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WO2024233251A2 - Anticorps monoclonaux humains qui améliorent pad4 pour une utilisation dans des maladies auto-immunes - Google Patents

Anticorps monoclonaux humains qui améliorent pad4 pour une utilisation dans des maladies auto-immunes Download PDF

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Publication number
WO2024233251A2
WO2024233251A2 PCT/US2024/027401 US2024027401W WO2024233251A2 WO 2024233251 A2 WO2024233251 A2 WO 2024233251A2 US 2024027401 W US2024027401 W US 2024027401W WO 2024233251 A2 WO2024233251 A2 WO 2024233251A2
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antibodies
antibody
pad4
binding
antigen
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WO2024233251A3 (fr
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Felipe Andrade
Erika DARRAH
Jing Shi
Antony Rosen
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Johns Hopkins University
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Johns Hopkins University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • FIELD FIELD
  • the present disclosure relates to monoclonal antibodies that enhance PAD4 activity in a subject, compositions comprising these antibodies, and methods of using these antibodies to develop treatments for disease, such as, for example, the treatment in autoimmune diseases.
  • STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0004] None.
  • BACKGROUND [0005] The examination of monoclonal antibodies, derived from cases in which patients with autoimmune diseases, has yielded crucial insights into the origins of the humoral response against self-antigens 1-5 and mechanisms of autoantibody pathogenicity 6-8 .
  • citrullinated proteins are major targets of the humoral response in RA has renewed interest in further characterizing the autoantibody repertoire in this disease 3,10-14 .
  • Citrullinated proteins are generated post-translationally by the peptidylarginine deiminases (PADs), a family of five calcium-dependent enzymes (termed PAD1-4 and 6) that convert arginine residues to citrulline residues 15 .
  • PADs peptidylarginine deiminases
  • PAD4 anti-citrullinated protein antibodies
  • ACPAs anti-citrullinated protein antibodies
  • C14935 anti-PAD4 antibodies that assist in the production of citrullinated antigens by enhancing the activity of PAD4 17 .
  • Antibody-mediated PAD4 activation is driven by a subgroup of anti-PAD4 antibodies initially recognized by their cross-reactivity with PAD3 17 .
  • PAD3/PAD4 cross- reactive antibodies enhance PAD4 activity by increasing the sensitivity of the enzyme to calcium.
  • ACPAs and antibodies to PAD4 may create a feed-forward loop that promotes effector functions via immune complex formation and autoantigen production.
  • the present disclosure relates to an isolated or purified monoclonal antibodies comprising: [0008] a heavy variable chain comprising SEQ ID NO:1 and a light variable chain comprising SEQ ID NO:2; [0009] a heavy variable chain comprising SEQ ID NO:3 and a light variable chain comprising SEQ ID NO:4; [0010] a heavy variable chain comprising SEQ ID NO:5 and a light variable chain comprising SEQ ID NO:6; [0011] a heavy variable chain comprising SEQ ID NO:7 and a light variable chain comprising SEQ ID NO:8; or [0012] a heavy variable chain comprising SEQ ID NO:9 and a light variable chain comprising SEQ ID NO:10.
  • the above described monoclonal antibody is a human monoclonal antibody or a derivative or variant thereof.
  • the monoclonal antibody is a humanized antibody or a derivative or variant thereof.
  • the monoclonal antibody is a chimeric antibody or a derivative or variant thereof.
  • the monoclonal antibody is a bi-specific antibody or a derivative or variant thereof.
  • the monoclonal antibody is an IgG antibody.
  • the present disclosure relates to a composition comprising at least one of the above monoclonal antibodies.
  • the composition of the present disclosure further comprises an excipient or at least one pharmaceutically acceptable carrier. Reference No.
  • the present disclosure relates to a kit comprising one or more of the above described compositions.
  • the present disclosure relates to a method of increasing PAD4 in a subject, the method comprising administering an effective amount of one of the above antibodies or compositions to a subject (such as a human, mouse, rat, rabbit, etc.).
  • a subject such as a human, mouse, rat, rabbit, etc.
  • the present disclosure relates to a method of screening for an agent that inhibits PAD4. In this aspect, the method comprises: [0019] a.
  • the agent to administered to the animal is a small molecule, a biologic, an antibody-drug conjugate, a peptide, a microRNA, a short interfering RNA, or any combinations thereof.
  • FIG. 1 shows the characterization of anti-PAD4 monoclonal antibodies derived from RA memory B cells.
  • FIG.1A Monoclonal antibodies with reactivity to PAD3/4 or PAD4 were screened by IP using [ 35 S]methionine-labeled PAD3 and PAD4 as antigens. Representative data from 12 of 32 monoclonal antibodies are shown.
  • FIG. 1B IP of radiolabelled P.
  • FIG. 1C Ig gene usage, mutation number and CDR3 amino acid sequences of monoclonal antibodies to PAD3/4 (clones 10, 16 and 17) and antibodies with reactivity only to PAD4 (clones 102 and 104).
  • FIG. 1D and FIG. 1E Venn diagrams show the number of SHM shared by clones 10, 16 and 17 in the heavy chain (FIG. 1D) and by clones Reference No.
  • FIG. 2 shows the importance of SHM in antigen recognition by human monoclonal antibodies to PAD4.
  • FIG.2A Antibody binding to PAD3/PAD4 was addressed by IP of radiolabeled substrates using mutated (SHM) and germline (GL) reverted anti-PAD4 antibodies (3.4 nM antibody/assay). Antibodies 10GL and 17GL are the same antibody (10/17GL).
  • FIG. 2B Anti-PAD4 antibodies and their germline variants were titrated from 0.4 pM to 685 nM against purified PAD4 by ELISA.
  • FIG. 2C Half maximal effective concentration (EC50) for anti-PAD4 binding were calculated from B (SD, standard deviation).
  • FIG. 2D The heavy and light chain (IgH and IgL, respectively) of SHM and GL reverted anti-PAD4 antibodies were combined to generate monoclonal antibodies (lanes 1-4). Irrelevant (IR) IgH and IgL from an antibody with no reactivity to PADs were used to produce anti-PAD4 hybrid antibodies (lanes 5- 8). Antibody binding to PAD3/PAD4 was addressed by IP (3.4 nM antibody/assay) using radiolabeled substrates. The experiments in FIG. 2A and FIG. 2D were performed on two separate occasions with similar results. [0025] FIG.
  • FIG. 3 shows the mutated and germline human anti-PAD4 antibodies are not polyreactive and have no cross-reactivity to citrullinated antigens.
  • FIG. 3A The specificity of mutated and GL anti-PAD4 monoclonal antibodies against ssDNA, dsDNA, LPS, insulin or cardiolipin was addressed by ELISA. The assays were performed in duplicate using serial dilutions of monoclonal antibody (stock 6.8 ⁇ M) or human SLE serum as positive control.
  • FIG. 3B Reactivity of SHM and GL anti-PAD4 monoclonal antibodies to cyclic citrullinated peptide 3 (CCP3) was addressed by ELISA. The assays were performed in duplicate. [0026] FIG.
  • FIG. 4 shows the functional characterization of mutated (SHM) and germline reverted (GL) anti-PAD4 monoclonal antibodies.
  • Histone H3 H3
  • PAD4 at 0.2 mM
  • 0.5 mM B
  • calcium in presence of mutated or GL anti-PAD4 antibodies (0.14 ⁇ M)
  • Clone 16 also included the analysis of antibodies containing mutated heavy chain (IgH) combined with GL light chain (IgL-GL) (lane 9) or an irrelevant IgL (IgL-IR) (lane 10), both with reactivity to PAD3 and PAD4.
