Patent Application
20210371519
Applicant hereby incorporates by reference a CRF sequence listing submitted herewith having a file name Sequence Listing 10738 748.txt, created on Sep. 9, 2019.
The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard abbreviations as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:
SEQ ID NO: 1 represents a nucleic acid sequence encoding a light chain variable region of a monovalent (my) anti-huFcεRIα monoclonal antibody (mAb);
SEQ ID NO: 2 represents a nucleic acid sequence encoding a heavy chain variable region of my anti-huFcεRIα mAb;
SEQ ID NO: 3 represents a light chain variable region of my anti-huFcεRIα mAb;
SEQ ID NO: 4 represents a heavy chain variable region of my anti-huFcεRIα mAb;
SEQ ID NO: 5 represents a light chain complementarity-determining region (CDR) 1 of my anti-huFcεRIα mAb;
SEQ ID NO: 5 represents a light chain complementarity-determining region (CDR) 1 of my anti-huFcεRIα mAb;
SEQ ID NO: 6 represents a light chain CDR2 of my anti-huFcεRIα mAb;
SEQ ID NO: 7 represents a light chain CDR3 of my anti-huFcεRIα mAb;
SEQ ID NO: 8 represents a heavy chain CDR1 of my anti-huFcεRIα mAb;
SEQ ID NO: 9 represents a heavy chain CDR2 of my anti-huFcεRIα mAb;
SEQ ID NO: 10 represents a heavy chain CDR3 of my anti-huFcεRIα mAb;
SEQ ID NO: 11 represents a full IE7 mAb light chain;
SEQ ID NO: 12 represents a full IE7 mAb heavy chain; and
SEQ ID NO: 13 represents a truncated heavy chain derived from the heavy chain of IE7 mAb, including a leader sequence that is cleaved during secretion.
Allergic disorders, including allergic rhinitis, asthma, atopic dermatitis, food allergy and anaphylaxis are an increasingly common cause of morbidity in developed countries. Anaphylaxis, in particular, is a life-threatening illness with an estimated lifetime prevalence of 1-2% in the United States. All of these allergic disorders are mediated, to some extent, by antigen activation of mast cells and basophils by the crosslinking of antigen-specific IgE bound to the alpha (a) chain of the high affinity IgE receptor, FcεRI. IgE/mast cell/basophil-mediated allergy and anaphylaxis can be severe and rapid in onset and can occur after multiple exposures or the initial exposure to an allergen. Foods, drugs, and insect venoms are common causes of IgE-mediated anaphylaxis. IgE-mediated allergy to these substances can have severe adverse effects on quality of life and on ability to treat disease and can be lethal.
Immunotherapeutic approaches for treatment of IgE-mediated allergy have been explored. Omalizumab is an anti-IgE monoclonal antibody that binds only to IgE that is not FcεR-associated; however, it is slow-acting and works poorly in patients with high IgE levels. An alternative approach is the use of monoclonal antibodies that target FcεRIα; however, there have been safety concerns with this approach.
Rapid desensitization (RD) is a process by which individuals who have IgE-mediated allergy to a specific antigen are made temporarily unresponsive to that antigen by exposing them to the antigen. RD was used to treat penicillin allergy as early as 1946. Drug RD is initiated by infusing the allergenic drug at a dose too small to induce a clinical reaction, then increasing it every 15-60 minutes until a full therapeutic dose is tolerated. Although RD with allergens can decrease the risk of subsequent anaphylaxis, this procedure is not without risk. It frequently has adverse effects, including systemic anaphylaxis, typically works for only a short time if not maintained by daily allergen exposure, and is allergen-specific.
A need persists for a safe, efficacious, convenient way to rapidly suppress IgE/basophil/mast cell-mediated disease without the drawbacks of antigen RD.
Some of the embodiments of the present disclosure are summarized below. Additional embodiments are described in the Detailed Description, Examples, Figures, and Claims sections of this disclosure. The description in each section of this disclosure is intended to be read in conjunction with the other sections. Furthermore, the various embodiments described in each section of this disclosure can be combined in various different ways, and all such combinations are intended to fall within the scope of the present invention.
While strong FcεRI crosslinking induces the mast cell/basophil activation and degranulation that causes allergic signs and symptoms, persistent, low-level crosslinking of FcεRI removes IgE from these cells and desensitizes them without inducing sufficient degranulation to cause allergy manifestations. The present disclosure shows that IgE-mediated allergy can safely be suppressed by a single administration of a recombinant monovalent Fab/Fc derivative anti-FcεRIα antibody, which persistently crosslinks FcεRI to a limited extent.
In one embodiment, a monovalent anti-huFcεRIα monoclonal antibody (mAb) is provided, comprising one light chain, one heavy chain, and one truncated heavy chain, wherein the truncated heavy chain lacks a variable domain and a CH1 domain, wherein the mAb specifically binds an epitope of FcεRIα. In some embodiments, the mAb does not compete with IgE for binding to FcεRIα. In some embodiments, the mAb binds to human FcεRIα.
In certain embodiments, the light chain is a kappa chain. The light chain can comprise a variable region encoded by SEQ ID NO: 1. The light chain can comprise a variable region having SEQ ID NO: 3. The light chain can comprise complementarity-determining region (CDR) 1 having the sequence QDINNY (SEQ ID NO: 5), CDR2 having the sequence RAN (SEQ ID NO: 6), and CDR3 having the sequence LLYNEFPWM (SEQ ID NO: 7).
In certain embodiments, the heavy chain is a gamma chain. In one embodiment, the heavy chain is a gamma subclass 4 heavy chain (IgG4). The heavy chain constant region may be human. The heavy chain can comprise a variable region encoded by SEQ ID NO: 2. The heavy chain can comprise a variable region having SEQ ID NO: 4. The heavy chain can comprise CDR1 having the sequence GFTFSFYG (SEQ ID NO: 8), CDR2 having the sequence ISGGGNYT (SEQ ID NO: 9), and CDR3 having the sequence VRAYYGNWNSY (SEQ ID NO: 10). One or both of the heavy chain and the truncated heavy chain can comprise one or more mutations in the constant region to promote dimerization of the heavy chain and the truncated heavy chain. In one embodiment, the heavy chain comprises a E365K mutation and/or a D399K mutation. In one embodiment, the truncated heavy chain comprises a K392D mutation and/or a K409D mutation. In some embodiments, the monovalent anti-huFcεRIα mAb is a chimeric antibody, in which the constant region of the heavy chain is from human IgG1 (γ1) and the constant region of the light chain is from human kappa chain.
