hFcεR1α-mFc ; SEQ ID NO: 313). Plates were then blocked with 0.5% BSA (w/v) for 1 hour at room temperature (RT). Plasma from allergic donors was diluted and total IgE was then captured on the recipient-coated surface. A constant amount of natural 0.1 nM biotin-labeled Bet v 1 (Indoor Biotechnologies, no NA-BV1-1) was premixed with anti-Bet v 1 antibodies, at a single concentration of 1 μg/ml or in dilutions in series starting from 10 μg/ml or 1 μg/ml of each antibody and incubated for 1 hour at room temperature to allow the Bet v 1-antibody interaction to reach equilibrium. The antibody-Bet v1 mixture was then added to the IgE-coated plate for 1 hour. The plates were subsequently washed and the amount of natural Bet v 1 bound to the plate was detected using horseradish peroxidase-conjugated streptavidin (Thermo Scientific, no. N200/QJ223091) at a dilution of 1:10,000 and incubated for 1 hour at room temperature. . Plates were then washed with PBS-T between each step of the ELISA protocol described above. To develop the colorimetric reaction, a TMB/H2O2 substrate (in Reagent A no. 51-2602KC + Reagent B, no. 51-2607KC from BD Pharmingen) was added to the plates and incubated for 20 minutes at RT. The reaction was stopped using 2 N sulfuric acid (H2SO4; VWR, on BDH3500-1). Absorbance was subsequently measured on a spectrophotometer (Victor, Perkin Elmer) at 450 nm. Percent blocking was calculated using the highest concentration of antibody used in each assay, as described below.

[00269] Twenty anti-Bet v 1 antibodies were tested as single antibodies for their ability to block the binding of Bet v 1 to plaque-captured IgE from human donor allergic plasma using the described ELISA. Individual monoclonal antibodies had the ability to partially block IgE binding to Bet v 1 by 8.5-64%, which underlines the polyclonality of IgEs. Seven antibodies showed blocking ranging from 36 to 64% at the highest antibody concentration tested at 10 μg/ml, as shown in Table 12.

[00270] Four anti-Bet v 1 antibodies, H4H17082P2, H4H17038P2, H4H16987P and H4H16992P were tested in a single-point blocking assay. A combination of H4H17082P2 and H4H16992P demonstrated greater than 90% blockade in seven of ten IgE donors. Three and four combinations of monoclonal antibodies demonstrated similar results and did not appear to add any additional blocking effect compared to the two-antibody combination of H4H17082P2 and H4H16992P, as shown in Table 13.

[00271] The four monoclonal antibodies, H4H17082P2, H4H17038P2, H4H16987P and H4H16992P, were subsequently tested as combinations of single monoclonal antibodies and 2, 3 and 4 in the dose response blocking assay with 3 samples of donor IgE. The results showed that the combination of anti-Bet v 1 monoclonal antibodies from H4H17082P2 and H4H16992P blocked the binding of Bet v 1 to allergen-specific IgE greater than 90% and close to baseline in 3 donors, as shown in Table 14. The combinations of 3 and 4 antibodies tested showed similar breadth and potency of blocking activity. As a positive control, purified polyclonal anti-Bet v1 mouse IgG demonstrated >90% blocking. TABLE 12: ANTI-BET V1 ANTIBODIES THAT BLOCK THE BINDING OF BET V1 TO ALLERGEN-SPECIFIC IGE

[00272] Allergy donor plasma was diluted 1:50 for these assays. TABLE 13: SINGLE ANTIBODIES AND COMBINATIONS OF ANTIBODIES THAT BLOCK BET V 1 WHICH BINDS ALLERGEN-SPECIFIC IGE

[00273] Allergy donor plasma was normalized to 1:20 (6 donors), 1:10 (3 donors) or 1:9 (1 donor) specific IgE Bet v 1 titer and then diluted 1:50 for this assay .TABLE 14: COMBINATIONS OF ANTIBODIES THAT BLOCK BET V 1 THAT BIND TO ALLERGEN-SPECIFIC IGE

EXAMPLE 6: MAPPING OF EPITOPES OF ANTIBET V 1 ANTIBODIES THAT BIND TO BET V 1 BY DEUTTERIUM HYDROGEN EXCHANGE (H/D)

[00274] To determine the amino acid residues of Bet v 1 [(amino acids M1-N60 from Uniprot P15494] with which H4H16992P2, H4H17082P2, H4H17038P2 and H4H16987P interact, H/D exchange epitope mapping was performed with mass spectrometry A general description of the H/D exchange method is presented in, for example, Ehring (1999) Analytical Biochemistry 267 (2): 252 to 259; and Engen and Smith (2001) Anal. Chem. 73: 256A-265A.

[00275] The HDX-MS experiments were performed on an integrated Waters HDX/MS platform, which consists of a Leaptec HDX PAL system for deuterium labeling, a Waters Acquity M-Class (auxiliary solvent manager) for digestion and loading of the sample, a Waters Acquity M-Class (binary solvent manager) for the column analytical gradient, and Synapt G2-Si mass spectrometer for peptide mass measurement.