  • H3 and PAD4 were incubated without (lane 1) or with calcium (lanes 2 and 3) in the absence (lane 1 and 2) or presence (lane 3) of an irrelevant antibody with no reactivity to PADs (clone 119). IgH and H3 were detected by Ponceau S staining. PAD4 and citrullinated H3 (cit-H3) were detected by Reference No. C14935 immunoblotting. The experiments were performed on three separate occasions with similar results. (FIG. 4C) Competition assays among different anti-PAD4 monoclonal antibodies were performed by measuring murinized antibody binding to PAD4 following incubation with increasing amounts of competing (human) antibodies (murinized:human antibody ratio from 1:0 to 1:100).
  • FIG. 5 shows that agonistic antibodies partially restore the activity of calcium-binding site 5 (Ca5) mutant PAD4. Histone H3 (H3) was incubated with wild type (WT) or PAD4 mutants at calcium-binding sites Ca3 and Ca5 (D 165 A), Ca5 (D 168 A), Ca3 and Ca4 (D 179 A) or Ca4 (D 388 A) in the presence of 10 mM CaCl 2 , with or without anti-PAD4 monoclonal antibodies.
  • FIG. 6 shows the genetic characteristics and functional features of RA-derived monoclonal antibodies to PAD4. Antibodies lineage trees were generated by comparison of Ig VH sequences. Monoclonal antibodies were derived from 2 patients with RA (RA-1 and RA-3). Clonally related antibodies were identified based on sequences derived from a single germline rearrangement characterized by the same V, D and J gene usage, CDR3 length and sequence.
  • FIG. 7 shows FACS analysis of mWasabi-PAD4 + memory B cells.
  • B cells pre-enriched by negative selection were stained with antibodies against human CD3, CD14, CD19, CD20, CD38, CD27 and IgM, as well as mWasabi-PAD4.
  • the gating strategy to identify CD3-CD14- CD19 + CD20 + CD27 + CD38-IgM- mWasabi-PAD4 + cells is shown from one healthy control and one representative patient with RA.
  • FIG. 8 shows germline reverted anti-PAD4 monoclonal antibodies have agonistic effect at high concentration.
  • Histone H3 H3 was citrullinated with PAD4 at 1 mM calcium in presence of mutated (0.14 nM) or increasing amounts of germline reverted (0.14 Reference No. C14935 ⁇ M, 0.46 ⁇ M or 1.4 ⁇ M) anti-PAD4 antibodies.
  • H3 and PAD4 were incubated without (lane 1) or with calcium (lane 2) in the absence (lanes 1 and 2) or presence (FIG.
  • FIG. 9 shows mutated, and germline reverted anti-PAD4 antibodies have no effect on PAD4 activity at high calcium concentrations. Histone H3 (H3) was citrullinated with PAD4 at 5 mM calcium in presence of mutated or germline anti-PAD4 antibodies.
  • FIG. 10 shows binding efficiency of murinized anti-PAD4 antibodies.
  • FIG. 10A Murinized anti-PAD4 antibodies were titrated from 0.4 pM to 685 nM against purified PAD4 by ELISA.
  • FIG. 11 shows characterization of immunodominant regions recognized by anti-PAD4 monoclonal antibodies.
  • PAD4 comprises an N-terminal domain (NTD, residues 1 to 300), further divided into subdomain 1 (NT1, amino acids 1 to 132) and subdomain 2 (NT2, amino acids 132 to 300), and a C-terminal domain (CTD, amino acids 301 to 663).
  • TTD N-terminal domain
  • CTD C-terminal domain
  • Truncated proteins generated for IP assays included the NTD, both subdomains, the CTD and NT2 plus the CTD (NT2+C).
  • FIG. 11C IP of IVTT-radiolabeled full length PAD4 (only in FIG. 11B) and truncated proteins (NTD, NT1, NT2, CTD and NT2+C) using anti-PAD4 monoclonal antibodies.
  • Immune complexes were formed by incubating the IVTT products with antibodies at 3.4 nM for 2 hrs (FIG. 11B) or at 34 nM overnight (FIG. 11C). IVTT products directly resolved by SDS–PAGE are shown to demonstrate loading. The experiments were performed on two separate occasions with similar results.
  • FIG. 12A shows the amino acid sequences of the monoclonal antibodies of the present disclosure. Reference No. C14935 [0035] FIG.
  • compositions comprising monoclonal antibodies as well as compositions comprising said antibodies, methods of using said antibodies to develop and screen for new treatments (e.g., therapeutic agents) for autoimmune disease and kits containing said antibodies and compositions.
  • compositions comprising monoclonal antibodies as well as compositions comprising said antibodies, methods of using said antibodies to develop and screen for new treatments (e.g., therapeutic agents) for autoimmune disease and kits containing said antibodies and compositions.
  • binding affinity refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., a binding protein, such as, an antibody) and its binding partner (e.g., an antigen).
  • binding affinity refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art. For example, the affinity of an antibody for an Reference No.
  • C14935 antigen or epitope of interest can be measured using any art-recognized assay.
  • Such methods include, for example, fluorescence activated cell sorting (FACS), separable beads (e.g., magnetic beads), antigen panning, and/or ELISA (see, e.g., Janeway et al. (eds.), Immunobiology, 5th ed., Garland Publishing, New York, N.Y., 2001).
  • an “Antibody” and “antibodies” as used herein refers to monoclonal antibodies, chimeric antibodies, multispecific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies such as, but not limited to, a bird (for example, a duck or a goose), a shark, a whale, and a mammal, including a non-primate (for example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, etc.) or a non-human primate (for example, a monkey, a chimpanzee, etc.), recombinant antibodies, chimeric antibodies, single-chain Fvs (“scFv”), single chain antibodies, single domain antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragment
  • Antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, namely, molecules that contain an analyte-binding site.
  • Immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA, and IgY), class (for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.
  • An “Antibody fragment” as used herein refers to a portion of an intact antibody comprising the antigen-binding site or variable region.
  • the portion does not include the constant heavy chain domains (i.e., CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody.
  • antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing the three CDRs of the light-chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing the three CDRs of the heavy chain variable region.
  • Autoimmune disease refers to disorders associated with an undesired immune response in a subject. Autoimmune diseases fall in two broad categories: organ-specific and systemic. Autoimmune diseases include, without limitation, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), type I diabetes mellitus, type diabetes mellitus, multiple sclerosis (MS), neuromyelitis optica, immune-mediated infertility such as premature ovarian failure, scleroderma, Sjogren's disease, vitiligo, alopecia (baldness), polyglandular, Grave's disease, hypothyroidism, polymyositis, pemphigus vulgaris, pemphigus foliaceus, inflammatory bowel disease including Crohn's disease and ulcerative colitis, autoimmune hepatitis including that associated with hepatitis B virus (HBV) and hepatitis C virus
  • RA rheumatoid arthritis
  • Autoimmune diseases may also include, without limitation, Hashimoto's thyroiditis, Type I and Type II autoimmune polyglandular syndromes, paraneoplastic pemphigus, bullus pemphigoid, dermatitis herpetiformis, linear IgA disease, epidermolysis bullosa acquisita, erythema nodosa, pemphigoid gestationis, cicatricial pemphigoid, mixed essential cryoglobulinemia, chronic bullous disease of childhood, hemolytic anemia, thrombocytopenic purpura, Goodpasture's syndrome, autoimmune neutropenia, myasthenia gravis, Eaton-Lambert myasthenic syndrome, stiff-man syndrome, acute disseminated encephalomyelitis, Guillain- Barre syndrome, chronic inflammatory demyelinating polyradiculoneuropathy, multifocal motor neuropathy with conduction block, chronic neuropathy with monoclonal gammopathy,
  • a “Binding protein” as used herein refers to a monomeric or multimeric protein that binds to and forms a complex with a binding partner, such as, for example, a polypeptide, an antigen, a chemical compound or other molecule, or a substrate of any kind.