Also provided is a pharmaceutical composition comprising the anti-FcεRIα monovalent mAb. In certain embodiments, the pharmaceutical composition is for use in treating an allergy or desensitizing a subject to an allergen. Also provided is the use of the anti-FcεRIα monovalent mAb in the manufacture of a medicament for the treatment of an allergy.
In another embodiment, a polynucleotide or combination of polynucleotides encoding the anti-FcεRIα monovalent mAb is provided. A vector and a host cell comprising the polynucleotide or combination of polynucleotides are also provided.
In another embodiment, provided are methods of desensitizing a subject to allergen, methods of treating an allergy, and methods of preventing an allergic reaction in a subject having an allergy. In some embodiments, the allergic reaction is anaphylaxis. The allergen can be, for example, selected from the group consisting of protein, polysaccharide, lipid, polynucleotide, food, pollen, mold spores, dust, animal dander, insect debris, blood serum, drugs, cosmetics, and combinations thereof. In certain embodiments, the allergen is a food allergen selected from the group consisting of eggs, milk, peanuts, tree nuts, soy, sesame, crustacean shellfish, and wheat.
In one embodiment, a method comprises administering to a subject with an allergy a plurality of doses of a first anti-FcεRIα mAb, wherein the doses are administered over a period of 1 to 4 days, and wherein each dose is lower than a level required to induce shock. In some embodiments, a dose of the first anti-FcεRIα mAb is administered every 1-4 hours. In some embodiments, each dose is the same as or higher than, for example double or triple, each immediately preceding dose. In certain embodiments, the method further comprises administering a plurality of doses of a second anti-FcεRIα mAb, wherein each dose is lower than a level required to induce shock. The second anti-FcεRIα mAb can be co-administered with the first anti-FcεRIα mAb, wherein the total dose of anti-FcεRIα mAb is lower than a level required to induce shock. The total dose of anti-FcεRIα mAb is the dose of the first anti-FcεRIα mAb plus the dose of the second anti-FcεRIα mAb. In some embodiments, each total dose of anti-FcεRIα mAb is the same as or higher than, for example double or triple, each immediately preceding total dose.
A variety of anti-FcεRIα mAbs can be used in the methods of desensitizing a subject to allergen, methods of treating an allergy, and methods of preventing an allergic reaction in a subject having an allergy. In some embodiments, the anti-FcεRIα mAb is a monovalent mAb. In some embodiments, the anti-FcεRIα mAb is IE7 or IB10. In methods comprising a first anti-FcεRIα mAb and a second anti-FcεRIα mAb, the first and second antibodies can each bind to a different epitope of FcεRIα. In one aspect, the first anti-FcεRIα mAb is IE7 and the second anti-FcεRIα mAb is D310.
Another method comprises administering to a subject with an allergy a first dose of a first anti-FcεRIα mAb at a dose that is lower than a level required to induce shock, wherein the first anti-FcεRIα mAb is a monovalent anti-huFcεRIα mAb comprising one light chain, one heavy chain, and one truncated heavy chain, wherein the truncated heavy chain lacks a variable domain and a CH1 domain, wherein the mAb specifically binds an epitope of FcεRIα, optionally the epitope that is bound by the mAb IE7. In some embodiments, the mAb does not compete with IgE for binding to FcεRIα. In some embodiments, the mAb binds to human FcεRIα.
In a particular embodiment, the method further comprises subsequently administering a second dose of the first anti-FcεRIα mAb at a dose that is lower than a level required to induce shock. The method can further comprise administering additional doses of the first anti-FcεRIα mAb every 1-4 weeks or every 1-6 months, wherein each additional dose is lower than a level required to induce shock.
In another embodiment, the method further comprises subsequently administering a first dose of a second anti-FcεRIα mAb at a dose that is lower than a level required to induce shock. The method can further comprise co-administering a second dose of the first anti-FcεRIα mAb with the first dose of the second anti-FcεRIα mAb, wherein the total dose of anti-FcεRIα mAb is lower than a level required to induce shock.
In certain embodiments, the first dose of the second anti-FcεRIα mAb is administered about 24 hours after the first dose of the first anti-FcεRIα mAb. Additional doses of the second anti-FcεRIα mAb or additional doses of the first anti-FcεRIα mAb and the second anti-FcεRIα mAb can be administered, for example, every 1-4 weeks, wherein each additional dose or total dose is lower than a level required to induce shock. These and other objects, features, embodiments, and advantages will become apparent to those of ordinary skill in the art from a reading of the following detailed description and the appended claims.
Described herein are monovalent (my) mAbs to FcεRIα. In particular, the present disclosure describes recombinant my Fab/Fc derivatives of anti-huFcεRIα mAbs that bind FcεRI, regardless of its association with IgE. As a result of their single Fab and complete Fc, these mAbs do not directly crosslink FcεRI, but induce persistent, low-level FcεRI crosslinking. The my mAbs of the present disclosure achieve safe and effective allergen-independent desensitization with a single administration. Also disclosed herein are methods of allergen desensitization, allergy treatment, and prevention of allergic reactions using an extended desensitization protocol and protocols using the my mAbs of the disclosure with or without subsequent injections of a divalent (dv) form of the same antibodies.
These my mAbs were tested in three different types of transgenic mice: (a) huFcεRIα mice, which have human (rather than mouse) FcεRIα; (b) huFcεRIα/IL-4RαF709 mice, which have human FcεRIα, as well as an IL-4RαF709 mutation that enhances the pro-allergic effects of IL-4 and IL-13; and (c) reNRGS mice, which are immunodeficient, hu IL-3-, GM-CSF- and SCF-producing mice that have developed large numbers of activated human mast cells following reconstitution with human cord blood cells. In some experiments, mice were pre-treated with a long-acting form of IL-4 (IL-4C) or IL-4C and the β-adrenergic receptor antagonist propranolol to increase their sensitivity to histamine and other mediators by a factor of approximately fifty.
