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WO2025117765A1 - Protective monoclonal antibodies to pseudomonas aeruginosa - Google Patents

Protective monoclonal antibodies to pseudomonas aeruginosa Download PDF

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Publication number
WO2025117765A1
WO2025117765A1 PCT/US2024/057782 US2024057782W WO2025117765A1 WO 2025117765 A1 WO2025117765 A1 WO 2025117765A1 US 2024057782 W US2024057782 W US 2024057782W WO 2025117765 A1 WO2025117765 A1 WO 2025117765A1
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WIPO (PCT)
Prior art keywords
seq
antibody
binding
binding protein
amino acid
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PCT/US2024/057782
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French (fr)
Inventor
Marion PEPPER PEW
Kennidy TAKEHARA
Christopher THOUVENEL
David J. Rawlings
Malika Rose HALE
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University of Washington
Seattle Childrens Hospital
Original Assignee
University of Washington
Seattle Childrens Hospital
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Publication of WO2025117765A1 publication Critical patent/WO2025117765A1/en
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1214—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Pseudomonadaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • 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

  • Pseudomonas aeruginosa is a ubiquitous, gram-negative bacteria responsible for significant morbidity and mortality in vulnerable individuals. Treatment is challenging because of intrinsic and acquired antibiotic resistance to most antibiotic drug classes. PA is one of the most common pathogens in severe healthcare associated infections. Due to the significant mortality caused by multi-drug-resistant strains and the lack of alternative therapies, new anti-pseudomonal therapeutics are urgently needed.
  • the present disclosure provides PA neutralizing binding proteins or antibodies, fragments thereof, or derivatives thereof.
  • the binding proteins of the present disclosure are specific for and bind to a wide range of epitopes present on the PA virulence factor, P. aeruginosa V-antigen (PcrV) antigen.
  • PcrV P. aeruginosa V-antigen
  • the binding proteins and/or antibodies and/or derivatives thereof disclosed herein exhibit protective, neutralizing activity against P. aeruginosa.
  • the binding proteins disclosed herein have a high affinity to a range of PcrV epitopes.
  • the binding proteins disclosed herein are effective in limiting a PA infection.
  • the present disclosure also provides variants of antibodies or binding proteins described herein. Variants can include those having one or more conservative amino acid substitutions or one or more non-conservative substitutions that do not adversely affect the binding of the antibody and/or binding protein with the PcrV antigen.
  • the present disclosure provides a binding protein comprising: a heavy chain variable domain (VH), the VH comprising a complementarity determining region (CDR), CDR3 amino acid sequence; and a human light chain variable domain (VL) comprising a CDR3 amino acid sequence.
  • VH heavy chain variable domain
  • CDR complementarity determining region
  • VL human light chain variable domain
  • the heavy chain variable domain (VH) CDR3 amino acid sequence is at least 85% identical to an amino acid sequence set forth in SEQ ID NO: 4, 11, 18, 25, 32, 39, 46, 53, 60, 67, 74, 81, 88, 95, 102, 109, 116, 123, 130, 137, 144, 151, 159, 167, 174, 181, 188, 195, 202, 209, 216, 223, 230, 237, 244, 251, 258, 265, 272, 279,
  • the human light chain variable domain (VL) CDR3 amino acid sequence is at least 85% identical to an amino acid sequence set forth in SEQ ID NO: 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 105, 112, 119, 126, 133, 140, 147, 155, 163, 170, 177, 184, 191, 198, 205, 212, 219, 226, 233, 240, 247, 254, 261, 268,
  • the binding protein is capable of specifically binding to an epitope on Pseudomonas aeruginosa V-antigen (PcrV).
  • the heavy chain variable domain comprises a CDR3 amino acid sequence of SEQ ID NO: 4, 11, 18, 25, 32, 39, 46, 53, 60, 67, 74, 81, 88, 95, 102, 109, 116, 123, 130, 137, 144, 151 , 159, 167, 174, 181, 188, 195, 202, 209, 216, 223, 230, 237, 244, 251, 258, 265, 272, 279, 286, 293, 300, 307, 314, 321, 328, 335, 342, 349, 356, 363, 370, 377, 385, 391, 398, 405, 412, 419, 426, 433,440, 447, 454, 461, 468, 475, 482, 489, 496, 503, 510, 517, 524, 531, 538, 545, 552, 559, 566, 573, 580, 587, 594,
  • the human light chain variable domain comprises a CDR3 amino acid sequence of SEQ ID NO: 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 105, 112, 119, 126, 133, 140, 147, 155, 163, 170, 177, 184, 191, 198, 205, 212, 219, 226, 233, 240, 247, 254, 261, 268, 275, 282, 289, 296, 303, 310, 317, 324,
  • the binding protein is capable of specifically binding to an epitope on Pseudomonas aeruginosa V-antigen (PcrV).
  • the VH has at least 85% sequence identity to a sequence set forth in an (a) in a pair below and the VL has at least 85% sequence identity to a sequence set forth in (b) of the same pair.
  • the pairs have the sequences set forth in:
  • SEQ ID NO: 259 (a) SEQ ID NO: 262 and (b) SEQ ID NO: 266; (a) SEQ ID NO: 269 and (b) SEQ ID NO: 273; (a) SEQ ID NO: 276 and (b) SEQ ID NO: 280; (a) SEQ ID NO: 283 and (b) SEQ ID NO: 287; (a) SEQ ID NO: 290 and (b) SEQ ID NO: 294; (a) SEQ ID NO: 297 and (b) SEQ ID NO: 301 ; (a) SEQ ID NO: 304 and (b) SEQ ID NO: 308; (a) SEQ ID NO: 311 and (b) SEQ ID NO: 315; (a) SEQ ID NO: 318 and (b) SEQ ID NO: 322; (a) SEQ ID NO: 325 and (b) SEQ ID NO: 329; (a) SEQ ID NO: 332 and (b) SEQ ID NO: 336;
  • SEQ ID NO: 406 (a) SEQ ID NO: 409 and (b) SEQ ID NO: 413; (a) SEQ ID NO: 416 and (b) SEQ ID NO: 420; (a) SEQ ID NO: 423 and (b) SEQ ID NO: 427; (a) SEQ ID NO: 430 and (b) SEQ ID NO: 434; (a) SEQ ID NO: 437 and (b) SEQ ID NO: 441; (a) SEQ ID NO: 444 and (b) SEQ ID NO: 448; (a) SEQ ID NO: 451 and (b) SEQ ID NO: 455; (a) SEQ ID NO: 458 and (b) SEQ ID NO: 462; (a) SEQ ID NO: 465 and (b) SEQ ID NO: 469; (a) SEQ ID NO: 472 and (b) SEQ ID NO: 476; (a) SEQ ID NO: 479 and (b) SEQ ID NO: 483;
  • SEQ ID NO: 714 (a) SEQ ID NO: 717 and (b) SEQ ID NO: 721; (a) SEQ ID NO: 724 and (b) SEQ ID NO: 728; (a) SEQ ID NO: 731 and (b) SEQ ID NO: 735; (a) SEQ ID NO: 738 and (b) SEQ ID NO: 742; (a) SEQ ID NO: 745 and (b) SEQ ID NO: 749; (a) SEQ ID NO: 752 and (b) SEQ ID NO: 756; (a) SEQ ID NO: 760 and (b) SEQ ID NO: 764; (a) SEQ ID NO: 767 and (b) SEQ ID NO: 771; (a) SEQ ID NO: 774 and (b) SEQ ID NO: 778; (a) SEQ ID NO: 781 and (b) SEQ ID NO: 785; (a) SEQ ID NO: 788 and (b) SEQ ID NO: 792;
  • SEQ ID NO: 862 (a) SEQ ID NO: 865 and (b) SEQ ID NO: 869; (a) SEQ ID NO: 872 and (b) SEQ ID NO: 876; (a) SEQ ID NO: 880 and (b) SEQ ID NO: 884; (a) SEQ ID NO: 887 and (b) SEQ ID NO: 889; (a) SEQ ID NO: 894 and (b) SEQ ID NO: 898; (a) SEQ ID NO: 901 and (b) SEQ ID NO: 905; (a) SEQ ID NO: 908 and (b) SEQ ID NO: 912; (a) SEQ ID NO: 915 and (b) SEQ ID NO: 919; (a) SEQ ID NO: 922 and (b) SEQ ID NO: 926; (a) SEQ ID NO: 929 and (b) SEQ ID NO: 933; (a) SEQ ID NO: 936 and (b) SEQ ID NO: 940;
  • SEQ ID NO: 1074 SEQ ID NO: 1077 and (b) SEQ ID NO: 1081; (a) SEQ ID NO: 1084 and (b) SEQ ID NO: 1088; (a) SEQ ID NO: 1091 and (b) SEQ ID NO: 1095; (a) SEQ ID NO: 1098 and (b) SEQ ID NO: 1102; (a) SEQ ID NO: 1105 and (b) SEQ ID NO: 1109; (a) SEQ ID NO: 1112 and (b) SEQ ID NO: 1116; (a) SEQ ID NO: 1119 and (b) SEQ ID NO: 1123; (a) SEQ ID NO: 1126 and (b) SEQ ID NO: 1130; (a) SEQ ID NO: 1133 and (b) SEQ ID NO: 1137; (a) SEQ ID NO: 1140 and (b) SEQ ID NO: 1144; (a) SEQ ID NO: 1147 and (b) SEQ ID NO: 1151; (a) SEQ ID NO:
  • variable heavy chain (VH) has at least 90% sequence identity to a sequence as set forth above in an (a) in a pair and the variable light chain (VL) has at least 90% sequence identity to the sequence as set forth above in the (b) of the same pair.
  • variable heavy chain (VH) has at least 95% sequence identity to a sequence as set forth above in an (a) in a pair and the variable light chain (VL) has at least 95% sequence identity to the sequence as set forth above in the (b) of the same pair.
  • variable heavy chain (VH) has at least 98% sequence identity to a sequence as set forth above in an (a) in a pair and the variable light chain (VL) has at least 98% sequence identity to the sequence as set forth above in the (b) of the same pair.
  • the binding protein comprises a set of CDRs comprising: (i) a HCDR1, HCDR2, HCDR3, comprising or contained within SEQ ID NO: 1; SEQ ID NO: 8; SEQ ID NO: 15; SEQ ID NO: 22; SEQ ID NO: 29; SEQ ID NO: 36; SEQ ID NO: 43; SEQ ID NO: 50; SEQ ID NO: 57; SEQ ID NO: 64; SEQ ID NO: 71 ; SEQ ID NO: 78; SEQ ID NO: 85; SEQ ID NO: 92; SEQ ID NO: 99; SEQ ID NO: 106; SEQ ID NO: 113; SEQ ID NO: 120; SEQ ID NO: 127; SEQ ID NO: 134; SEQ ID NO: 141; SEQ ID NO: 148; SEQ ID NO: 156; SEQ ID NO: 164; SEQ ID NO: 171; SEQ ID NO: 178; SEQ ID NO: 185; SEQ ID NO:
  • the present disclosure provides a composition
  • a composition comprising an isolated human monoclonal antibody or derivative thereof which binds to a specific epitope of Pseudomonas aeruginosa V-antigen (PcrV).
  • the composition comprises: a heavy chain variable domain (VH); a light chain variable domain (VL); and a pharmaceutically acceptable carrier.
  • the antibody or derivative thereof is selected from: an antibody or derivative thereof wherein at least one variable region has at least 85% sequence identity to a sequence set forth in and selected from the group consisting of: SEQ ID NO: 1 ; SEQ ID NO: 8; SEQ ID NO: 15; SEQ ID NO: 22; SEQ ID NO: 29; SEQ ID NO: 36; SEQ ID NO: 43; SEQ ID NO: 50; SEQ ID NO: 57; SEQ ID NO: 64; SEQ ID NO: 71 ; SEQ ID NO: 78; SEQ ID NO: 85; SEQ ID NO: 92; SEQ ID NO: 99; SEQ ID NO: 106; SEQ ID NO: 113; SEQ ID NO: 120; SEQ ID NO: 127; SEQ ID NO: 134; SEQ ID NO: 141; SEQ ID NO: 148; SEQ ID NO: 156; SEQ ID NO: 164; SEQ ID NO: 171; SEQ ID NO: 178; SEQ ID NO: 1 ; SEQ ID NO
  • the antibody or derivative thereof is selected from: an antibody or derivative thereof where at least one variable region is a sequence set forth in and selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 8; SEQ ID NO: 15; SEQ ID NO: 22; SEQ ID NO: 29; SEQ ID NO: 36; SEQ ID NO: 43; SEQ ID NO: 50; SEQ ID NO: 57; SEQ ID NO: 64; SEQ ID NO: 71 ; SEQ ID NO: 78; SEQ ID NO: 85; SEQ ID NO: 92; SEQ ID NO: 99; SEQ ID NO: 106; SEQ ID NO: 113; SEQ ID NO: 120; SEQ ID NO: 127; SEQ ID NO: 134; SEQ ID NO: 141; SEQ ID NO: 148; SEQ ID NO: 156; SEQ ID NO: 164; SEQ ID NO: 171 ; SEQ ID NO: 178; SEQ ID NO: 185; SEQ ID NO: 1; SEQ ID NO:
  • the antibody or derivative thereof is selected from: an antibody or derivative thereof comprising from one to three heavy chain CDR sequences and/or from one to three light chain CDR sequences selected from the group consisting of sequences as set forth in SEQ ID NO: 2, 3, 4, 6, 7, 9, 10, 11, 13, 14, 16, 17, 18, 20, 21, 23, 24, 25, 27, 28, 30, 31, 32, 34, 35, 37, 38, 39, 41, 42, 44, 45, 46, 48, 49, 51, 52, 53, 55, 56, 58, 59, 60, 62, 63, 65, 66, 67, 69, 70, 72, 73, 74, 76, 77, 79.