  • a binding protein specifically binds a binding partner.
  • Binding proteins include antibodies, as well as antigen- binding fragments thereof and other various forms and derivatives thereof as are known in the art and described herein below, and other molecules comprising one or more antigen-binding domains that bind to an antigen molecule or a particular site (epitope) on the antigen molecule.
  • a binding protein includes, but is not limited to, an antibody a tetrameric immunoglobulin, an IgG molecule, an IgG1 molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a human antibody, an affinity matured antibody, and fragments of any such antibodies that retain the ability to bind to an antigen.
  • a “Bispecific antibody” as used herein refers to a full-length antibody that is generated by quadroma technology (see Milstein et al., Nature, 305(5934): 537-540 (1983)), by chemical conjugation of two different monoclonal antibodies (see, Staerz et al., Nature, 314(6012): 628- 631 (1985)), or by knob-into-hole or similar approaches, which introduce mutations in the Fc region (see Holliger et al., Proc. Natl. Acad. Sci. USA, 90(14): 6444-6448 (1993)), resulting in multiple different immunoglobulin species of which only one is the functional bispecific antibody.
  • a bispecific antibody binds one antigen (or epitope) on one of its two binding arms (one pair of HC/LC), and binds a different antigen (or epitope) on its second arm (a different pair of HC/LC).
  • a bispecific antibody has two distinct antigen-binding arms (in both specificity and CDR sequences) and is monovalent for each antigen to which it binds to.
  • Bispecific antibodies can be made using the techniques as described in Brinkmann et al., MABS, 9(2):182-212 (2017), the contents of which are herein incorporated by reference.
  • a “CDR” as used herein refers to the “complementarity determining region” within an antibody variable sequence.
  • CDR1 There are three CDRs in each of the variable regions of the heavy chain and the light chain. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted "CDR1", “CDR2”, and “CDR3", for each of the variable regions.
  • CDR set refers to a group of three CDRs that occur in a single variable region that binds the antigen.
  • An antigen-binding site therefore, may include six CDRs, comprising the CDR set from each of a heavy and a light chain variable region.
  • a polypeptide comprising a single CDR, (e.g., a CDR1, CDR2, or CDR3) may be referred to as a “molecular recognition Reference No. C14935 unit”.
  • a “chimeric antibody” as used herein refers to a molecule in which different portions of the antibody are derived from different immunoglobulin molecules such as antibodies having a variable region derived from a non-human antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art.
  • Chimeric antibodies comprising one or more CDRs from a non-human species and framework Reference No. C14935 regions from a human immunoglobulin molecule can be produced using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; International Publication No. WO 91/09967; and U.S. Pat. Nos.
  • a “Derivative” as used herein refers to an antibody or antigen-binding fragment thereof that immunospecifically binds to an antigen, but which comprises, one, two, three, four, five or more amino acid substitutions, additions, deletions or modifications relative to a “parental” (or wild-type) molecule. Such amino acid substitutions or additions may introduce naturally occurring (i.e., DNA-encoded) or non-naturally occurring amino acid residues.
  • “derivative” encompasses, for example, chimeric or humanized variants of any of antibodies 1.3, 4.5 or 7.8, as well as variants having altered CH1, hinge, CH2, CH3 or CH4 regions, so as to form, for example antibodies, etc., having variant Fc regions that exhibit enhanced or impaired effector or binding characteristics.
  • “derivative” additionally encompasses non-amino acid modifications, for example, amino acids that may be glycosylated (e.g., have altered mannose, 2-N-acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-glycolneuraminic acid, etc.
  • the altered carbohydrate modifications modulate one or more of the following: solubilization of the antibody, facilitation of subcellular transport and secretion of the antibody, promotion of antibody assembly, conformational integrity, and antibody-mediated effector function.
  • the altered carbohydrate modifications enhance antibody mediated effector function relative to the antibody lacking the carbohydrate modification.
  • Carbohydrate modifications that lead to altered antibody mediated effector function are well known in the art (for example, see Shields, R. L. et al.
  • DVDs may be monospecific, i.e., capable of binding one antigen (or one specific epitope), or multispecific, i.e., capable of binding two or more antigens (i.e., two or more epitopes of the same target antigen molecule or two or more epitopes of different target antigens).
  • a preferred DVD binding protein comprises two heavy chain DVD polypeptides and two light chain DVD polypeptides and is referred to as a “DVD immunoglobulin” or “DVD-Ig.” Such a DVD-Ig binding protein is thus tetrameric and reminiscent of an IgG molecule but provides more antigen binding sites than an IgG molecule.
  • each half of a tetrameric DVD-Ig molecule is reminiscent of one half of an IgG molecule and comprises a heavy chain DVD polypeptide and a light chain DVD polypeptide, but unlike a pair of heavy and light chains of an IgG molecule that provides a single antigen binding domain, a pair of heavy and light chains of a DVD-Ig provide two or more antigen binding sites.
  • Reference No. C14935 Each antigen binding site of a DVD-Ig binding protein may be derived from a donor ("parental") monoclonal antibody and thus comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) with a total of six CDRs involved in antigen binding per antigen binding site.
  • a DVD-Ig binding protein that binds two different epitopes comprises an antigen binding site derived from a first parental monoclonal antibody and an antigen binding site of a second parental monoclonal antibody.
  • a preferred example of such DVD-Ig molecules comprises a heavy chain that comprises the structural formula VD1-(X1)n-VD2-C- (X2)n, wherein VD1 is a first heavy chain variable domain, VD2 is a second heavy chain variable domain, C is a heavy chain constant domain, X1 is a linker with the proviso that it is not CH1, X2 is an Fc region, and n is 0 or 1, but preferably 1; and a light chain that comprises the structural formula VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first light chain variable domain, VD2 is a second light chain variable domain, C is a light chain constant domain, X1 is a linker with the proviso that it is not CH1, and X2 does not comprise an Fc region; and n is 0 or 1, but preferably 1.
  • Such a DVD-Ig may comprise two such heavy chains and two such light chains, wherein each chain comprises variable domains linked in tandem without an intervening constant region between variable regions, wherein a heavy chain and a light chain associate to form tandem functional antigen binding sites, and a pair of heavy and light chains may associate with another pair of heavy and light chains to form a tetrameric binding protein with four functional antigen binding sites.
  • a DVD-Ig molecule may comprise heavy and light chains that each comprise three variable domains (VD1, VD2, VD3) linked in tandem without an intervening constant region between variable domains, wherein a pair of heavy and light chains may associate to form three antigen binding sites, and wherein a pair of heavy and light chains may associate with another pair of heavy and light chains to form a tetrameric binding protein with six antigen binding sites.
  • VD1, VD2, VD3 variable domains linked in tandem without an intervening constant region between variable domains
  • a pair of heavy and light chains may associate to form three antigen binding sites
  • a pair of heavy and light chains may associate with another pair of heavy and light chains to form a tetrameric binding protein with six antigen binding sites.
