Patent Application
20240083986
The present invention is related to human antibodies and antigen-binding fragments of human antibodies that bind to the birch pollen allergen, Bet v 1, therapeutic compositions comprising the antibodies, and methods of using the antibodies.
An official copy of the sequence listing is submitted concurrently with the specification electronically via Patent Center. The contents of the electronic sequence listing (10301US03_Sequence_Listing_ST26.xml; Size 386,848 bytes; and Date of Creation: Aug. 8, 2023) is part of the specification and is herein incorporated by reference in its entirety.
Birch is the predominant trigger in 23% of US and 14% of European allergy patients (Datamonitor report on Allergic Rhinitis, July 2010), and the main cause of type 1 allergies in the spring across Europe, North America, Russia, and Australia (Breiteneder et al., EMBO J. 1989, 8(7):1935-8). Bet v 1 protein is a major birch allergen identified in pollen from Betula verrucosa (European white birch tree, also synonymous with Betula pendula), and is responsible for IgE binding in more than 95% of birch pollen allergic patients (Breiteneder, supra). Bet v 1 is a small, 7-stranded anti-parallel β sheet with three α helices and a known crystalline 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 relates to peptides derived from trees of the Fagales order.
Sixty percent of birch pollen allergic patients 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 which travel by air up to 100 yards from the tree. Symptoms of birch pollen allergy can range from mild rhinitis and conjunctivitis to life-threatening asthmatic responses.
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, the 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 cross-linking of several IgE molecules and triggering the release of histamine and other inflammatory mediators causing the various allergic symptoms.
The treatment for allergies includes steroids for suppressing the immune activity and bronchial dilators for relieving asthma symptoms. Desensitization therapy is also used for severely allergic patients. Peptide vaccine combinations have been tested for desensitizing individuals to particular allergens, e.g. Bet v 1 (See U.S. Pat. No. 9,017,689). Antibodies have been proposed as a treatment for allergies, since they may be able to block the entry of allergenic molecules into the mucosal tissues, or may bind the allergen before it has the opportunity to bind to the IgE bound to the high affinity receptor on mast cells or basophils, thus preventing the release of histamine and other inflammatory mediators from these cells.
U.S. Pat. No. 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 the mucosal tissue. U.S. Pat. No. 6,849,259 describes the use of allergen-specific antibodies to inhibit allergic inflammation in an in vivo mouse model of allergy. Milk-based and egg-based antibody systems have been described. For example, US2003/0003133A1 discloses using milk as a carrier for allergens for inducing 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 use of a molecule that inhibits the ability of the allergen to bind to mast cells were described in WO1994/024164A2. Other antibodies to Bet v 1 were mentioned in U.S. 2010/0034812.
The present invention is directed toward overcoming one or more of the problems discussed above.
Provided herein are fully human monoclonal antibodies and antigen-binding fragments thereof that bind birch pollen, e.g. natural Bet v 1, Betula pendula birch pollen extract (BPE), Betula nigra BPE, or Betula populifolia BPE. The antibodies can be useful to bind the Bet v 1 allergen in vivo following exposure of a sensitized patient to the birch allergen, and as such, may act to either promote clearance of natural Bet v 1, Betula pendula birch pollen extract (BPE), Betula nigra BPE, or Betula populifolia BPE or to block the binding of the allergen to pre-formed IgE on the surface of mast cells or basophils. By doing so, the antibodies described herein can prevent the release of histamine or other inflammatory mediators from mast cells or basophils, thereby preventing or diminishing the untoward effects observed in patients sensitized to the 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, the antibodies may be capable of preventing even more serious in vivo complications associated with exposure to the birch pollen allergen in sensitized individuals, such as asthmatic responses, anaphylaxis, or even death.
The antibodies provided herein can be full-length, for example, an IgG1 or and IgG4 antibody, or may comprise only an antigen-binding portion, for example, a Fab, F(ab′)2, or scFv fragment, and can be modified to affect functionality, e.g., to eliminate residual effector functions (Reddy et al., 2000, J. Immunol. 164: 1925-1933).
A first aspect of the invention provides an isolated human monoclonal antibody or antigen-binding fragment thereof that binds natural Bet v 1, Betula pendula birch pollen extract (BPE), Betula nigra BPE, and/or Betula populifolia BPE.
In one embodiment, the isolated human monoclonal antibody or antigen-binding fragment thereof inhibits natural Bet v 1, Betula pendula BPE, Betula nigra BPE, or Betula populifolia BPE binding to allergen specific IgE.
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 antigen-binding fragment 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 a KD equal ranging from about 0.66 nM to about 27.9 nM.
In one embodiment, the isolated human antibody is a fully human monoclonal antibody.
In one embodiment, the isolated human monoclonal antibody or antigen-binding fragment thereof cross-reacts with one or more allergens selected from the group consisting of Aln g1, Cor a1, Car b1, Que a1, Api g2, Api g1, Dau c1, Mal d1, Ost c1, Fag s1, and Cas s1. Such allergens can also be termed PR-10 proteins, or so-called pathogenesis-related (PR) proteins. Additional PR-10 proteins (Bet v 1 family members) also include Act c 8 and Act d 8 (kiwi), Ara h 8 (peanut), Pru ar 1 (apricot), Pru av 1 (cherry), Pru p 1 (peach), Pyr c 1 (pear), Gly m 4 (soybean), 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.
In one embodiment, the antibody or antigen-binding fragment thereof cross-reacts with one or more allergens selected from the group consisting of Aln g1, Mal d1, Api g1, Car b1, and Cor a1.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof comprises the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within any one of the heavy chain variable region (HCVR) sequences selected from the group consisting of SEQ ID NOs: 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 within any one of the light chain variable region (LCVR) sequences selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, and 298. Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, e.g., the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., (1997), J. Mol. Biol. 273:927-948; and Martin et al., (1989), Proc. Natl. Acad. Sci. USA 86:9268-9272. Public databases are also available for identifying CDR sequences within an antibody.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof comprises the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within any one of the heavy chain variable region (HCVR) sequences selected from the group consisting of SEQ ID NOs: 114, 146, 98, and 290; and the three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within any one of the light chain variable region (LCVR) sequences selected from the group consisting of SEQ ID NOs: 122, 154, 106, and 298.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof comprises a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 282, and 290.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof comprises a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 114, 146, 98, and 290.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof comprises a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, and 298.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof comprises a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 122, 154, 106, and 298.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof comprises: (a) a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 282, and 290; and (b) a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, and 298.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof comprises: (a) a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 114, 146, 98, and 290; and (b) a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 122, 154, 106, and 298.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof comprises:
In one embodiment, the isolated human antibody or antigen-binding fragment thereof comprises a HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/266, 282/266, and 290/298.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof comprises a HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 114/122, 146/154, 98/106, and 290/298.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof which binds to Bet v 1 comprises a HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 146/154 and 290/298.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof which 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 to 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.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof which binds to Bet v 1 interacts with amino acid residues ranging from about position 23 to about position 44 of SEQ ID NO: 306.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof which binds to Bet v 1 interacts with amino acid residues ranging from about position 23 to about position 43 of SEQ ID NO: 306.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof which binds to Bet v 1 interacts with amino acid residues ranging from about position 44 to about position 70 of SEQ ID NO: 306.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof which binds to Bet v 1 interacts with amino acid residues ranging from about position 44 to about position 56 of SEQ ID NO: 306.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof which binds to Bet v 1 interacts with amino acid residues ranging from about position 2 to about position 19 of SEQ ID NO: 306.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof which binds to Bet v 1 interacts with amino acid residues ranging from about position 57 to about position 70 of SEQ ID NO: 306.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof which binds to Bet v 1 interacts with amino acid residues ranging from about position 57 to about position 66 of SEQ ID NO: 306.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof which binds to Bet v 1 interacts with amino acid residues ranging from about position 81 to about position 96 of SEQ ID NO: 306.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof which binds to Bet v 1 interacts with amino acid residues ranging from about position 81 to about position 89 of SEQ ID NO: 306.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof which 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 NOs: 307, 308, 309, 310, or 311 can be extended by 1 to 5 amino acids, or 5 to 10 amino acids, on either the C-terminal end or 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.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof which binds to Bet v 1 interacts with SEQ ID NO: 307.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof which binds to Bet v 1 interacts with SEQ ID NO: 308.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof which binds to Bet v 1 interacts with SEQ ID NO: 309.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof which binds to Bet v 1 interacts with SEQ ID NO: 310.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof which binds to Bet v 1 interacts with SEQ ID NO: 311.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof which binds to Bet v 1 interacts with SEQ ID NO: 315.
In one embodiment, the isolated human antibody or antigen binding fragment thereof which binds to Bet v 1 interacts with at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 307, 308, 309, 310, 311, and 315 and comprises an HCVR/LCVR sequence pair selected from the group consisting of SEQ ID NOs: 114/122, 146/154, 98/106, and 290/298.
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 light chain variable region of SEQ ID NO: 154.
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 light chain variable region of SEQ ID NO: 298.
In one embodiment, the human antibody or antigen binding fragment thereof that binds Bet v 1 comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQ ID NO: 148, 150, and 152, respectively and LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 156, 158, and 160, respectively.
In one embodiment, the human antibody or antigen binding fragment thereof that binds to Bet v 1 comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQ ID NO: 292, 294, and 296, respectively and LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 300, 302, and 304, respectively.
In one embodiment, the human antibody or antigen binding fragment thereof that binds to Bet v 1 comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQ ID NO: 4, 6, and 8, respectively and LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 12, 14, and 16, respectively.
In one embodiment, the human antibody or antigen binding fragment thereof that binds to Bet v 1 comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQ ID NO: 20, 22, and 24, respectively and LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 28, 30, and 32, respectively.
In one embodiment, the human antibody or antigen binding fragment thereof that binds to Bet v 1 comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQ ID NO: 36, 38, and 40, respectively and LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 44, 46, and 48, respectively.
In one embodiment, the human antibody or antigen binding fragment thereof that binds to Bet v 1 comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQ ID NO: 52, 54, and 56, respectively and LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 60, 62, and 64, respectively.
In one embodiment, the human antibody or antigen binding fragment thereof that binds to Bet v 1 comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQ ID NO: 68, 70, and 72, respectively and LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 76, 78, and 80, respectively.
In one embodiment, the human antibody or antigen binding fragment thereof that binds to Bet v 1 comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQ ID NO: 84, 86, and 88, respectively and LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 92, 94, and 96, respectively.
In one embodiment, the human antibody or antigen binding fragment thereof that binds to Bet v 1 comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQ ID NO: 100, 102, and 104, respectively and LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 108, 110, and 112, respectively.
In one embodiment, the human antibody or antigen binding fragment thereof that binds to Bet v 1 comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQ ID NO: 116, 118, and 120, respectively and LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 124, 126, and 128, respectively.
In one embodiment, the human antibody or antigen binding fragment thereof that binds to Bet v 1 comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQ ID NO: 132, 134, and 136, respectively and LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 140, 142, and 144, respectively.
In one embodiment, the human antibody or antigen binding fragment thereof that binds to Bet v 1 comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQ ID NO: 164, 166, and 168, respectively and LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 172, 174, and 176, respectively.
In one embodiment, the human antibody or antigen binding fragment thereof that binds to Bet v 1 comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQ ID NO: 180, 182, and 184, respectively and LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 188, 190, and 192, respectively.
In one embodiment, the human antibody or antigen binding fragment thereof that binds to Bet v 1 comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQ ID NO: 196, 198, and 200, respectively and LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 204, 206, and 208, respectively.
In one embodiment, the human antibody or antigen binding fragment thereof that binds to Bet v 1 comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQ ID NO: 212, 214, and 216, respectively and LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 220, 222, and 224, respectively.