  • the antibody or derivative thereof is selected from: an antibody comprising a means for binding the Pseudomonas aeruginosa V-antigen (PcrV) antigen.
  • the antibody comprises (a) means for binding the PcrV antigen and (b) an immunoglobulin Fc region.
  • Exemplified structures for binding the PcrV antigen include the heavy and light chain variable region pairs and corresponding CDR sets shown in Table 1 and Table 2 of the present disclosure.
  • the binding protein comprises a set of CDRs having the sequences as set forth in Table 1 and Table 2 of the present disclosure.
  • the CDRs are grafted into a human IgG acceptor antibody framework or an IgM acceptor antibody framework.
  • the acceptor framework comprises an Fc region of the IgG or the IgM linked to the variable heavy chain.
  • the Fc region is a variant Fc region, wherein the variant Fc region comprises one or more amino acid substitutions, insertions, or deletions that alter an effector function relative to a naturally occurring Fc region.
  • the binding protein is a genetically engineered intact antibody comprising a variant Fc region.
  • the variant Fc region comprises one or more amino acid substitutions that alter an effector function relative to a naturally-occurring Fc region.
  • the binding protein is a genetically engineered intact antibody or antibody fragment or a derivative thereof.
  • the binding protein is formatted into a multimer, a single chain variable fragment (scFv), or an antibody conjugate.
  • composition comprising a binding protein of the present disclosure and a pharmaceutically acceptable carrier, diluent, or excipient.
  • the present disclosure also provides a polynucleotide encoding a binding protein of the present disclosure.
  • the polynucleotide is codon optimized.
  • the present disclosure provides an expression vector comprising the polynucleotide encoding a binding protein of the present disclosure.
  • the polynucleotide is operably linked to an expression control sequence.
  • the expression vector is capable of delivering the polynucleotide to a host cell.
  • a recombinant host cell comprising the polynucleotide encoding a binding protein of the present disclosure.
  • a recombinant host cell comprising the expression vector comprising the polynucleotide encoding a binding protein of the present disclosure.
  • the host cell is capable of expressing the encoded binding protein.
  • composition comprising: (a) means for binding a specific epitope of Pseudomonas aeruginosa V-antigen (PcrV); and (b) a pharmaceutically acceptable carrier.
  • PcrV Pseudomonas aeruginosa V-antigen
  • the present disclosure provides a method of treating and/or preventing a disease.
  • the method comprises administering to a subject in need thereof a therapeutically effective amount of a binding protein, or a composition disclosed herein.
  • the subject is a human.
  • the disease is an infectious disease.
  • the disease is an infectious disease caused by Pseudomonas aeruginosa.
  • the Pseudomonas aeruginosa infection is associated with an inflammatory lung disease.
  • the inflammatory lung disease is cystic fibrosis (CF).
  • the administration comprises intravenous administration.
  • the method further comprises administering another therapeutic agent and/or therapy to the subject.
  • the present disclosure provides a method of immunizing a subject against a pathogen.
  • the method comprises administering to the subject in need thereof an effective amount of a binding protein, a composition, a polynucleotide, an expression vector, or a recombinant host cell, disclosed herein.
  • a method of treating and/or preventing a disease comprising administering to a subject in need thereof a therapeutically effective amount of a composition disclosed herein.
  • a method of immunizing a subject against Pseudomonas aeruginosa comprises administering to the subject in need thereof an effective amount of a composition discloses herein.
  • the present disclosure provides a method of detecting the presence of Pseudomonas aeruginosa V-antigen (PcrV), or a cell expressing PcrV, in a sample.
  • the sample is a biological sample.
  • the method comprises contacting the sample with a binding protein or an isolated human monoclonal antibody disclosed herein.
  • the sample is a biological sample.
  • FIGS. 1A-1C show PcrV-specific B cells in subjects with cystic fibrosis (CF).
  • CF cystic fibrosis
  • FIGS. 1A-1C show PcrV-specific B cells in subjects with cystic fibrosis (CF).
  • FIG. 1A Schematic of primary human B cells binding to PcrV tetramer reagent and decoy (FIG. 1A, Left). Representative flow plot for B cells after enrichment (FIG. 1A, Right). Cells binding only to the PcrV tetramer are indicated with the box.
  • FIG. 2 shows tetramer-specific class-switched B cells in mice immunized with PcrV.
  • FIGS. 3A-3D show generation of anti-PcrV mAbs derived from B cells isolated from a chronically PA infected CF donor.
  • Percentage of somatic hypermutation (SHM) detected in B cell receptor (BCR) sequences from cells of the indicated phenotype for CF donor 1 (FIG. 3A).
  • ELIS As showing PcrV binding using supernatants derived from 293T cells transfected with IgM expression plasmids containing the indicated BCR sequences (FIG. 3B) Area under the curve (AUC) for representative ELISAs of purified antibodies 408 and 411 expressed alternatively as IgM vs. IgG (FIG. 3C).
  • mAbs derived from CF subject B cells are labeled with the CF prefix for clarity (FIG. 3D).
  • FIGS. 4A-4C show CF-subject derived, germline, anti-PcrV-specific mAbs exhibit robust anti-PA activity in an in vivo mouse pneumonia model.
  • FIG. 4A Schematic illustrating the experimental PA infection and mAb delivery protocol (FIG. 4A). Bacterial load in mouse lungs at 48 h post-infection for mice that received a 60 pg (FIG. 4B) or 20 pg (FIG. 4C) intranasal dose of off-target, control IgGl mAb (ctl), indicated anti-PcrV mAbs, or diluent alone (none).
  • FIGS. 5A-5C show high affinity anti-PcrV mAbs derived from memory B cells isolated from CF Donor 2. Somatic hypermutation (%SHM) rates in BCR sequences from individual B cells with the indicated surface phenotype isolated from CF donor 2. Each circle represents a singly sorted cell (FIG. 5A). Heatmap showing paired heavy (x-axis) and light (y-axis) V gene families for 31 MBCs in donor 3 (FIG. 5B). The grey scale depicts SHM for the heavy chains in each pairing. Clone numbers for BCRs to be expressed as mAbs are included to the left of their corresponding box.
  • FIGS. 7A-7C show B cell receptor (BCR) sequencing of PcrV-tetramer- specific B cells derived from 3 CF donors. Percentage of somatic hypermutation (SHM) detected in BCR sequences from cells of the indicated phenotype in chronically PA infected, CF donor 3 (FIG. 7A). Each circle represents a singly sorted cell. Histograms show the number of unique BCR sequences obtained for each V gene family (y-axis) for heavy and light (kappa or lambda) chains (FIG. 7B). Data from each CF donor is shown in a separate panel of graphs (with Donor number indicated at top).
  • SHM somatic hypermutation
  • the bars for the V gene families used by in vivo-tested mAbs are colored as: blue-green (CF 411) and orange (CF 408).
  • Heatmap showing pairings of heavy- (x-axis) and light (y-axis) chain V gene families where full-length, high quality V region sequence was attained (FIG. 7C). For each heavy/light chain pair, the percentage of heavy chain sequence which differs from the germline sequence is depicted by the grey scale .
  • FIG. 8 shows transfectant supernatant screen of 12 MBC-derived mAbs.
  • ELISA assessing PcrV binding for supernatants from 293T cells transfected with IgG expression plasmids. Numbers (430-442) indicate the BCR sequences identified from individual, PcrV- specific, MBCs from CF donor 2.
  • Supernatant for mAb 411 IgG (dotted line; isolated from CF Donor 1) is included as a benchmark (positive control).
  • a or “an” entity refers to one or more of that entity, for example, “a binding protein or a binding molecule,” is understood to represent one or more binding proteins or binding molecules.
  • the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
  • “about” or “comprising essentially of’ mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” or “comprising essentially of’ can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, “about” or “comprising essentially of” can mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values are provided in the application and claims, unless otherwise stated, the meaning of “about” or “comprising essentially of’ should be assumed to be within an acceptable error range for that particular value.
  • any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • binding protein refers in its broadest sense to a molecule that specifically binds to a receptor, e.g., an epitope or an antigenic determinant.
  • a binding protein can comprise one of more “antigen binding domains” described herein.
  • a non-limiting example of a binding protein is an antibody, a derivative, or fragment thereof that retains antigen- specific binding.
  • epitope includes any molecular determinant capable of specific binding to a binding protein, e.g., an antibody.
  • an epitope can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain aspects, can have three-dimensional structural characteristics, and or specific charge characteristics.
  • An epitope is a region of a target that is bound by a binding protein.
  • binding domain or “binding fragment,” or “antigen binding domain” or “antigen binding fragment” may be used interchangeably and refer to a region of a binding protein that is necessary and sufficient to specifically bind to an epitope of the antigen.
  • an “Fv,” e.g., a variable heavy chain (VH) and variable light chain (VL) of an antibody, either as two separate polypeptide subunits or as a single chain, is considered to be a “binding domain.”
  • Other binding domains include, without limitation, the variable heavy chain (VH) of an antibody derived from a camelid species, or six immunoglobulin complementarity determining regions (CDRs) expressed in a fibronectin scaffold.
  • a “binding protein” as described herein can include one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more “antigen binding domains.”
  • polypeptide is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds).
  • polypeptide refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product.
  • polypeptides peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of “polypeptide,” and the term “polypeptide” can be used instead of, or interchangeably with any of these terms.
  • polypeptide is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, and derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids.
  • a polypeptide can be derived from a biological source or produced by recombinant technology but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.
  • an “isolated” polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required.
  • an isolated polypeptide can be removed from its native or natural environment.
  • Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated as disclosed herein, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.
  • a “conservative amino acid substitution” is one in which one amino acid is replaced with another amino acid having a similar side chain.
  • Families of amino acids having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g.
  • aspartic acid glutamic acid
  • uncharged polar side chains e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine.
  • substitution of a phenylalanine for a tyrosine is a conservative substitution.
  • conservative substitutions in the sequences of the polypeptides and antibodies of the present disclosure do not abrogate the binding of the polypeptide or antibody containing the amino acid sequence, to the antigen to which the binding molecule binds.
  • Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen-binding are well-known in the art (see, e.g., Brummell et al , Biochem. 32: 1 180-1 187 (1993); Kobayashi et al , Protein Eng. 12(10): 879-884 (1999); and Burks et al, Proc. Natl. Acad. Sci. USA 94:.412-417 (1997)).
  • polynucleotide is intended to encompass a singular nucleic acid as well as plural nucleic acids and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA), cDNA, or plasmid DNA (pDNA).
  • a polynucleotide can comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)).
  • PNA peptide nucleic acids
  • nucleic acid or “nucleic acid sequence” refer to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.
  • an “isolated” nucleic acid or polynucleotide is intended any form of the nucleic acid or polynucleotide that is separated from its native environment.
  • gel-purified polynucleotide, or a recombinant polynucleotide encoding a polypeptide contained in a vector would be considered to be “isolated.”
  • a polynucleotide segment e.g., a PCR product, which has been engineered to have restriction sites for cloning is considered to be “isolated.”
  • binding protein encompasses full-sized antibodies as well as antigenbinding subunits, fragments, variants, analogs, or derivatives thereof, e.g., isolated monoclonal antibody molecules, engineered antibody molecules or fragments that bind antigen in a manner similar to antibody molecules, but which use a different scaffold.
  • antibody and “immunoglobulin” can be used interchangeably herein.
  • the term “antibody” as referred to herein includes whole antibodies and any antigen-binding fragment (i.e., “antigen-binding portion”) or single chains thereof.