  • a DVD-Ig binding protein not only binds the same target molecules bound by its parental monoclonal antibodies, but also possesses one or more desirable properties of one or more of its parental monoclonal antibodies. Preferably, such an additional property is Reference No.
  • C14935 an antibody parameter of one or more of the parental monoclonal antibodies.
  • Antibody parameters that may be contributed to a DVD-Ig binding protein from one or more of its parental monoclonal antibodies include, but are not limited to, antigen specificity, antigen affinity, potency, biological function, epitope recognition, protein stability, protein solubility, production efficiency, immunogenicity, pharmacokinetics, bioavailability, tissue cross reactivity, and orthologous antigen binding.
  • a “Fragment antigen-binding fragment” or “Fab fragment” as used herein refers to a fragment of an antibody that binds to antigens and that contains one antigen-binding site, one complete light chain, and part of one heavy chain.
  • a Fab is a monovalent fragment consisting of the VL, VH, CL and CH1 domains.
  • a Fab is composed of one constant and one variable domain of each of the heavy and the light chain.
  • the variable domain contains the paratope (the antigen- binding site), comprising a set of complementarity determining regions, at the amino terminal end of the monomer.
  • Each arm of the Y thus binds an epitope on the antigen.
  • Fab fragments can be generated such as has been described in the art, e.g., using the enzyme papain, which can be used to cleave an immunoglobulin monomer into two Fab fragments and an Fc fragment, or can be produced by recombinant means.
  • F(ab') 2 fragment refers to antibodies generated by pepsin digestion of whole IgG antibodies to remove most of the Fc region while leaving intact some of the hinge region.
  • F(ab')2 fragments have two antigen-binding F(ab) portions linked together by disulfide bonds, and therefore are divalent with a molecular weight of about 110 kDa.
  • Divalent antibody fragments are smaller than whole IgG molecules and enable a better penetration into tissue thus facilitating better antigen recognition in immunohistochemistry.
  • the use of F(ab')2 fragments also avoids unspecific binding to Fc receptor on live cells or to Protein A/G.
  • F(ab') 2 fragments can both bind and precipitate antigens.
  • a “Framework” (FR) or “Framework sequence” as used herein may mean the remaining sequences of a variable region minus the CDRs. Because the exact definition of a CDR sequence can be determined by different systems (for example, see above), the meaning of a framework sequence is subject to correspondingly different interpretations.
  • the six CDRs also divide the framework regions on the light chain and the heavy chain into four sub-regions (FR1, FR2, FR3, and FR4) on each chain, in which CDR1 is positioned between FR1 and FR2, CDR2 between Reference No. C14935 FR2 and FR3, and CDR3 between FR3 and FR4.
  • a framework region represents the combined FRs within the variable region of a single, naturally occurring immunoglobulin chain.
  • a FR represents one of the four sub-regions, and FRs represents two or more of the four sub-regions constituting a framework region.
  • Human heavy chain and light chain FR sequences are known in the art that can be used as heavy chain and light chain "acceptor" framework sequences (or simply, "acceptor” sequences) to humanize a non-human antibody using techniques known in the art.
  • human heavy chain and light chain acceptor sequences are selected from the framework sequences listed in publicly available databases such as V-base or in the international ImMunoGeneTics® (IMGT®) information system,
  • a “Functional antigen binding site” as used herein may mean a site on a binding protein (e.g., an antibody) that is capable of binding a target antigen.
  • the antigen binding affinity of the antigen binding site may not be as strong as the parent binding protein, e.g., parent antibody, from which the antigen binding site is derived, but the ability to bind antigen must be measurable using any one of a variety of methods known for evaluating protein, e.g., antibody, binding to an antigen.
  • a “human antibody’ as used herein refers to antibodies developed in a transgenic animal (e.g., mouse or rat) that have been genetically engineered with the human immunoglobulin locus (in contrast to humanized mAbs which are initially generated in wild type animal (e.g., mouse or rat) with a native genome bearing the mouse immunoglobulin locus).
  • Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences (see U.S. Pat. Nos.
  • Human antibodies can be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.
  • the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells.
  • the human variable region, constant region, and diversity region may be introduced into mouse embryonic Reference No. C14935 stem cells in addition to the human heavy and light chain genes.
  • the mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production.
  • the modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies.
  • the transgenic mice are immunized using conventional methodologies with a selected antigen, e.g., all or a portion of any of the polypeptides of antibodies A-E, described herein.
  • Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology (see, e.g., U.S. Pat. No. 5,916,771).
  • the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation.
  • it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93, which is incorporated herein by reference in its entirety).
  • a “humanized antibody” as used herein refers to an immunoglobulin comprising a human framework region and one or more CDR's from a non-human (usually a transgenic animal such as a mouse or rat) immunoglobulin.
  • the non-human immunoglobulin providing the CDR's is called the “donor” and the human immunoglobulin providing the framework is called the “acceptor.”
  • Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, preferably about 95% or more identical.
  • all parts of a humanized immunoglobulin, except possibly the CDR's are substantially identical to corresponding parts of natural human immunoglobulin Reference No.
  • a humanized antibody is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin.
  • a humanized antibody would not encompass a typical chimeric antibody, because, e.g., the entire variable region of a chimeric antibody is non-human.
  • the donor antibody has been “humanized,” by the process of “humanization,” because the resultant humanized antibody is expected to bind to the same antigen as the donor antibody that provides the CDR's.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit, or a non-human primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit, or a non-human primate having the desired specificity, affinity, and capacity.
  • FR Framework Region residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin that immunospecifically binds to an Fc RIIB polypeptide, that has been altered by the introduction of amino acid residue substitutions, deletions, or additions (i.e., mutations).
  • Fc immunoglobulin constant region
  • the percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity.
  • the residues of the single sequence are included in the denominator but not the numerator of the calculation.
  • immunogen and “antigen” are used interchangeably herein and refer to any molecule, compound, or substance that induces an immune response in an animal (e.g., a mammal).
  • An “immune response” can entail, for example, antibody production and/or the activation of immune effector cells.
  • An antigen in the context of the disclosure can comprise any subunit, fragment, or epitope of any proteinaceous or non-proteinaceous (e.g., carbohydrate or lipid) molecule that provokes an immune response in a mammal.
  • epitope is meant a sequence of an antigen that is recognized by a binding protein, an antibody or an antigen receptor.
  • an epitope is a region of an antigen that is specifically bound by an antibody.
  • an epitope may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups.
  • an epitope may have specific three-dimensional structural characteristics (e.g., a “conformational” epitope) and/or specific charge characteristics.
  • the antigen can be a protein or peptide of viral, bacterial, parasitic, fungal, protozoan, prion, cellular, or extracellular origin, which provokes an immune response in a mammal, preferably leading to protective immunity.
  • An “Isolated polynucleotide” as used herein may mean a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or a combination thereof) that, by virtue of its origin, the isolated polynucleotide is not associated with all or a portion of a polynucleotide with which the “isolated polynucleotide” is found in nature; is operably linked to a polynucleotide that it is not linked to in nature; or does not occur in nature as part of a larger sequence.
  • a “Monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen (e.g., although cross-reactivity or shared reactivity may occur). Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological.
  • Methods for producing monoclonal antibodies are well known in the art.