In one embodiment, the human antibody or antigen binding fragment thereof that binds to Bet v 1 comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQ ID NO: 228, 230, and 232, respectively and LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 236, 238, and 234, respectively.
In one embodiment, the human antibody or antigen binding fragment thereof that binds to Bet v 1 comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQ ID NO: 244, 246, and 248, respectively and LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 252, 254, and 256, respectively.
In one embodiment, the human antibody or antigen binding fragment thereof that binds to Bet v 1 comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQ ID NO: 260, 262, and 264, respectively and LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 268, 270, and 272, respectively.
In one embodiment, the human antibody or antigen binding fragment thereof that binds to Bet v 1 comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQ ID NO: 276, 278, and 280, respectively and LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 268, 270, and 272, respectively.
In one embodiment, the human antibody or antigen binding fragment thereof that binds to Bet v 1 comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQ ID NO: 284, 286, and 288, respectively and LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 268, 270, and 272, respectively.
In one embodiment, the invention provides a fully human monoclonal antibody or antigen-binding fragment thereof that binds to natural Bet v 1, Betula pendula birch BPE, Betula nigra BPE, or Betula populifolia BPE, wherein the antibody or fragment thereof exhibits one or more of the following characteristics: (i) comprises a HCVR having 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 having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (ii) comprises a LCVR having 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 thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (iii) comprises a HCDR3 domain having 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 thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a LCDR3 domain having 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 thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (iv) comprises a HCDR1 domain having 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 thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a HCDR2 domain having 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 thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, and 300, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a LCDR2 domain having 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 thereof having 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, Betula pendula birch BPE, Betula nigra BPE, or Betula populifolia BPE.
In one embodiment, a “reference antibody” may include, for example, antibodies having a combination of heavy chain and light chain amino acid sequence pairs selected from the group consisting of 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/266, 282/266, and 290/298.
The invention encompasses antibodies having a modified glycosylation pattern. In some applications, modification to remove undesirable glycosylation sites may be useful, or e.g., removal of a fucose moiety to increase antibody dependent cellular cytotoxicity (ADCC) function (see Shield et al., 2002, JBC 277: 26733). In other applications, modification of galactosylation can be made in order to modify complement dependent cytotoxicity (CDC).
A second aspect provides an isolated antibody or antigen-binding fragment thereof that competes for specific binding to natural Bet v 1, Betula pendula birch BPE, Betula nigra BPE, or Betula populifolia BPE with an antibody or antigen-binding fragment 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 NOs: 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 NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, and 298.
One embodiment provides an isolated antibody or antigen-binding fragment thereof that competes for specific binding to natural Bet v 1, Betula pendula birch BPE, Betula nigra BPE, or Betula populifolia BPE with an antibody or antigen-binding fragment 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 NOs: 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 NOs: 122, 154, 106, and 298.
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, Betula pendula birch BPE, Betula nigra BPE, or Betula populifolia BPE with an antibody or antigen-binding fragment comprising the heavy and light chain CDRs contained within heavy and light chain sequence pairs selected from the group consisting of SEQ ID NOs: 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.
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 heavy chain variable region (HCVR), wherein the HCVR has an amino acid sequence selected from the group consisting of SEQ ID NOs: 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 NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, and 298.
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 heavy chain variable region (HCVR), wherein the HCVR has an amino acid sequence selected from the group consisting of SEQ ID NOs: 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 NOs: 122, 154, 106, and 298.
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 within heavy and light chain sequence pairs selected from the group consisting of SEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/266, 282/266, and 290/298.
In a fourth aspect, the invention provides nucleic acid molecules encoding Bet v 1 antibodies or fragments thereof. Recombinant expression vectors carrying such nucleic acids, and host cells into which such vectors have been introduced, are also contemplated herein, as are methods of producing the antibodies by culturing the host cells under conditions permitting production of the antibodies, and recovering the antibodies produced.
In one embodiment, provided herein are nucleic acid molecules encoding a human monoclonal antibody or fragment thereof that binds to natural Bet v 1, Betula pendula BPE, Betula nigra BPE, or Betula populifolia BPE.
In one embodiment, provided herein is an antibody or fragment thereof comprising a HCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, 17, 33, 49, 65, 81, 97, 113, 129, 145, 161, 177, 193, 209, 225, 241, 257, 273, 281, and 289, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereof.
In one embodiment, the HCVR is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 113, 145, 257, and 289.
In one embodiment, the antibody or fragment thereof further comprises a LCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, 25, 41, 57, 73, 89, 105, 121, 137, 153, 169, 185, 201, 217, 233, 249, 265, and 297, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereof.
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.
In one embodiment, provided herein is an antibody or antigen-binding fragment of an antibody comprising a 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 having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a 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 thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
In one embodiment, provided herein is an antibody or fragment thereof further comprising a HCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, 19, 35, 51, 67, 83, 99, 115, 131, 147, 163, 179, 195, 211, 227, 243, 259, 275, 283, and 291, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a 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 thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a 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 thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a 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 thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
A fifth aspect provides a pharmaceutical composition comprising a therapeutically effective amount of one or more isolated human antibodies or antigen-binding fragments thereof that bind natural Bet v 1, Betula pendula birch BPE, Betula nigra BPE, or Betula populifolia BPE, together with one or more pharmaceutically acceptable excipients.
In one embodiment, the pharmaceutical composition 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.
In one embodiment, the pharmaceutical composition comprises:
In one embodiment, the pharmaceutical composition comprises:
In one embodiment, the pharmaceutical composition comprises:
In one embodiment, the pharmaceutical composition comprises:
In one embodiment, the pharmaceutical composition comprises:
In one embodiment, the pharmaceutical composition comprises:
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, comprising HCVR/LCVR amino acid sequence pairs selected from the group consisting of SEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/266, 282/266, and 290/298; and one or more pharmaceutically acceptable excipients.
In one embodiment, the pharmaceutical composition comprises three or more isolated human monoclonal antibodies or antigen-binding fragments thereof that bind to Bet v 1, comprising HCVR/LCVR amino acid sequence pairs selected from the group consisting of SEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/266, 282/266, and 290/298; and one or more pharmaceutically acceptable excipients.
In one embodiment, the pharmaceutical composition comprises four isolated human monoclonal antibodies that bind to Bet v 1, or antigen-binding fragments thereof, wherein the human antibodies or antigen-binding fragments thereof comprise the HCVR/LCVR amino acid sequence pairs of SEQ ID NOs: 114/122, 146/154, 98/106, and 290/298; and one or more pharmaceutically acceptable excipients.
In one embodiment, the pharmaceutical composition comprises the antibody designated H4H16992P having the HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 146/154; the antibody designated H4H17082P2 having the HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 290/298; and the antibody designated H4H17038P2 having the HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 98/106.
In one embodiment, the pharmaceutical composition comprises the antibody designated H4H16992P having the HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 146/154; the antibody designated H4H17082P2 having the HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 290/298; the antibody designated H4H17038P2 having the HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 98/106; and the antibody designated H4H16987P having the HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 114/122.
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, wherein the first antibody or fragment thereof interacts with amino acid residues ranging from about position 23 to about position 44 of SEQ ID NO: 306, 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 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.
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, 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 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 amino acid residues ranging from about position 44 to about position 56 of SEQ ID NO: 306.
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, 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.
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 further isolated human monoclonal antibodies, or antigen-binding fragments 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 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.
In one embodiment, the one or more further isolated human monoclonal antibodies or fragments thereof interacts 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 further isolated human monoclonal antibodies interacts with a different amino acid sequence than the first isolated human monoclonal antibody.
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 further isolated human monoclonal antibodies, or antigen-binding fragments 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 further antibodies or fragments thereof interact with an amino acid sequence selected from the group consisting of SEQ ID NOs: 308, 309, 310, 311, and 315.
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 pharmaceutically acceptable excipients, wherein 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 NOs: 146/154 and 290/298, respectively.
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 pharmaceutically acceptable excipients, wherein 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 NOs: 146/154, 290/298, and 98/106, respectively.
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 pharmaceutically acceptable excipients, wherein 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 NOs: 146/154, 290/298, 98/106, and 114/122, respectively.
In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of an isolated human monoclonal antibody or antigen-binding fragment thereof that binds 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 ci, Fag s1, and Cas s1. In some embodiments, the antibody or antigen-binding fragment thereof cross-reacts with one or more allergens selected from the group consisting of Aln g1, Mal d1, Api g1, Car b1, and Cor a1.
In one embodiment, the 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.
The second therapeutic agent may be a small molecule drug, a protein/polypeptide, an antibody, a nucleic acid molecule, such as an anti-sense molecule, or a siRNA. The second therapeutic agent may be synthetic or naturally derived.
The second therapeutic agent may 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 on mast cells or basophils. A second therapeutic agent may also be any agent that is used as standard of care in treating an allergic response to any allergen. Such second therapeutic agent may be an antihistamine, epinephrine, a decongestant, a corticosteroid, or a peptide vaccine.
In certain embodiments, the second therapeutic agent may be an agent that helps to counteract or reduce any possible side effect(s) associated with the antibody or antigen-binding fragment of an antibody of the invention, if such side effect(s) should occur.
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 concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures, including, for example in combination with an allergen-specific immunotherapy (SIT) regimen where the antibodies are administered before or during SIT. The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics 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 (for example, an antibody may be administered concurrently with another agent used to treat the same disorder), or they may achieve different effects (e.g., control of any adverse effects). As used herein, additional therapeutic agents that are normally administered to treat or prevent a particular disease, or condition, are appropriate for the disease, or condition, being treated.
When multiple therapeutics are co-administered, dosages may be adjusted accordingly, as is recognized in the pertinent art.
A sixth aspect provides a method for treating a patient who demonstrates a sensitivity to, 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 treating at least one symptom or complication associated with a sensitivity to, or allergic reaction against a Fagales protein, a 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 antigen-binding fragments thereof that bind to natural Bet v 1, Betula pendula BPE, Betula nigra BPE, or Betula populifolia BPE, or a pharmaceutical composition comprising an effective amount of one or more isolated human monoclonal antibodies or fragments thereof that bind to natural Bet v 1, Betula pendula BPE, Betula nigra BPE, or Betula populifolia BPE, to a patient in need thereof, wherein the sensitivity to, or an allergic reaction against, a Fagales protein, a Fagales related allergen, birch pollen or an extract thereof, or Bet v 1 protein is either prevented, or lessened in severity and/or duration, or at least one symptom or complication associated with the sensitivity to, or allergic reaction against, a Fagales protein, a Fagales related allergen, 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 the sensitivity to or allergic reaction against, a Fagales protein, a Fagales related-protein, birch pollen or an extract thereof, or Bet v 1 protein is reduced following administration of one or more of the isolated human monoclonal antibodies or fragments thereof that bind to natural Bet v 1, Betula pendula BPE, Betula nigra BPE, or Betula populifolia BPE, or following administration of a composition comprising any one or more of the foregoing antibodies.
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.
In some embodiments, the 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, a Fagales related allergen, birch pollen or an extract thereof, or Bet v 1 protein.
In some embodiments, the method further comprises administering an effective amount of a second therapeutic agent useful for diminishing 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 can be selected from the group consisting of a corticosteroid, a bronchial dilator, an antihistamine, epinephrine, a decongestant, another different antibody to Bet v 1 and a peptide vaccine.
In some embodiments, the method further comprises treating the patient with an allergen-specific immunotherapy (SIT) regimen just after or concurrent with the antibodies or fragments thereof or the pharmaceutical composition comprising the antibodies.