  • a “whole antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three or four domains, depending on antibody isotype (specifically, CHI, CH2, and CH3 in the case of IgG, IgA, and IgD, or CHI, CH2, CH3, and CH4 in the case of IgM and IgE).
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • 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 regions (FR).
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may 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.
  • An antibody (or a fragment, variant, or derivative thereof as disclosed herein) includes at least the variable domain of a heavy chain or at least the variable domains of a heavy chain and a light chain.
  • antibody encompasses anything ranging from a small antigen- binding fragment of an antibody to a full sized antibody, e.g., an IgG antibody that includes two complete heavy chains and two complete light chains, an IgA antibody that includes four complete heavy chains and four complete light chains and optionally includes a J chain and/or a secretory component, or an IgM antibody that includes ten or twelve complete heavy chains and ten or twelve complete light chains and optionally includes a J chain.
  • immunoglobulin comprises various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (y, p, a, 5, s) with some subclasses among them (e.g., yl-y4 or 1- a.2). It is the nature of this chain that determines the “isotype” of the antibody as IgG, IgM, IgA IgG, or IgE, respectively.
  • immunoglobulin subclasses e.g., IgGi, IgG2, IgG3, IgG4, IgAi, IgA?, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these immunoglobulins are readily discernible to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of this disclosure.
  • Light chains are classified as either kappa or lambda (K, X). Each heavy chain class can be bound with either a kappa or lambda light chain.
  • the light and heavy chains are covalently bonded to each other, and the “tail” portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are expressed, e.g., by hybridomas, B cells or genetically engineered host cells.
  • the amino acid sequences run from an N- terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.
  • the basic structure of certain antibodies, e.g., IgG antibodies includes two heavy chain subunits and two light chain subunits covalently connected via disulfide bonds to form a “Y” structure.”
  • Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide.
  • CDR complementarity determining region
  • engineered molecules such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigenbinding fragment,” as used herein.
  • SMIPs small modular immunopharmaceuticals
  • the six “complementarity determining regions” or “CDRs” present in an antigen-binding domain or antigen-binding fragment are short, non-contiguous sequences of amino acids that are specifically positioned to form the binding domain as the antibody assumes its three-dimensional configuration in an aqueous environment.
  • the remainder of the amino acids in the binding domain referred to as “framework” regions, show less inter- molecular variability.
  • the framework regions largely adopt a P-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the P-sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions.
  • the binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the protein/molecule to its cognate epitope.
  • CDR complementarity determining region
  • a binding protein comprising an antigen-binding fragment of an antibody will typically comprise at least one variable domain.
  • the variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences.
  • the VH and VL domains may be situated relative to one another in any suitable arrangement.
  • the variable region may be dimeric and contain VH — VH, VH — VL or VL — VL dimers.
  • the antigen-binding fragment may contain a monomeric VH or VL domain.
  • an antigen-binding fragment of a binding protein may contain at least one variable domain covalently linked to at least one constant domain.
  • variable and constant domains that may be found within an antigen-binding fragment of the present disclosure include: (i) VH— CHI ; (ii) VH— CH2; (iii) VH— CH3; (iv) VH— CH1-CH2; (v) VH— CH1-CH2-C H3; (vi) VH— CH2-CH3; (vii) VH— CL; (viii) VL— CHI ; (ix) VL— CH2; (x) VL— CH3; (xi) VL— CH1-CH2; (xii) VL— CH1-CH2-CH3; (xiii) VL— CH2-CH3; and (xiv) VL — CL.
  • an antigen-binding fragment of the present disclosure may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).
  • antigen-binding fragments of the present disclosure may be monospecific or multispecific (e.g., bispecific).
  • a multispecific antigenbinding fragment will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen.
  • Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of the present disclosure using routine techniques available in the art.
  • a binding protein e.g., an antibody or fragment, variant, or derivative thereof binds to an epitope via its antigenbinding domain or antigen-binding fragment, and that the binding entails some complementarity between the antigen binding- fragment and the epitope.
  • a binding protein is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen binding domain more readily than it would bind to a random, unrelated epitope.
  • the term “specificity” is used herein to qualify the relative affinity by which certain binding proteins bind to a certain epitope.
  • binding protein “A” can be deemed to have a higher specificity for a given epitope than binding protein “B,” or binding protein “A” can be said to bind to epitope “C” with a higher specificity than it has for related epitope “D.”
  • affinity refers to a measure of the strength of the binding of an individual epitope with one or more binding domains, e.g., of an immunoglobulin molecule. See, e.g., Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) at pages 27-28.
  • a binding protein e.g., an antibody or fragment, variant, or derivative thereof of the present disclosure can be said to bind a target antigen with an off rate (k(off)) of less than or equal to 5 X 10" 2 sec -1 , 10" 2 sec -1 , 5 X 10" 3 sec -1 , 10' 3 sec -1 , 5 X 10" 4 sec -1 , 10"
  • a binding protein e.g., an antibody or antigen-binding fragment, variant, or derivative disclosed herein can be said to bind a target antigen with an on rate (k(on)) of greater than or equal to 10 3 M -1 sec -1 , 5 X 10 3 M -1 sec -1 , 10 4 M -1 sec -1 , 5 X 10 4 M -1 sec -1 , 10 5 M -1 sec -1 ,
  • a binding protein of the present disclosure can also be described or specified in terms of their binding affinity to an antigen.
  • a binding protein can bind to an antigen with a dissociation constant or KD no greater than 5 x 10" 2 M, 10" 2 M, 5 x IO’ 3 M, IO’ 3 M, 5 x 10’ 4 M, IO’ 4 M, 5 x 10’ 5 M, 10’ 5 M, 5 x IO’ 6 M, IO’ 6 M, 5 x 10’ 7 M, IO’ 7 M, 5 x 10’ 8 M, 10’ s M, 5 x IO’ 9 M, IO 9 M, 5 x IO' 10 M, IO’ 10 M, 5 x 10’ 1 1 M, 10’ 11 M, 5 x IO 12 M, 10‘ 12 M, 5 x 10’ 13 M, 10‘ 13 M, 5 x 10 14 M, 10 14 M, 5 x 10‘ 15 M, or W 15 M.
  • a binding protein of the present disclosure e.g., an antibody or fragment, variant, or derivative thereof including single-chain antibodies or other antigen-binding fragments can exist alone or in combination with one or more of the following: hinge region, CHI, CH2, CH3, or CH4 domains, J chain, or secretory component. Also included are antigen- binding fragments that can include any combination of variable region(s) with one or more of a hinge region, CHI, CH2, CH3, or CH4 domains, a J chain, or a secretory component.
  • the binding protein comprises an anti-PA PcrV antibody.
  • the antibody is a human antibody.
  • the term “human antibody,” as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.
  • the term “human antibody,” as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • the binding proteins of the disclosure may, in some embodiments, comprise anti-PA PcrV recombinant human antibodies.
  • the term “recombinant human antibody,” as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences.
  • such recombinant human antibodies are subjected to in vitro mutagenesis and thus the amino acid sequences of the Vn and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • human antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and can in some instances express endogenous immunoglobulins and some not, as described, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.
  • the term “heavy chain subunit” includes amino acid sequences derived from an immunoglobulin heavy chain,
  • a binding protein e.g., an antibody comprising a heavy chain subunit can include at least one of: a heavy chain variable (VH) domain, a CHI domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, or a variant or fragment thereof.
  • a binding protein e.g., an antibody or fragment, variant, or derivative thereof can include without limitation, in addition to a VH domain:, a CHI domain; a CHI domain, a hinge, and a CH2 domain; a CHI domain and a CH3 domain; a CHI domain, a hinge, and a CH3 domain; or a CHI domain, a hinge domain, a CH2 domain, and a CH3 domain.
  • a binding protein, e.g., an antibody or fragment, variant, or derivative thereof can include, in addition to a VH domain, a CH3 domain and a CH4 domain; or a CH3 domain, a CH4 domain, and a J chain.
  • a binding protein for use in the disclosure can lack certain constant region portions, e.g., all or part of a CH2 domain. It will be understood by one of ordinary skill in the art that these domains (e.g., the heavy chain subunit) can be modified such that they vary in amino acid sequence from the original immunoglobulin molecule.
  • the term “light chain subunit” includes amino acid sequences derived from an immunoglobulin light chain.
  • the light chain subunit includes at least a light chain variable domain (VL), and can further include a CL (e.g., CK or C) domain.
  • VL light chain variable domain
  • CL e.g., CK or C
  • expression refers to a process by which a gene produces a biochemical, for example, a polypeptide.
  • the process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient expression and stable expression. It includes without limitation transcription of the gene into RNA, e.g., messenger RNA (mRNA), and the translation of such mRNA into polypeptide(s). If the final desired product is a biochemical, expression includes the creation of that biochemical and any precursors.
  • RNA messenger RNA
  • a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide that is translated from a transcript.
  • Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.
  • the present disclosure also provides polynucleotides encoding the binding proteins, fragments, and derivatives thereof, disclosed herein, as well as expression vectors comprising such polynucleotides, and host cells comprising such expression vectors. Additionally, provided are methods of production of at least one binding protein, a fragment, or a derivative, thereof of the present disclosure by recombinant techniques, as is well known in the art. See, e.g., Sambrook, et al., supra; Ausubel, et al., supra, each entirely incorporated herein by reference.
  • DNAs encoding partial or full-length light and heavy chains can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma that expresses the antibody of interest) and the DNAs can be inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences.
  • operatively linked is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene.
  • the expression vector and expression control sequences are chosen to be compatible with the expression host cell used.
  • the antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors or, more typically, both genes are inserted into the same expression vector.
  • the polynucleotide encoding the binding protein is inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites, or blunt end ligation if no restriction sites are present).
  • the recombinant expression vector can encode a signal peptide that facilitates secretion of the binding protein from a host cell.
  • the signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).
  • the expression vectors of the present disclosure carry regulatory sequences that control the expression of the binding protein in a host cell.
  • the term “regulatory sequence” is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes.
  • Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter (AdIVILP) and polyoma.
  • CMV cytomegalovirus
  • SV40 Simian Virus 40
  • AdIVILP adenovirus major late promoter
  • nonviral regulatory sequences may be used, such as the ubiquitin promoter or 13-globin promoter.
  • regulatory elements composed of sequences from different sources such as the SRa, promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8:466-472).
  • the expression vectors of the disclosure may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes.
  • the selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al.).
  • the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced.
  • Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
  • DHFR dihydrofolate reductase
  • Preferred mammalian host cells for expressing the binding proteins of the present disclosure include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mot Biol. 1.59:601-621), NSO myeloma cells, COS cells and SP2 cells.
  • another preferred expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036, and EP 338,841.
  • 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 or slow the progression of an existing diagnosed pathologic condition or disorder.
  • Terms such as “prevent,” “prevention,” “avoid,” “deterrence” and the like refer to prophylactic or preventative measures that prevent the development of an undiagnosed targeted pathologic condition or disorder.
  • “those in need of treatment” can include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented.
  • subject or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired.
  • Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, swine, cows, bears, and so on.
  • a “therapeutically effective amount” is a dose or an amount sufficient to provide a useful therapeutic or prophylactic effect against a particular disease or disease condition.
  • an effective amount is sufficient to achieve one or more of the following therapeutic effects: reduce the ability of the bacteria or virus to propagate in the patient or reduce the amount of bacteria or viral load in the patient.
  • an effective amount is sufficient to achieve one or more of the following prophylactic effects: a reduced susceptibility to a bacterial or viral infection or a reduced ability of an infecting bacterium or virus to establish persistent infection.
  • a therapeutically effective amount or an effective amount of the compositions disclosed herein includes an amount sufficient to generate an immune response useful to provide a therapeutic or prophylactic effect against a particular disease or disease condition.
  • the dose or amount of a composition of the present disclosure administered to a subject may vary depending upon the age and the size of the subject, conditions, route of administration, and the like.
  • the preferred dose is typically calculated according to body weight or body surface area.
  • intravenously administer the antibody of the present disclosure normally at a single dose of about 0.01 to about 20 mg/kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight.
  • the frequency and the duration of the treatment can be adjusted.
  • Effective dosages and schedules for administering of the compositions of the present disclosure may be determined empirically; for example, progress can be monitored by periodic assessment, and the dose adjusted accordingly. Moreover, interspecies scaling of dosages can be performed using well-known methods in the art (e.g., Mordenti et al., Pharmaceut Res 8: 1351 (1991)).
  • compositions of the disclosure e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing an antibody or other therapeutic protein of the invention, receptor mediated endocytosis (see, e.g., Wu et al., J Biol Chem 262:4429-4432 (1987)).