This disclosure demonstrates: (1) that humanized mice can be safely protected against IgE-mediated anaphylaxis and food allergy by rapid desensitization with anti-huFcεRIα mAb or by a single injection of the my Fab/Fc of this mAb; (2) that desensitization is characterized by decreased mast cell responsiveness to FcεRI crosslinking, loss of most IgE, and some loss of FcεRI; (3) that rapid desensitization with anti-FcεRIα mAb is safer and lasts longer than rapid desensitization with a relevant allergen; (4) that the greater the sensitivity of mice to FcεRI crosslinking, the longer it takes to safely and rapidly desensitize them; (5) that desensitization can be maintained by repeated injections of anti-FcεRIα mAb; (6) that FcεRI crosslinking by my anti-FcεRIα mAbs is mediated by the simultaneous binding of these mAbs to mast cell/basophil FcεRI and to FcγRs; (7) the greater safety of my over dv anti-FcεRIα mAbs for single dose desensitization of mast cells and suppression of IgE-mediated allergy; (8) the importance of deaggregating my anti-FcεRIα mAbs that will be used for desensitization; (9) the contribution of avidity for FcγRs to induction of anaphylaxis and suppression of IgE-mediated disease by my anti-FcεRIα mAbs; (10) the ability to decrease induction of anaphylaxis by a my form of the anti-huFcεRIα mAb, IE7, by producing a variant that has huIgG4, rather than huIgG1 heavy chain constant regions, consistent with the lower avidity of huIgG4 than huIgG1 for FcγRs; (11) that suppression of IgE-mediated anaphylaxis is not entirely due to removal of IgE from mast cells; and (12) that desensitization by my IE7 is accompanied by selective loss of some mast cell signaling molecules: SYK, phospho-SYK, and phospho-SHIP, but neither phospho-AKT nor phospho-ERK.
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 subject matter is related.
As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably.
Furthermore, “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).
Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint for a range that includes other individual values provided herein. For example, a set of values such as 1, 2, 3, 8, 9, and 10 is also a disclosure of a range of numbers from 1-10. Where a numeric term is preceded by “about,” the term includes the stated number and values ±10% of the stated number. The headings provided herein are not limitations of the various aspects or embodiments of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.
Amino acids are referred to herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter codes. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation, and nucleic acid sequences are written left to right in 5′ to 3′ orientation.
Wherever embodiments are described with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of” and/or “consisting essentially of” are included.
“FcεRI” refers to the high-affinity receptor for the Fc region of the epsilon heavy chain of immunoglobulin E (IgE). FcεRT is constitutively expressed on mast cells and basophils, and can be induced in eosinophils. FcεRI is comprised of an alpha chain, a beta chain, and two gamma chains. The IgE binding site of FcεRI is on the alpha chain. An “anti-FcεRIα antibody” binds specifically to the alpha chain of FcεRT. Some anti-FcεRIα antibodies compete with IgE for binding to FcεRIα. Other anti-FcεRIα antibodies do not compete with IgE for binding to FcεRIα.
The term “antibody” refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule. The terms “antibody” or “immunoglobulin” are used interchangeably herein.
A typical IgG antibody comprises at least two γ heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each γ chain is comprised of a heavy chain variable region (VH) and a γ heavy chain constant region. The γ heavy chain constant region is comprised of three domains, CH1, CH2, and CH3. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, Cl. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g. effector cells) and the first component (Clq) of the classical complement system.
For purposes of the present disclosure, a “monovalent” molecule contains a single antigen-binding region. A “bivalent” or “divalent” molecule contains two antigen-binding regions, which each may bind the same or different antigen molecules. When each of the antigen binding regions of a bivalent molecule binds the same antigen, it is “mono-specific.” When each of the antigen-binding regions of a bivalent molecule binds a different antigen or a different epitope on the same antigen, it is “bi-specific.” For example, native antibodies are bivalent mono-specific molecules. The terms “bivalent,” “divalent,” and the abbreviation “dv” are used herein interchangeably.
Antibodies can be of any the five major classes (isotypes) of immunoglobulins: IgA, IgD, IgE, IgG and IgM, or subclasses thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu respectively. Mutated subclasses of immunoglobulins, for example, IgG2σ, are also included in the scope of the disclosure. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. There are two classes of mammalian light chains, lambda and kappa.
The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity-determining regions (CDRs), interspersed with regions that are more conserved, termed framework (FW) regions. The CDRs in each chain are held together in close proximity by the FW regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. Each VH and VL is composed of three CDRs and four FWs, arranged from amino-terminus to carboxy-terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4.
There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., J. Molec. Biol. 273:927-948 (1997)). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs.
The amino acid position numbering as in Kabat, refers to the numbering system used for heavy chain variable domains or light chain variable domains (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain). Using this numbering system, the actual linear amino acid sequence can contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FW or CDR of the variable domain. For example, a heavy chain variable domain can include a single amino acid insert (residue 52σ, according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82σ, 82b, and 82c, etc., according to Kabat) after heavy chain FW residue 82.
The Kabat numbering of residues can be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence. Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. See Table 1.
IMGT (ImMunoGeneTics) also provides a numbering system for the immunoglobulin variable regions, including the CDRs. See, e.g., Lefranc, M. P. et al., Dev. Comp. Immunol. 27: 55-77 (2003). The IMGT numbering system was based on an alignment of more than 5,000 sequences, structural data, and characterization of hypervariable loops and allows for easy comparison of the variable and CDR regions for all species. According to the IMGT numbering schema VH-CDR1 is at positions 26 to 35, VH-CDR2 is at positions 51 to 57, VH-CDR3 is at positions 93 to 102, VL-CDR1 is at positions 27 to 32, VL-CDR2 is at positions 50 to 52, and VL-CDR3 is at positions 89 to 97. As used throughout the specification, the CDR sequences described correspond to the IMGT numbering system.