In one embodiment, the invention provides a method for treating a Fagales allergic patient who demonstrates a sensitivity to, or an allergic reaction against, one or more Fagales allergens, or Fagales related allergens, or for treating at least one symptom or complication associated with a sensitivity to, or allergic reaction against 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 Bet v 1, or a pharmaceutical composition comprising an effective amount of one or more isolated human monoclonal antibodies or fragments thereof that bind to Bet v 1, to a patient in need thereof, wherein the sensitivity to, or an allergic reaction against, a Fagales allergen, or Fagales related allergen, is either prevented, or lessened in severity and/or duration, or at least one symptom or complication associated with the sensitivity to, or allergic reaction against, a Fagales allergen, or Fagales related allergen, is prevented, or ameliorated, or that the frequency and/or duration of, or the severity of the 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 Bet v 1, or following administration of a composition comprising any one or more of the foregoing antibodies.
In some embodiments, the one or more Fagales allergens is selected from the group consisting of Bet v 1, Aln g1, Cor a1, Car b1, and Que a1.
In one embodiment, the invention provides a pharmaceutical composition comprising one or more of the antibodies described herein that bind natural Bet v 1, Betula pendula BPE, Betula nigra BPE, and Betula populifolia BPE for use in treating a patient who demonstrates a sensitivity to, or an allergic reaction against, a Fagales protein, birch pollen or an extract thereof, or Bet v 1 protein, or for treating at least one symptom or complication associated with a sensitivity to, or allergic reaction against, a Fagales protein, birch pollen or an extract thereof, or Bet v 1 protein, wherein the sensitivity to, or an allergic reaction against, a Fagales protein, birch pollen or an extract thereof, or Bet v 1 protein is either prevented, or lessened in severity and/or duration, or at least one symptom or complication associated with the sensitivity to, or allergic reaction against, a Fagales protein, 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 the sensitivity to or allergic reaction against, a Fagales protein, birch pollen or an extract thereof, or Bet v 1 protein is reduced.
In one embodiment, the invention provides for use of a pharmaceutical composition comprising one or more of the antibodies of the invention that binds to Bet v 1 in the manufacture of a medicament for use in treating a patient who demonstrates a sensitivity to, or an allergic reaction against, birch pollen or an extract thereof, or to Bet v 1 protein, or for treating at least one symptom or complication associated with a sensitivity to, or allergic reaction against, birch pollen or an extract thereof, or to Bet v 1 protein, wherein the sensitivity to, or an allergic reaction against, birch pollen or an extract thereof, or to Bet v 1 protein is either prevented, or lessened in severity and/or duration, or at least one symptom or complication associated with the sensitivity to, or allergic reaction against, birch pollen or an extract thereof, or to Bet v 1 protein is prevented, or ameliorated, or that the frequency and/or duration of, or the severity of the sensitivity to or allergic reaction against, birch pollen or an extract thereof, or to Bet v 1 protein is reduced.
In one embodiment, the invention provides use of a pharmaceutical composition as described above, wherein the composition is administered in combination with a second therapeutic agent useful for diminishing an allergic reaction to a Fagales protein, birch pollen or an extract thereof, or Bet v 1 protein. In one embodiment, the invention provides for 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 peptide vaccine.
In certain embodiments, the antibodies of the invention that bind to Bet v 1 may be capable of reducing, minimizing, or preventing at least one symptom in a patient sensitive to birch pollen or Bet v 1, such as sneezing, congestion, nasal blockage, coughing, wheezing, bronchoconstriction, rhinitis, or conjunctivitis.
In one embodiment, the antibodies of the invention that bind to Bet v 1, or a composition comprising one or more antibodies of the invention may be used to prevent more serious in vivo complications associated with an allergy to Bet v 1, including asthmatic responses, anaphylactic shock, or even death resulting from anaphylaxis.
In one embodiment, the pharmaceutical composition is administered to the patient in combination with a second therapeutic agent.
In another embodiment, the second therapeutic agent is selected from the group consisting of an antihistamine, epinephrine, a decongestant, a corticosteroid, another different antibody to Bet v 1, a peptide vaccine and any other palliative therapy useful for reducing the severity of the allergic reaction or for ameliorating at least one symptom associated with the allergic reaction.
In yet another embodiment, the pharmaceutical composition is administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures, including, for example, administered prior to or concurrently with an allergen-specific immunotherapy (SIT) regimen. In some aspects, use of a SIT regimen together with the antibodies provided herein provides a synergistic effect.
In one embodiment, a method is provided for enhancing the efficacy and/or safety of an allergen-specific immunotherapy (SIT) regimen, the method comprising administering an effective amount of one or more isolated human monoclonal antibodies or antigen-binding fragments thereof, as provided herein, or a pharmaceutical composition comprising an effective amount of one or more isolated human monoclonal antibodies or fragments thereof, to a patient in need thereof just prior to or concurrent with the SIT regimen, wherein the severity of an allergic reaction to the SIT regimen is mitigated. In some embodiments, the SIT regimen comprises an up-dosing phase followed by a maintenance phase. In some embodiments, the SIT regimen is a rush SIT regimen.
In an additional aspect, a method is provided 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, the method comprising administering a pharmaceutical composition described herein to a patient in need thereof.
In some embodiments, the BPE is selected from the group consisting of natural Bet v 1, Betula pendula BPE, Betula nigra BPE, and Betula populifolia BPE.
Other embodiments will become apparent from a review of the ensuing detailed description.
Before the present compositions and methods are described, it is to 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 is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term “about,” when used in reference to a particular 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.).
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are now described.
The term “Bet v 1” as used herein, refers to at least one Bet v 1 protein, either in natural/native form, or recombinantly produced. 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 a 7 stranded anti-parallel β-sheet (β1-β7), two short α-helices (α1 and α2) connecting β1 and β2, a long C-terminal α-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 mutant Bet v 1, SEQ ID NO: 312, comprises G2-N160 of Uniprot P15494 with a S85A substitution and contains a Myc-Myc-hexahistidine tag. The Bet v 1 amino acid sequence from Uniprot: P15494, i.e. SEQ ID NO: 314, can also refer to Bet v 1.
“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 “homologous sequence of SEQ ID NO: 306 or SEQ ID NO: 314”, as used herein, refers to a polypeptide that has an identity to SEQ ID NO: 306 or SEQ ID NO: 314 which is greater than 70%, preferably greater than 80%, more preferably greater than 90%, and even more preferably greater than 95%.
The term “Bet v 1 fragment” as used herein, refers to a polypeptide comprising or alternatively consisting of, at least one antigenic site of Bet v 1. In one embodiment, the term “Bet v 1 fragment” as used herein, refers to a polypeptide comprising or alternatively consisting of at least two antigenic sites of Bet v 1. 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 NOs: 306, 307, 308, 309, 310, 311, and 315. In one embodiment, any of the Bet v 1 fragments are capable of inducing the production of antibodies in vivo that specifically bind to naturally occurring Bet v 1, or to recombinantly produced Bet v 1.
The term “antibody”, as used herein, means any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., Bet v 1). The term “antibody”, as used herein, is intended to refer to immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds (i.e., “full antibody molecules”), as well as multimers thereof (e.g. IgM) or antigen-binding fragments thereof. Each heavy chain is comprised of a heavy chain variable region (“HCVR” or “VH”) and a heavy chain constant region (comprised of domains CH1, CH2 and CH3). Each light chain is comprised of a light chain variable region (“LCVR or “VL”) and a light chain constant region (CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the invention, the FRs of the antibody (or antigen binding fragment thereof) may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.
Substitution of one or more CDR residues or omission of one or more CDRs is also possible. Antibodies have been described in the scientific literature in which one or two CDRs can be dispensed with 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 about one fifth to one third of CDR residues actually contact the antigen. Padlan also found many antibodies in which one or two CDRs had no amino acids in contact with an antigen (see also, Vajdos et al., 2002, J Mol Biol 320:415-428).
CDR residues not contacting antigen can be identified based on previous studies (for example residues H60-H65 in CDRH2 are often not required), from regions of Kabat CDRs lying outside Chothia CDRs, by molecular modeling and/or empirically. If a CDR or residue(s) thereof is omitted, it is usually substituted with an amino acid occupying the corresponding position in another human antibody sequence or a consensus of such sequences. Positions for substitution within CDRs and amino acids to substitute can also be selected empirically. Empirical substitutions can be conservative or non-conservative substitutions.
The 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 framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The present invention includes antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as “germline mutations”). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and/or CDR residues within the VH and/or VL domains are mutated back to the 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, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived).
Furthermore, the antibodies of the present invention may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ 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 property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present invention.
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 having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein.
The term “human antibody”, as used herein, is intended to include non-naturally occurring human antibodies. The term includes antibodies that are recombinantly produced in a non-human mammal, or in cells of a non-human mammal. The term is not intended to include antibodies isolated from or generated in a human subject.
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. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. In certain embodiments, such 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, while related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
Human antibodies can exist in two forms that are 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 inter-chain disulfide bonds and a molecule of about 75-80 kDa is formed composed of a covalently coupled light and heavy chain (half-antibody). These forms have been extremely difficult to separate, even after affinity purification.
The frequency of appearance of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype 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 typically observed using a human IgG1 hinge. The instant invention encompasses antibodies having one or more mutations in the hinge, CH2 or CH3 region, which may be desirable, for example, in production, to improve the yield of the desired antibody form.
As used herein, the expression “antigen-binding molecule” means a protein, polypeptide or molecular complex comprising or consisting of at least one complementarity determining region (CDR) that alone, or in combination with one or more additional CDRs and/or framework regions (FRs), specifically binds to a particular antigen. In certain embodiments, an antigen-binding molecule is an antibody or a fragment of an antibody, as those terms are defined elsewhere herein.
The phrase “specifically binds” or “binds specifically to” or the like, means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1×10−6 M or less (e.g., a smaller KD denotes a 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 have been identified by surface plasmon resonance, e.g., BIACORE™, which bind specifically to Bet v 1.
The phrase “high affinity” antibody refers to those monoclonal antibodies having a binding affinity to Bet v 1, expressed as KD, of at least 10−8 M; preferably 10−9 M; more preferably 10−10M, even more preferably 10−11 M, even more preferably 10−12 M, as measured by surface plasmon resonance, e.g., BIACORE™ or solution-affinity ELISA.
By the term “slow off rate”, “Koff” or “kd” is meant an antibody that dissociates from Bet v 1, with a rate constant of 1×10−3 s−1 or less, preferably 1×10−4 s−1 or less, as determined by surface plasmon resonance, e.g., BIACORE™.
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 obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds 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 to Bet v 1.
The specific embodiments, antibody or antibody fragments of the invention may be conjugated to a therapeutic moiety (“immunoconjugate”), such as a corticosteroid, a second anti-Bet v 1 antibody, or epinephrine, a vaccine, or any other therapeutic moiety useful for treating an allergic response to Bet v 1.
The antibodies of the invention may 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 naturally produced, 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 been subjected to 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.
According to certain embodiments, an isolated antibody may be substantially free of other antibodies (Abs) having different antigenic specificities (e.g., an isolated antibody that specifically binds Bet v 1, or a fragment thereof, is substantially free of Abs that specifically bind antigens other than Fagales antigens, or in some aspects, other than Bet v 1.
A “blocking antibody” or a “neutralizing antibody”, as used herein (or an “antibody that neutralizes Bet v 1 activity”), is intended to refer to an antibody, or an antigen binding portion thereof, whose binding to Bet v 1 results in inhibition of at least one biological activity of Bet v 1. For example, an antibody of the invention may aid 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 Bet v 1 biological activity by one or more of several standard in vitro or in vivo assays (such as a passive cutaneous anaphylaxis assay, as described herein) or other in vivo assays known in the art (for example, other animal models to look at protection from challenge with Bet v 1 following administration of one or more of the antibodies described herein).
The term “surface plasmon resonance”, as used herein, refers to an optical phenomenon that allows for the analysis of real-time biomolecular interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).
The term “KD”, as used herein, is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.