  • the binding proteins, antibodies and other therapeutically active compositions of the present disclosure may also be delivered by gene therapy techniques. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • epithelial or mucocutaneous linings e.g., oral mucosa, rectal and intestinal mucosa, etc.
  • Administration can be systemic or local.
  • a pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe.
  • a pen delivery device readily has applications in delivering a pharmaceutical composition of the present invention.
  • Such a pen delivery device can be reusable or disposable.
  • a reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused.
  • a disposable pen delivery device there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.
  • Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition as discussed herein. Examples include, but are not limited to AUTOPENTM (Owen Mumford, Inc., Woodstock, UK), DISETRONICTM pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25TM pen, HUMALOGTM pen, HUMALIN 70/30TM pen (Eli Lilly and Co., Indianapolis, IN), NOVOPENTM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIORTM (Novo Nordisk, Copenhagen, Denmark), BDTM pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPENTM, OPTIPEN PROTM, OPTIPEN STARLETTM, and OPTICLIKTM (sanofi-aventis, Frankfurt, Germany), to name only a few.
  • Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but are not limited to the SOLOSTARTM pen (sanofi-aventis), the FLEXPENTM (Novo Nordisk), and the KWIKPENTM (Eli Lilly), the SURECLICKTM Autoinjector (Amgen, Thousand Oaks, CA), the PENLETTM (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), and the HUMIRATM Pen (Abbott Labs, Abbott Park IL), to name only a few.
  • the pharmaceutical composition can be delivered in a controlled release system.
  • a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987)).
  • polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida.
  • a controlled release system can be placed in proximity of the composition’s target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, Science 249:1527-1533 (1990).
  • the injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known.
  • the injectable preparations may be prepared, e.g., by dissolving, suspending, or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections.
  • aqueous medium for injections there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents.
  • the binding proteins, antibodies, and other therapeutically active compositions of the present disclosure can be administered with a further therapeutic agent.
  • the further therapeutic agent can be administered prior to, concurrently, or after the administration of the binding proteins, antibodies, and other therapeutically active compositions of the present disclosure.
  • Antimicrobial monoclonal antibodies may fill a widening gap as alternatives, or adjuncts, to traditional antibiotics.
  • Traditional methods of monoclonal antibody (mAb) development require extensive labor in the early stages, from immunization of mice with a recombinant protein to screening hundreds of hybridomas.
  • mouse-derived antibodies must be iteratively re-humanized in vitro and/or modified into antibody fragments (Fab) which lack effector functions and are subject to rapid renal clearance.
  • Fab antibody fragments
  • fully in vitro strategies like phage display can enable the use of human variable domains, the high-throughput screening processes are vulnerable to bottleneck effects and drift, do not select against autoreactivity or immunogenicity, and do not screen paired VH/VL sequences within the context of full-length paired chains.
  • An alternative approach that has also sought to maximize throughput in screening for high affinity anti-PcrV binders from a library of llama-derived nanobodies shares similar advantages and drawbacks.
  • mAbs Monoclonal antibodies that bind to key PA virulence factors have shown promise in animal models.
  • P. aeruginosa V-antigen P. aeruginosa V-antigen (PcrV)
  • PcrV P. aeruginosa V-antigen
  • LcrV LcrV
  • T3S type III secretion system
  • PcrV facilitates the integration of pore-forming proteins into the eukaryotic cell membrane and is required for translocation of cytotoxins into the host cell.
  • CF cystic fibrosis
  • CFTR cystic fibrosis transmembrane conductance regulator
  • the PcrV- specific B cells that give rise to antibody- secreting cells have not been previously studied.
  • the present disclosure shows that individuals with CFhave more PcrV- specific circulating B cells.
  • the present disclosure utilized single-cell sequencing to identify PcrV-specific paired-chain BCR sequences in CF subjects. From BCR sequences derived from CF plasmablasts and MBCs, novel anti-PcrV mAbs, including several that confer robust protection against a virulent strain of PA in an in vivo pneumonia challenge model, were generated.
  • the present disclosure addresses the unmet need in the art by generating multiple anti-PcrV mAbs directly from the BCR variable regions of antigenspecific B cells derived from CF donors.
  • the present disclosure provides human anti-PcrV mAbs that improve immunogenicity risk and pharmacokinetics, while maintaining robust protective activity.
  • the antibodies are derived from a broad range of memory B cell subsets including IgG, IgM, and IgA memory cells. Notably, it is shown that these antibodies exhibit protective, neutralizing activity against P. aeruginosa. They exhibit high affinity to a range of PcrV epitopes and limit bacterial infection.
  • human MBC-derived mAbs are the product of evolved B cell development and maturation processes that encompass tremendous diversity and include selection against auto-reactivity and, for cells that have exited GCs, pre-optimized binding to antigen via affinity maturation.
  • human antigen- specific B cells may be broadly considered as an underutilized high-yield resource in the critical endeavor to discover new treatments for infectious disease.
  • Naturally occurring antibody structural units disclosed herein include a tetramer.
  • Each tetramer includes two pairs of polypeptide chains, each pair having one light chain and one heavy chain.
  • the amino-terminal portion of each chain includes a variable region that is responsible for antigen recognition and epitope binding.
  • the variable regions exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions (CDRs).
  • the CDRs from the two chains of each pair are aligned by the framework regions, which enables binding to a specific epitope.
  • both light and heavy chain variable regions include the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • IMGT Manso T, Folch G, Giudicelli V, Jabado-Michaloud J, Kushwaha A, Nguefack Ngoune V, Georga M, Papadaki A, Debbagh C, Pegorier P, Bertignac M, Hadi- Saljoqi S, Chentli I, Cherouali K, Aouinti S, El Hamwi A, Albani A, Elazami Elhassani M, Viart B, Goret A, Tran A, Sanou G, Rollin M, Duroux P, Kossida S., IMGT® databases, related tools and web resources through three main axes of research and development. Nucleic Acids Res. 2022 Jan 7;50(Dl):D1262-D1272.), as described in more detail below.
  • each chain defines a constant region, which can be responsible for effector function particularly in the heavy chain (the Fc).
  • effector functions include: Clq binding and complement dependent cytotoxicity (CDC); antibody dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B-cell receptors); and B-cell activation.
  • variable and constant regions are joined by a “J” region of amino acids, with the heavy chain also including a “D” region of amino acids. See, e.g., Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)).
  • Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody’s isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • IgG has several subclasses, including IgGl , lgG2, lgG3, and lgG4.
  • IgM has subclasses including IgMl and lgM2.
  • IgA is similarly subdivided into subclasses including IgAl and lgA2.
  • IgG causes opsonization and cellular cytotoxicity and crosses the placenta
  • IgA functions on the mucosal surface
  • IgM is most effective in complement fixation
  • IgE mediates degranulation of mast cells and basophils.
  • the function of IgD is still not well understood.
  • Resting B cells which are immunocompetent but not yet activated, express IgM and IgD. Once activated and committed to secrete antibodies these B cells can express any of the five isotypes.
  • the heavy chain isotypes of IgG, IgA, IgM, IgD and IgE are respectively designated the y, a, p, 8, and 8 chains.
  • Antibodies and antibody-like molecules that can multimerize have emerged as promising drug candidates in the fields of, e.g., immuno-oncology and infectious diseases allowing for improved specificity, improved avidity, and the ability to bind to multiple binding targets. See, e.g., U.S. Patent Nos. 9,951 ,134, 10,400,038, and 9,938,347, U.S. Patent Application Publication Nos. US20190100597A1, US20180118814A1, US20180118816A1, US20190185570A1 , and US20180265596A1, and PCT Publication Nos.
  • antibodies bind epitopes on antigens.
  • antigen refers to a molecule or a portion of a molecule capable of being bound by an antibody.
  • An epitope is a region of an antigen that is bound by the variable region of an antibody.
  • Epitope determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups, and can have specific three- dimensional structural characteristics, and/or specific charge characteristics.
  • the antigen is a protein or peptide
  • the epitope includes specific amino acids within that protein or peptide that contact the variable region of an antibody.
  • an epitope denotes the binding site on the PcrV protein bound by a corresponding variable region of an antibody.
  • the variable region either binds to a linear epitope, (e.g., an epitope including a stretch of 5 to 12 consecutive amino acids), or the variable region binds to a three-dimensional structure formed by the spatial arrangement of several short stretches of the protein target.
  • Three-dimensional epitopes recognized by a variable region e.g., by the epitope recognition site or paratope of an antibody or antibody fragment, can be thought of as three-dimensional surface features of an epitope molecule.
  • an epitope can be considered to have two levels: (i) the “covered patch” which can be thought of as the shadow an antibody variable region would cast on the antigen to which it binds; and (ii) the individual participating side chains and backbone residues that facilitate binding. Binding is then due to the aggregate of ionic interactions, hydrogen bonds, and hydrophobic interactions.
  • binding protein and/or “antibody” includes (in addition to antibodies having two full-length heavy chains and two full-length light chains as described above) variants, derivatives, and fragments thereof, examples of which are described below.
  • antibodies or binding proteins can include monoclonal antibodies, human antibodies, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, multi- specific antibodies, polyclonal antibodies, linear antibodies, minibodies, domain antibodies, synthetic antibodies, chimeric antibodies, antibody fusions, and fragments thereof, respectively.
  • antibodies can include oligomers or multiplexed versions of antibodies.
  • a monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies including the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.
  • monoclonal antibodies can be made by a variety of techniques, including the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci.
  • a chimeric antibody is a monoclonal antibody constructed from variable regions derived from a different organism from the constant regions.
  • a chimeric antibody can contain variable regions from a murine source and constant regions derived from the intended host source (e.g., human; for a review, see Morrison and Oi 1989, Advances in Immunology 44: 65-92).
  • a “human antibody” is one which includes an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibodyencoding sequences.
  • the strategy of “humanizing” involves sequence comparison between the non-human and human antibody variable domain sequences to determine whether specific amino acid substitutions from a non-human to human consensus is appropriate (Jones et al., 1986, Nature 321 : 522-526).
  • Fully human mAbs can be produced using genetically engineered mouse strains which possess an immune system whereby the mouse antibody genes have been inactivated and in turn replaced with a repertoire of functional human antibody genes, leaving other components of the mouse immune system unchanged.
  • Such genetically engineered mice allow for the natural in vivo immune response and affinity maturation process, resulting in high affinity, fully human monoclonal antibodies.
  • This technology is well known in the art and is fully detailed in various publications, including but not limited to U.S. Pat. Nos. 5,939,598; 6,075,181 ; 6,114,598; 6,150,584 and related family members; as well as U.S. Pat. Nos.
  • a “human consensus framework” is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. The subgroup of sequences can be a subgroup as in IMGT (supra).
  • antibodies and/or binding proteins disclosed herein include the following sequences, and functional variants thereof as described below:
  • CDR sequences provided above are based on IMGT numbering (Lefranc M P et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 January; 27(l):55-77 (“IMGT” numbering scheme)).
  • Definitive delineation of a CDR and identification of residues including the binding site of an antibody can be accomplished by solving the structure of the antibody and/or solving the structure of the antibody-epitope complex. In particular embodiments, this can be accomplished by methods such as X-ray crystallography. Alternatively, CDRs are determined by comparison to known antibodies (linear sequence) and without resorting to solving a crystal structure. [0121] In addition to IMGT, CDR sets can be based on Kabat numbering (Kabat et al. (1991), “Sequences of Proteins of Immunological Interest.'’ 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.
  • a co-crystal structure of the Fab (antibody fragment) bound to the target can be determined.
  • Software programs and bioinformatical tools, such as ABodyBuilder, Paratome, and LlamaMagic can also be used to determine CDR and FR sequences.
  • the Kabat numbering scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering.
  • the Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. Similar to the IMGT scheme, the AHo scheme can skip numbers in the sequential residue numbering.
  • antibodies disclosed herein are expressed as IgA, IgG, or IgM isotypes.
  • Antibodies and/or binding proteins disclosed herein can be utilized to prepare various forms of relevant binding domain molecules.
  • particular embodiments can include binding fragments of an antibody, e.g., Fv, Fab, Fab’, F(ab’)2, and single chain Fv fragments (scFvs) or any biologically effective fragments of an immunoglobulin that bind specifically to an epitope described herein.
  • an antibody fragment is used.
  • An “antibody fragment” denotes a portion of a complete or full-length antibody that retains the ability to bind to an epitope.
  • Antibody fragments can be made by various techniques, including proteolytic digestion of an intact antibody as well as production by recombinant host-cells (e.g., mammalian suspension cell lines, E. coli or phage).
  • Antibody fragments can be screened for their binding properties in the same manner as intact antibodies. Examples of antibody fragments include Fv, scFv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; and linear antibodies.