  • monoclonal antibodies can be produced by: (1) fusion of sensitized lymphocytes and myelomas from different sources to produce continuous antibody-producing cell lines; (2) in vitro viral transformation of sensitized lymphocytes to form continuous antibody-producing cells; or (3) hybrid fusion of sensitized lymphocytes and continuous B lymphocyte cell lines (R.K Nakamura, “Monoclonal Antibodies: Methods and Clinical Lab Applications”, Clin. Physiol. Biochem, 1(2-5):160-72 (1983)).
  • a multivalent binding protein is preferably engineered to have three or more antigen binding sites and is generally not a naturally occurring antibody.
  • multispecific binding protein refers to a binding protein that can bind two or more related or unrelated targets, including a binding protein capable of binding two or more different epitopes of the same target molecule.
  • a “peptide” or “polypeptide” as used herein refers to a linked sequence of two or more amino acids linked by peptide bonds. Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies.
  • the terms “polypeptide” and “protein” are used interchangeably herein. Reference No.
  • a “Recombinant antibody” and “recombinant antibodies” as used herein refers to antibodies prepared by one or more steps, including cloning nucleic acid sequences encoding all or a part of one or more monoclonal antibodies into an appropriate expression vector by recombinant techniques and subsequently expressing the antibody in an appropriate host cell.
  • the terms include, but are not limited to, recombinantly produced monoclonal antibodies, chimeric antibodies, humanized antibodies (fully or partially humanized), multi-specific or multi-valent structures formed from antibody fragments, bifunctional antibodies, heteroconjugate Abs, DVD-Ig®s, and other antibodies as described in herein (Dual-variable domain immunoglobulins and methods for making them are described in Wu, C., et al., Nature Biotechnology, 25:1290-1297 (2007)).
  • an antibody or other entity e.g., antigen binding domain
  • an antibody or other entity e.g., antigen binding domain
  • affinity which is substantially higher means affinity that is high enough to enable detection of an antigen or epitope which is distinguished from entities using a desired assay or measurement apparatus.
  • binding affinity having a binding constant (Ka) of at least 10 7 M -1 (e.g., >10 7 M -1 , >10 8 M -1 , >10 9 M -1 , >10 10 M -1 , >10 11 M- 1, >10 12 M -1 , >10 13 M -1 , etc.).
  • Ka binding constant
  • an antibody is capable of binding different antigens so long as the different antigens comprise that particular epitope.
  • homologous proteins from different species may comprise the same epitope.
  • a “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as Reference No.
  • patient may include either adults or juveniles (e.g., children).
  • patient may mean any living organism, preferably a mammal (e.g., human or non- human) that may benefit from the administration of compositions contemplated herein.
  • mammals include, but are not limited to, any member of the Mammalian class: humans, non- human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like.
  • non- mammals examples include, but are not limited to, birds, fish, and the like.
  • the mammal is a human.
  • “treat,” “treating,” and the like means a slowing, stopping, or reversing of progression of a disease or disorder when provided a peptide or composition described herein to an appropriate subject. The term also includes a reversing of the progression of such a disease or disorder to a point of eliminating or greatly reducing the disease.
  • treating means an application or administration of the peptides or compositions described herein to a subject, where the subject has a disease or a symptom of a disease, where the purpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease or symptoms of the disease.
  • a “Variant” as used herein refers to and/or describes a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity.
  • biological activity include the ability to be bound by a specific antibody or to promote an immune response.
  • Variant is also used herein to describe a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity.
  • a conservative substitution of an amino acid i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree, and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge.
  • Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. Substitutions may be performed with amino acids having hydrophilicity values within ⁇ 2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.
  • the present invention relates to one or more monoclonal antibodies that can be used to enhance or increase PAD4 in a subject (e.g., a human, a mouse, a rat, a rabbit, a dog, a cat, etc.).
  • a subject e.g., a human, a mouse, a rat, a rabbit, a dog, a cat, etc.
  • the monoclonal antibodies are human monoclonal antibodies, Reference No.
  • EIVMTQSPSSLSASVGDRVTITCRASQGIRNELGWYQQKPGKAPRLLIYAASSL QSGVPSRFSGSGSGTDFTLTIGSLQPEDFATYYCLQDNNYPRTFGQGTKVDIK (SEQ ID NO:2).
  • Clone 10 (Antibody B) [0084] 10 heavy chain [0085] EVQLVQSGAEVKKPGSSVKVSCLASGGTFNNYAVSWVRQAPGQGLEWMGGI IPMFDIAKYPQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCATSYFRQWLTWDWG QGTLVTVSSA (SEQ ID NO:3).
  • Clone 104 (Antibody E) [0099] 104 heavy chain [00100] EVQLVESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYS GSTNYNPSLKSRVSISVDTSKNQFSLRLSSVTAADTAVYYCAGHFRSSSSAKEYFHHWG QGTLVTVSSA (SEQ ID NO:9).
  • the present invention relates to monoclonal antibodies having the below SEQ ID NOS. as well as any antigen binding fragments thereof comprising: [00110] A) a heavy variable chain comprising SEQ ID NO:1 and a light variable chain comprising SEQ ID NO:2; [00111] B) a heavy variable chain comprising SEQ ID NO:3 and a light variable chain comprising SEQ ID NO:4; [00112] C) a heavy variable chain comprising SEQ ID NO:5 and a light variable chain comprising SEQ ID NO:6; [00113] D) a heavy variable chain comprising SEQ ID NO:7 and a light variable chain comprising SEQ ID NO:8; or [00114] E) a heavy variable chain comprising SEQ ID NO:9 and a light variable chain comprising SEQ ID NO:10.
  • the monoclonal antibodies of the present disclosure are a human antibody, a humanized antibody, a chimeric antibody or any derivative or variant thereof.
  • the monoclonal antibodies are bispecific antibodies or any derivative or variant thereof.
  • the antibody is a fragment selected from the group consisting of Fab, Fab-C, Fab’-SH, Fv, scFv, and (Fab’)2 fragments.
  • the monoclonal antibodies of the present disclosure are human IgG antibodies.
  • the antibodies of the present disclosure may be produced by any method known in the art useful for the production of polypeptides, e.g., in vitro synthesis, recombinant DNA production, and the like.
  • any of antibodies A-E can be produced by recombinant DNA technology.
  • any of antibodies A-E may be produced using recombinant Reference No. C14935 immunoglobulin expression technology.
  • the recombinant production of immunoglobulin molecules, including humanized antibodies are described in U.S. Pat. No. 4,816,397, U.S. Pat. Nos. 6,331,415 and 4,816,567, U.K. patent GB 2,188,638, and U.K. patent GB 2,209,757.
  • an exemplary process for the production of the recombinant chimeric antibodies for any of antibodies A-E can include the following: a) constructing, by conventional molecular biology methods, an expression vector that encodes and expresses an antibody heavy chain in which the CDRs and variable region of any of monoclonal antibodies A-E are fused to an Fc region derived from a human immunoglobulin, thereby producing a vector for the expression of a chimeric antibody heavy chain; b) constructing, by conventional molecular biology methods, an expression vector that encodes and expresses an antibody light chain of any of monoclonal antibodies A-E, thereby producing a vector for the expression of chimeric antibody light chain; c) transferring the expression vectors to a host cell by conventional molecular biology methods to produce a transfected host cell for the expression of chimeric antibodies; and d) culturing the transfected cell by conventional cell culture techniques so as to produce chimeric antibodies.
  • Recombinant humanized antibodies of any of antibodies A-E can also be produced using techniques known in the art.