The term “epitope” refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. The term “epitope” also refers to a site on an antigen to which B and/or T cells respond. It also refers to a region of an antigen that is bound by an antibody. Epitopes may be either linear or conformational. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics. Epitopes may also be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.
The term “substantial identity” or “substantially identical,” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with 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 algorithm of sequence identity, such as FASTA, BLAST or GAP, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.
As applied to polypeptides, the term “substantial similarity” or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 90% sequence identity, even more preferably at least 95%, 98% or 99% sequence identity. Preferably, residue positions, which are not identical, differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. (See, e.g., Pearson, 1994, Methods Mol. Biol. 24: 307-331). 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 acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992 Science 256: 1443-45). A “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.
Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as GAP and BESTFIT which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can 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 percent sequence identity of the regions of the best overlap between the 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 computer program BLAST, especially BLASTP or TBLASTN, using default parameters. (See, e.g., Altschul et al., 1990, J. Mol. Biol. 215: 403-410 and 1997 Nucleic Acids Res. 25:3389-3402).
By the phrase “therapeutically effective amount” is meant an amount that produces the desired effect for which it is administered. The exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).
The antibodies of the invention may be used to “desensitize” a Fagales allergic individual. The term to “desensitize” is defined herein as 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 (to a level less than that which the Fagales allergic individual would otherwise experience), or to a Fagales related allergen. The term “desensitize” is further defined herein as to decrease the allergic-reactivity of an individual to PR-10 proteins including Act c 8 and Act d 8 (kiwi), Ara h 8 (peanut), Pru ar 1 (apricot), Pru av 1 (cherry), Pru p 1 (peach), Pyr c 1 (pear), Gly m 4 (soybean), Vig r 1 (mung bean), Sola I 4 (tomato), Cuc m 3 (melon), Rub i 1 (raspberry), and Fra a 1 (strawberry).
Trees belonging to the order of Fagales are the source of spring allergy symptoms, and the major birch allergen, Bet v 1 is responsible for IgE binding in more than 95% of birch pollen allergic patients (Breiteneder, EMBO J. 1989, 8(7):1935-8). Birch is the predominant trigger in 23% of US and 14% of European allergy patients. (DataMonitor report on Allergic Rhinitis, July, 2010). The severity of the symptoms in individuals who demonstrate a sensitivity to birch pollen ranges from a relatively mild rhinitis and conjunctivitis to a potentially life-threatening asthmatic condition. It has been shown that greater than 60% of patients who are 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).
Fagales trees show a distinct geographical distribution where birch and alder are endemic in the northern parts of Europe and North America, while hazel, hornbeam and oak prefer a warmer climate, thus populating rather the southern parts of these continents. Co-populations of all five species are frequently found in temperate climate zones 5. (Spieksma F T M. Regional European pollen calendars. D'Amato G, Spieksma F T M, Bonini S, editors. Allergenic pollen and pollinosis in Europe. Hoboken, NJ: Wiley-Blackwell; 1991. pp. 49-65) Several Betulaceae trees including alder, hazel and hombeam have the potential to initiate sensitization to Bet v 1-like allergens in susceptible individuals resulting in the production of highly cross-reactive IgE antibodies. (Hauser, et al., 2011, Clin Exp Allergy. 41: 1804-14)
The Fagales order allergens, or Fagales allergens, as defined herein include birch pollen (Bet v 1), alder pollen (Aln g1 and Aln g4), hazel pollen (Cor a1, Cor a2, Cor a8, Cor a9, Cor a10, Cor all, Cor a12, Cor a13, and Cor a14), hornbeam pollen (Car b1), hop-hornbeam pollen (Ost c1), chestnut pollen (Cas s1, Cas s5, Cas s8, and Cas s9), beech pollen (Fag 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 like or related to Bet v 1, i.e. Fagales related allergens. These allergens are also expressed in nuts of the Fagales trees, and in the fruits of unrelated trees belonging to the order Rosales. Cross-reactivity of these allergens may prompt symptoms of food allergy in pollen allergic patients. Exemplary cross-reactive food allergies include apple, cherry, apricot, pear, medicago, pea, soybean, tomato, celery, carrot, and asparagus. (Allergens and Allergen Immunotherapy: Subcutaneous, Sublingual, and Oral, 5′ Edition. Richard F. Lockey, Dennis K. Ledford, editors. CRC Press, Taylor & Francis Group, London, N Y, 2014. pp. 114, 118-119).
As used herein, the phrase “Fagales allergen” includes Fagales related allergens. In some embodiments, the “Fagales related allergen” is defined as a protein having an overall sequence homology with Bet v 1 of at least about 35%, or a sequence homology with epitope 1 of Bet v 1 of at least about 50%, or a sequence homology with epitope 2 of Bet v 1 of at least about 40%, or a sequence homology with epitope 3 of Bet v 1 of at least about 25%, or a sequence homology with epitope 4 of Bet v 1 of at least about 15%. In some embodiments, the “Fagales related allergen” is defined as a protein to which the anti-Bet v 1 antibodies cross-react, including allergens from the Rosales order.
Immunoglobulin E (IgE) is responsible for type 1 hypersensitivity, which manifests itself in allergic rhinitis, allergic conjunctivitis, hay fever, allergic 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, the 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 cross-linking of several IgE molecules and triggering the release of histamine and other inflammatory mediators causing the various allergic symptoms.
The treatment for birch pollen allergies includes desensitization therapy, which involves repeated injections with increasing dosages of either a crude birch pollen extract, or short peptides derived from Bet v 1. Insufficiencies of allergen specific immunotherapy include long treatment duration resulting in patient compliance issues, 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 is dependent on composition and quality of extract, and treatment is contraindicated in patients with severe asthma/food allergies due to risk of IgE mediated severe adverse events. Accordingly, there is a need in the field of birch pollen allergy treatment for alternative strategies for treating patients sensitive to Fagales allergens, in particular Bet v 1.
Antibodies have been proposed as a general treatment strategy for allergies, since they may be able to block the entry of allergenic molecules into the mucosal tissues, or may bind the allergen before it has the opportunity to bind to the IgE bound to the high affinity receptor on mast cells or basophils, thus preventing the release of histamine and other inflammatory mediators from these cells. U.S. Pat. No. 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 the mucosal tissue. U.S. Pat. No. 6,849,259 describes the use of allergen-specific antibodies to inhibit allergic inflammation in an in vivo mouse model of allergy. Milk-based and egg-based antibody systems have been described. For example, US20030003133A1 discloses using milk as a carrier for allergens for inducing 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 use of a molecule that inhibits the ability of the allergen to bind to mast cells were described in WO1994/024164A2. Other antibodies to Bet v 1 were mentioned in U.S. 2010/0034812.
The fully human antibodies described herein demonstrate specific binding to Bet v 1 and may be useful for treating patients suffering from birch pollen allergies, in particular, in patients who demonstrate sensitivity to the Bet v 1 allergen. The use of such antibodies may be an effective means of treating patients suffering from allergies to pollen from Fagales trees, or they may be used to prevent a heightened response to Bet v 1 upon secondary exposure, or the accompanying symptoms associated with the allergy, or may be used to lessen the severity and/or the duration of the allergic response associated with a primary exposure to birch pollen allergen or with the recurrence of the symptoms upon secondary exposure. They may be used alone or as adjunct therapy with other therapeutic moieties or modalities known in the art for treating such allergies, such as, but not limited to, treatment with corticosteroids or epinephrine. They may be used in conjunction with a second or third different antibody specific for Bet v 1. They may be used with allergen-specific immunotherapy (SIT). In some embodiments, the combination with SIT results is synergistically effective.
Unlike desensitization therapy, treatment with the antibodies described herein can provide effective relief within about 2 weeks of starting treatment, or within about 10 days or about 8 days of starting treatment. In combination with exposure to Bet v 1 protein or peptides, or one or more additional Fagales allergens, treatment with the fully human antibodies described herein can not only block an allergic reaction, but can more effectively or synergistically desensitize patients suffering from allergies to pollen from Fagales trees.
In certain embodiments, the antibodies of the invention are obtained from mice immunized with a primary immunogen, such as natural Bet v 1, which may be purchased commercially (e.g., from Indoor Biotechnologies, VA, #NA-BV1-1), or may be produced recombinantly. The full-length amino acid sequence of Bet v 1 is shown as SEQ ID NO: 306. The full-length Bet v 1 amino acid sequence may also be found in SEQ ID NO: 314, from Uniprot: P15494.
The immunogen may be a biologically active and/or immunogenic fragment of natural, native, or recombinantly produced Bet v 1, or DNA encoding the active fragment thereof. The fragment may be derived from either the N-terminal or C-terminal of Bet v 1, or from any site within the Bet v 1 amino acid sequence.
Unless specifically indicated otherwise, the term “antibody,” as used herein, shall be understood to encompass antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., “full antibody molecules”) as well as antigen-binding fragments thereof. 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 obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds 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 specifically bind to Bet v 1. An antibody fragment may include a Fab fragment, a F(ab′)2 fragment, a Fv fragment, a dAb fragment, a fragment containing a CDR, or an isolated CDR. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and (optionally) constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.
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 (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the 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 constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment,” as used herein.
An antigen-binding fragment of an antibody will typically comprise 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 having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another 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.
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 variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present invention include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (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 either directly linked to one another or may be linked by a full or partial hinge or linker region. 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 linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the present invention may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).
Methods for generating human antibodies in transgenic mice are known in the art. Any such known methods can be used in the context of the present invention to make human antibodies that specifically bind to Bet v 1.
Using VELOCIMMUNE™ technology (see, for example, U.S. Pat. No. 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 having a human variable region and a mouse constant region. The VELOCIMMUNE® technology involves generation of a transgenic mouse having a genome comprising human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces an antibody comprising a human variable region and a mouse constant region in response to antigenic stimulation. The DNA encoding the variable regions of the heavy and light chains of the antibody are isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing the fully human antibody.
Generally, a VELOCIMMUNE® mouse is challenged with the antigen of interest, and lymphatic cells (such as B-cells) are recovered from the mice that express antibodies. The lymphatic cells may be fused with a myeloma cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the variable regions of the heavy chain and light chain may be isolated and linked to desirable isotypic constant regions of the heavy chain and light chain. Such an antibody protein may be produced in a cell, such as a CHO cell. Alternatively, DNA encoding the antigen-specific chimeric antibodies or the variable domains of the light and heavy chains may be isolated directly from antigen-specific lymphocytes.
Initially, high affinity chimeric antibodies are isolated having a human variable region and a mouse constant region. As in the experimental section below, the antibodies are characterized 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. While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region.
In general, the antibodies of the instant invention possess very high affinities, typically possessing KD of from about 10−12 through about 10−9 M, when measured by binding to antigen either immobilized on solid phase or in solution phase. The mouse constant regions are replaced with desired human constant regions to generate the fully human antibodies of the invention. While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region.
The anti-Bet v 1 antibodies and antibody fragments of the present invention encompass proteins having amino acid sequences that vary from those of the described antibodies, but that retain the ability to bind Bet v 1. Such variant antibodies and antibody fragments comprise one or more additions, deletions, or substitutions of amino acids when compared to parent 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 additions, deletions, or substitutions of nucleotides when 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.
Two antigen-binding proteins, or antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single does or multiple dose. Some antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product studied.
In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.
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 diminished effectiveness, as compared to continued therapy without such switching.
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.
Bioequivalence may be demonstrated by in vivo and/or in vitro methods. Bioequivalence measures include, e.g., (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 human in vivo 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 safety, efficacy, or bioavailability or bioequivalence of an antibody.
Bioequivalent variants of the antibodies of the invention may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity. For example, cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other contexts, bioequivalent antibodies may include antibody variants comprising amino acid changes, which modify the glycosylation characteristics of the antibodies, e.g., mutations that eliminate or remove glycosylation.