  • a single chain variable fragment is a fusion protein of the variable regions of the heavy and light chains of immunoglobulins connected with a short linker peptide.
  • Fv fragments include the VL and VH domains of a single arm of an antibody but lack the constant regions.
  • the two domains of the Fv fragment, VL and VH are coded by separate genes, they can be joined, using, for example, recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (single chain Fv (scFv)).
  • Linker sequences that are used to connect the VL and VH of an scFv are generally five to 35 amino acids in length.
  • a VL-VH linker includes from five to 35, ten to 30 amino acids or from 15 to 25 amino acids. Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies.
  • Linker sequences of scFv are commonly Gly-Ser linkers.
  • a linker is an amino acid sequence which can provide flexibility and room for conformational movement between the binding domains of a LAM. Any appropriate linker may be used.
  • Examples of linkers can be found in Chen et al., Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357-1369. Linkers can be flexible, rigid, or semi-rigid, depending on the desired functional domain presentation to a target.
  • Commonly used flexible linkers include linker sequence with the amino acids glycine and serine (Gly-Ser linkers).
  • the linker sequence includes sets of glycine and serine repeats such as from one to ten repeats of (GlyxSery) n , wherein x and y are independently an integer from 0 to 10 provided that x and y are not both 0 and wherein n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) (SEQ ID NO: 1586).
  • Linkers that include one or more antibody hinge regions and/or immunoglobulin heavy chain constant regions, such as CH3 alone or a CH2CH3 sequence can also be used.
  • antibody -based binding domain formats include scFv-based grababodies and soluble VH domain antibodies. These antibodies form binding regions using only heavy chain variable regions. See, for example, Jespers et al., Nat. Biotechnol. 22: 1161, 2004; Cortez-Retamozo et al., Cancer Res. 64:2853, 2004; Baral et al., Nature Med. 12:580, 2006; and Barthelemy et al., J. Biol. Chem. 283:3639, 2008.
  • a Fab fragment is a monovalent antibody fragment including VL, VH, CL and CHI domains.
  • a F(ab’)2 fragment is a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region.
  • Diabodies include two epitope-binding sites that may be bivalent. See, for example, EP 0404097; WO1993/01161; and Holliger, et al., Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993.
  • Dual affinity retargeting antibodies (DARTTM; based on the diabody format but featuring a C- terminal disulfide bridge for additional stabilization (Moore et al., Blood 117:4542-51, 2011) can also be used.
  • Antibody fragments can also include isolated CDRs. For a review of antibody fragments, see Hudson, et al., Nat. Med. 9: 129-134, 2003.
  • variants of antibodies and/or binding proteins described herein are also included.
  • Variants can include those having one or more conservative amino acid substitutions or one or more non-conservative substitutions that do not adversely affect the binding of the protein.
  • a conservative amino acid substitution may not substantially change the structural characteristics of the reference sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the reference sequence or disrupt other types of secondary structure that characterizes the reference sequence).
  • Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden & J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al., Nature, 354: 105 (1991).
  • conservative amino acid substitutions see the Closing Paragraphs section of this disclosure.
  • a VL region can be derived from or based on a disclosed VL and can include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the VL regions disclosed herein.
  • one or more e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the VL regions disclosed herein.
  • An insertion, deletion or substitution may be anywhere in the VL region, including at the amino- or carboxy-terminus or both ends of this region, provided that each CDR includes zero changes or at most one, two, or three changes, and provided an antibody including the modified VL region can still specifically bind its target epitope with an affinity similar to the wild type binding domain.
  • a VH region can be derived from or based on a disclosed VH and can include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the VH regions disclosed herein.
  • one or more e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the VH regions disclosed herein.
  • An insertion, deletion or substitution may be anywhere in the VH region, including at the amino- or carboxy-terminus or both ends of this region, provided that each CDR includes zero changes or at most one, two, or three changes and provided an antibody including the modified VH region can still specifically bind its target epitope with an affinity similar to the wild type binding domain.
  • a variant includes or is a sequence that has at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to an antibody sequence disclosed herein.
  • a variant includes or is a sequence that has at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to a light chain variable region (VL) and/or to a heavy chain variable region ( VH), or both, wherein each CDR includes zero changes or at most one, two, or three changes, from the reference antibody disclosed herein or fragment or derivative thereof that specifically binds a PcrV epitope as described herein.
  • VL light chain variable region
  • VH heavy chain variable region
  • one or more amino acid modifications may be introduced into the Fc region of an antibody, thereby generating an Fc region variant.
  • the Fc region variant may include a human Fc region sequence (e.g. , a human IgG 1 , lgG2, lgG3 or lgG4 Fc region) including an amino acid modification (e.g., a substitution) at one or more amino acid positions. Numerous Fc variants are described below that provide administration benefits.
  • variants have been modified from a reference sequence to produce an administration benefit.
  • exemplary administration benefits can include (1) reduced susceptibility to proteolysis, (2) reduced susceptibility to oxidation, (3) altered binding affinity for forming protein complexes, (4) altered binding affinities, (5) reduced immunogenicity; and/or (6) extended half-live. While the disclosure below describes these modifications in terms of their application to antibodies and/or binding proteins disclosed herein, when applicable to another particular PcrV binding domain format (e.g., an scFv, bispecific antibodies), the modifications can also be applied to these other formats.
  • PcrV binding domain format e.g., an scFv, bispecific antibodies
  • alterations are made in the Fc region that result in altered Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551 , WO 99/51642, and Idusogie et al., J. Immunol. 164: 4178-4184, 2000.
  • CDC Complement Dependent Cytotoxicity
  • cysteine engineered antibodies e.g., “thioMAbs,” in which one or more residues of an antibody are substituted with cysteine residues.
  • the substituted residues occur at accessible sites of the antibody.
  • reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further below.
  • residue 5400 (EU numbering) of the heavy chain Fc region is selected.
  • Cysteine engineered antibodies may be generated as described, e.g., in U.S. Pat. No. 7,521 ,541.
  • Antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region.
  • the amount of fucose in such antibody may be from 1 % to 80%, from 1 % to 65%, from 5% to 65% or from 20% to 40%.
  • the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI- TOF mass spectrometry, as described in WO 2008/077546, for example.
  • Asn297 refers to the asparagine residue located at position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located ⁇ 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., WG2000/61739; WO 2001/29246; W02002/031140; US2002/0164328; W02003/085119; W02003/084570; US2003/0115614; US2003/0157108; US2004/0093621 ;
  • Examples of cell lines capable of producing defucosylated antibodies include Led 3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545, 1986, and knockout cell lines, such as alpha- 1 ,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614, 2004; Kanda et al., Biotechnol. Bioeng., 94(4):680-688, 2006; and W02003/085107).
  • modified antibodies include those wherein one or more amino acids have been replaced with a non- amino acid component, or where the amino acid has been conjugated to a functional group or a functional group has been otherwise associated with an amino acid.
  • the modified amino acid may be, e.g., a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, or an amino acid conjugated to an organic derivatizing agent.
  • Amino acid(s) can be modified, for example, co-translationally or post-translationally during recombinant production (e.g., N-linked glycosylation at N-X-S/T motifs during expression in mammalian cells) or modified by synthetic means.
  • the modified amino acid can be within the sequence or at the terminal end of a sequence. Modifications also include nitrited constructs.
  • variants include glycosylation variants wherein the number and/or type of glycosylation site has been altered compared to the amino acid sequences of a reference sequence.
  • glycosylation variants include a greater or a lesser number of N-linked glycosylation sites than the reference sequence.
  • An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X can be any amino acid residue except proline.
  • the substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions which eliminate this sequence will remove an existing N-linked carbohydrate chain.
  • N-linked carbohydrate chains wherein one or more N-linked glycosylation sites (e.g., those that are naturally occurring) are eliminated and one or more new N-linked sites are created.
  • Additional antibody variants include cysteine variants wherein one or more cysteine residues are deleted from or substituted for another amino acid (e.g., serine) as compared to the reference sequence. These cysteine variants can be useful when antibodies must be refolded into a biologically active conformation such as after the isolation of insoluble inclusion bodies. These cysteine variants generally have fewer cysteine residues than the reference sequence, and typically have an even number to minimize interactions resulting from unpaired cysteines.
  • PEGylation particularly is a process by which polyethylene glycol (PEG) polymer chains are covalently conjugated to other molecules such as proteins.
  • PEG polyethylene glycol
  • Several methods of PEGylating proteins have been reported in the literature. For example, N- hydroxy succinimide (NHS)-PEG was used to PEGylate the free amine groups of lysine residues and N-terminus of proteins; PEGs bearing aldehyde groups have been used to PEGylate the amino-termini of proteins in the presence of a reducing reagent; PEGs with maleimide functional groups have been used for selectively PEGylating the free thiol groups of cysteine residues in proteins; and site-specific PEGylation of acetylphenylalanine residues can be performed.
  • NHS N- hydroxy succinimide
  • PEGylation can also decrease protein aggregation (Suzuki et al., Biochem. Bioph. Acta 788:248, 1984), alter protein immunogenicity (Abuchowski et al., J. Biol. Chem. 252: 3582, 1977), and increase protein solubility as described, for example, in PCT Publication No. WO 92/16221).
  • PEGs are commercially available (Nektar Advanced PEGylation Catalog 2005-2006; and NOF DDS Catalogue Ver 7. 1), which are suitable for producing proteins with targeted circulating half-lives.
  • active PEGs include mPEG succinimidyl succinate, mPEG succinimidyl carbonate, and PEG aldehydes, such as mPEG- propionaldehyde.
  • the antibody can include an Fc polypeptide that includes amino acid alterations that extend the in vivo half-life of the antibody that contains the altered Fc polypeptide as compared to the half-life of a similar antibody containing the same Fc polypeptide without the amino acid alterations.
  • Fc polypeptide amino acid alterations can include M252Y, T252L, T253S, S254T, T254F, T256E, T256N, N286/D/E/Q, E294delta, T307P, Q31 IV, A379V, S383N, M428L, N434S, N434A, N434Y, and/or R435H.
  • J chain mutations that extend half-life include Y102A and R106A/E. These mutations are described in Braathen et al., Journal of Immunology, (2007) 178(3) 1589-1597.
  • the R435H mutation is described in more detail in Stapleton et al., Nat. Comm. (2011)2:599.
  • the N434A mutation is described in more detail in Shields et al., J. Biol. Chem. (2001) 276: 6591-604.
  • the E294delta mutation is described in more detail in Monnet et al., Mabs (2014) 6:422-36 and Bas et al., J. Immunol. (2019) 202:1582094.
  • M428L/N434S is a pair of mutations that increase the half-life of antibodies in serum, as described in Zalevsky et al., Nature Biotechnology 28, 157-159, 2010.
  • M252Y/S254T/T256E are a trio of mutations described in Dall’acqua et al., J. Immunol. (2002) 169: 5171-80.
  • T252L/T253S/T254F is a trio described in Ghetie et al., Nat. Biotechnol. (1997) 15:637-40.
  • Additional combinations include E294delta/T307P/N434Y (Monnet et al., Mabs (2014) 6:422-36) and T256N/A379V/S383N/N434Y (Monnet et al., Mabs (2014) 6:422-36).
  • the N286/D/E/Q and Q31 IV mutations are described in Booth et al., mAbs, (2016) 10(7) 1098-1110.
  • any substitution at one of the following amino acid positions in an Fc polypeptide can be considered an Fc alteration that extends half-life: 250, 251, 252, 253, 254, 256, 286, 294, 259, 307, 308, 311 , 332, 378, 379, 380, 383,428, 430, 434, 435, 436.
  • Each of these alterations or combinations of these alterations can be used to extend the halflife of an antibody as described herein.
  • PcrV-specific antibodies of the present disclosure can be multimers.
  • multimers include at least two binding domains. Examples of multimers include dimers, trimers, tetramers, pentamers, hexamers, and higher order binding molecules.
  • the binding domains of a multimer can all bind the same epitope.
  • the binding domains of a multimer can bind the same antigen, but distinct epitopes on the antigen.
  • the binding domains of a multimer can bind different antigens.
  • Multi- specific antibodies are examples of multimers and include bispecific, trispecific, or tetraspecific antibodies.
  • PcrV bispecific antibodies bind at least two epitopes wherein at least one of the epitopes is located on PcrV antigen.
  • PcrV trispecific antibodies bind at least 3 epitopes, wherein at least one of the epitopes is located on PcrV antigen, and so on.
  • a dimer includes a bispecific antibody.
  • a trimer includes a trispecific antibody.
  • Dimers can be prepared as full-length antibodies or antibody fragments (for example, F(ab’)2 bispecific antibodies).