A “monoclonal antibody” (mAb) refers to a homogeneous antibody population that is involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants. The term “monoclonal” can apply to both intact and full-length monoclonal antibodies, as well as to antibody fragments (such as Fab, Fab′, F(ab′)2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, “monoclonal antibody” refers to such antibodies made in any number of ways including, but not limited to, by hybridoma, phage selection, recombinant expression, and transgenic animals.
The term “humanized antibody” refers to an antibody derived from a non-human (e.g., murine) immunoglobulin, which has been engineered to contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies are human immunoglobulins in which residues from the complementary determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, or hamster) that have the desired specificity, affinity, and capability (Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536). In some instances, the Fv framework region (FW) residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and capability.
Humanized antibodies can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or capability. In general, humanized antibodies will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. Humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Pat. Nos. 5,225,539 and 5,639,641.
The term “human antibody” means an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art. The definition of a human antibody includes intact or full-length antibodies comprising at least one human heavy and/or light chain polypeptide such as, for example, an antibody comprising murine light chain and human heavy chain polypeptides.
The term “chimeric antibodies” refers to antibodies wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of mammals (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability while the constant regions are homologous to the sequences in antibodies derived from another (usually human) to avoid eliciting an immune response in that species.
The term “antigen-binding fragment” refers to a portion of an intact antibody comprising the complementarity determining variable regions of the antibody. Fragments of a full-length antibody can be an antigen-binding fragment of an antibody. Examples of antibody fragments include, but are not limited to Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, single chain antibodies (e.g., ScFvs), and multispecific antibodies formed from antibody fragments.
An “isolated” molecule is one that is in a form not found in nature, including those which have been purified.
A “polynucleotide,” as used herein can include one or more “nucleic acids,” “nucleic acid molecules,” or “nucleic acid sequences,” and refers to a polymer of nucleotides of any length, and includes DNA and RNA. The polynucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.
The term “vector” means a construct, which is capable of delivering and, in some embodiments expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.
The terms “inhibit,” “block,” and “suppress” are used interchangeably and refer to any statistically significant decrease in occurrence or activity, including full blocking of the occurrence or activity. For example, “inhibition” can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in activity or occurrence. An “inhibitor” is a molecule, factor, or substance that produces a statistically significant decrease in the occurrence or activity of a process, pathway, or molecule.
An “active agent” is an ingredient that is intended to furnish pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease, or to affect the structure or any function of a subject's body. The active agent can be in association with one or more other ingredients, and can be, but is not necessarily, in a finished dosage form.
An “effective amount” of an active agent is an amount sufficient to carry out a specifically stated purpose. In embodiments, an effective amount of an active agent is an amount that is sufficient to desensitize a subject to an allergen without inducing anaphylactic shock.
The term “pharmaceutical composition” refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective and which contains no additional components that are unacceptably toxic to a subject to which the composition would be administered. Such composition can be sterile and can comprise a pharmaceutically acceptable carrier, such as physiological saline. Suitable pharmaceutical compositions can comprise one or more of a buffer (e.g. acetate, phosphate, or citrate buffer), a surfactant (e.g. polysorbate), a stabilizing agent (e.g. human albumin, polyol, or amino acid), a preservative (e.g. benzyl alcohol or sodium benzoate), an absorption promoter to enhance bioavailability and/or other conventional solubilizing or dispersing agents. Choice of excipients depends upon dosage form, the active agent to be delivered, and the disease or disorder to be treated or prevented.
“Equal doses” are two or more doses of an active agent, wherein each subsequent dose is the same as, or equal to, the first dose of the active agent. Each dose administered in the methods of the present disclosure is lower than a level required to induce shock in the subject to whom the dose is administered.
“Sequentially escalating doses” are two or more doses of an active agent, wherein the second dose is greater than the first, the third is greater than the second, and so forth, such that the subsequent dose is greater than the immediately preceding dose. Each subsequent dose can be larger by a determined amount or can be characterized in relation to the first dose or the immediately preceding dose, such as each subsequent dose is doubled or tripled in relation to the first dose or in relation to the immediately preceding dose.
“Shock” in a subject is a clinical diagnosis that is typically characterized by change in the subject's temperature, changes in circulatory function, pallor, sweating, weak pulse, and/or very low blood pressure.
An “allergen” is a substance that produces an immune response; such responses can include hypersensitivities and allergies. The allergen can, for example, be a protein, can be from food or can be from the environmental surroundings. Allergens may be naturally occurring or of synthetic origin and include, among other things, pollen, mold spores, dust, animal dander, insect debris, foods, blood serum, drugs, and cosmetics. Common food allergens include eggs, milk, peanuts, tree nuts, soy, sesame, crustacean shellfish, and wheat.
A “subject” or “individual” or “animal” or “patient” or “mammal,” is any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, sports animals, and laboratory animals including, e.g., humans, non-human primates, canines, felines, porcines, bovines, equines, rats, mice, rabbits, and the like.
Terms such as “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder. Thus, those in need of treatment include those already suffering from the disorder. In certain embodiments, a subject is successfully “treated” for a disease or disorder according to the methods provided herein if the patient shows, e.g., total, partial, or transient alleviation or elimination of symptoms associated with the condition, disease, or disorder; diminishment of the extent of the condition, disease, or disorder; stabilization (i.e., not worsening) of the condition, disease, or disorder; delay in onset or slowing of progression of the condition, disease or disorder; amelioration of the condition, disease, or disorder, including partial or total remission; and/or prolonged survival, as compared to expected survival if not receiving treatment. In the context of the present disclosure, “desensitization” to an allergen is synonymous with treating an allergic disorder and with preventing or reducing an allergic reaction to an allergen. One example of treatment is reduction or amelioration of symptoms associated with an allergic reaction, such as anaphylaxis, allergic rhinitis, asthma, atopic dermatitis, hives, and/or itching.