In general, the antibodies of the present invention may function by binding to Bet v 1, a fragment of Bet v 1, or to Bet v 1 and one or more Fagales allergens or Fagales related allergens.
In certain embodiments, the antibodies of the present invention may bind to an epitope or fragment located within the Bet v 1 protein, for example, an epitope or fragment 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 may bind to at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 307, 308, 309, 310, 311, and 315, wherein an epitope sequence can be extended by 1 to 5 amino acids, or about 5 to about 10 amino acids on either the C-terminal end or the N-terminal end.
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 basophil activation 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.
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 a HCVR having 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 having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (ii) comprises a LCVR having 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 thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (iii) comprises a HCDR3 domain having 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 thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a LCDR3 domain having 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 thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (iv) comprises a HCDR1 domain having 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 thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a HCDR2 domain having 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 thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, and 300, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a LCDR2 domain having 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 thereof having 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 from about 10−8 to about 10−11; (vi) demonstrates efficacy in at least one animal model of anaphylaxis or inflammation; or (vii) competes with a reference antibody for binding to Bet v 1.
In one embodiment, the invention provides for the use of a combination of two or more fully human antibodies of the invention, or fragments thereof, for preparation of a composition, wherein the antibodies bind to Bet v 1, and wherein each antibody or fragment thereof contained within the composition exhibits one or more of the following characteristics: (i) comprises a HCVR having 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 having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (ii) comprises a LCVR having 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 thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (iii) comprises a HCDR3 domain having 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 thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a LCDR3 domain having 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 thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (iv) comprises a HCDR1 domain having 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 thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a HCDR2 domain having 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 thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, and 300, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a LCDR2 domain having 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 thereof having 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 from about 10−8 to about 10−11; (vi) demonstrates efficacy in at least one animal model of anaphylaxis or inflammation; or (vii) competes with a reference antibody for binding to Bet v 1.
Certain Bet v 1 antibodies of the present invention, when used alone, or in combination, are able to bind to and neutralize 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 to and neutralize the activity of Bet v 1 may be measured using any standard method known to those skilled in the art, including binding assays, or neutralization of activity (e.g., protection from anaphylaxis) assays, as described herein.
Non-limiting, exemplary in vitro assays for measuring binding activity are illustrated in Example 3, herein. In Example 3, the binding affinities and kinetic constants of human anti-Bet v 1 antibodies were determined by surface plasmon resonance.
The Bet v 1 proteins or peptides may be modified to include addition or substitution of certain residues for tagging or for purposes of conjugation to carrier molecules, such as, KLH. For example, a cysteine may be added at either the N terminal or C terminal end of a peptide, or a linker sequence may be added to prepare the peptide for conjugation to, for example, KLH for immunization. The antibodies specific for Bet v 1 may contain no additional labels or moieties, or they may contain an N-terminal or C-terminal label or moiety. In one embodiment, the label or moiety is biotin. In a binding assay, the location of a label (if any) may determine the orientation of the peptide relative to the surface upon which the peptide is bound. For example, if a surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented such that the C-terminal portion of the peptide will be distal to the surface.
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 may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. Epitopes may be either 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 circumstance, an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.
Provided herein are anti-Bet v 1 antibodies which interact with one or more amino acids found within the Bet v 1 molecule including, e.g., any fragment of Bet v 1 shown in SEQ ID NO: 306, or within comparable regions of a recombinantly produced Bet v 1 protein. The epitope to which the 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 within 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. Further exemplary contiguous sequences include at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 307, 308, 309, 310, 311, and 315, wherein such sequence can be extended by about 1 to about 5 amino acids, or about 5 to about 10 amino acids, on either the C-terminal end or 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 (e.g. a conformational epitope).
Various techniques known to persons of ordinary skill 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 that described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY). Other methods include alanine scanning mutational analysis, peptide blot analysis (Reineke (2004) Methods Mol Biol 248:443-63), peptide cleavage analysis crystallographic studies and NMR analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer (2000) Protein Science 9: 487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antibody interacts is hydrogen/deuterium exchange detected by mass spectrometry. In general terms, the hydrogen/deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antibody to the deuterium-labeled protein. Next, the protein/antibody complex is transferred to water and exchangeable protons within amino acids that are protected by the antibody complex undergo deuterium-to-hydrogen back-exchange at a slower rate than exchangeable protons within amino acids that are not part of the interface. As a result, amino acids that form part of the protein/antibody interface may retain deuterium and therefore exhibit relatively higher mass compared to amino acids not included in the interface. After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues which correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A. X-ray crystallography of the antigen/antibody complex may also be used for epitope mapping purposes.
Modification-Assisted Profiling (MAP), also known as Antigen Structure-based Antibody Profiling (ASAP) is a method that categorizes large numbers 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 (US 2004/0101920). Each category may reflect a unique epitope either distinctly different from or partially overlapping with epitope represented by another category. This technology allows rapid filtering of genetically identical antibodies, such that characterization can be focused on genetically distinct antibodies. When applied to hybridoma screening, MAP may facilitate identification of rare hybridoma clones that produce monoclonal antibodies having the desired characteristics. MAP may be used to sort the antibodies of the invention into groups of antibodies binding different epitopes.
In certain embodiments, the anti-Bet v 1 antibodies or antigen-binding fragments thereof bind an epitope within Bet v 1, in natural or native form, as exemplified in SEQ ID NO: 306 or SEQ ID NO: 314 (Bet v 1 amino acid sequence from Uniprot: P15494), or recombinantly produced, or to 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 NOs: 307, 308, 309, 310, 311 and 315.
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 having 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 Bet v 1 fragment with any of the specific exemplary antibodies described herein in Table 1, or an antibody having the CDR sequences of any of the exemplary antibodies described in Table 1.
One can easily determine whether an antibody binds to the same epitope as, or competes for binding with, a reference anti-Bet v 1 antibody by using routine methods known in the art. For example, to determine if a test antibody binds to the same epitope as a reference anti-Bet v 1 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. If the test antibody is able to bind to Bet v 1 following saturation binding with the reference anti-Bet v 1 antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-Bet v 1 antibody. On the other hand, if the test antibody is not able to bind to the Bet v 1 molecule following saturation binding with the reference anti-Bet v 1 antibody, then the test antibody may bind to the same epitope as the epitope bound by the reference anti-Bet v 1 antibody of the invention.
To determine if an antibody competes for binding with a reference anti-Bet v 1 antibody, the above-described binding methodology is performed in two orientations: In a first orientation, the reference antibody is allowed to bind to a Bet v 1 molecule under saturating conditions followed by assessment of 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 saturating conditions followed by assessment of binding of the reference antibody to the Bet v 1 molecule. If, in both orientations, only the first (saturating) antibody is capable of binding to the Bet v 1 molecule, then it is concluded that the test antibody and the reference antibody compete for binding to Bet v 1. As will be appreciated by a person of ordinary skill 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 reference antibody by binding an overlapping or adjacent epitope.
Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) 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 competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990 50:1495-1502). 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.
Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out 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 if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art.
The invention encompasses a human anti-Bet v 1 monoclonal antibody conjugated to a therapeutic moiety (“immunoconjugate”), such as an agent that is capable of reducing the severity of an allergic response to the Bet v 1 allergen, or in an environment where Fagales trees are present, or to ameliorate at least one symptom associated with exposure to birch pollen or to the Bet v 1 allergen, including rhinitis, conjunctivitis, or breathing difficulties, or the severity thereof. Such an agent may be a corticosteroid, a second different antibody to Bet v 1, or a vaccine. The type of therapeutic moiety that may 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 conjugate an agent appropriate 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.
The invention provides therapeutic compositions comprising the anti-Bet v 1 antibodies or antigen-binding fragments thereof of the present invention. The administration of therapeutic compositions in accordance with the invention will be administered via a suitable route including, but not limited to, intravenously, subcutaneously, intramuscularly, intranasally, with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (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 52:238-311.
The dose of antibody may vary depending upon the age and the 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 for treating the rhinitis or conjunctivitis associated with exposure to pollen from a Fagales tree, or birch pollen, or Bet v 1, in an individual having a sensitivity to Bet v 1, or for preventing an anaphylactic response to the Fagales allergen, or for lessening the severity of the allergic response, it is advantageous to intravenously administer the antibody of the present invention normally at a single dose of about 0.01 to about 30 mg/kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. In certain embodiments, the antibody or antigen-binding fragment thereof of the invention can 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 a plurality of subsequent doses of the antibody or antigen-binding fragment thereof in an amount that can be approximately the same or less than that of the initial dose, wherein the subsequent doses are separated by at least 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.
Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, 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 together with other biologically active agents. Administration can be systemic or local.
The pharmaceutical composition can be also delivered in a vesicle, in particular a liposome (see, for example, Langer, 1990, Science 249: 1527-1533).
In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose.
The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule.
A pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present invention. Such a pen delivery device can 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 readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled 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.
Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, but certainly are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (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 only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but certainly are not limited to the SOLOSTAR™ pen (sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.) and the HUMIRA™ Pen (Abbott Labs, Abbott Park, IL), to name only a few.
Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit 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 aforesaid antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, it is preferred that the aforesaid antibody is contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms.
Due to their 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 to an environment containing the Bet v 1 protein, or at least one symptom associated with the allergic response, such as itchy eyes, conjunctivitis, rhinitis, wheezing, breathing difficulties, or for preventing a secondary response to the Bet v 1 allergen, including a more serious anaphylactic response, or for lessening the severity, duration, and/or frequency of symptoms following reexposure to the Fagales allergen. Accordingly, it is envisioned that the antibodies of the present invention may be used prophylactically or therapeutically.
In yet a further embodiment of the invention the present antibodies are used for the preparation of a pharmaceutical composition for treating patients suffering from a 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 for reducing the severity of primary exposure to Bet v 1, or for reducing the severity, duration of, and/or number of allergic responses to Bet v 1. In a further embodiment of the invention the present antibodies are used as adjunct therapy with any other agent useful for treating Fagales allergens, including corticosteroids, vaccines, allergen specific immunotherapy (SIT), or any other palliative therapy known to those skilled in the art.
Combination therapies may include an anti-Bet v 1 antibody of the invention and any additional therapeutic agent that may be advantageously combined with an antibody of the invention, or with a biologically active fragment of an antibody of the invention.
For example, a second therapeutic agent may be employed to aid in reducing the 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 Fagales trees are present and blooming, such as a corticosteroid. The antibodies may also be used in conjunction with other therapies, such as a vaccine specific for the Bet v 1 allergen. The additional therapeutically active component(s) may be administered prior to, concurrent with, or after the administration of the anti-Bet v 1 antibody of the present invention. For purposes of the present disclosure, such administration regimens are considered the administration of an anti-Bet v 1 antibody “in combination with” a second therapeutically active component.
As used herein, the expressions “allergen-specific immunotherapy”, “specific immunotherapy”, “SIT”, “SIT regimen”, and the like, refer to the repeated administration of an allergen to a patient over time as means for 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 administration of increased amounts of allergen. In certain instances, the SIT regimen comprises at least two consecutive phases: (1) an up-dosing phase, and (2) a maintenance phase. In the up-dosing phase, increasing doses of allergen are administered until an effective and safe dose is achieved. The dose that is established at the end of the up-dosing phase is then administered to the patient throughout the course of the maintenance phase. The duration of the up-dosing phase can be several weeks or several months. In certain embodiments, however, the up-dosing phase is of substantially shorter duration (e.g., less than one week, less than 6 days, less than 5 days, less than 4 days, less than 3 days, or less than 2 days). SIT regimens comprising an up-dosing phase of less than 5 days are sometimes referred to as “Rush” immunotherapy or “Rush SIT”. The maintenance phase of an SIT regimen can last several weeks, several months, several years, or indefinitely.