[00276] 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 the mixture of all 4 anti-Bet antibodies v 1 at a 1:1 molar ratio was incubated with 56.2 µl of D2O labeling solution for various time points (e.g., Undeuterated control = 0 second, labeled for 1 minute, 5 minutes, 10 minutes, and 20 minutes). Deuteration was quenched by transferring 50 μl of the 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 minutes. The quenched sample was then injected into a Waters HDX Manager for online pepsin/protease XIII digestion. Digested peptides were captured on a 1.7 μm, 2.1 × 5 mm VanGuard ACQUITY UPLC BEH C18 precolumn at 0°C and eluted onto an ACQUITY UPLC BEH C18 1.7 μm, 1.0 x 50 mm for 9 minutes gradient separation from 5% to 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 at a cone voltage of 37 V, scan time of 0.5 seconds, and mass/charge range of 50–1700 Th.

[00277] For the identification of Bet v 1 peptides, LC-MS E data from an undated sample was processed and searched in the database, including Bet v 1, and its random sequence using the Waters ProteinLynx Global Server software (PLGS). The identified peptides were imported into DynamX software and filtered by two criteria: 1) minimum products per amino acid: 0.3 and 2) replication file limit: 3. DynamX software automatically determined the deuterium uptake of each peptide based in retention time and high mass accuracy (<10ppm) at multiple points in time, with three repetitions each time.

[00278] Using the online pepsin/protease XIII column coupled to MSE data acquisition, a total of 36 Bet v 1 peptides were reproducibly identified in the absence or presence of the antibody, representing 91.2% sequence coverage. Peptides with significantly reduced deuteration uptake when bound to antibody combinations H4H16992P2, H4H17082P2, H4H17038P2, H4H16987P, and 4 are illustrated in Figures 1 to 4 and 5, respectively. The recorded peptide mass corresponds to the average mass value of the MH + centroid of three repeats of Bet v 1 in complex with anti-Bet v 1 antibody or antibodies.

[00279] As shown in Figure 1, peptides corresponding to amino acids 23-43 (FILDGDNLFPKVAPQAISSVE, SEQ ID NO: 307) showed a slower deuteration rate in the presence of H4H16992P.

[00280] As shown in Figure 2, peptides corresponding to amino acids 44-56 (NIEGNGGPGTIKK, SEQ ID NO: 308) showed a slower deuteration rate in the presence of H4H17082P.

[00281] As shown in Figure 3, peptides corresponding to amino acids 2-19 (GVFNYETETTSVIPAARL, SEQ ID NO: 309) showed a slower deuteration rate in the presence of H4H17038P2.

[00282] As shown in Figure 4, peptides corresponding to amino acids 57-70 (ISFPEGFPFKYVKD, SEQ ID NO: 310) and 81-96 (KYNYSVIEGGPIGDTL, SEQ ID NO: 315) showed a slower deuteration rate in the presence of H4H16987P .

[00283] As shown in Figure 5, the 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) showed slower deuteration rates in the presence of the combination of 4 anti-Bet v 1 antibodies ( H4H16992P, H4H17082P, H4H17038P2 and H4H16987P).

[00284] Furthermore, a modest level of protection was observed for the 23-43 peptide in the presence of H4H17082P, H4H17038P2 and H4H16987P, identifying that this region could represent a secondary epitope for these monoclonal antibodies.

EXAMPLE 7: EFFECT OF ANTI-BET V 1 ANTIBODIES IN THE IN VIVO MODEL OF PASSIVE CUTANEOUS ANAPHYLAXIS (PCA)

[00285] To determine the effectiveness of the anti-Bet v 1 antibodies of the invention to block allergen-induced mast cell degranulation, the in vivo model of passive cutaneous anaphylaxis (PCA) was used. This model involves intradermal injection of an allergen-specific antiserum into a local area of the skin, followed by intravenous injection of an antigen along with a dye. The allergic reaction causes capillary dilation and increased vascular permeability at the site of sensitization, resulting in preferential accumulation of dye at that site. The dye can be extracted from the tissue and quantified spectrophotometrically. Dye extravasation in tissue sensitized with test antiserum can then be compared to extravasation from tissue sensitized with a non-relevant antiserum.

[00286] Antisera were generated for use in the assay by immunizing Balb/c mice with 5 μg of natural Bet v 1 protein (Indoor Biotechnologies, no NA-BV1-1) in a solution of 1 mg/ml alum in buffered saline with 1X phosphate (PBS) on day 0. One week later (day 7), sensitized mice were stimulated with 5μg of natural Bet v 1 protein in a solution of 1 mg/ml alum in 1X PBS. Two weeks after augmentation, mice were subjected to an intranasal airway challenge with 0.5 μg of natural 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. The total IgE concentration in the isolated antisera was determined using an OptEIA™ ELISA kit (BD Biosciences, no. 555248) according to the manufacturer's instructions. The final concentration of Bet v 1 antisera was diluted to 2,600 ng/ml IgE in PBS.

[00287] The antisera used as a negative control in the assay were generated by immunizing Balb/c mice with 5 μg of natural Fel d 1 protein purified from cat hair extracts (Indoor Biotechnologies, no. LTN-FD1-1) in a 1 mg/ml alum solution. PBS on day 0. Mice were stimulated with 5 μg of Fel d 1 protein in a solution of 1 mg/ml alum in PBS on days 14 and 21. One week after the final boost (day 28), mice were sacrificed and serum was collected. The total IgE concentration in the isolated antisera was determined using an OptEIA™ ELISA kit (BD Biosciences, no. 555248) according to the manufacturer's instructions. The final concentration of antisera was diluted to 2,500 ng/ml IgE in PBS.