The methods, uses, and compositions described herein comprise anti-FcεRIα mAbs. Examples of suitable mAbs include, for example, AER-37 (CRA-1), 15.1, IE7, and D310.
In a specific embodiment, the anti-FcεRIα mAb is IE7. A biological material deposit of the IE7 hybridoma was made on Jun. 29, 2018 under the terms of the Budapest Treaty as ATCC® Patent Deposit Designation PTA-125115 with the American Type Culture Collection (ATCC®), P.O. Box 1549, Manassas, Va., 20108, USA.
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NISWVRQTPDKKLEWV
YYPDSVKGRFTISRDNAMNTLYLQMNSLKSEDTAMYYC
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In another embodiment, the anti-FcεRIα mAb is IB10. A biological material deposit of the IB10 hybridoma was made on Jun. 29, 2018 under the terms of the Budapest Treaty as ATCC® Patent Deposit Designation PTA-125116 with the American Type Culture Collection (ATCC®), P.O. Box 1549, Manassas, Va., 20108, USA.
In embodiments, the anti-FcεRIα mAb is a monovalent mAb comprising one light chain, one heavy chain, and one truncated heavy chain, wherein the truncated heavy chain lacks a variable domain and a CH1 domain.
In some embodiments, the my mAb is derived from IE7.
In certain embodiments, the light chain is a kappa light chain and comprises a light chain variable region encoded by SED ID NO: 1 or SEQ ID NO: 3. In embodiments, the my mAb comprises complementarity-determining region (CDR) 1 having the sequence QDINNY (SEQ ID NO: 5), CDR2 having the sequence RAN (SEQ ID NO: 6), and CDR3 having the sequence LLYNEFPWM (SEQ ID NO: 7) of IE7. In embodiments, the my mAB comprises the light chain of IE7 (SEQ ID NO: 11).
In certain embodiments, the heavy chain is a gamma chain. In embodiments, the heavy chain is a gamma subclass 4 heavy chain (IgG4). The heavy chain constant region is preferably human. In one embodiment, the heavy chain comprises a variable region encoded by SEQ ID NO: 2. In another embodiment, the heavy chain comprises a variable region of IE7 having SEQ ID NO: 4. In a specific embodiment, the heavy chain comprises CDR1 having the sequence GFTFSFYG (SEQ ID NO: 8), CDR2 having the sequence ISGGGNYT (SEQ ID NO: 9), and CDR3 having the sequence VRAYYGNWNSY (SEQ ID NO: 10). One or both of the heavy chain and the truncated heavy chain can comprise one or more mutations in the constant region to promote dimerization of the heavy chain and the truncated heavy chain. In one embodiment, the heavy chain comprises a E365K mutation and/or a D399K mutation. In one embodiment, the truncated heavy chain comprises a K392D mutation and/or a K409D mutation. In embodiments, the monovalent anti-huFcεRIα mAb is a chimeric antibody, in which the constant region of the heavy chain is from human IgG1 (γ1) and the constant region of the light chain is from human kappa chain.
In another specific embodiment, the my mAb comprises the heavy chain of IE7 (SEQ ID NO: 12). In another specific embodiment, the my mAb comprises a truncated heavy chain derived from the heavy chain of IE7, having SEQ ID NO: 13: METDTLLLWVLLLWVPGSTGDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, which includes a leader sequence that is cleaved during secretion.
In embodiments, the epitope is the same epitope that is bound by the mAb IE7. In embodiments, the mAb does not compete with IgE for binding to FcεRIα. In some embodiments, the mAb binds to human FcεRIα.
In one embodiment, a pharmaceutical composition comprising an anti-FcεRIα monovalent mAb of the present disclosure is provided. In certain embodiments, the pharmaceutical composition is for use in treating an allergy or desensitizing a subject to an allergen. In embodiments, the pharmaceutical composition comprises an anti-FcεRIα monovalent mAb of the present disclosure and a pharmaceutical carrier. In embodiments, the pharmaceutical composition is formulated for intravenous or subcutaneous administration.
In another embodiment, a method of use of an anti-FcεRIα monovalent mAb as described herein in the manufacture of a medicament for the treatment of an allergy is provided.
In another embodiment, a polynucleotide or combination of polynucleotides encoding the anti-FcεRIα monovalent mAb are provided.
In another embodiment, a vector comprising the polynucleotide or combination of polynucleotides is provided.
In another embodiment, a host cell comprising the polynucleotide or combination of polynucleotides is provided.
Methods of Use
In embodiments, desensitization to an allergen is accomplished by administering an anti-FcεRIα mAb. Various anti-FcεRIα mAbs are suitable for use in the presently disclosed methods. Examples of suitable mAbs include, but are not limited to, AER-37 (CRA-1), 15.1, IE7, and IB10.
In embodiments, a method of desensitizing a subject to allergen is provided, the method comprising administering to a subject with an allergy a plurality of doses of a first anti-FcεRIα mAb, wherein the doses are administered over a period of 1 to 4 days, and wherein each dose is lower than a level required to induce shock, thereby desensitizing the subject to the allergen.
In embodiments, the allergen is selected from the group consisting of protein, polysaccharide, lipid, polynucleotide, food, pollen, mold spores, dust, animal dander, insect debris, blood serum, drugs, cosmetics, and combinations thereof. In certain embodiments, the allergen is a food allergen selected from the group consisting of eggs, milk, peanuts, tree nuts, soy, sesame, crustacean shellfish, and wheat.
In embodiments, the first anti-FcεRIα mAb is a monovalent mAb. In a specific embodiment, the first anti-FcεRIα mAb is IE7. In another specific embodiment, the first anti-FcεRIα mAb is IB10.
In one embodiment, the methods disclosed herein comprise administration of a first anti-FcεRIα mAb followed by repeated equal or escalating doses of the first anti-FcεRIα mAb. In a specific embodiment, each succeeding dose is double, (i.e., 2×) the immediately preceding dose.