According to certain embodiments of the present invention, multiple doses of one or more anti-Bet v 1 antibodies (an antibody combination) may be administered to a patient over a defined time course. The methods according to this aspect of the invention comprise sequentially administering to a patient multiple doses of an antibody, antibody combination. As used herein, “sequentially administering” means that each dose of an antibody or antibody combination is administered to the patient at a different point in 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.
The terms “initial dose,” “secondary doses,” and “tertiary doses,” refer to the temporal sequence of administration of an antibody or antibody combination provided herein. Thus, the “initial dose” is the dose which is administered at the beginning of the treatment regimen (also referred to as the “baseline dose”); the “secondary doses” are the doses which are administered after the initial dose; and the “tertiary doses” are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses may all contain the same amount of an antibody or antibody combination but generally may differ from one another in terms of frequency of administration. In certain embodiments, however, the amount of an antibody or antibody combination, 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 (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as “loading doses” followed by subsequent doses that are administered on a less frequent basis (e.g., “maintenance doses”).
In one exemplary embodiment of the present invention, each secondary and/or tertiary dose is administered 1 to 26 (e.g., 1, 1½, 2, 2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½, 13, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½, 20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more) weeks after the immediately preceding dose. The phrase “the immediately preceding dose,” as used herein, means, in a sequence of multiple administrations, the dose of an antibody or antibody combination, which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.
The 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 antibody combination. 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.
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. Similarly, 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 at which the secondary and/or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.
The anti-Bet v 1 antibodies of the present invention may also be used to detect and/or measure Bet v 1 in a sample, e.g., for diagnostic purposes. It is envisioned that confirmation of an allergic response thought to be caused by Bet v 1 may be made by measuring the presence of either Bet v 1 through use of any one or more of the antibodies of the invention. Exemplary diagnostic assays for Bet v 1 may comprise, e.g., 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 detectable label or reporter molecule or used as a capture ligand to selectively isolate Bet v 1 protein from patient samples. Alternatively, an unlabeled anti-Bet v 1 antibody can be used in diagnostic applications in combination with a secondary antibody which is itself detectably labeled. The detectable label or reporter molecule can 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, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure Bet v 1 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).
Samples that can be used in Bet v 1 diagnostic assays according to the present invention include any tissue or fluid sample obtainable from a patient, which contains detectable quantities of Bet v 1 protein, or fragments thereof, under normal or pathological conditions. Generally, levels of Bet v 1 in a particular sample obtained from a healthy/non-allergic patient (e.g., a patient not afflicted with 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 against the levels of Bet v 1 measured in samples obtained from individuals suspected of having a sensitivity to Bet v 1 in birch pollen, or symptoms associated with such condition.
While the invention has been particularly shown and described with reference to a number of embodiments, it would be understood by those skilled in the art that changes in the form and details may be made to 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.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure 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 regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
An immunogen comprising any one 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 may be purchased commercially (e.g., from Stallergenes Greer, Lenoir, NC, #XP527D3A25), or isolated from birch pollen (See, for example, Buters, et al. (2012), Atomospheric Environment 55:496-505), or which may be produced recombinantly (See GenBank accession number P 15494 for the full length amino acid sequence of Bet v 1), or fragments of the Bet v 1 protein, followed by immunization with a secondary immunogen, or with an immunogenically active fragment of the natural protein. Various constructs may be prepared using portions of the Bet v 1 protein known to those skilled in the art. These constructs may 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 NOs: 307, 308, 309, 310, 311, and 315, or fragments thereof, may be used as immunogens.
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 the active fragment thereof. The fragment may be derived from the N-terminal or C-terminal portion of Bet v 1.
In certain embodiments, the recombinant Bet v 1 protein constructs used in the studies described herein may also include a C-terminal tag (myc-myc-hexahistidine tag) as indicated below. In other embodiments, the recombinant Bet v 1 protein construct includes amino acids G2 through N160 of Uniprot P15494. In some embodiments, the construct comprises an S85A substitution. 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.
In certain embodiments, the immunogen may be a Bet v 1 fragment or fusion protein that comprises 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).
In certain embodiments, antibodies that bind specifically to Bet v 1 may be prepared using fragments of the above-noted regions, or peptides that extend beyond the designated regions by about 5 to about 20 amino acid residues from either, or both, the N or C terminal ends of the regions described herein. In certain embodiments, any combination of the above-noted regions or fragments thereof may be used in the preparation of Bet v 1 specific antibodies. In certain embodiments, any one or more of the above-noted regions of Bet v 1, or fragments thereof may be used for preparing monospecific, bispecific, or multispecific antibodies.
The full-length 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 human immunoglobulin heavy and kappa light chain variable regions.
Anti-Bet v 1 antibodies were isolated directly from antigen-positive B cells without fusion to myeloma cells, as described in U.S. Pat. No. 7,582,298. Using this method, several fully human anti-Bet v 1 antibodies (i.e., antibodies possessing human variable domains and human constant domains) were obtained; exemplary antibodies generated in this manner were designated as follows: H4H16943P, H4H16946P, H4H16950P, H4H16960P, H4H16967P, H4H16971P, H4H16979P, H4H16987P, H4H16991P, H4H16992P, H4H17001P, H4H17015P, H4H17027P, H4H17028P, H4H17031P, H4H17033P, H4H17038P2, H4H17045P2, H4H17067P2, and H4H17082P2.
The biological properties of the exemplary antibodies generated in accordance with the methods of this Example are described in detail in the Examples set forth below.
Table 1a provides the amino acid sequence identifiers of the heavy and light chain variable regions 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 v 1 antibodies.
Antibodies are typically referred to herein according to the following nomenclature: Fc prefix (e.g. “H4H,” “H2M,” etc.), followed by a numerical identifier (e.g. “16943,” “17001,” etc., as shown in Table 1), followed by a “P” or “P2” suffix. The H4H and H2M prefixes on the antibody designations used herein indicate the particular Fc region isotype of the antibody. Thus, according to this nomenclature, an antibody may be referred to herein as, e.g., “H4H16943P”, etc., as the “H4H” designates the antibody has a human IgG4 Fc (all variable regions are fully human as denoted by the first ‘H’ in the antibody designation). As will be appreciated by a person of ordinary skill in the art, an antibody having a particular Fc isotype can be converted to an antibody with a different Fc isotype (e.g., an antibody with a human IgG1 Fc can be converted to an antibody with a human IgG4, etc.), but in any event, the variable domains (including the CDRs)—which are indicated by the numerical identifiers shown in Table 1—will remain the same, and the binding properties to antigen are expected to be identical or substantially similar regardless of the nature of the Fc domain.
Table 1c provides the amino acid sequence identifiers of the full length heavy and light chains of selected anti-Bet v 1 antibodies.
Equilibrium dissociation constants (KD) for natural Bet v 1 binding to purified anti-Bet v 1 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 surfactant Tween-20, pH 7.4 (HBS-ET) running buffer at 25° C. and 37° C. The Biacore CM5 sensor surface was first derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (GE, #BR-1008-39) to subsequently capture anti-Bet v 1 monoclonal antibodies. Different concentrations of natural Bet v 1 (Indoor, Cat #NA-BV1-1) or CHO produced recombinant Bet v 1 containing a S85A mutation with a C-terminal myc-myc hexahistidine tag (mutant Bet v 1-MMH; SEQ ID NO: 312) reagent were first prepared in HBS-ET running buffer (100 nM−1.23 nM; serially diluted by 3-fold) and were injected over anti-human Fc captured anti-Bet v 1 monoclonal antibody surface for 4 minutes at a flow rate of 30 μL/minute, while the dissociation of monoclonal antibody bound Bet v 1 reagent was monitored for 10 minutes in HBS-ET running buffer. Association (ka) and dissociation (kd) rate constants were determined by fitting the real-time binding sensorgrams to a 1:1 binding model with mass transport limitation using Scrubber 2.0c curve-fitting software. Binding dissociation equilibrium constants (KD) and dissociative half-lives (t½) were calculated from the kinetic rate constants as:
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 Table 2 through Table 5.
At 25° C., all of the 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. At 37° C., all of the 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 both 25° C. and 37° C. the isotype control antibody did not demonstrate any measurable binding to natural Bet v 1.
At both 25° C. and 37° C., 3 out of 20 anti-Bet v 1 monoclonal antibodies of the invention did not bind to mutant Bet v 1-MMH. At 25° C., 17 out of 20 ani-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 out of 20 anti-Bet v 1 monoclonal antibodies of the invention bound to mutant Bet v 1-MMH with KD values ranging from 655 pM to 106 nM, as shown in Table 5. At both 25° C. and 37° C. the isotype control antibody did not demonstrate any measurable binding to mutant Bet v 1-MMH.
Equilibrium dissociation constants (KD) for different related allergens binding to purified anti-Bet v 1 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 surfactant Tween-20, pH 7.4 (HBS-ET) running buffer at 25° C. The Biacore CM5 sensor surface was first derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (GE, #BR-1008-39) to capture the anti-Bet v 1 monoclonal antibodies. Binding studies were performed on the following related allergens: Alder (Aln g 1, MyBiosource, Cat #MBS1041484), Apple (Mal d 1, MyBiosource, Cat #MBS1224919), Carrot (Dau c 1.2, MyBiosource, Cat #MBS1212920), Celery (Api g 1, MyBiosource, Cat #MBS1171376), Celery (Api g 2, MyBiosource, Cat #MBS1047880), European Hornbeam (Car b 1 isoform 1A & 1B, MyBiosource, Cat #MBS1200018), European Hornbeam (Car b 1 isoform 2, MyBiosource, Cat #MBS1043940), Hazel (Cor A 1, MyBiosource, Cat #MBS5304600), and White Oak (Que a 1, MyBiosource, Cat #MBS1258822). Different concentrations of the related allergens were prepared in HBS-ET running buffer (100 nM−6.25 nM; serially diluted by 4-fold) and then were injected over anti-human Fc captured anti-Bet v 1 monoclonal antibody surface for 3 minutes at a flow rate of 30 μL/minute, while the dissociation of monoclonal antibody bound allergens was monitored for 8 minutes in HBS-ET running buffer. Association (ka) and dissociation (kd) rate constants were determined by fitting the real-time binding sensorgrams to a 1:1 binding model with mass transport limitation using Scrubber 2.0c curve-fitting software. Binding dissociation equilibrium constants (KD) and dissociative half-lives (t½) were calculated from the kinetic rate constants as:
Binding kinetics parameters for related allergens to different anti-Bet v 1 monoclonal antibodies of the invention at 25° C. are shown in Table 6 through Table 11.
As shown in Table 6 at 25° C., nine of the 20 anti-Bet v 1 antibodies of the invention demonstrated measurable binding to Aln g 1 with KD values ranging from 1.03 nM to 175 nM. The other 11 antibodies did not demonstrate any measurable binding to Aln g 1 under the tested conditions.
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 did not demonstrate any measurable binding to Mal d 1 under the tested conditions.
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 did not demonstrate any measurable binding to Api g 1 under the tested conditions.
As shown in Table 9 at 25° C., eight of the 20 of the anti-Bet v 1 antibodies of the invention demonstrated measurable binding to Car b 1 isoform 1A & 1B with KD values ranging from 1.2 nM to 380 nM. The other 12 antibodies did not demonstrate any measurable binding to Car b 1 isoform 1A & 1B under the tested conditions.
As shown in Table 10 at 25° C., 14 of the 20 of the anti-Bet v 1 antibodies of the invention demonstrated measurable binding to Car b 1 isoform 2 with KD values ranging from 335 pM to 564 nM. The other 6 antibodies did not demonstrate any measurable binding to Car b 1 isoform 2 under the tested conditions.
As shown in Table 11 at 25° C., one of the anti-Bet v 1 antibodies of the invention demonstrated measurable binding to Cor A 1 with a KD value of 396 nM. The other 19 antibodies did not demonstrate any measurable binding to Cor A 1 under the tested conditions.