  • an exemplary process for the production of recombinant humanized antibodies of any of antibodies A-E can include the following: a) constructing, by conventional molecular biology methods, an expression vector that encodes and expresses a heavy chain of any of antibodies A-E in which the CDRs and a minimal portion of the variable region framework that are required to retain donor antibody binding specificity are derived from a non-human immunoglobulin, such as any of monoclonal antibodies A-E, and the remainder of the antibody is derived from a human immunoglobulin, thereby producing a vector for the expression of a humanized antibody heavy chain; b) Reference No.
  • C14935 constructing, by conventional molecular biology methods, an expression vector that encodes and expresses an antibody light chain in which the CDRs and a minimal portion of the variable region framework that are required to retain donor antibody binding specificity are derived from a non-human immunoglobulin, such as any of monoclonal antibodies A-E, and the remainder of the antibody is derived from a human immunoglobulin, thereby producing a vector for the expression of humanized antibody light chain; c) transferring the expression vectors to a host cell by conventional molecular biology methods to produce a transfected host cell for the expression of humanized antibodies; and d) culturing the transfected cell by conventional cell culture techniques so as to produce humanized antibodies.
  • a non-human immunoglobulin such as any of monoclonal antibodies A-E
  • host cells may be co- transfected with such expression vectors, which may contain different selectable markers but, with the exception of the heavy and light chain coding sequences, are preferably identical.
  • This procedure provides for equal expression of heavy and light chain polypeptides.
  • a single vector may be used which encodes both heavy and light chain polypeptides.
  • the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA or both.
  • the host cell used to express any of the recombinant monoclonal antibodies A-E can be either a bacterial cell such as Escherichia coli, or more preferably a eukaryotic cell (e.g., a Chinese hamster ovary (CHO) cell or a HEK-293 cell).
  • a eukaryotic cell e.g., a Chinese hamster ovary (CHO) cell or a HEK-293 cell.
  • the choice of expression vector is dependent upon the choice of host cell, and may be selected so as to have the desired expression and regulatory characteristics in the selected host cell.
  • Other cell lines that may be used include, but are not limited to, CHO-K1, NSO, and PER.C6 (Crucell, Leiden, Netherlands).
  • any of the above-described antibodies can be used to generate antiidiotype antibodies using techniques well known to those skilled in the art (see, e.g., Greenspan, N. S. et al. (1989) “Idiotypes: Structure and Immunogenicity,” FASEB J. 7:437-444; and Nisinoff, A. (1991) “Idiotypes: Concepts and Applications,” J. Immunol. 147(8):2429-2438).
  • the binding properties of any of the above antibodies can, if desired, be further improved by screening for variants that exhibit such desired characteristics. For example, such antibodies can be generated using various phage display methods known in the art.
  • phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
  • phage can be utilized to display antigen binding domains, such as Fab and Fv or disulfide-bond Reference No. C14935 stabilized Fv, expressed from a repertoire or combinatorial antibody library (e.g., human or murine).
  • Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage, including fd and M13.
  • the antigen binding domains are expressed as a recombinantly fused protein to either the phage gene III or gene VIII protein.
  • phage display methods that can be used to make the immunoglobulins, or fragments thereof, of the present disclosure include those disclosed in Brinkman, U. et al. (1995) “Phage Display Of Disulfide-Stabilized Fv Fragments,” J. Immunol. Methods, 182:41-50, 1995; Ames, R. S. et al. (1995) “Conversion Of Murine Fabs Isolated From A Combinatorial Phage Display Library To Full Length Immunoglobulins,” J. Immunol. Methods, 184:177-186; Kettleborough, C. A.
  • the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including humanized antibodies, or any other desired fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below.
  • techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art (such as those disclosed in PCT Publication WO 92/22324; Mullinax, R. L. et al.
  • Phage display technology can be used to increase the affinity of any of antibodies A-E. This technique would be useful in obtaining high affinity antibodies that could be used in the disclosed combinatorial methods.
  • This technology referred to as affinity maturation, employs mutagenesis or CDR walking and re-selection using such receptors or ligands (or their extracellular domains) or an antigenic fragment thereof to identify antibodies that bind with higher affinity to the antigen when compared with the initial or parental antibody (See, e.g., Glaser, S. M. et al. (1992) “Antibody Engineering By Codon-Based Mutagenesis In A Filamentous Phage Vector System,” J. Immunol. 149:3903-3913).
  • Libraries can be constructed consisting of a pool of variant clones each of which differs by a single amino acid alteration in a single CDR and which contain variants representing each possible amino acid substitution for each CDR residue.
  • Mutants with increased binding affinity for the antigen can be screened by contacting the immobilized mutants with labeled antigen. Any screening method known in the art can be used to identify mutant antibodies with increased avidity to the antigen (e.g., ELISA) (see, e.g., Wu, H. et al.
  • affinity maturation employs mutagenesis or “CDR walking” and re-selection uses the target antigen or an antigenic fragment thereof to identify antibodies having CDRs that bind with higher (or lower) affinity to the antigen when compared with the initial or parental antibody (see, e.g., Glaser, S. M. et al. (1992) “Antibody Engineering By Codon-Based Mutagenesis In A Filamentous Phage Vector System,” J. Immunol. 149:3903- 3913). Mutagenizing entire codons rather than single nucleotides results in a semi-randomized repertoire of amino acid mutations.
  • Libraries can be constructed consisting of a pool of variant clones each of which differs by a single amino acid alteration in a single CDR and which contain variants representing each possible amino acid substitution for each CDR residue.
  • Mutants with increased (or decreased) binding affinity for the antigen can be screened by contacting the immobilized mutants with labeled antigen. Any screening method known in the art can be used to identify mutant antibodies with increased (or decreased) avidity to the antigen (e.g., ELISA) (see, Wu, H. et al. (1998) “Stepwise In Vitro Affinity Maturation Of Vitaxin, An Alphav Beta3- Specific Humanized Mab,” Proc. Natl. Acad. Sci.
  • a humanized antibody is a derivative.
  • Such a humanized antibody comprises amino acid residue substitutions, deletions or additions in one or more non-human CDRs.
  • the humanized antibody derivative may have substantially the same binding, better binding, or worse binding when compared to a non-derivative humanized antibody.
  • one, two, three, four, or five amino acid residues of the CDR have been substituted, deleted or added (i.e., mutated).
  • a derivative antibody or antibody fragment may be modified by chemical modifications using techniques known to those of skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc.
  • an antibody derivative will possess a similar or identical function as the parental antibody.
  • an antibody derivative will exhibit an altered activity relative to the parental antibody.
  • a derivative antibody (or fragment thereof) can bind to its epitope more tightly or be more resistant to proteolysis than the parental antibody.
  • Derivatized antibodies may be used to alter the half-lives (e.g., serum half-lives) of parental antibodies in a mammal, preferably a human. Preferably such alteration will result in a half-life of greater than 15 days, preferably greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months.
  • the increased half-lives of the humanized antibodies of the present disclosure or fragments thereof in a mammal, preferably a human results in a higher serum titer of said antibodies or antibody fragments in the mammal, and thus, reduces the frequency of the administration of said Reference No.
  • Antibodies or fragments thereof having increased in vivo half- lives can be generated by techniques known to those of skill in the art. For example, antibodies or fragments thereof with increased in vivo half-lives can be generated by modifying (e.g., substituting, deleting or adding) amino acid residues identified as involved in the interaction between the Fc domain and the FcRn receptor.