In another embodiment, administration of the plurality of doses of the first anti-FcεRIα mAb further comprises administering a plurality of doses of a second anti-FcεRIα mAb, wherein each dose of the second anti-FcεRIα mAb is lower than a level required to induce shock. The first anti-FcεRIα mAb and second anti-FcεRIα mAb may be administered together (i.e., co-administered). In embodiments, the total dose of anti-FcεRIα mAb co-administered is lower than a level required to induce shock. When first and second anti-FcεRIα mAbs are co-administered, each total dose of anti-FcεRIα mAb may be the same as, or higher than, each immediately preceding total dose. In a specific embodiment, each succeeding total dose is double (i.e., 2×) the immediately preceding total dose.
In embodiments, one or both of the first and second anti-FcεRIα mAbs are monovalent mAbs. In a specific embodiment, the first anti-FcεRIα mAb and the second anti-FcεRIα mAb each bind to a different epitope of FcεRIα. In a very specific embodiment, the first anti-FcεRIα mAb is IE7 and the second anti-FcεRIα mAb is IB10.
In embodiments, any of the methods disclosed herein comprise administration of a first anti-FcεRIα mAb and optionally a second anti-FcεRIα mAb in intervals, namely, at intervals such as 30 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24 hours, or at varying intervals. In a specific embodiment, the first anti-FcεRIα mAb is administered every 1-4 hours. In another specific embodiment, the first anti-FcεRIα mAb and the second anti-FcεRIα mAb are co-administered every 1-4 hours.
Administration of one or more anti-FcεRIα mAbs of the presently disclosed methods comprises from 4-40 administrations over a period of 1, 2, 3, or 4 days. For instance, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses can be administered per day. The doses can be provided at intervals such as 30 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24 hours, or at varying intervals.
In embodiments, the methods of the disclosure comprise administration of a single dose of the my anti-FcεRIα mAb disclosed herein.
Methods of the disclosure can further comprise additional doses of one or more anti-FcεRIα mAbs at intervals of 1, 2, 3, 4, 6, 8, or 12 weeks or of 1, 2, 3, 4, 5, or 6 months following the last repeated equal or escalating dose of the anti-FcεRIα mAb, or following the single dose of the my anti-FcεRIα mAb.
Each dose of the anti-FcεRIα mAb is lower than a level required to induce shock in the subject. In one embodiment, the dose can be about ½ the level required to induce shock in the subject. In another embodiment, the dose can be less than about ½ the level required to induce shock in the subject, for example, about ⅓, ¼, ⅕, ⅙, 1/7, ⅛, 1/10, 1/16, 1/20, or 1/25 the level required to induce shock in the subject.
In another embodiment, a method of desensitizing a subject to an allergen is provided, the method comprising administering to a subject with an allergy a first dose of a first anti-FcεRIα mAb at a dose that is lower than a level required to induce shock, wherein the first anti-FcεRIα mAb is a monovalent anti-FcεRIα mAb according to the present disclosure, thereby desensitizing the subject to the allergen.
In embodiments, the method further comprises subsequently administering a second dose of the first anti-FcεRIα mAb at a dose that is lower than a level required to induce shock. In embodiments, the method further comprises administering additional doses of the first anti-FcεRIα mAb every 1-4 weeks, wherein each additional dose is lower than a level required to induce shock.
In another embodiment, the method further comprises subsequently administering a first dose of a second anti-FcεRIα mAb at a dose that is lower than a level required to induce shock. Optionally, the method comprises co-administering a second dose of the first anti-FcεRIα mAb with the first dose of the second anti-FcεRIα mAb, wherein the total dose of anti-FcεRIα mAb is lower than a level required to induce shock. In embodiments, the first dose of the second anti-FcεRIα mAb is administered about 24 hours after the first dose of the first anti-FcεRIα mAb.
In embodiments, the method further comprises administering additional doses of the second anti-FcεRIα mAb every 1-4 weeks, wherein each additional dose is lower than a level required to induce shock. In another embodiment, the method further comprises co-administering additional doses of the first anti-FcεRIα mAb and the second anti-FcεRIα mAb every 1-4 weeks, wherein each additional total dose of anti-FcεRIα mAb is lower than a level required to induce shock.
Methods of treating an allergy and methods of preventing an allergic reaction in a subject having an allergy are provided, comprising administration of one or more anti-FcεRIα mAbs as described herein, and according to the dosing schedules described herein. In specific embodiments, the allergic reaction is anaphylaxis.
Embodiments of the present disclosure are further defined by reference to the following non-limiting examples. It will be apparent to those skilled in the art that various modifications, both to materials and methods, can be practiced without departing from the scope of the present disclosure.
The present inventors previously reported that mice can be rapidly desensitized (RD′ d) with MAR-1, a hamster IgG mAb that binds mouse FcεRIα that is not associated with IgE. See Khodoun et al., J. Allergy Clin. Immunol. 133:1555-1564 (2013). Although a single large dose of MAR-1 caused anaphylaxis, administering MAR-1 by a RD approach did not induce anaphylaxis. RD with MAR-1 rapidly induced short-lived mast cell anergy. Continued treatment with MAR-1 removed all IgE from mast cells over several days. Once RD with MAR-1 was performed, mast cell loss of IgE could be safely maintained by repeated, large doses of MAR-1. RD with MAR-1 completely suppressed IgE-mediated anaphylaxis in passive and active sensitization models, suppressed established IgE-mediated food allergy, and was antigen-non-specific. In addition, RD with MAR-1 was safer and its effects longer-lasting than rapid desensitization with antigen in mice that had been sensitized to that antigen by active immunization.