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 did not demonstrate any measurable binding to any of the allergens tested.
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 Bet v 1 binding to plate-captured IgE from allergic human donor plasma/sera was determined using an ELISA. Antibodies were tested either alone or in polyclonal mixes. For the assay, microtiter plates were coated overnight at 4° C. with human FcεR1α (the high affinity receptor for IgE) extracellular domain protein 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 over the receptor-coated surface. A constant amount of 0.1 nM of biotin labeled natural Bet v 1 (Indoor Biotechnologies, #NA-BV1-1) was pre-mixed with anti-Bet v 1 antibodies, at a single concentration of 1 μg/mL or in serial dilutions starting from either 10 μg/mL or 1 μg/mL of each antibody and incubated for 1 hour at RT to allow Bet v 1-antibody interaction to reach equilibrium. The antibody-Bet v 1 mixture was then added to the IgE-coated plate for 1 hour. Plates were subsequently washed and the amount of natural Bet v 1 bound to plate was detected using streptavidin conjugated to horseradish peroxidase (Thermo Scientific, #N200/QJ223091) at a 1:10,000 dilution and incubated for 1 hour at RT. Plates were then washed with PBS-T in between each step of the ELISA protocol described above. To develop the colorimetric reaction, TMB/H2O2 substrate (BD Pharmingen Reagent A #51-2602KC+Reagent B, #51-2607KC) was added to the plates and incubated for 20 minutes at RT. The reaction was stopped using 2 N sulfuric acid (H2SO4; VWR, #BDH3500-1). The absorbance was subsequently measured on a spectrophotometer (Victor, Perkin Elmer) at 450 nm. The percent blocking was calculated using the highest antibody concentration used in each assay as described below.
Twenty anti-Bet v 1 antibodies were tested as single antibodies for their ability to block Bet v 1 binding to plate-captured IgE from allergic human donor plasma using the ELISA described. Individual monoclonal antibodies were able to partially block IgE binding to Bet v 1 by 8.5-64%, which underlines the polyclonality of the IgEs. Seven antibodies showed blocking ranging from 36-64% at the highest antibody concentration tested of 10 μg/mL, as shown in Table 12.
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% blocking in seven out of ten IgE donors. Three and four monoclonal antibody combinations demonstrated similar results and did not appear to add any additional blocking effect as compared to the two-antibody combination of H4H17082P2 and H4H16992P, as shown in Table 13.
The four monoclonal antibodies, H4H17082P2, H4H17038P2, H4H16987P, and H4H16992P, were subsequently tested as single and 2, 3 and 4 monoclonal antibody combinations in dose response blocking assay with 3 donor IgE samples. The results showed that the anti-Bet v 1 two monoclonal antibody combination of H4H17082P2 and H4H16992P blocked Bet v 1 binding to allergen specific IgE greater than 90% and close to baseline in 3 donors, as shown in Table 14. The tested 3- and 4-antibody combinations showed similar breadth and potency of blocking activity. As positive control, purified mouse anti-Bet v 1 polyclonal IgG demonstrated >90% blocking.
To determine the amino acid residues of Bet v 1 [(amino acids M1-N160 of Uniprot P15494] with which H4H16992P2, H4H17082P2, H4H17038P2, and H4H16987P interact, a H/D exchange epitope mapping with mass spectrometry study was performed. A general description of the H/D exchange method is set forth in e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; and Engen and Smith (2001) Anal. Chem. 73:256A-265A.
The HDX-MS experiments were performed on an integrated Waters HDX/MS platform, consisting of a Leaptec HDX PAL system for the deuterium labeling, a Waters Acquity M-Class (Auxiliary solvent manager) for the sample digestion and loading, a Waters Acquity M-Class (Binary solvent manager) for the analytical column gradient, and Synapt G2-Si mass spectrometer for peptic peptide mass measurement.
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, Catalog #NA-BV1-1, 28 pmol/μL), Bet v 1 premixed with each antibody, or the mixture of all 4 anti-Bet v 1 antibodies in a 1:1 molar ratio was incubated with 56.2 μL D2O labeling solution for various time-points (e.g., Undeuterated control=0 second, labeled for 1 minute, 5 minutes, 10 minutes and 20 minutes). The deuteration was quenched by transferring 50 μL of the sample to 50 μL of pre-chilled 0.2 M TCEP, 6 M guanidine chloride in 100 mM phosphate buffer, pH 2.5 (quench 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. The digested peptides were trapped onto an ACQUITY UPLC BEH C18 1.7-μm, 2.1×5 mm VanGuard pre-column at 0° C. and eluted to an analytical column ACQUITY UPLC BEH C18 1.7-μm, 1.0×50 mm for a 9-minute gradient separation of 5%-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.
For the identification of the peptides from Bet v 1, LC-MSE data from undeuterated sample were processed and searched against the database including Bet v land its randomized sequence via Waters ProteinLynx Global Server (PLGS) software. The identified peptides were imported to DynamX software and filtered by two criteria: 1) minimum products per amino acid: 0.3, and 2) replication file threshold: 3. DynamX software then automatically determined deuterium uptake of each peptide based on retention time and high mass accuracy (<10 ppm) across multiple time points with 3 replicates at each time.
Using the online pepsin/protease XIII column coupled with MSE data acquisition, a total of 36 peptides from Bet v 1 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 H4H16992P2, H4H17082P2, H4H17038P2, H4H16987P and 4 antibody combinations are illustrated in
As shown in
As shown in
As shown in
As shown in
As shown in
In addition, a modest level of protection was observed for peptide 23-43 in the presence of H4H17082P, H4H17038P2 and H4H16987P, identifying that this region could represent a secondary epitope for these monoclonal antibodies.
To determine the efficacy of anti-Bet v 1 antibodies of the invention for blocking allergen induced mast cell degranulation the passive cutaneous anaphylaxis (PCA) in vivo model was used. This model involves intradermal injection of an allergen-specific antiserum into a local area on the skin followed by intravenous injection of an antigen along with a dye. The allergic reaction causes capillary dilatation and increased vascular permeability at the site of sensitization, resulting in preferential accumulation of dye at this site. The dye can be extracted from the tissue and quantitated spectrophotometrically. Dye extravasation into tissue sensitized with test antiserum can then be compared to extravasation into tissue sensitized with a non-relevant antiserum.
Antisera were generated for use in the assay by immunizing Balb/c mice with 5 μg of natural Bet v 1 protein (Indoor Biotechnologies, #NA-BV1-1) in a solution of 1 mg/mL of alum in 1× phosphate buffered saline (PBS) on day 0. One week later (day 7), sensitized mice were boosted with 5 μg natural Bet v 1 protein in a solution of 1 mg/mL alum in 1×PBS. Two weeks after the boost, mice were subjected to an intranasal airway challenge with 0.5 μg of natural Bet v 1 protein in 20 μL 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, #555248) according to the manufacturer's instructions. The final concentration of Bet v 1 antisera was diluted to 2600 ng/mL of IgE in PBS.
Antisera used as a negative control in the assay was generated by immunizing Balb/c mice with 5 μg natural Fel d 1 protein purified from cat hair extract (Indoor Biotechnologies, #LTN-FD1-1) in a solution of 1 mg/mL of alum in PBS on day 0. Mice were boosted with 5 μg 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, #555248) according to the manufacturer's instructions. The final concentration of antisera was diluted to 2500 ng/mL of IgE in PBS.
For the PCA assays, groups of Balb/c mice (n≥5 per experiment) were first subcutaneously injected with either an isotype control antibody, an anti-Bet v 1 antibody, or a combination of anti-Bet v 1 antibodies at a dose of 1 mg/kg (total antibody dose). Three days after antibody administration, 10 μL of either 1.5 ng Bet v 1 antiserum or 3 ng negative control anti-serum were injected into the right and left ears of mice in each group, respectively. Twenty-four hours after the local administration of allergen-specific antisera, mice were challenged by intravenous injection (100 μL per mouse) of a solution of 1 μg/mL of natural Bet v 1 dissolved in PBS containing 0.5% (w/v) Evan's blue dye (Sigma Aldrich, #E2129). One hour after antigen challenge, mice were sacrificed, their ears were excised, placed in 1 mL formamide, and subsequently incubated for 3 days from 50-56° C. to extract the Evan's blue dye. The ear tissue was then removed from the formamide, blotted to remove excess liquid and weighed. Two-hundred microliter aliquots of each formamide extract were transferred to 96 well plates in duplicate and their absorbance was then measured at 620 nm. The optical density measured was converted to Evan's blue dye concentration using a standard curve and represented as ng of Evan's blue dye per mg tissue. Mean values±the standard deviation are shown in Table 15 for each group. Mean difference as compared to isotype control was calculated using Bonferroni's multiple comparisons test in GraphPad Prism.
As shown in Table 15, in the first study, the single anti-Bet v 1 antibody, H4H17082P2, did not demonstrate a significant reduction of dye extravasation compared to isotype control. In contrast in 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 of dye extravasation as 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 v 1 antibodies of the invention (H4H16992P and H4H17082P2) again demonstrated significant reductions of dye extravasation as compared to the isotype control treatment, with a reduction of 41.09 ng/mg. As was previously demonstrated in Study 1, in Study 3 the single anti-Bet v 1 antibody, H4H17082P2, did not demonstrate a significant reduction of dye extravasation compared to isotype control. However, another single antibody, H4H16992P, demonstrated a significant reduction of dye extravasation compared to isotype control, with a reduction of 25.86 ng/mg. From the studies conducted the single antibody H4H16992P, the two-antibody combination of H4H17082P2+H4H16992P, as well as the four-antibody combination of H4H17082P2+H4H16992P+H4H17038P2+H4H16987P were able to block mast cell degranulation as indicated by a significant reduction of dye extravasation compared to isotype control in the passive cutaneous anaphylaxis in vivo model as determined by two-way ANOVA with Bonferroni's post test. The H4H17082P2 antibody alone was not able to block mast cell degranulation as indicated by an increase in dye extravasation compared to isotype in two of the studies conducted. The number of mice used per group (n) is noted within parentheses in the table.
Anti-Bet v 1 antibodies provided herein were tested for efficacy in blocking allergen induced mast cell degranulation in the passive cutaneous anaphylaxis (PCA) in vivo model. Allergen-specific antiserum is transdermally injected into a local area on the skin followed by intravenous injection of an antigen along with a dye. The allergic reaction causes capillary dilatation and increased vascular permeability at the site of sensitization, resulting in preferential accumulation of dye at this site. The dye can be extracted from the tissue and quantitated spectrophotometrically. Dye extravasation into tissue sensitized with test antiserum can then be compared to extravasation into tissue sensitized with a non-relevant antiserum.
Antisera to natural Bet v 1, Betula pendula (also known as Betula verrucosa) birch pollen extract (BPE), Betula nigra BPE, and Betula populifolia BPE were generated for use in this assay by immunizing Balb/c mice with 5 μg of natural Bet v 1 protein (Indoor Biotechnologies, Catalog #NA-BV1-1 Lot 36164) or 5 μg of BPE; pendula (Stallargenes Greer, Catalog #XP527D3A25 Lot #277329), nigra (Stallargenes Greer Catalog #XP79D3A25 Lot #285077) or populifolia (Stallargenes Greer Catalog #XP80D3A2.5 Lot #273622) in a solution of 1 mg/ml of alum in 1× phosphate buffered saline (PBS) on day 0. One week later (day 7), sensitized mice were boosted 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 1×PBS. Two weeks after the boost, mice were subjected to an intranasal airway challenge with 0.5 μg natural Bet v 1 protein or 0.5 μg of the respective birch pollen extract in 20 μL of 1×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 lots was determined using an OptEIA™ ELISA kit (BD Biosciences, #555248) according to the manufacturer's instructions. The final concentration of Bet v 1 antisera was diluted to 2500 ng/mL of IgE in 1×PBS and the final concentration of the birch pollen extract antisera lots were diluted to 3000 ng/mL for pendula, 1900 ng/mL for nigra and 3700 ng/mL for populifolia.