  • F(ab’)2 bispecific antibodies For example, WO 1996/016673 describes a bispecific ErbB2/ Fc gamma RIH antibody; US Pat. No. 5,837,234 describes a bispecific ErbB2/ Fc gamma R1 antibody; WO 1998/002463 describes a bispecific ErbB2/Fc alpha antibody; and US 5,821 ,337 describes a bispecific ErbB2/ CD3 antibody.
  • Some additional exemplary dimers or bispecific antibodies have two heavy chains (each having three heavy chain CDRs, followed by (N-terminal to C-terminal) a CHI domain, a hinge, a CH2 domain, and a CH3 domain), and two immunoglobulin light chains that confer antigen-binding specificity through association with each heavy chain.
  • additional architectures are envisioned, including dimers or bispecific antibodies in which the light chain(s) associate with each heavy chain but do not (or minimally) contribute to antigen-binding specificity, or that can bind one or more of the epitopes bound by the heavy chain antigen-binding regions, or that can associate with each heavy chain and enable binding of one or both of the heavy chains to one or both epitopes.
  • IgA and IgM antibodies have the ability to multimerize.
  • IgA as the major class of antibody present in the mucosal secretions of most mammals, represents a key first line of defense against invasion by inhaled and ingested pathogens.
  • IgA is also found at significant concentrations in the serum of many species, where it functions as a second line of defense mediating elimination of pathogens that have breached the mucosal surface.
  • Receptors specific for the Fc region of IgA, FcaR are key mediators of IgA effector function.
  • Native IgA is a tetrameric protein including two identical light chains (K or A) and two identical heavy chains.
  • IgA similarly, to IgG, contains three constant domains (CA1-CA3), with a hinge region between the CAI and CA2 domains.
  • the main difference between IgA I and lgA2 resides in the hinge region that lies between the two Fab arms and the Fc region.
  • IgAl has an extended hinge region due to the insertion of a duplicated stretch of amino acids, which is absent in lgA2.
  • Both forms of IgA have the capacity to form dimers, in which two monomer units, each including two heavy chains and light chains, are arranged in an end-to-end configuration stabilized by disulfide bridges and incorporation of a J-chain. J-chains are also part of IgM pentamers and are discussed in more detail below.
  • Dimeric IgA produced locally at mucosal sites, is transported across the epithelial cell boundary and out into the secretions by interaction with the polymeric immunoglobulin receptor (plgR). During this process the plgR is cleaved and the major fragment, termed secretory component (SC), becomes covalently attached to the IgA dimer.
  • plgR polymeric immunoglobulin receptor
  • Both IgA and IgM possess an 18-amino acid extension in the C terminus called the “tail piece” (tp).
  • the IgA and IgM tail piece is highly conserved among various animal species. The conserved penultimate cysteine residue in the IgA and IgM tail pieces has been demonstrated to be involved in multimerization by forming a disulfide bond between heavy chains to permit formation of a multimer. Both tail pieces contain an N-linked carbohydrate addition site, the presence of which is required for dimer formation in IgA and J-chain incorporation and pentamer formation in IgM.
  • the structure and composition of the N-linked carbohydrates in the tail pieces differ, suggesting differences in the accessibility of the glycans to processing by glycosyltransferases.
  • the IgA (atp) and IgM (ptp) tail pieces differ at seven amino acid positions.
  • the human IgAl constant region typically includes the amino acid sequence: ASPTSPKVFPLSLCSTQPDGNWIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQ DASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPS TPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKS AVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGN TFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTW ASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAG KPTHVNVSVVMAEVDGTCY (SEQ ID NO: 1252).
  • the human CAI domain extends from amino acid 6 to amino acid 98; the human IgAl hinge region extends from amino acid 102 to amino acid 124, the human CA2 domain extends from amino acid 125 to amino acid 219, the human CA3 domain extends from amino acid 228 to amino acid 330, and the tailpiece extends from amino acid 331 to amino acid 352.
  • the human lgA2 constant region typically includes the amino acid sequence ASPTSPKVFPLSLDSTPQDGNWVACLVQGFFPQEPLSVTWSESGQNVTARNFPPS QDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCH PRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDL CGCYSVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLP PPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGT TTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSV VMAEVDGTCY(SEQ ID NO: 1253).
  • the human CAI domain extends from amino acid 6 to amino acid 98
  • the human lgA2 hinge region extends from amino acid 102 to amino acid 111
  • the human CA2 domain extends from amino acid 113 to amino acid 206
  • the human CA3 domain extends from amino acid 215 to amino acid 317
  • the tailpiece extends from amino acid 318 to amino acid 340.
  • two IgA binding units can form a complex with two additional polypeptide chains, the J-chain (e.g., the mature human J chain) and the secretory component to form a bivalent secretory IgA (slgA)-derived binding molecule.
  • a multimerizing dimeric IgA-derived binding molecule typically includes IgA constant regions that include at least the CA3 and tailpiece domains.
  • An engineered IgA heavy chain constant region can additionally include a CA2 domain or a fragment thereof, an IgA hinge region or fragment thereof, a CAI domain or a fragment thereof, and/or other IgA (or other immunoglobulin, e.g., IgG) heavy chain domains, including, e.g., an IgG hinge region.
  • a binding molecule as provided herein can include a complete IgA heavy chain constant domain (e.g., SEQ ID NO: 1252 or SEQ ID NO: 1253), or a variant, derivative, or analog thereof.
  • scFv dimers or diabodies may also be used, rather than whole antibodies.
  • Diabodies and scFv can be constructed without an Fc region, using only variable domains (usually including the variable domain components from both light and heavy chains of the source antibody), potentially reducing the effects of anti-idiotypic reaction.
  • Other forms of bispecific antibodies include the single chain “Janusins” described in Traunecker et al. (Embo lournal, 10, 3655-3659, 1991).
  • Dimers e.g., bispecific antibodies
  • US Patent No. 8,921 ,528 and US Patent Publication No. 2014/0308285.
  • dimers are similarly well-known in the art.
  • traditional production of full-length antibodies such as IgA antibodies
  • traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (see, for example, Millstein et al. Nature 305:37-39, 1983).
  • Similar procedures are disclosed in, for example, WO 1993/008829, Traunecker et al., EMBO I. 10:3655- 3659, 1991 and Holliger & Winter, Current Opinion Biotechnol. 4, 446-449 (1993).
  • dimers can be prepared using chemical linkage.
  • Brennan et al. (Science 229: 81, 1985) describes a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab’)2 fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize vicinal di thiols and prevent intermolecular disulfide formation.
  • the Fab’ fragments generated then are converted to thionitrobenzoate (TNB) derivatives.
  • One of the Fab’-TNB derivatives then is reconverted to the Fab’ -thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab’-TNB derivative to form the dimer.
  • Particular embodiments include IgM immunoglobulin constant region domains that allow the binding portion of molecules provided herein to readily multimerize into pentamers or hexamers.
  • IgM constant regions include IgM constant regions (or variants thereof). These embodiments have the ability to form hexamers, or in association with a J-chain, form pentamers.
  • Embodiments with an IgM constant region typically include at least the Cp4-tp domains of the IgM constant region but can include heavy chain constant region domains from other antibody isotypes, e.g., IgG, from the same species or from a different species.
  • one or more constant region domains can be deleted so long as the IgM antibody is capable of forming hexamers and/or pentamers.
  • an IgM antibody can be, e.g., a hybrid IgM/IgG antibody or can be a “multimerizing fragment” of an IgM-derived binding molecule.
  • a pentameric or hexameric IgM antibody described in this disclosure typically includes at least the Cp4 and/or tailpiece domains (also referred to herein collectively as Cp4-tp).
  • a “multimerizing fragment” of an IgM heavy chain constant region thus includes at least the Cp4-tp domains.
  • An IgM heavy chain constant region can additionally include a Cp3 domain or a fragment thereof, a Cp2 domain or a fragment thereof, a Cpl domain or a fragment thereof, and/or other IgM heavy chain domains.
  • IgM monomers form a complex with a J-chain to form a native IgM molecule.
  • the J-chain is considered to facilitate polymerization of chains before IgM is secreted from antibody producing cells.
  • Sequences for the human IGJ gene are known in the art, for example, (IGMT Accession: J00256, X86355, M25625, AJ879487).
  • the J chain establishes the disulfide bridges between IgM antibodies to form multimeric structures such as pentamers. See, for example, Sorensen et al. International Immunology, (2000), pages 19-27.
  • the Kabat numbering system for the human IgM constant domain can be found in Kabat, et. al. “Tabulation and Analysis of Amino acid and nucleic acid Sequences of Precursors, V- Regions, C-Regions, J-Chain, T-Cell Receptors for Antigen, T-Cell Surface Antigens, b-2 Microglobulins, Major Histocompatibility Antigens, Thy-I, Complement, C-Reactive Protein, Thymopoietin, Integrins, Post-gamma Globulin, a-2 Macroglobulins, and Other Related Proteins,” U.S. Dept of Health and Human Services (1991).
  • IgM constant regions can be numbered sequentially (i.e., amino acid #1 starting with the first amino acid of the constant region) or by using the Kabat numbering scheme.
  • a “full length IgM antibody heavy chain” is a polypeptide that includes, in N- terminal to C- terminal direction, an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CMl or Cpl), an antibody heavy chain constant domain 2 (CM2 or Cp2), an antibody heavy chain constant domain 3 (CM3 or Cp3), and an antibody heavy chain constant domain 4 (CM4 or Cp4) that can include a tailpiece, as indicated above.
  • VH antibody heavy chain variable domain
  • CMl or Cpl an antibody heavy chain constant domain 1
  • CM2 or Cp2 an antibody heavy chain constant domain 2
  • CM3 or Cp3 an antibody heavy chain constant domain 3
  • CM4 or Cp4 antibody heavy chain constant domain 4
  • each binding unit of a multimeric binding molecule as provided herein includes two IgM heavy chain constant regions or multimerizing fragments or variants thereof, each including at least an IgM Cp4 domain and an IgM tail piece domain.
  • the IgM heavy chain constant regions can each further include an IgM Cp3 domain situated N-terminal to the IgM Cp4 and IgM tail piece domains.
  • the IgM heavy chain constant regions can each further include an IgM Cp2 domain situated N-terminal to the IgM Cp3 domain.
  • Exemplary multimeric binding molecules provided herein include human IgM constant regions that include the wild-type human Cp2, Cp3, and Cp4-TP domains as follows: VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVT TDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMC VPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHT NISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALH RPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVSEEEWNTGETY
  • each IgM constant region can include, instead of, or in addition to an IgM Cp2 domain, an IgG hinge region or functional variant thereof situated N-terminal to the IgM Cp3 domain.
  • An exemplary variant human IgGl hinge region amino acid sequence in which the cysteine at position 6 is substituted with serine is VEPKSSDKTHTCPPCPAP (SEQ ID NO: 1255).
  • An exemplary IgM constant region of this type includes the variant human IgGl hinge region fused to a multimerizing fragment of the human IgM constant region including the Cp3, Cp4, and TP domains, and includes the amino acid sequence: VEPKSSDKTHTCPPCPAPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSV TISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDL PSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQW MQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEA LPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 1256).
  • the human IgM constant region typically includes the amino acid sequence GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFP SVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPP KVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQA EAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDT AIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHP NATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYL LPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQA PGRYFAHSILTVSEEEWNTGETYTCWAHEA
  • the human Cpl region ranges from amino acid 5 to amino acid 102; the human Cp2 region ranges from amino acid 114 to amino acid 205, the human Cp3 region ranges from amino acid 224 to amino acid 319, the Cp4 region ranges from amino acid 329 to amino acid 430, and the tailpiece ranges from amino acid 431 to amino acid 453.
  • an IgM heavy chain constant region includes the sequence:GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDI SSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPL PVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGV TTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSM CVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKT HTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVA LHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTS APMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVA
  • five IgM binding units can form a complex with a J-chain to form a pentameric IgM antibody.
  • the precursor form of the human J-chain includes: MKNHLLFWGVLAVFIKAVHVKAQEDERIVLVDNKCKCARITSRIIRSSEDPNEDI VERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSN ICDEDSATETCYTYDRNKC YTAVVPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 1259).
  • the signal peptide extends from amino acid 1 to amino acid 22 of SEQ ID NO: 1259 and the mature human J-chain extends from amino acid 23 to amino acid 159 of SEQ ID NO: 1259.
  • the mature human J-chain includes the amino acid sequence QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRT RFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAV VPLVYGGETKMVETALTPD ACYPD (SEQ ID NO: 1260).