[00288] For PCA assays, groups of Balb/c mice (n>5 per experiment) were first injected subcutaneously with an isotype control antibody, an anti-Bet v1 antibody, or a combination of anti-Bet antibodies. Bet v 1 at a dose of 1 mg/kg (total antibody dose). Three days after antibody administration, 10 μl of 1.5 ng Bet v 1 antiserum or 3 ng negative control antiserum were injected into the right and left ears of mice in each group, respectively. Twenty-four hours after administration of allergen-specific antisera, mice were challenged by intravenous injection (100 μl per mouse) of a 1 μg/ml solution of natural Bet v 1 dissolved in PBS containing 0.5% (w/w /v) Evan blue dye (Sigma Aldrich, no. E2129). One hour after antigen challenge, the mice were sacrificed, their ears were excised, placed in 1 ml of formamide and subsequently incubated for 3 days at 50 to 56°C to extract the Evan blue dye. The ear tissue was then removed from the formamide, transferred to remove excess liquid, and weighed. Two hundred microliter aliquots of each formamide extract were transferred to 96-well plates in duplicate and their absorbance was then measured at 620 nm. The measured optical density was converted to Evan blue dye concentration using a standard curve and represented as ng of Evan blue dye per mg of tissue. Mean values ± standard deviation are shown in Table 15 for each group. The mean difference compared to the isotype control was calculated using the Bonferroni multiple comparisons test in GraphPad Prism.

[00289] As shown in Table 15, in the first study, the single anti-Bet v 1 antibody, H4H17082P2, did not demonstrate a significant reduction in dye extravasation compared to the isotype control. Unlike Study 2, the combination of two anti-Bet v 1 antibodies of the invention (H4H16992P and H4H17082P2) and the combination of four anti-Bet v 1 antibodies of the invention (H4H16992P, H4H17082P2, H4H17038P2 and H4H16987P) demonstrated significant reductions in extravasation. of dye compared to the isotype control treatment, with reductions of 35.26 and 36.49 ng/mg, respectively. Similarly, in Study 3, the combination of two anti-Bet v1 antibodies of the invention (H4H16992P and H4H17082P2) again demonstrated significant reductions in dye extravasation compared to the isotype control treatment, with a reduction of 41.09 ng/mg. As previously demonstrated in Study 1, in Study 3 the single anti-Bet v1 antibody, H4H17082P2, did not demonstrate a significant reduction in dye extravasation compared to the isotype control. However, another single antibody, H4H16992P, demonstrated a significant reduction in dye extravasation compared to the isotype control, with a reduction of 25.86 ng/mg. From the studies carried out, the single antibody H4H16992P, the combination of two antibodies of H4H17082P2 + H4H16992P and the combination of four antibodies of H4H17082P2 + H4H16992P + H4H17038P2 + H4H16987P had the ability to block mast cell degranulation, as indicated by a significant reduction in dye extravasation compared to isotype control in the in vivo passive cutaneous anaphylaxis model, as determined by two-way ANOVA with Bonferroni post-test. The H4H17082P2 antibody alone was unable to block mast cell degranulation, as indicated by an increase in dye extravasation compared to isotype in two of the studies performed. The number of mice used per group (n) is indicated in parentheses in the table. TABLE 15: EFFECT OF ANTI-BET V 1 ANTIBODIES IN THE IN VIVO MODEL OF PASSIVE CUTANEOUS ANAPHYLAXIS.

[00290] Total antibody concentration of 1mg/kg used for all antibody treatment groups * P≤0.05, *** P≤0.001, *** P≤0.0001n = number of mice in each group

EXAMPLE 8: EFFECT OF ANTI-BET V 1 ANTIBODIES AGAINST THREE DIFFERENT BIRCH POLLEN EXTRACTS IN THE IN VIVO MODEL OF PASSIVE CUTANEOUS ANAPHYLAXIS (PCA)

[00291] The anti-Bet v 1 antibodies provided here were tested for efficacy in blocking allergen-induced mast cell degranulation in the in vivo model of passive cutaneous anaphylaxis (PCA). Allergen-specific antiserum is injected transdermally into a local area of the skin, followed by intravenous injection of an antigen along with a dye. The allergic reaction causes capillary dilation and increased vascular permeability at the site of sensitization, resulting in preferential accumulation of dye at that site. The dye can be extracted from the tissue and quantified spectrophotometrically. Dye extravasation in tissue sensitized with test antiserum can then be compared to extravasation from tissue sensitized with a non-relevant antiserum.

[00292] Antisera to natural Bet v 1, birch pollen extract (BPE) from Betula Pendulda (also known as Betula verrucosa), BPE from Betula nigra and BPE from Betula populifolia were generated for use in this assay by immunizing Balb/c mice with 5 μg of natural Bet v 1 protein (Internal Biotechnologies, catalog no. NA-BV1-1, lot 36164) or 5 μg of BPE; pendula (Stallargenes Greer, Cat no XP527D3A25 lot no 277329), nigra (Stallargenes Greer Cat no XP79D3A25 lot no 285077) or populifolia (Stallargenes Greer Cat no XP80D3A2. 5 lot no 273622) in a solution of 1mg/ml alum in 1X phosphate buffered saline (PBS) on day 0. One week later (day 7), sensitized mice were stimulated with 5 μg of natural Bet v 1 protein or 5 μg of the respective BPE (pendula, nigra, or populifolia) in a solution of 1 mg/ml alum in 1X PBS. Two weeks after augmentation, mice were subjected to an intranasal airway challenge with 0.5μg of natural Bet v 1 protein or 0.5μg of the respective birch pollen extract in 20 μl of 1X PBS on days 21, 24, and 28. The mice were then sacrificed on day 31 and serum was collected. The total IgE concentration in the batches of isolated antisera was determined using an OptEIA™ ELISA kit (BD Biosciences, no. 555248) according to the manufacturer's instructions. The final concentration of Bet v 1 antisera was diluted to 2,500 ng/ml IgE in 1X PBS and the final concentration of batches of birch pollen extract antisera was diluted to 3,000 ng/ml for pendula, 1,900 ng/ml for nigra and 3,700 ng/ml for populifolia.