To determine if this approach could be adapted to humans, the studies used (1) anti-human (hu) FcεRIα mAbs and (2) mice whose mast cells and basophils express huFcεRIα. For the first purpose, hybridoma 15.1 was obtained, which produces a mouse IgG1 mAb that, like MAR-1, competes with IgE for binding to FcεRIα. AER-37 (also called CRA-1) was purchased, a mouse IgG2b mAb that binds huFcεRIα regardless of its association with IgE. Mouse IgG1 mAbs to huFcεRIα was produced by immunizing FcεRIα-deficient BALB/c mice with huFcεRIα. One of these mAbs, IE7, does not compete with IgE (or 15.1) for binding to huFcεRIα but competes with AER-37; another of these mAbs, D310, has the opposite characteristics. See U.S. Pat. No. 10,086,005. For the second purpose, acquired BALB/c mice that lack mouse FcεRIα and express a hu FcεRIα transgene were acquired. In these “huFcεRIα mice,” a functional receptor is formed that includes huFcεRIα chain, mouse FcεRIβ chain, and mouse FcεRIγ chains. NSGS and NRGS mice were reconstituted, which lack T, B, and NK cells, and express transgenic huSCF, hulL-3 and huGM-CSF, with T cell-depleted human cord blood cells. Stem cells in the donor cell population generate hu B cells, T cells, basophils, mast cells and myeloid cells in these “reNSGS” and “reNRGS” mice in 2-3 months. (NRGS mice have non-functional RAG1 alleles, instead of the SCID alleles in NSGS mice.)
Initial studies with mAbs 15.1 and AER-37 in huFcεRIα mice showed that a single large dose of either mAb induces anaphylaxis, with AER-37 causing more severe disease (
Anti-huFcεRIα mAb RD of actively sensitized huFcεRIα/F709 mice and passively sensitized reNSGS and reNRGS mice was more difficult than RD of passively sensitized huFcεRIα mice. Challenge of reNSGS mice with TNP-BSA (after mouse IgE anti-TNP mAb sensitization) or with only 200 ng of anti-huIgE mAb induced fatal anaphylaxis (
To determine if slower, more gentle desensitization could more safely and completely protect reNRGS and huFcεRIα/F709 mice, investigators first determined whether huFcεRIα mice could be fully desensitized by repeated injection of very low doses of the anti-huFcεRIα mAb, IE7, instead of injecting escalating doses of this mAb. Six hourly 400 ng doses of IE7 removed nearly all IgE and most FcεRI from mast cells in huFcεRIα mice and blocked the anaphylactic response to injection of 200 μg of IE7 (
The success in huFcεRIα mice led the present investigators to try a 3-day desensitization protocol in reNRGS mice that treated them with repeated low doses of IE7, followed by increasing doses of IE7, then IB10 (a mouse IgG1 mAb that, like 15.1, competes with IgE for binding to huFcεRIα). This approach did not cause hypothermia during desensitization, removed >98% of IgE from peritoneal and splenic mast cells and 91% of FcεRIα from splenic mast cells, and protected reNRGS mice against IgE-mediated, antigen-induced anaphylaxis (
The greater loss of IgE than FcεRI by mast cells in anti-FcεRIα mAb-treated mice has been consistent and is particularly marked when mice are treated for a limited time with a low dose of AER-37 or IE7 (
Because IgE-mediated mast cell/basophil degranulation requires FcεRI crosslinking, injecting mice with sufficient my anti-FcεRIα mAb might saturate the FcεRIα epitopes recognized by that mAb without crosslinking FcεRI sufficiently to induce mast cell degranulation. If so, a my mAb might delay the binding of subsequently injected dv mAb to the same epitope sufficiently for the dv mAb to cause desensitization without degranulation. Alternatively, my anti-FcεRIα mAb might indirectly crosslink basophil and mast cell FcεRI, through other mechanisms, which could cause these cells to become less responsive to FcεRI crosslinking (i.e.; to become anergic) and remove some or all of their IgE. As a third possibility, my anti-FcεRIα mAb might be retained on the basophil/mast cell surface long enough to mediate complement-mediated cytotoxicity or antibody-dependent cell mediated cytotoxicity (ADCC).
To evaluate these possibilities, a my version of IE7 composed of one κ chain, one complete heavy chain that has huγ1 constant and hinge regions, and one truncated huγ1 chain that lacks V and CH1 regions was designed and produced (
Injection of huFcεRIα mice i.v. with 3-100 μg of my huIgG1 IE7 failed to induce hypothermia or any detectable change in mouse behavior (
huFcεRIα mice treated with 50 or 100 μg of my IE7, followed by 100 μg of dv IE7, were completely protected against anaphylaxis when sensitized with mouse IgE anti-TNP mAb and challenged i.v. with TNP-ovalbumin (OVA) (
Surprisingly, treating IgE anti-TNP mAb-sensitized huFcεRIα mice with 100 of my huIgG1 IE7, without subsequent dv huIgG1 IE7, protected them nearly as well as 100 μg of dv huIgG1 IE7 against TNP-OVA challenge (
Both my and dv huIgG1 IE7 modestly reduced mast cell percentages in the peritoneum in this experiment (but had no effect on peritoneal mast cell percentages in other experiments) and depleted blood basophils, but did not decrease percentages of BM mast cells or basophils, although they considerably reduced IgE on both basophil populations (
Taken together, these results support the conclusion that the my anti-huFcεRIα mAb is safe and efficacious because it simultaneously binds FcεRI and FcγRs. This produces slow but persistent FcεRI crosslinking, which is insufficient to induce substantial rapid mast cell degranulation, but sufficient to suppress mast cell signaling and remove mast cell IgE. The surprising result that my huIgG1 IE7 is more effective than the dv form of this Ab at depleting blood basophils (
To determine whether desensitization to one FcεRIα epitope prevents an anaphylactic response to a second FcεRIα epitope, huFcεRIα mice (4/group) were desensitized by i.v. injection of 100 μg of my huIgG1 IE7, followed the next day by i.v. injection of 100 μg of dv huIgG1 IE7. Neither injection caused a temperature drop. One day after the last IE7 injection, all mice were injected i.v. with 100 μg of mAb IB10, which binds to an epitope of huFcεRIα that is not bound or blocked by IE7. Hypothermia was not observed in desensitized mice (