  • the humanized antibodies of any of antibodies A- E can be engineered to increase biological half-lives (see, e.g. U.S. Pat. No. 6,277,375).
  • Antibodies or fragments thereof with increased in vivo half-lives can be generated by attaching to said antibodies or antibody fragments polymer molecules such as high molecular weight polyethyleneglycol (PEG).
  • PEG polymer molecules
  • PEG can be attached to said antibodies or antibody fragments with or without a multifunctional linker either through site-specific conjugation of the PEG to the N- or C-terminus of said antibodies or antibody fragments or via epsilon-amino groups present on lysine residues.
  • Linear or branched polymer derivatization that results in minimal loss of biological activity will be used.
  • the degree of conjugation will be closely monitored by SDS- PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibodies.
  • Unreacted PEG can be separated from antibody-PEG conjugates by, e.g., size exclusion or ion- exchange chromatography.
  • bispecific antibodies can be made using the techniques as described in Brinkmann et al., MABS, 9(2):182-212 (2017), the contents of which are herein incorporated by reference.
  • the present invention comprises a monoclonal antibody comprising:’ [00131] A) a heavy variable chain comprising SEQ ID NO:1 and a light variable chain comprising SEQ ID NO:2; [00132] B) a heavy variable chain comprising SEQ ID NO:3 and a light variable chain comprising SEQ ID NO:4; [00133] C) a heavy variable chain comprising SEQ ID NO:5 and a light variable chain comprising SEQ ID NO:6; [00134] D) a heavy variable chain comprising SEQ ID NO:7 and a light variable chain comprising SEQ ID NO:8; or [00135] E) a heavy variable chain comprising SEQ ID NO:9 and a light variable chain comprising SEQ ID NO:10 and at least one excipient or pharmaceutically acceptable carrier.
  • the present disclosure relates to monoclonal antibodies having the below SEQ ID NOS. as well as any antigen binding fragments thereof comprising: [00137] A) a heavy variable chain having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identity to SEQ ID NO:1 and a light variable chain having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identity to SEQ ID NO:2; [00138] B) a heavy variable chain having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identity to SEQ ID NO:3 and a light variable chain having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or
  • compositions of the present invention will be readily apparent to those skilled in the art. Techniques and formulations may be found, for example, in Remington’s Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).
  • pharmaceutically acceptable carrier means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material, surfactant, cyclodextrins or Reference No. C14935 formulation auxiliary of any type.
  • materials which can serve as pharmaceutically acceptable carriers are sugars such as, but not limited to, lactose, glucose and sucrose; starches such as, but not limited to, corn starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as, but not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; surfactants such as, but not limited to, cremophor EL, cremophor RH 60, Solutol HS 15 and polysorbate 80; cyclodextrins such as, but not limited to, alpha-CD, beta-CD, gamma-CD, HP-beta-CD, SBE-beta-CD;
  • compositions may be in a variety of forms, suitable, for example, for systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implants, or parenteral injections) or topical administration (e.g., dermal, pulmonary, nasal, aural, ocular, liposome delivery systems, or iontophoresis).
  • systemic administration e.g., oral, rectal, nasal, sublingual, buccal, implants, or parenteral injections
  • topical administration e.g., dermal, pulmonary, nasal, aural, ocular, liposome delivery systems, or iontophoresis
  • the composition may be inhaled.
  • Methods of Use Also disclosed herein are methods of using the disclosed monoclonal antibodies and compositions.
  • the monoclonal antibodies described herein can be used to screen and/or develop new therapies or therapeutic agents that inhibit PAD4.
  • the monoclonal antibodies herein can be used to develop animal models, such as, for example, mouse, rat, rabbit, goat, dog, cat, etc. More specifically, the antibodies of Reference No. C14935 the present disclosure can be administered to an animal to increase the amount or level of PAD4 in the animal. Once the levels of PAD4 in the animal are increased, the animal can be administered one more therapeutic agents to screen for new therapies.
  • kits comprising one or more of the antibodies (e.g., compositions) as disclosed herein.
  • kits can also comprise other agents (e.g., pharmaceutical agents) and/or products co-packaged, co-formulated, and/or co-delivered with other components.
  • the kits can also comprise instructions for using the components of the kit.
  • the instructions are relevant materials or methodologies pertaining to the kit. The materials may include any combination of the following: background information, list of components, brief or detailed protocols for using the monoclonal antibodies, troubleshooting, references, technical support, and any other related documents. Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation. [00148] It is understood that the disclosed kits can be employed in connection with the disclosed methods.
  • kits may further contain containers or devices for use with the methods disclosed herein.
  • the kits can comprise delivery devices (e.g., syringes).
  • delivery devices e.g., syringes.
  • the kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Individual member components of the kits may be physically packaged together or separately.
  • the present disclosure has multiple aspects, illustrated by the following non-limiting examples. EXAMPLES Example 1: Preparation of Antibodies Reference No. C14935 [00151] PADs cloning and expression. Plasmids encoding human PAD2, PAD3, PAD4 and P. gingivalis PAD were previously described 34,35 .
  • DNA sequences encoding specific PAD4 domains were generated by PCR using PAD4 plasmid as template and then cloned into pcDNA3.1 (Invitrogen). Plasmids encoding N-terminal His 6 -T7-tagged PAD4 calcium-binding site mutants were generated by site-directed mutagenesis using PAD4 cloned into pET28a(+) (Novagen) as template. cDNA for the monomeric green fluorescent protein mWasabi (Allele Biotech) was cloned in frame with PAD4 into pET28a(+), generating a N-terminal His 6 -T7- mWasabi-tagged PAD4 fusion protein.
  • Plasmids were used to generate [ 35 S]methionine-labeled proteins by in vitro coupled transcription/translation (IVTT) (Promega). N-terminal His6-T7- tagged proteins were expressed in BL21-Gold (DE3) cells (Agilent) and purified using Ni-NTA agarose (Qiagen). [00152] Single anti-PAD4 memory B cell sorting.
  • B cells were enriched from whole blood using RosetteSep human B cell enrichment cocktail (Stemcell Technologies) according to the manufacturer’s instructions and further stained with LIVE/DEAD Fixable Blue Dead Cell Stain Kit (Thermo Fisher Scientific), PE-CF594 mouse anti-human CD19, BV421 mouse anti-human CD20, BV605 mouse anti-human CD14, APC-H7 mouse anti-human CD27, PE-CyTM7 mouse anti-human CD38, APC mouse anti-human IgM, BV510 mouse anti-human CD3 (BD Biosciences) and mWasabi-PAD4.
  • Single cells CD3-CD14-CD19 + CD20 + CD27 + CD38-IgM- with high binding to mWasabi-PAD4 were sorted into 96-well PCR plates containing 10 ⁇ l/well of 10 mM Tris pH 8.0 and 8 U RNasin Ribonuclease Inhibitor (Promega).
  • Cloning and Ig sequence analysis Single cell cDNA was generated using SuperScript VILO Master Mix (Thermo Fisher Scientific). IgH and IgL ( ⁇ or ⁇ ) variable regions were amplified using the cDNA by nested PCR, using previously described primers and PCR conditions.
  • IVTT-radiolabelled proteins were diluted in IP buffer (1%NP-40/PBS) and incubated with 3.4 or 34 nM of purified monoclonal antibody as indicated in the figure legends. After 1 hr at room temperature (RT), immune complexes were purified using Protein A agarose beads (Pierce). Samples were washed in IP buffer and immunoprecipitates visualized by SDS-PAGE/sodium salicylate autoradiography. [00157] Citrullination assay.