[00293] The antisera used as a negative control in this assay were generated by immunizing Balb/c mice with 5μg of natural Fel d 1 protein purified from cat hair extracts (Indoor Biotechnologies, Cat no LTN-FD1-1, batch no 36099) in a solution of 1mg/ml alum in 1X PBS. Mice were stimulated with 5 μg of Fel d 1 protein in a solution of 1 mg/ml alum in 1X PBS on days 14 and 21. One week after the final boost (day 28), mice were sacrificed and serum was collected. . The total IgE concentration in the isolated antisera was determined using an OptEIA™ ELISA kit (BD Biosciences, no. 555248) according to the manufacturer's instructions. The final concentration of antisera was diluted to 4800 ng/ml IgE in PBS.

[00294] For PCA assays, groups of Balb/c mice (n>4 per experiment, repeated three times) were first injected subcutaneously with an isotype control antibody or with a combination of two anti-Bet v antibodies. 1 at a dose of 1mg/kg (total dose of antibody). Three days after antibody administration, 10 μl of 1ng Bet v 1 antisera, 25 ng Betula pendula antiserum, 25 ng Betula nigra antiserum, or 25 ng Betula populifolia antiserum were injected into the right ear of mice in the designated groups. Left ears were administered 1 ng or 25 ng of Fel d 1 (negative control) to match the antiserum concentration of the corresponding right ear. Twenty-four hours after local administration of allergen-specific antisera, mice were challenged by intravenous injection (100 μl per mouse) with a 1 μg/ml solution of natural Bet v 1 (Cat no NA-BV1-1, batch 36164) or 1 μg/ml of the respective BPE (Stallargenes Greer, Cat lot no. XP527D3A25 no. 277329, Cat lot no. XP79D3A25 no. 285077 and Cat lot no. in w/v) of Evan blue dye (Sigma Aldrich, no. E2129). One hour after antigen challenge, the mice were sacrificed, the ears were excised, placed in 1 ml of formamide and subsequently incubated for 3 days at 50°C to extract the Evan blue dye. The ear tissue was then removed from the formamide, transferred to remove excess liquid, and weighed. Two hundred microliter aliquots of each formamide extract were transferred to 96-well plates in duplicate. The absorbance of the resulting supernatants was measured at 620 nm. The measured optical density was converted to Evan blue dye concentration using a standard curve and is represented as ng of Evan blue dye per mg of ear tissue. Mean values ± standard deviation are shown in Table 1 for each group. The mean difference compared to the isotype control was calculated using the Bonferroni multiple comparisons test in GraphPad Prism.

[00295] Table 16 demonstrates the effectiveness of the combination of two anti-Bet v 1 antibodies, H4H16992P and H4H17082P2, indicated by a significant reduction in dye extravasation when compared to the isotype control in all groups tested. As shown, the combination of two anti-Bet v 1 monoclonal antibodies from H4H17082P2/H4H16992P blocks mast cell degranulation in the passive cutaneous in vivo model against sensitization and subsequent challenge with natural Bet v 1 compared to isotype control, demonstrating a significant reduction in extravasation dye of 88.34. Similarly, reduced dye extravasation is also observed in the H4H17082P2/H4H16992P-treated groups for all three birch pollen extracts, compared to the respective 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 mean difference relative to the isotype control was calculated by two-way ANOVA with Bonferroni post-test. The number of mice used per group (n) is noted in parentheses in the tables. TABLE 16: EFFECT OF ANTI-BET V 1 ANTIBODIES IN THE IN VIVO MODEL OF PASSIVE CUTANEOUS ANAPHYLAXIS (PCA)

[00296] Total antibody concentration of 1mg/kg used for all antibody treatment groups* P≤0.05, *** P≤0.001, *** P≤0.0001 n = number of mice per group