  • J-chain refers to the J-chain of native sequence IgM or IgA antibodies of any animal species. When specified, it can also refer to any functional fragment thereof, derivative thereof, and/or variant thereof. Functional fragments, derivatives and variants retain the multimerizing functionality of the reference sequence and also include at least 90% or 95% sequence identity to the reference sequence.
  • An antibody binding domain can be introduced into the J-chain at any location that allows the binding of the binding domain to its binding target without interfering with J-chain function or the function of an associated IgA, IgM, or hybrid IgG antibody. Insertion locations include at or near the C- terminus, at or near the N-terminus or at an internal location that, based on the three- dimensional structure of the J-chain, is accessible.
  • the human lgG2 Fc region includes the amino acid sequence: PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTFRWSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PMLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1261).
  • the human CH2 region extends from amino acid 10 to amino acid 107 and the human CH3 region extends from amino acid 116 to amino acid 212.
  • the human lgG3 Fc region includes the amino acid sequence: PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVQFKWYVDGVEV HNAKTKPREEQFNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKT KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTT PPMLDSDGSFFLYSKLTVDKSRWQ QGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 1262).
  • the human CH2 region extends from amino acid 1 1 to amino acid 108 and the human CH3 region extends from amino acid 117 to amino acid 212.
  • the human lgG4 Fc region includes the amino acid sequence: PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 1263).
  • the human CH2 region extends from amino acid 1 to amino acid 111 and the human CH3 region extends from amino acid 112 to amino acid 218.
  • Particular embodiments include an IgG Fc region with an IgA or IgM tailpiece inserted into the IgG Fc region or fused to the C-terminus. J-chains can also be inserted or fused.
  • the PcrV-specific antibodies and/or binding proteins disclosed herein may be conjugated to an additional molecule.
  • exemplary molecules may include immunotoxins, drugs, radioisotopes, and detectable labels.
  • any of the antibodies and/or binding proteins described herein in any exemplary format can be formulated alone or in combination into compositions for administration to subjects. Salts and/or pro-drugs of the antibodies can also be used.
  • a pharmaceutically acceptable salt includes any salt that retains the activity of the antibody and is acceptable for pharmaceutical use.
  • a pharmaceutically acceptable salt also refers to any salt which may form in vivo as a result of administration of an acid, another salt, or a prodrug which is converted into an acid or salt.
  • a prodrug includes an active ingredient which is converted to a therapeutically active compound after administration, such as by cleavage or by hydrolysis of a biologically labile group.
  • the compositions include antibodies and/or binding proteins of at least 0.1 % w/v or w/w of the composition; at least 1 % w/v or w/w of composition; at least 10% w/v or w/w of composition; at least 20% w/v or w/w of composition; at least 30% w/v or w/w of composition; at least 40% w/v or w/w of composition; at least 50% w/v or w/w of composition; at least 60% w/v or w/w of composition; at least 70% w/v or w/w of composition; at least 80% w/v or w/w of composition; at least 90% w/v or w/w of composition; at least 95% w/v or w/w of composition; or at least 99% w/v or w/w of composition.
  • Exemplary generally used pharmaceutically acceptable carriers include any and all absorption delaying agents, antioxidants, binders, buffering agents, bulking agents or fillers, chelating agents, coatings, disintegration agents, dispersion media, gels, isotonic agents, lubricants, preservatives, salts, solvents or co-solvents, stabilizers, surfactants, and/or delivery vehicles.
  • antioxidants include ascorbic acid, methionine, and vitamin E.
  • Exemplary buffering agents include citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and/or trimethylamine salts.
  • An exemplary chelating agent is EDTA (ethylene-diamine-tetra-acetic acid).
  • Exemplary isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.
  • Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, and benzalkonium halides.
  • Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the antibodies or helps to prevent denaturation or adherence to the container wall.
  • Typical stabilizers can include polyhydric sugar alcohols, amino acids, organic sugars or sugar alcohols, PEG, amino acid polymers, sulfur-containing reducing agents, low molecular weight polypeptides (i.e., ⁇ 10 residues), proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins, hydrophilic polymers, monosaccharides, disaccharides, trisaccharides, and polysaccharides.
  • Cell-based compositions e.g., cells genetically modified to express a binding domain of an antibody disclosed herein, are also contemplated.
  • compositions disclosed herein can be formulated for administration by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion.
  • the compositions disclosed herein can further be formulated for intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral, sublingual, and/or subcutaneous administration.
  • compositions can be formulated as aqueous solutions, such as in buffers including Hanks’ solution, Ringer’s solution, or physiological saline.
  • the aqueous solutions can include formulatory agents such as suspending, stabilizing, and/or dispersing agents.
  • the formulation can be in lyophilized and/or powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • compositions can be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like.
  • Compositions can be formulated as an aerosol.
  • the aerosol is provided as part of an anhydrous, liquid, or dry powder inhaler. Aerosol sprays from pressurized packs or nebulizers can also be used with a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • compositions can also be formulated as depot preparations.
  • Depot preparations can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • compositions can be formulated as sustained-release systems utilizing semipermeable matrices of solid polymers including at least one type of antibody.
  • sustained-release materials have been established and are well known by those of ordinary skill in the art.
  • compositions disclosed herein can advantageously include any other pharmaceutically acceptable carriers which include those that do not produce significantly adverse, allergic, or other untoward reactions that outweigh the benefit of administration.
  • exemplary pharmaceutically acceptable carriers and formulations are disclosed in Remington’ s Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990.
  • formulations can be prepared to meet sterility, pyrogenicity, general safety, and purity standards as required by U.S. FDA Office of Biological Standards and/or other relevant foreign regulatory agencies.
  • Methods disclosed herein include treating subjects (e.g., humans, veterinary animals (dogs, cats, reptiles, birds) livestock (e.g., horses, cattle, goats, pigs, chickens) and research animals (e.g., monkeys, rats, mice, fish) with compositions disclosed herein. Treating subjects includes delivering therapeutically effective amounts. Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments, and/or therapeutic treatments.
  • an “effective amount” is the amount of a composition necessary to result in a desired physiological change in the subject. Effective amounts are often administered for research purposes. Effective amounts disclosed herein can cause a statistically- significant effect in an animal model or in vitro assay relevant to the assessment of an infection’s development, progression, and/or resolution.
  • prophylactic treatment includes a treatment administered to a subject who does not display signs or symptoms of an infection or displays only early signs or symptoms of an infection such that treatment is administered for the purpose of diminishing or decreasing the risk of developing the infection further.
  • a prophylactic treatment functions as a preventative treatment against an infection.
  • prophylactic treatments reduce, delay, or prevent the worsening of an infection.
  • a “therapeutic treatment” includes a treatment administered to a subject who displays symptoms or signs of an infection and is administered to the subject for the purpose of diminishing or eliminating those signs or symptoms of the infection.
  • the therapeutic treatment can reduce, control, or eliminate the presence or activity of the infection and/or reduce control or eliminate side effects of the infection.
  • prophylactic treatment or therapeutic treatment are not mutually exclusive, and in particular embodiments, administered dosages may accomplish more than one treatment type.
  • therapeutically effective amounts can be initially estimated based on results from in vitro assays and/or animal model studies. Such information can be used to more accurately determine useful doses in subjects of interest.
  • the actual dose amount administered to a particular subject can be determined by a physician, veterinarian or researcher taking into account parameters such as physical and physiological factors including target, body weight, severity of condition, type of infection, stage of infection, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.
  • Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly).
  • a treatment regimen e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly.
  • Biological samples can include any biological sample obtained from a subject from which B cells or B cell progenitor cells can be isolated and include bone marrow, spleen, lymph node, blood, e.g., peripheral blood, tissue biopsies or samples, surgical specimens, fine needle aspirates, autopsy material, and the like.
  • a biological sample refers to a sample isolated from a subject, such as a peripheral blood sample, which is then further processed, for example, by cell sorting (e.g., magnetic sorting and/or FACS), to obtain a population of antigen-specific IgM, IgG, or IgA memory B cells.
  • cell sorting e.g., magnetic sorting and/or FACS
  • a biological sample including IgM, IgG, or IgA memory B cells refers to an in vitro or ex vivo culture of expanded antigen- specific IgM, IgG, or IgA memory B cells.
  • Such a sample is enriched for antigen- specific IgM, IgG, or IgA memory B cells relative to the proportion of such cells that might occur in, e.g., a blood sample from a subject exposed to the antigen.
  • methods disclosed herein include a method of making an antibody construct including a PcrV antigen-binding domain from a memory B cell that specifically binds to a PcrV antigen of interest.
  • the method includes: (a) isolating a population of IgM, IgG, or IgA expressing memory B cells from a biological sample; (b) amplifying heavy chain and light chain variable domain sequences from the memory B cell population of step (a); (c) ligating heavy chain variable domain sequences amplified in step (b) into a heavy chain expression vector sequence and ligating light chain variable domain sequences amplified in step (b) into a light chain expression vector sequence; (d) introducing one or more vectors encoding heavy chain and light chain expression vector sequences of step (c) to a cell and culturing the cell under conditions that permit expression of antibody polypeptides from the expression vector sequences; (e) contacting antibody polypeptides expressed by the cell with an anti
  • the method further includes, prior to step (e), collecting cell culture medium from cells of step (d).
  • step (e) includes contacting antibody polypeptides with the PcrV antigen of interest immobilized on a solid support.
  • methods include sorting PcrV-specific IgM, IgG, or IgA memory B cells (MBCs), including: contacting a biological sample obtained from a subject infected with Pseudomonas aeruginosa with a tetramer (or other multimer) including a PcrV antigen; and sorting a cell population including PcrV-specific IgM, IgG, or IgA MBCs based on binding to the tetramer (or other multimer).
  • the method further includes generating tetramers or other multimers including PcrV antigens prior to the contacting step.
  • the methods further include a step of sequencing one or more BCRs, or at least the PcrV antigen-binding domains thereof, expressed by the cell population including IgM, IgG, or IgA MBCs.
  • Methods of sequencing are known in the art. See, for example, Schwartz et al. (2014). J Immunol 193, 3492-3502.
  • the methods further include a step of cloning the one or more BCRs and expressing the one or more BCRs as recombinant PcrV-specific antibodies or antigen-binding fragments thereof. See, for example, Tiller et al. (2009) J Immunol Methods 350, 183-193.
  • a multimer of a PcrV antigen of interest can be formed by labeling the PcrV antigen with a labeling system that allows multimerization and detection by flow cytometry.
  • a labeling system that allows multimerization and detection by flow cytometry.
  • PE phycoerythrin
  • tagged (e.g., His tagged) recombinant PcrV antigen can be produced and purified using the tag.
  • the purified recombinant antigen can be biotinylated and tetramerized with streptavidin-PE (e.g., from Prozyme, Agilent Technologies, Santa Clara, CA) as described in Taylor et al. (2012).
  • B cells binding PcrV antigen in the tetramers can be enriched using anti-PE magnetic microbeads.
  • a decoy reagent to gate out non-antigen-specific B cells e.g., B cells binding to non-antigen tetramer components such as PE, streptavidin, and biotin epitopes
  • B cells binding to non-antigen tetramer components such as PE, streptavidin, and biotin epitopes
  • AF647 Alexa Fluor 647
  • AF647 protein labeling kit ThermoFisher Scientific, Waltham, MA
  • splenocytes can first be stained with a decoy reagent to exclude cells binding other components of the tetramer and then with an antigen PE tetramer (Taylor et al. (2012). J Exp Med 209, 2065-2077).
  • Anti-PE coated magnetic beads can be used to enrich both decoy-specific and PcrV antigen-specific B cells, which can subsequently be stained with antibodies for analysis by multiparameter flow cytometry.
  • antibody panels can be used that allow visualization of all stages of mature B2 B cell differentiation.
  • Control experiments can be conducted using B cells with BCRs that do not bind the PcrV antigen tetramer and to assess that they are not activated non-specifically by PcrV antigen.
  • compositions including a population of PcrV antigen-specific IgM, IgG, or IgA memory B cells bound via their B cell receptors to PcrV antigen immobilized on a solid support.
  • the PcrV antigen immobilized on the solid support includes a multimer construct including the PcrV antigen.
  • compositions described herein selectively bind the PcrV antigen of interest.
  • Methods of measuring binding of a polypeptide to an antigen are known in the art (e.g., enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immune absorbent spot (ELISPOT) assay).
  • ELISA enzyme-linked immunosorbent assay
  • ELISPOT enzyme-linked immune absorbent spot
  • Another aspect of the present disclosure describes a method of detecting the presence of a PA PcrV antigen in a solution or on a cell.
  • the method involves providing a binding protein or an antibody described herein to the solution or cell and measuring the ability of the protein to bind to the PA PcrV antigen in the solution or cell. Measurements can be quantitative or qualitative.