Investigators compared the safety of my and dv anti-FcεRIα mAbs for single dose desensitization of mast cells and suppression of IgE-mediated allergy. Mice were treated with IL-4C (a long-acting form of IL-4) and the β-adrenergic receptor blocker propranolol to make them highly sensitive to development of anaphylaxis, then challenged with dv or my huIgG1 IE7. Rectal temperatures were followed for 1 hour. Results indicate that desensitization with my anti-FcεRIα mAb provides a safety advantage over desensitization with the dv form of the same mAb (
The effect of deaggregating the my anti-FcεRIα mAbs used for desensitization was investigated. IL-4RαF709 mice have a mutation in their IL-4 receptor that increases their sensitivity to IL-4. When bred onto huFcεRIα mice, this mutation allows the induction of egg allergy. In this experiment, egg-allergic huFcεRIα/IL-4RαF709 mice were injected i.v. with 100 μg of non-deaggregated (˜95% monomer, as determined by analytical ultracentrifugation) or gel filtration-deaggregated (>99.5% monomer) or control IgG mAb and followed for 1 hour for development of hypothermia. Mice injected with deaggregated my huIgG1 IE7 were evaluated the next day for development of hypothermia and diarrhea following oral gavage (o.g.) with egg, and for mast cell degranulation (serum MMCP1 level) and loss of mast cell IgE during 4 hours after o.g. challenge with egg. Results are shown in
In another experiment, deaggregated or non-deaggregated my huIgG1 IE7 was added to cultured cells of the LAD2 human mast cell line at concentrations ranging from 25 ng/mL to 400 ng/mL. The percent of mast cell degranulation was determined by hexosaminidase release (
Investigators explored whether my anti-FcεRIα mAb can crosslink FcεRI by simultaneously binding through its V region antigen-binding site to FcεRI and through its Fc region to FcγRs on another cell (
To determine whether investigators could optimize safety and efficacy of my anti-FcεRIα mAbs by “tuning” my IE7 avidity for FcγRs, huFcεRIα/IL-4RαF709 mice were sensitized with IgE anti-TNP mAb, IL-4C, and propranolol, injected with deaggregated my huIgG1 IE7 or my huIgG4 IE7, and challenged the next day i.v. with TNP-OVA. Mice were followed for development of hypothermia after injection of the my mAb and after challenge with the antigen TNP-OVA. Results demonstrate that decreasing my IE7 avidity for FcγRs by replacing huIgG1 with huIgG4 heavy chain constant regions prevents direct induction of anaphylaxis and reduces protection against IgE-mediated anaphylaxis (
In another experiment, egg-allergic huFcεRIα/IL-4RαF709 mice were injected i.v. with 100 μg of deaggregated my huIgG4 IE7 or isotype control mAb, then challenged o.g. with 100 mg of egg the next day and 3 and 7 days after that. Mice were evaluated for development of hypothermia after injection of my huIgG4 IE7 and for hypothermia and diarrhea after each o.g. challenge. Mice were also evaluated for mast cell degranulation (serum MMCP1 level) 4 hours after the first o.g. challenge. The results demonstrate that my huIgG4 IE7 can safely desensitize egg-allergic mice that are highly sensitive to the development of anaphylaxis (
To study dose-dependent effects of my huIgG4 IE7 on mast cells and IgE-mediated anaphylaxis, huFcεRIα/IL-4RαF709 mice were injected i.v. with IgE anti-TNP mAb and IL-4C. Twelve hours later, mice were injected i.v. with 0-400 μg of deaggregated my huIgG4 IE7 and followed for 60 minutes for development of hypothermia. Twelve hours after that, mice were injected i.v. with TNP-OVA and followed for 60 minutes for development of hypothermia and survival. One hour after TNP-OVA injection, peritoneal mast cells were obtained and analyzed by flow cytometry for percent of mast cells among peritoneal wash-out cells and for surface IgE on these cells. Results are shown in
In summary, consistent with the lower avidity of huIgG4 than huIgG1 for FcγRs, induction of anaphylaxis is decreased by administration of my IE7 having huIgG4 heavy chain constant regions, compared with the huIgG1 form.
To study the relationship between suppression of anaphylaxis and suppression of mast cell IgE expression by my huIgG1 IE7, IgE anti-TNP mAb-sensitized huFcεRIα mice were given a single i.v. injection of 100 μg of my huIgG1 IE7. Mice were followed for 48 hours for (i) hypothermia response to i.v. TNP-OVA, (ii) percent of mast cells among peritoneal lavage cells, (iii) serum levels of my huIgG1 IE7, (iv) IgE on peritoneal mast cells, (v) mast cell FcεRI that was not occupied by IgE, and (vi) mast cell degranulation 2 hours post i.v. TNP-OVA challenge. Results show that a single dose of my huIgG1 IE7 desensitizes mice for at least 48 hours; that this treatment has little effect on peritoneal mast cell number; that my huIgG1 IE7 has a relatively long in vivo half-life; that desensitization persists despite the reacquisition of IgE by mast cells; that the loss of mast cell IgE exceeds the loss of mast cell FcεRI; and that mast cell degranulation over the 2 hours after challenge is less dramatic than the suppression of hypothermia (
To determine the effect of my huIgG1 IE7 on FcεRI signaling, huFcεRIα mice were sensitized with IgE anti-TNP mAb, treated with my huIgG1 IE7 or control mAb, and challenged 1 day later i.p. with TNP-OVA. Peritoneal mast cells were obtained 5 minutes after challenge and stained for phospho-SYK, total SYK, phospho-SHIP, phospho-AKT, and phospho-ERK. Expression of these markers on mast cells (large, granular c-kit+ cells) was evaluated by flow cytometry. My huIgG1 IE7 reduced expression of SYK, phospho-SYK, and phospho-SHIP, but not of phospho-AKT or phospho-ERK.
The foregoing description of the specific embodiments will so fully reveal the general nature of the disclosed subject matter that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance. The disclosed subject matter is further described by the following claims.
The present application claims priority to U.S. Provisional Application Ser. No. 62/734,676, filed Sep. 21, 2018, and U.S. Provisional Application Ser. No. 62/871,964, filed Jul. 9, 2019, each of which is incorporated herein by reference in its entirety.
This invention was made with government support under 1R01AI113162 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/052132 | 9/20/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62734676 | Sep 2018 | US | |
| 62871964 | Jul 2019 | US |