EXAMPLE 9: CROSS COMPETITION BETWEEN ANTI-BET V 1 MONOCLONAL ANTIBODIES

[00297] Binding competition within a panel of anti-Bet v1 monoclonal antibodies was determined using a real-time label-free biolayer interferometry assay on the Octet HTX biosensor platform (Pall ForteBio Corp.). The entire experiment was carried out at 25°C in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% v/v Tween-20 surfactant, 1 mg/ml BSA buffer, pH 7.4 ( HBS-EBT) with the plate shaking at a speed of 1000 rpm. To evaluate whether two antibodies had the ability to compete with each other for binding to their respective epitopes in the recombinant mutant Bet v 1 expressed with a C-terminal myc-myc-hexahistidine tag (Bet v 1 mutant-MMH; SEQ ID NO: 312 ), about ~0.21 nm of Bet v 1 mutant-MMH was first captured on the tips of Octet biosensors coated with anti-Penta-His antibody (Fortebio Inc, no. 18-5122) by submerging the biosensor tips for 90 seconds in wells containing 5 μg/ml Bet v 1 mutant-MMH solution. The antigen-captured biosensor tips were then saturated with the first anti-Bet monoclonal antibody v 1 (referred to as mAb-1) by dipping into wells containing 50 μg/ml mAb-1 solution for 4 minutes. The biosensor tips were then dipped into wells containing 50 μg/ml solution of the second anti-Bet monoclonal antibody v 1 (referred to as mAb-2) for 3 minutes. The biosensor tips were washed in HBS-EBT buffer between all steps of the experiment. The real-time binding response was monitored throughout the course of the experiment and the binding response at the end of each step was recorded. The binding response of mAb-2 to Bet v 1 mutant-MMH pre-complexed with mAb-1 was compared and the competitive/non-competitive behavior of different anti-Bet v 1 monoclonal antibodies was determined as shown in Table 17.

[00298] Three of 20 anti-Bet v 1 monoclonal antibodies did not bind to mutant Bet v 1-MMH and cross-competition data were inconclusive. TABLE 17 CROSS COMPETITION AMONG ANTI-BET V 1 MONOCLONAL ANTIBODIES

EXAMPLE 10. THE ABILITY OF ANTI-BET V1 ANTIBODY COMBINATIONS TO BLOCK BET V1-INDUCED MAST CELL DEGRANULATION IN A HUMANIZED MUSE PCA MODEL

[00299] To explore the polyclonality of the allergen-specific IgE response across human birch allergic individuals, a humanized FcεR1α mouse model was used to facilitate the binding of human IgE to FcεR1α on the surface of mouse mast cells. Since human IgE may not bind to mouse FcεR1α, a genetically modified mouse was created, in which an endogenous mouse FcεR1α was replaced with the corresponding human FcεR1α sequence and denoted as FcεR1αhu/hu. FcεR1αhu/hu mice were validated for use in this model, demonstrating surface expression of human FcεR1α and the ability to respond to allergen: IgE activation in the Passive Cutaneous Anaphylaxis (PCA) model in a manner comparable to wild-type mice. . This PCA model involves intradermal injection of human allergy sera into a local area on the skin, followed by intravenous injection of the relevant allergen in conjunction with a dye. The allergic reaction causes capillary dilation and increased vascular permeability at the site of sensitization, resulting in preferential accumulation of dye at that site. The dye can be extracted from the tissue and quantified spectrophotometrically. Dye extravasation in tissue sensitized with test antiserum is compared to extravasation from tissue sensitized with non-allergic human sera.

METHODS

[00300] To determine the effect of anti-Bet v 1 antibodies on mast cell degranulation in this model, humanized FcεR1α mice received a subcutaneous injection of isotype control antibody or anti-Bet v 1 antibody combinations on day 1. For each donor human, two independent experiments were performed, n = 5 mice per group with combined data. The groups consist of no monoclonal antibody negative control, isotype negative control, REGN5713 + REGN5715 anti-Bet v 1 antibody double treatment group and REGN5713 + REGN5714 + REGN5715 triple anti-Bet v 1 antibody treatment group. The total or combined concentration of antibodies was 1 mg/kg or an IgG4 isotype control antibody (anti-IL6R α as a negative isotype control). Three days later, serum from birch-allergic patients or serum from birch-allergic patients (negative control) was injected intradermally (DI) into the right and left ears, respectively, allowing allergen-specific IgE to bind to FcεRI on mast cells. . To ensure that the same amount of allergen-specific IgE from each donor was used in the experiment, each antiserum injection was normalized to Bet v 1 ImmunoCAP®-specific IgE of 10 KU a/l.

[00301] Twenty-four hours after local administration of allergen-specific antibodies, mice were challenged by IV injection of 1μg of Bet v 1 diluted in PBS containing 0.5% Evan blue dye. One hour after allergen challenge, mice were sacrificed. Evan blue dye was extracted from ear tissue and quantified spectrophotometrically using a standard curve. (See Figure 6 for diagram of the protocol used) The reduction in Evan blue dye extravasation was averaged by subtracting the Evan blue dye concentration (normalized by ear tissue weight) for the ear administered by allergic serum from birch serum from the antibody-treated group, B (mAb, i), from the isotype control antibody-treated group, B (isotype, med). This number was divided by the difference between B (isotype, avg) and the dye concentration in the ear administered by non-allergic serum from the antibody-treated group [N (mAb, i)] and multiplied by 100 to obtain the overall average percentage reduction in dye extravasation (% reduction). The equation is shown below: % reduction (average) = 100 * [B (isotype, average) - B (mAb, i)]/[B (isotype, average) - N (mAb, i)]

[00302] An increase in the percentage reduction in dye leakage in the anti-Bet v 1 antibody treated group compared to the negative isotype control group is a measure of the effectiveness of the Bet v 1 antibody or antibody combinations in blocking the mast cell degranulation.