Antisera used as a negative control in this assay was generated by immunizing Balb/c mice with 5 μg natural Fel d 1 protein purified from cat hair extract (Indoor Biotechnologies, Catalog #LTN-FD1-1, Lot #36099) in a solution of 1 mg/mL of alum in 1×PBS. Mice were boosted with 5 μg Fel d 1 protein in a solution of 1 mg/mL alum in 1×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, #555248) according to the manufacturer's instructions. The final concentration of antisera was diluted to 4800 ng/mL of IgE in PBS.
For the PCA assays, groups of Balb/c mice (n≥4 per experiment, repeated three times) were first subcutaneously injected with either an isotype control antibody or a combination of two anti-Bet v 1 antibodies at a dose of 1 mg/kg (total antibody dose). Three days after antibody administration, 10 μL of either ing Bet v 1 antisera, 25 ng Betula pendula antiserum, 25 ng Betula nigra antiserum, or 25 ng Betula populifolia antiserum was injected into the right ear of mice in assigned groups. Left ears were administered ing or 25 ng Fel d 1 (negative control) to match antiserum concentration of the corresponding right ear. Twenty-four hours after the local administration of allergen-specific antisera, mice were challenged by intravenous injection (100 μL per mouse) with a solution of 1 μg/mL natural Bet v 1 (Catalog #NA-BV1-1, Lot 36164) or 1 μg/mL of the respective BPE (Stallargenes Greer, Catalog #XP527D3A25 Lot #277329, Catalog #XP79D3A25 Lot #285077, and Catalog #XP80D3A2.5 Lot #273622) dissolved in 1×PBS containing 0.5% (w/v) Evan's blue dye (Sigma Aldrich, #E2129). One hour after antigen challenge, mice were sacrificed, ears were excised, placed in 1 mL formamide and subsequently incubated for 3 days at 50° C. to extract the Evan's blue dye. The ear tissue was then removed from the formamide, blotted to remove excess liquid and weighed. Two-hundred microliter aliquots of each formamide extract were transferred to 96 well plates in duplicate. Absorbance of the resulting supernatants was measured at 620 nm. The optical density measured was converted to Evan's blue dye concentration using a standard curve and is represented as ng of Evan's blue dye per mg ear tissue. Mean values±the standard deviation are shown in Table 1 for each group. Mean difference as compared to isotype control was calculated using Bonferroni's multiple comparisons test in GraphPad Prism.
Table 16 demonstrates efficacy of the combination of two anti-Bet v 1 antibodies, H4H16992P and H4H17082P2, indicated by a significant reduction of dye extravasation when compared to isotype control in all groups tested. As shown, the two anti-Bet v 1 monoclonal antibody combination of 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 the isotype control, demonstrating a significant reduction in dye extravasation of 88.34. Similarly, reduction of dye extravasation is also observed in H4H17082P2/H4H16992P treated groups for all three birch pollen extracts as compared to 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 as compared to isotype control was calculated by two-way ANOVA with Bonferroni's post test. The number of mice used per group (n) is noted within parentheses in the tables.
pendula
nigra
populifolia
Binding competition within a panel of anti-Bet v 1 monoclonal antibodies was determined using a real time, label-free bio-layer interferometry assay on the Octet HTX biosensor platform (Pall ForteBio Corp.). The entire experiment was performed at 25° C. in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v/v Surfactant Tween-20, 1 mg/mL BSA, pH 7.4 (HBS-EBT) buffer with the plate shaking at the speed of 1000 rpm. To assess whether 2 antibodies were able to compete with one another for binding to their respective epitopes on the recombinant mutant Bet v 1 expressed with a C-terminal myc-myc-hexahistidine tag (mutant Bet v 1-MMH; SEQ ID NO: 312), around ˜0.21 nm of mutant Bet v 1-MMH was first captured onto anti-Penta-His antibody coated Octet biosensor tips (Fortebio Inc, #18-5122) by submerging the biosensor tips for 90 seconds in wells containing 5 μg/mL solution of mutant Bet v 1-MMH. The antigen captured biosensor tips were then saturated with the first anti-Bet v 1 monoclonal antibody (referred to as mAb-1) by dipping into wells containing 50 μg/mL solution of mAb-1 for 4 minutes. The biosensor tips were then dipped into wells containing 50 μg/mL solution of second anti-Bet v 1 monoclonal antibody (referred to as mAb-2) for 3 minutes. The biosensor tips were washed in HBS-EBT buffer between every step of the experiment. The real-time binding response was monitored during the entire course of the experiment and the binding response at the end of every step was recorded. The response of mAb-2 binding to mutant Bet v 1-MMH pre-complexed with mAb-1 was compared and competitive/non-competitive behavior of different anti-Bet v 1 monoclonal antibodies was determined as shown in Table 17.
Three out of 20 anti-Bet v 1 monoclonal antibodies did not bind to mutant Bet v 1-MMH and cross-competition data was found to be inconclusive.
To explore the polyclonality of the allergen-specific IgE response across human birch allergic individuals, a humanized FcεR1α mouse model was utilized to facilitate binding of human IgE to FcεR1α on the surface of mouse mast cells. Since human IgE cannot bind mouse FcεR1α, a genetically modified mouse was created where endogenous mouse FcεR1α was replaced by the corresponding human FcεR1α sequence and denoted as FcεR1αhu/hu. The FcεR1αhu/hu mice were validated for use in this model by 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 allergic human sera into a local area on the skin followed by intravenous injection of relevant allergen along with a dye. The allergic reaction causes capillary dilatation and increased vascular permeability at the site of sensitization, resulting in preferential accumulation of dye at this site. The dye can be extracted from the tissue and quantitated spectrophotometrically. Dye extravasation into tissue sensitized with test antiserum is compared to extravasation into tissue sensitized with a non-allergic human sera.
To determine the effect of anti-Bet v 1 antibodies on mast cell degranulation in this model, humanized FcεR1a mice received a subcutaneous injection of isotype control antibody or anti-Bet v 1 antibody combinations on day 1. For each human donor, two independent experiments were performed, n=5 mice per group with data combined. Groups consist of no monoclonal antibody negative control, isotype negative control, REGN5713+REGN5715 dual anti-Bet v 1 antibody treatment group and REGN5713+REGN5714+REGN5715 triple anti-Bet v 1 antibody treatment group. The total or combined antibody concentration was 1 mg/kg or an IgG4 isotype control antibody (anti-IL6Rα as a negative isotype control). Three days later, serum from birch allergic patients or serum from non-birch allergic patients (negative control) was injected intradermally (ID) into the right and left ears, respectively, allowing allergen-specific IgE to bind 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 a Bet v 1-specific IgE ImmunoCAP® of 10 KUa/L.
Twenty-four hours after local administration of allergen-specific antibodies, mice were challenged by IV injection of 1 μg Bet v 1 diluted in PBS containing 0.5% Evan's blue dye. One hour after allergen challenge, mice were sacrificed. Evan's blue dye was extracted from ear tissue and quantitated spectrophotometrically using a standard curve. (See
% Reduction(average)=100*[B(isotype,avg)−B(mAb,i)]/[B(isotype,avg)−N(mAb,i)]
An increase in the percent reduction in dye leakage in the anti-Bet v1 antibody treated group compared to the negative isotype control group is a measure of effectiveness of the Bet v 1 antibody or antibody combinations in blocking mast cell degranulation.
In this model, the combined use of anti-Bet v 1 antibodies designated H4H16992P (also referred to as REGN5713), H4H17038P2 (also referred to as REGN5714) and H4H17082P2 (also referred to as REGN5715) demonstrated maximal blocking of the IgE mediated response when using IgE containing sera from 3/3 birch allergic donors (See
The human IgE response was explored by testing the effect of various combinations of the anti-Bet v 1 antibodies H4H16992P (also referred to as REGN5713), H4H17038P2 (also referred to as REGN5714) and H4H17082P2 (also referred to as REGN5715) on inhibiting basophil activation using samples from 8 birch allergic individuals. More specifically, to assess FcεR engagement and activation, basophils were tested in a functional phosphoflow based 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, 93-103.
Blood was drawn from birch allergic patients (n=8) and PBMCs isolated by density centrifugation on a Ficoll layer, washed, resuspended and plated as single points in a 96-well format. In parallel, a 2× 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 constant dose (final concentration 100 pM) of purified natural Bet v 1. The cells were stimulated and subsequently stained with an antibody cocktail containing pErk-Alexa 488, CD123-BUV395 and HLA-DR-APC antibodies. Following staining, data was acquired using an LSR-Fortessa instrument and analyzed by calculating the MFI of phosphorylated Erk staining within the basophil gate. Percent Max Inhibition was calculated as: 100−((100×Maximum Antibody Response)/Isotype Response). Maximum antibody response was the average Median Fluorescence Intensity (MFI) of phosphorylated Erk in the top three doses of antibody in the dose response curve (plateau of the curve) minus the baseline MFI (average of replicate unstimulated samples), and isotype response is the average of all the MFI values in the dose response of a Regeneron produced isotype control antibody (REGN1945 anti-Fel d 1 IgG4P) minus the baseline MFI.
Basophils from all 8-birch pollen-allergic individuals responded to Bet v 1 stimulation with varying intensities. See
This experiment was performed to ensure that the binding epitopes of three select Bet v 1 monoclonal antibodies were unique and that, irrespective of the order of monoclonal antibody binding, no steric hindrance was exhibited upon simultaneous binding of the three antibodies. Order dependent competition between the three Bet v 1 monoclonal antibodies was also assessed.
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 Surfactant Tween-20, pH7.4 (HBS-ET). The antibodies were immobilized on different surfaces of CM5 sensor using EDC/NHS chemistry to achieve immobilization levels of 5000-13,000 RU. REGN1945 (Fel d 1 monoclonal antibody) was also immobilized as the negative control. Natural Bet v 1 (nBet v 1), 10 nM or 20 nM, was injected over different Bet v 1 monoclonal antibody immobilized sensor surfaces for 10-12 seconds followed by sequential injection of different Bet v 1 monoclonal antibodies for 6 minutes at 15 L/min.
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) represents no competition. All three Bet v 1 monoclonal antibodies included in this example were able to simultaneously bind to nBet v 1 and binding response was not affected by the order in which the antibodies were added.
Data represents average of at least 3 independent injections of Bet v 1 monoclonal antibodies over the complex of nBet v 1 and immobilized Bet v 1 monoclonal antibody
Regardless of the order of antibody binding to Bet v 1, there was no competition impeding the simultaneous binding of all three antibodies, suggesting that REGN5713, REGN5714, and REGN5715 bind to non-overlapping, distinct epitopes.
This application is a continuation of U.S. patent application Ser. No. 17/006,599, filed on Aug. 28, 2020, which is a divisional of U.S. patent application Ser. No. 15/994,294, filed May 31, 2018, which claims priority under 35 U.S.C. 119 (e) to U.S. Provisional Patent Application Ser. No. 62/513,872, filed Jun. 1, 2017; U.S. Provisional Application Ser. No. 62/571,696, filed Oct. 12, 2017; and U.S. Provisional Application Ser. No. 62/662,165, filed Apr. 24, 2018, all of which are herein specifically incorporated by reference in their entireties.
| Number | Date | Country | |
|---|---|---|---|
| 62513872 | Jun 2017 | US | |
| 62571696 | Oct 2017 | US | |
| 62662165 | Apr 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15994294 | May 2018 | US |
| Child | 17006599 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17006599 | Aug 2020 | US |
| Child | 18232238 | US |