  • Monoclonal antibodies (0.14 ⁇ M, 0.46 ⁇ M or 1.4 ⁇ M), 0.09 ⁇ M PAD4 (wild type or mutants) and 2.2 ⁇ M histone H3.1 (New England Biolabs) were mixed in 100 mM Tris pH 7.5/1 mM dithiothreitol (DTT) and increasing amounts of calcium. After incubation for 2 hrs at 37 o C, the mixtures were boiled in SDS sample buffer, separated by electrophoresis, transferred to nitrocellulose, stained with Ponceau S and analyzed by immunoblotting with antibodies to citrullinated histone H3 (citrulline R2 + R8 + R17, Abcam) and PAD4 (Sigma).
  • Antibodies were screened for reactivity against previously defined polyreactive antigens, including ssDNA, dsDNA, cardiolipin, insulin and LPS.
  • ssDNA or dsDNA Sigma at 5 ⁇ g/mL in 10 mM 1- methylimidazole/50 mM N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide were coated on covalink plates (ThermoFisher) for 5 hrs at 50°C.
  • Monoclonal antibodies or SLE serum were diluted in 1% BSA/PBS/0.05% tween and assayed in duplicate.
  • HRP- conjugated anti-human IgG at 100 ng/mL was used as a secondary antibody.
  • the reactivity of Reference No. C14935 anti-PAD4 antibodies to citrullinated antigen was determined using the QUANTA Lite® CCP3 IgG assay (Inova Diagnostics).
  • Anti-PAD4 ELISA assay Maxisorp plates (ThermoFisher) were coated with 66 nM human recombinant PAD4 in 0.1 M carbonate buffer pH 9.6. Plates were blocked with 3% BSA/PBS.
  • Antibodies were diluted in 1% BSA/PBS/0.05% tween and assayed in duplicate. After antibody incubation for 1 hr, the plate was washed and HRP-conjugated anti-human IgG or HRP-conjugated anti-mouse IgG at 100 ng/mL was used as a secondary antibody. The EC 50 values were calculated from linear regressions. The average EC50 was determined from two independent experiments. [00160] Cross-competition ELISA. Human anti-PAD4 monoclonal antibodies were murinized by cloning the variable region into the pFUSE-mIgG2a-Fc vector (InvivoGen).
  • Human recombinant PAD4 was coated on ELISA plates at 66 nM in 0.1 M carbonate buffer pH 9.6 and the plates blocked with 3% BSA in PBS. After washing, the plates were incubated in the absence or presence of human anti-PAD4 monoclonal antibodies (competing antibody, from 0.25 nM to 548 nM) in 1% BSA/PBS/0.05% tween. After 1 hour at RT, murinized antibodies (5.5 nM) were directly added into the wells and the plate was further incubated for 1 hour at RT. After washing, HRP-conjugated anti-mouse IgG was used to detect murinized antibodies.
  • Example 2 Use of PAD4-tagged with the monomeric fluorescent protein mWasabi and single cell cloning technology to produce monoclonal antibodies. Materials and Methods [00161] Patients. All samples were collected after obtaining signed informed consent in accordance with the Johns Hopkins Institutional Review Board. Patients with RA were recruited from the Johns Hopkins Arthritis Center and screened for anti-PAD4 and anti-PAD3/4XR antibodies in serum by immunoprecipitation (IP) as previously described.
  • IP immunoprecipitation
  • Peripheral blood anti-PAD4 single memory B cells were purified from three patients who had with high titer anti- PAD4 and anti-PAD3/4XR antibodies were used to generate monoclonal antibodies as per Example 1.
  • Results Reference No. C14935 [00162] Sequence analysis of anti-PAD4 antibodies cloned from single memory B cells.
  • Single anti-PAD4 memory B cells were isolated by flow cytometry using recombinant PAD4 tagged with the monomeric fluorescent protein mWasabi (FIG. 7). In total, 44 single memory B cells with high binding to mWasabi-PAD4 were collected from three patients with RA.
  • Heavy chain (IgH) and corresponding light chain (IgL) variable gene sequences were amplified from 32 cells and used to generate IgG1 monoclonal antibodies.
  • the antibodies were screened by IP (Fig. 1A), identifying five monoclonal antibodies with specificity to PAD4. Clones 10, 16 and 17 derived from patient #1 were cross-reactive with PAD3, while clones 102 and 104 from patient #3 only recognized PAD4 (anti-PAD4-only antibodies). No antibodies to PAD4 were isolated from patient #2. None of the anti-PAD4 antibodies recognized PAD2 or the PAD from Porphyromonas gingivalis (PPAD) (Fig.1B).
  • PPAD Porphyromonas gingivalis
  • both mutated and reverted anti-PAD4 antibodies showed no polyreactivity to self-antigens (ssDNA, dsDNA, insulin and cardiolipin) or LPS (FIG. 3A).
  • the monoclonal antibodies showed no reactivity to cyclic citrullinated peptides (CCPs) (FIG. 3B), confirming that they were developed independently of ACPAs.
  • CCPs cyclic citrullinated peptides
  • PAD4 consists of a C-terminal domain and an N- terminal domain that is divided into subdomains 1 and 2 (FIG. 11A) 26 . Under standard conditions used to IP PAD4, none of the antibodies showed binding to any domain (FIG. 11B).
  • PAD4 has 5 calcium-binding sites designated Ca1-Ca5. Ca1 and Ca2 in the C-terminal domain are required for catalysis, while Ca3-Ca5 are likely relevant to regulate enzyme activity 26 . Aspartic acid residues that coordinate calcium-binding at Ca3 and Ca5 (D 165 ), Ca3 and Ca4 (D 179 ), only Ca4 (D 388 ) and only Ca5 (D 168 ) were changed to alanine. 27 Then, the PAD4 mutants were used to test the functional activity of the antibodies. None of the PAD4 mutants were active in the absence of anti-PAD4 antibodies (FIG. 5).
  • Ca5 is positioned near the surface of subdomain 2 at the interface between the N- and C-terminal domains in a region shown to be important for full activation of the related enzyme PAD2 26,33 . This location may allow Ca5 to be accessible to functional autoantibody binding and potentially to other regulatory molecules. Although pharmacologic PAD inhibition has been focused on blocking the catalytic site in the enzyme, the finding that autoantibodies can modulate PAD4 activity by regulating Ca5 offers an additional target for inhibitor development. Molecules designed to interact with Ca5 could potentially block PAD4 hyperactivation with a limited effect on physiologic functions that require lower enzyme activity.
  • Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis- specific autoantibodies. J Clin Invest 1998;101:273-81. 11. Corsiero E, Bombardieri M, Carlotti E et al. Single cell cloning and recombinant monoclonal antibodies generation from RA synovial B cells reveal frequent targeting of citrullinated histones of NETs. Ann Rheum Dis 2016;75:1866-75. 12. Li S, Yu Y, Yue Y et al. Autoantibodies From Single Circulating Plasmablasts React With Citrullinated Antigens and Porphyromonas gingivalis in Rheumatoid Arthritis.

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Abstract

L'invention concerne des anticorps et des compositions comprenant des anticorps monoclonaux qui peuvent être utilisés pour améliorer l'activité de PAD4 chez un sujet.
PCT/US2024/027401 2023-05-05 2024-05-02 Anticorps monoclonaux humains qui améliorent pad4 pour une utilisation dans des maladies auto-immunes Pending WO2024233251A2 (fr)

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