RESULTS

[00303] In this model, the combined use of anti-Bet v 1 antibodies designated H4H16992P (also known as REGN5713), H4H17038P2 (also known as REGN5714) and H4H17082P2 (also known as REGN5715) demonstrated maximal blockade of the IgE-mediated response when using IgE-containing sera from 3/3 birch allergic donors (see Figure 7). Using sera from birch allergic donors 25609, H4H16992P, H4H17038P2 and H4H17082P2 when combined exhibited 95% blockade of mast cell degranulation compared to isotype control (mean difference -42.04 +/- 6.9 (p < 0. 0001)) and combined use of H4H16992P and H4H17082P2 exhibited 93% blockade of mast cell degranulation compared to isotype control (mean difference -41.74 +/- 3.7 (p < 0.0001)). Using sera from birch allergic donors 23658, H4H16992P, H4H17038P2 and H4H17082P2, when combined exhibited 90% blockade of mast cell degranulation compared to the isotype control (mean difference -53.67+/-7.1 (p <0 .0001)) and H4H16992P combined with H4H17082P2 exhibited 74% blockade of mast cell degranulation compared to the isotype control (mean difference -44.58 +/- 11.4 (p < 0.0001)). Finally, the use of birch allergic donor sera 25414, H4H16992P, H4H17038P2 and H4H17082P2 when combined exhibited 92% blockade of mast cell degranulation compared to the isotype control (mean difference -39.72 +/- 7.5 (p < 0.0001)) and H4H16992P combined with H4H17082 exhibited 80% blockade of mast cell degranulation compared to the isotype control (mean difference -34.27 +/- 7.8 (p < 0.0001)) .

EXAMPLE 11. THE ABILITY OF ANTI-BET V 1 ANTIBODY COMBINATIONS TO BLOCK ACTIVATION OF BASOPHILS IN THE PHOSPHATE-ERK PHOSPHOFLUX ASSAY

[00304] The human IgE response was explored by testing the effect of various combinations of the anti-Bet v 1 antibodies H4H16992P (also known as REGN5713), H4H17038P2 (also known as REGN5714) and H4H17082P2 (also known as REGN5715) in inhibiting activation of basophils using samples from 8 birch allergic individuals. More specifically, to assess FcεR engagement and activation, basophils were tested in a phosphoflow-based functional assay that measures phosphorylation of the kinase ERK, a proximal readout of basophil activation and degranulation (Liu, Y. et al. (2007), J Exp Med 204, 93 to 103.

METHODS

[00305] Blood was collected from birch allergic patients (n = 8) and PBMCs isolated by density centrifugation on a Ficoll layer, washed, resuspended and plated as single spots in a 96-well format. In parallel, a 2X stimulation plate was prepared that included a dose response of purified Bet v 1 as well as dose responses of anti-Bet v 1 antibodies and antibody combinations (2.56 pM-200 nM) mixed with a dose constant (final concentration 100 pM) of purified natural Bet v 1. Cells were stimulated and subsequently stained with an antibody cocktail containing pErk-Alexa 488, CD123-BUV395 and HLA-DR-APC antibodies. After staining, data were acquired using an LSR-Fortessa instrument and analyzed by calculating the MFI of phosphorylated Erk staining within the basophil gate. The maximum percentage of inhibition was calculated as: 100 - ((100 x maximum antibody response)/isotype response). The maximum antibody response was the Mean Fluorescence Intensity (MFI) of phosphorylated Erk at the three main antibody doses on the dose response curve (curve plateau) minus the baseline IMF (average of replicate unstimulated samples). and the isotype response is the average of all MFI values in the dose response of an isotype control antibody produced by Regeneron (REGN1945 anti-Fel d 1 IgG4P) minus the baseline MFI.

RESULTS

[00306] Basophils from all individuals allergic to pollen from 8 birch trees responded to Bet v 1 stimulation with varying intensities. See Figure 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 magnitude of inhibition in 6/8 donors. Notably, individual antibodies, when tested separately, showed a high degree of variability in the ability to impact 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% blockade in only 1/8 donors tested.

EXAMPLE 12: DETERMINATION OF THE SIMULTANEOUS BINDING OF THREE MONOCLONAL ANTIBODIES ANTI-BET V 1 TO BET V 1 NATURAL

[00307] This experiment was performed to ensure that the binding epitopes of three selected Bet v 1 monoclonal antibodies were unique and that, regardless of the binding order of the monoclonal antibody, no steric hindrance was exhibited upon simultaneous binding of the three antibodies. The order-dependent competition between the three Bet v 1 monoclonal antibodies was also evaluated.

[00308] Simultaneous binding of three anti-Bet v 1 monoclonal antibodies to the same Bet v 1 was determined using a real-time label-free surface plasmon resonance-based Biacore 3000 biosensor platform (GE Healthcare.). The entire experiment was performed at 25°C in running buffer containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v/v Tween-20 surfactant, pH 7.4 (HBS-ET). Antibodies were immobilized on different surfaces of the CM5 sensor using EDC/NHS chemistry to achieve immobilization levels of 5,000-13,000 RU. REGN1945 (Fel d 1 monoclonal antibody) was also immobilized as a negative control. Natural Bet v 1 (nBet v 1), 10 nM or 20 nM, was injected onto different sensory surfaces immobilized with Bet v 1 monoclonal antibodies for 10 to 12 seconds, followed by sequential injection of different Bet v 1 monoclonal antibodies for 6 minutes at 15 μl/min.

[00309] The binding of different Bet v 1 monoclonal antibodies to nBet v 1 bound to a 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 (Resonance Unit) indicates that no binding was observed when the Bet v 1 monoclonal antibody was injected, while a higher binding signal (greater than 2 RU) does not represent competition. All three Bet v 1 monoclonal antibodies included in this example had the ability to bind simultaneously to nBet v 1 and the binding response was not affected by the order in which the antibodies were added. TABLE 18: SIMULANEOUS COMPETITION ANTI-BET V 1 ANTIBODIES

[00310] The data represents the average of at least 3 independent injections of Bet v 1 monoclonal antibodies onto the complex of nBet v 1 and immobilized Bet v 1 monoclonal antibody

SUMMARY

[00311] Regardless of the order of antibody binding to Bet v 1, there was no competition preventing the simultaneous binding of the three antibodies, suggesting that REGN5713, REGN5714 and REGN5715 bind to distinct and non-overlapping epitopes.

Claims (15)

1. Pharmaceutical composition characterized by the fact that it comprises a therapeutically effective amount of two isolated monoclonal antibodies or antigen-binding fragments thereof that bind to natural Bet v 1 or BPE, together with one or more pharmaceutically acceptable excipients, wherein the two isolated monoclonal antibodies do not compete for binding to natural Bet v 1 or BPE, the two isolated monoclonal antibodies comprising: (a) a heavy chain complementarity determining region (HCDR)1 comprising the amino acid sequence of SEQ ID NO: 292; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 294; an HCDR3 comprising the amino acid sequence of SEQ ID NO: 296; a light chain complementarity determining region (LCDR)1 comprising the amino acid sequence of SEQ ID NO: 300; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 302; and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 304, and (b) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 148; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 150; an HCDR3 comprising the amino acid sequence of SEQ ID NO: 152; a light chain complementarity determining region (LCDR)1 comprising the amino acid sequence of SEQ ID NO: 156; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 158; and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 160. 2. Pharmaceutical composition characterized by comprising a therapeutically effective amount of two isolated monoclonal antibodies or antigen-binding fragments thereof that bind to natural Bet v 1 or BPE, together with one or more pharmaceutically acceptable excipients, wherein the two monoclonal antibodies isolates do not compete for binding to natural Bet v 1 or BPE, the two isolated monoclonal antibodies comprising: (a) a heavy chain complementarity determining region (HCDR)1 comprising the amino acid sequence of SEQ ID NO: 292; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 294; an HCDR3 comprising the amino acid sequence of SEQ ID NO: 296; a light chain complementarity determining region (LCDR)1 comprising the amino acid sequence of SEQ ID NO: 300; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 302; and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 304, and (c) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 100; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 102; an HCDR3 comprising the amino acid sequence of SEQ ID NO: 104; an LCDR1 comprising the amino acid sequence of SEQ ID NO: 108; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 110; and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 112. 3. Pharmaceutical composition characterized by comprising a therapeutically effective amount of two isolated monoclonal antibodies or antigen-binding fragments thereof that bind to natural Bet v 1 or BPE, together with one or more pharmaceutically acceptable excipients, wherein the two monoclonal antibodies isolates do not compete for binding to natural Bet v 1 or BPE, the two isolated monoclonal antibodies comprising: (a) a heavy chain complementarity determining region (HCDR)1 comprising the amino acid sequence of SEQ ID NO: 292; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 294; an HCDR3 comprising the amino acid sequence of SEQ ID NO: 296; a light chain complementarity determining region (LCDR)1 comprising the amino acid sequence of SEQ ID NO: 300; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 302; and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 304, and (d) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 116; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 118; an HCDR3 comprising the amino acid sequence of SEQ ID NO: 120; an LCDR1 comprising the amino acid sequence of SEQ ID NO: 124; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 126; and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 128. 4. Pharmaceutical composition characterized by comprising a therapeutically effective amount of two isolated monoclonal antibodies or antigen-binding fragments thereof that bind to natural Bet v 1 or BPE, together with one or more pharmaceutically acceptable excipients, wherein the two monoclonal antibodies isolates do not compete for binding to natural Bet v 1 or BPE, the two isolated monoclonal antibodies comprising: (b) a heavy chain complementarity determining region (HCDR)1 comprising the amino acid sequence of SEQ ID NO: 148; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 150; an HCDR3 comprising the amino acid sequence of SEQ ID NO: 152; a light chain complementarity determining region (LCDR)1 comprising the amino acid sequence of SEQ ID NO: 156; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 158; and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 160; and (c) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 100; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 102; an HCDR3 comprising the amino acid sequence of SEQ ID NO: 104; an LCDR1 comprising the amino acid sequence of SEQ ID NO: 108; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 110; and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 112. 5. Pharmaceutical composition characterized by comprising a therapeutically effective amount of two isolated monoclonal antibodies or antigen-binding fragments thereof that bind to natural Bet v 1 or BPE, together with one or more pharmaceutically acceptable excipients, wherein the two monoclonal antibodies isolates do not compete for binding to natural Bet v 1 or BPE, the two isolated monoclonal antibodies comprising: (b) a heavy chain complementarity determining region (HCDR)1 comprising the amino acid sequence of SEQ ID NO: 148; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 150; an HCDR3 comprising the amino acid sequence of SEQ ID NO: 152; a light chain complementarity determining region (LCDR)1 comprising the amino acid sequence of SEQ ID NO: 156; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 158; and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 160; and (d) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 116; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 118; an HCDR3 comprising the amino acid sequence of SEQ ID NO: 120; an LCDR1 comprising the amino acid sequence of SEQ ID NO: 124; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 126; and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 128. 6. Pharmaceutical composition characterized by comprising a therapeutically effective amount of two isolated monoclonal antibodies or antigen-binding fragments thereof that bind to natural Bet v 1 or BPE, together with one or more pharmaceutically acceptable excipients, wherein the two monoclonal antibodies isolates do not compete for binding to natural Bet v 1 or BPE, the two isolated monoclonal antibodies comprising: (c) a heavy chain complementarity determining region (HCDR)1 comprising the amino acid sequence of SEQ ID NO: 100; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 102; an HCDR3 comprising the amino acid sequence of SEQ ID NO: 104; an LCDR1 comprising the amino acid sequence of SEQ ID NO: 108; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 110; and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 112; and (d) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 116; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 118; an HCDR3 comprising the amino acid sequence of SEQ ID NO: 120; an LCDR1 comprising the amino acid sequence of SEQ ID NO: 124; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 126; and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 128. 7. Pharmaceutical composition according to claim 1, characterized by the fact that it further comprises a third isolated monoclonal antibody or antigen-binding fragment thereof, which binds to natural Bet v 1 or BPE, comprising: (c) a HCDR1 comprising the amino acid sequence of SEQ ID NO: 100; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 102; an HCDR3 comprising the amino acid sequence of SEQ ID NO: 104; an LCDR1 comprising the amino acid sequence of SEQ ID NO: 108; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 110; and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 112. 8. Pharmaceutical composition according to claim 1, characterized by the fact that it further comprises a third isolated monoclonal antibody or antigen-binding fragment thereof, which binds to natural Bet v 1 or BPE, comprising: (d) a HCDR1 comprising the amino acid sequence of SEQ ID NO: 116; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 118; an HCDR3 comprising the amino acid sequence of SEQ ID NO: 120; an LCDR1 comprising the amino acid sequence of SEQ ID NO: 124; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 126; and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 128. 9. Pharmaceutical composition according to claim 2, characterized by the fact that it further comprises a third isolated monoclonal antibody or antigen-binding fragment thereof, which binds to natural Bet v 1 or BPE, comprising: (d) a HCDR1 comprising the amino acid sequence of SEQ ID NO: 116; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 118; an HCDR3 comprising the amino acid sequence of SEQ ID NO: 120; an LCDR1 comprising the amino acid sequence of SEQ ID NO: 124; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 126; and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 128. 10. Pharmaceutical composition according to claim 4, characterized by the fact that it further comprises a third isolated monoclonal antibody or antigen-binding fragment thereof, which binds to natural Bet v 1 or BPE, comprising: (d) a HCDR1 comprising the amino acid sequence of SEQ ID NO: 116; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 118; an HCDR3 comprising the amino acid sequence of SEQ ID NO: 120; an LCDR1 comprising the amino acid sequence of SEQ ID NO: 124; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 126; and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 128. 11. Pharmaceutical composition according to claim 7, characterized by the fact that it further comprises a fourth isolated monoclonal antibody or antigen-binding fragment thereof, which binds to natural Bet v 1 or BPE, comprising: (d) a HCDR1 comprising the amino acid sequence of SEQ ID NO: 116; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 118; an HCDR3 comprising the amino acid sequence of SEQ ID NO: 120; an LCDR1 comprising the amino acid sequence of SEQ ID NO: 124; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 126; and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 128. 12. Use of the pharmaceutical composition as defined in any one of claims 1 to 11, characterized by the fact that it is in the manufacture of a medicament for preventing or reducing mast cell degranulation or blocking basophil activation associated with natural Bet v 1 sensitization, Betula pendula BPE, Betula nigra BPE or Betula populifolia BPE. 13. Use of the pharmaceutical composition as defined in any one of claims 1 to 11, characterized by the fact that it is in the manufacture of a medicament for use in treating a patient who demonstrates a sensitivity or an allergic reaction against a Fagales protein, a Fagales-related 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 reaction to a Fagales protein, Fagales-related allergen, birch pollen birch or its extract or Bet v 1 protein, wherein sensitivity or an allergic reaction to a Fagales protein, a Fagales-related allergen, birch pollen or an extract thereof, or the Bet v 1 protein is prevented or decreased in severity and/or duration, or at least one symptom or complication associated with sensitivity or allergic reaction to a Fagales protein, Fagales-related allergen, birch pollen or an extract thereof, or Bet v 1 protein is prevented or ameliorated or that the frequency the concentration and/or duration or severity of the sensitivity or allergic reaction against a Fagales protein, a Fagales-related allergen, birch pollen or an extract thereof or Bet v 1 protein is reduced. 14. Use of the pharmaceutical composition as defined in any one of claims 1 to 11, characterized in that the composition is administered in combination with a second therapeutic agent useful for decreasing an allergic reaction to a Fagales protein, Fagales-related allergen, pollen of birch or an extract thereof, or Bet Protein v 1. 15. Use of the pharmaceutical composition as defined in any one of claims 1 to 11, characterized by the fact that it is for the manufacture of a medicament to increase the efficacy and/or safety of an allergen-specific immunotherapy (SIT) regimen for a patient in need of the same immediately before or at the same time as the SIT regimen, whereby the severity of an allergic reaction to the SIT regimen is mitigated.