  • Another aspect of the present disclosure describes a cell line producing a binding protein disclosed herein.
  • PBMCs Peripheral mononuclear blood cells
  • STMELL Technologies SepMate-50 PBMC Isolation Tubes
  • Non-CF donor samples were provided by BloodWorks Northwest from regular blood donors or from non-mobilized healthy donors through the Fred Hutch Hematopoietic Cell Procurement and Processing core.
  • PcrV Recombinant PcrV was expressed using E. coli transformed with low copy plasmid containing the PcrV CDS (GenBank: AF010149.1) and a 10 X N terminal His tag and purified from filtered bacterial supernatant on a His affinity column.
  • PcrV was biotinylated using EZ-Link Sulfo-NHS-LC Biotinylation Kit (ThermoFisher). The production of antigen-specific B cell tetramer reagents has been previously described in some detail.
  • biotinylated PcrV was co-incubated with streptavidin-fluorophore (SA-PE or SA-APC; Agilent).
  • SA-PE or SA-APC streptavidin-fluorophore
  • Decoy tetramers are produced by tetramerization of an irrelevant protein with a matched conjugated fluorophore (e.g., PE- Cy7 for use in experiments requiring the PE-conjugated PcrV tetramer).
  • cDNA was amplified from singly sorted B cells using SMART-Seq v4 (Takara Bio) at half reaction volumes.
  • Initial BCR sequencing for donor 1 followed protocols previously described in detail in Hale, Malika et al. “IgM antibodies derived from memory B cells are potent cross-variant neutralizers of SARS-CoV-2.”
  • each light chain was cloned into vectors of its isotype, K or X, following the manufacturer’s protocol for in-fusion cloning (Takara Bio). All heavy chains were similarly cloned into IgGl and IgM plasmids in parallel. Concordance with the parental cDNA was confirmed by Sanger sequencing of the cloned plasmids.
  • V2L2MD IgG and IgM mAbs heavy and light chain sequences were synthesized as a gBlock (IDT) by introducing mutations to match the amino acid sequence for the anti-PcrV VH/VL in gremubamab (INN 10909; 17) in the closest human VH and VL nucleotide sequences.
  • the resultant V(D)J sequences were then synthesized as a gBlock (IDT) and then cloned into expression plasmids using the same methods described for generation of our human B cell BCR sequences.
  • IgG mAbs were produced in HEK-293T cells (ATCC) by co-transfection of heavy- and light-chains in polyethylenimine as previously described in Thouvenel, Christopher D et al. “Multimeric antibodies from antigen- specific human IgM-i- memory B cells restrict Plasmodium parasites.” The Journal of experimental medicine vol. 218,4 (2021): e20200942. doi:10.1084/jem.20200942, incorporated herein by reference. Production of IgM mAbs was carried out as described in Hale et al., using the same human J chain plasmid.
  • mice All animal procedures were conducted according to the guidelines of the Emory University Institutional Animal Care and Use Committee (IACUC), under approved protocol number PROTO 201700441. The study was carried out in strict accordance with established guidelines and policies at Emory University School of Medicine, and recommendations in the Guide for Care and Use of Laboratory Animals, as well as local, state, and federal laws. Eight- to ten- week-old female BALB/c mice (Jackson Laboratories, Bar Harbor, ME) were anesthetized by intraperitoneal injection of 0.2 ml of a cocktail of ketamine (100 mg/ml) and xylazine (5 mg/ml) and intranasally instilled with approximately 10 5 CFU P.
  • IACUC Emory University Institutional Animal Care and Use Committee
  • aeruginosa PA103 in 10-20 pL of PBS.
  • monoclonal antibodies or PBS were delivered via the same route in a 20 pL volume.
  • Mice were euthanized at 24 hours post-infection, and whole lungs were collected aseptically, weighed, and homogenized for 20 seconds in 1 ml of PBS, followed by serial dilution onto DifcoTM Pseudomonas isolation agar, and plated for CFU enumeration.
  • PcrV-specific B cells are enriched in CF individuals
  • Peripheral blood samples were obtained from a cohort of 14 young adults with CF who had received a swab test for PA as part of routine outpatient care. As B cells specific for any single antigen are rare, tetramer-bound cells were enriched via an anti- fluorophore magnetic column prior to analysis. After enrichment, PcrV-specific cells made up > 5% of B cells in 7 of 14 (50%) of CF donors, while 0 of 14 (0%) samples obtained from non-CF donors (isolated from a local blood bank) exhibited binding (FIG. IB). Similar differences between CF vs. non-CF donors were obtained when the number of PcrV-specific B cells in each sample was normalized to lymphocyte count (FIG. 1C).
  • the light chain for BCR 421 contains several mutations in CDR1. A strong binding by germline antibody sequences 408 and 411 was observed. The source cells for 411 and 421, but not 408, expressed a surface marker suggestive of a plasmablast/plasma cell phenotype (CD38 + ).
  • the inventors have previously found that for mAbs that target divergent pathogens, including SARS-CoV-2 and Plasmodium falciparum, multimerized antibodies (e.g., pentameric IgM) have enhanced binding and protection properties in vitro.
  • multimerized antibodies e.g., pentameric IgM
  • IgGl isotype
  • the activity of these mAbs in both the IgM and IgGl formats was next compared and expression plasmids containing the heavy chain variable regions upstream of the gammal constant region to enable expression as IgGl mAbs were generated.
  • V2L2MD is a heavily engineered anti-PcrV mAb that was generated by phage display binding optimization from a pre-cursor candidate identified by hybridoma screening from PcrV-immunized, human-variable region transgenic mice.
  • the clinical candidate bi-specific, gremubamab consists of the paired heavy-and light chain variable regions of V2L2MD fused to an anti-polys accharide (Psi) single chain variable fragment (scFv) and the human IgGl constant region.
  • V2L2MD anti-PcrV variable regions
  • FIG. 4A a challenge model of pneumonia was utilized to test the anti-PA activity of candidate mAbs.
  • Mice treated with a single, 60 pg intranasal dose of anti-PcrV IgG exhibited an ⁇ 2 log reduction in burden of bacteria in the lungs at 48 h in comparison with an off-target control mAb or vehicle (PBS) only (FIG. 4B).
  • PBS off-target control mAb or vehicle
  • FIG. 4C A similarly dramatic reduction in lung bacteria load was also achieved when the experiment was repeated with a 3 -fold lower dose of anti-PcrV IgG.
  • the CF-derived mAbs performed equivalently to the positive control V2L2MD mAb at both doses. While assessment of lung bacterial burden requires sacrifice of the treated mice, doses achieving similar reductions in lung bacterial burdens have resulted in 100% survival in cohorts of mice in prior studies of V2L2MD.
  • peripheral blood samples from 2 additional donors with CF were obtained: Donor 2, had previously tested positive for PA but was negative on most recent testing, while Donor 3, like Donor 1, was chronically infected.
  • Donor 2 had previously tested positive for PA but was negative on most recent testing, while Donor 3, like Donor 1, was chronically infected.
  • single cell BCR sequencing of PcrV-specific B cells from each donor was performed. Strikingly, Donor 2, had abundant PcrV-specific MBCs after tetramer enrichment. Of the ⁇ 40 sequences from PcrV-specific cells identified by surface phenotype as likely MBCs (CD21 + CD27 + ), many were somatically hypermutated (FIG. 5A). In contrast, Donor 3 had fewer MBCs and fewer somatically hypermutated cells (FIG. 7A).
  • BCRs 435 and 442 were the most striking binders in the transfection screen. Because the initial screen did not adequately control for antibody concentration in the supernatant, antibodies that are less efficiently expressed by 293T cells might appear to be poor binders.
  • 5 MBC- derived BCRs were chosen for production as purified IgG mAbs.
  • this panel of purified mAbs was evaluated at matched concentrations (FIG. 5C), 2 of 5 of MBC-derived mAbs matched or exceeded PcrV binding exhibited by the protective, donor 1 (plasmablast derived) mAb, 411. Further, 4 of 5 (including the IgA-derived mAb, 439) matched or exceeded the other protective donor- 1 derived protective mAb, 408 IgG.
  • CF MBC-derived mAbs 437 originally derived from an IgG MBC
  • 439 derived from an IgA MBC
  • 442 derived from an IgM MBC
  • each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component.
  • the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.”
  • the transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts.
  • the transitional phrase “consisting of’ excludes any element, step, ingredient, or component not specified.
  • the transition phrase “consisting essentially of’ limits the scope of the embodiment to the specified elements, steps, ingredients, or components and to those that do not materially affect the embodiment. A material effect would cause a statistically significant reduction in the ability to obtain a claimed effect according to a relevant experimental method described in the current disclosure.
  • the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ⁇ 20% of the stated value; ⁇ 19% of the stated value; ⁇ 18% of the stated value; ⁇ 17% of the stated value; ⁇ 16% of the stated value; ⁇ 15% of the stated value; ⁇ 14% of the stated value; ⁇ 13% of the stated value; ⁇ 12% of the stated value; ⁇ 11% of the stated value; ⁇ 10% of the stated value; ⁇ 9% of the stated value; ⁇ 8% of the stated value; ⁇ 7% of the stated value; ⁇ 6% of the stated value; ⁇ 5% of the stated value; ⁇ 4% of the stated value; ⁇ 3% of the stated value; ⁇ 2% of the stated value; or ⁇ 1% of the stated value.

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Abstract

The present disclosure provides Pseudomonas aeruginosa (PA) neutralizing binding proteins or antibodies or derivatives thereof. The binding proteins of the present disclosure are specific for and bind to a wide range of epitopes present on the PA virulence factor, Pseudomonas aeruginosa V-antigen (PcrV) antigen. In an embodiment, the binding proteins or antibodies or derivatives thereof disclosed herein exhibit protective, neutralizing activity against P. aeruginosa. The binding proteins or antibodies or derivatives thereof disclosed herein have a high affinity to a wide range of PcrV epitopes. In an embodiment, the binding proteins disclosed herein are effective in limiting a PA infection.

Description

PROTECTIVE MONOCLONAL ANTIBODIES TO PSEUDOMONAS AERUGINOSA
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 63/605,366, filed December 1, 2023.
STATEMENT REGARDING SEQUENCE LISTING
[0002] The Sequence Listing XML associated with this application is provided in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the sequence listing is 3915-P1318WO_UW_SL.xml. The XML file is 1,436,278 bytes; was created on November 13, 2024; and is being submitted electronically via Patent Center with the filing of the specification.
BACKGROUND
[0003] Pseudomonas aeruginosa (PA) is a ubiquitous, gram-negative bacteria responsible for significant morbidity and mortality in vulnerable individuals. Treatment is challenging because of intrinsic and acquired antibiotic resistance to most antibiotic drug classes. PA is one of the most common pathogens in severe healthcare associated infections. Due to the significant mortality caused by multi-drug-resistant strains and the lack of alternative therapies, new anti-pseudomonal therapeutics are urgently needed.
SUMMARY
[0004] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
[0005] The present disclosure provides PA neutralizing binding proteins or antibodies, fragments thereof, or derivatives thereof. The binding proteins of the present disclosure are specific for and bind to a wide range of epitopes present on the PA virulence factor, P. aeruginosa V-antigen (PcrV) antigen. In an embodiment, the binding proteins and/or antibodies and/or derivatives thereof disclosed herein exhibit protective, neutralizing activity against P. aeruginosa. In an embodiment, the binding proteins disclosed herein have a high affinity to a range of PcrV epitopes. In an embodiment, the binding proteins disclosed herein are effective in limiting a PA infection. [0006] The present disclosure also provides variants of antibodies or binding proteins described herein. Variants can include those having one or more conservative amino acid substitutions or one or more non-conservative substitutions that do not adversely affect the binding of the antibody and/or binding protein with the PcrV antigen.
[0007] In one aspect, the present disclosure provides a binding protein comprising: a heavy chain variable domain (VH), the VH comprising a complementarity determining region (CDR), CDR3 amino acid sequence; and a human light chain variable domain (VL) comprising a CDR3 amino acid sequence.
[0008] In some embodiments, the heavy chain variable domain (VH) CDR3 amino acid sequence is at least 85% identical to an amino acid sequence set forth in SEQ ID NO: 4, 11, 18, 25, 32, 39, 46, 53, 60, 67, 74, 81, 88, 95, 102, 109, 116, 123, 130, 137, 144, 151, 159, 167, 174, 181, 188, 195, 202, 209, 216, 223, 230, 237, 244, 251, 258, 265, 272, 279,
286, 293, 300, 307, 314, 321, 328, 335, 342, 349, 356, 363, 370, 377, 385, 391, 398, 405,
412, 419, 426, 433,440, 447, 454, 461, 468, 475, 482, 489, 496, 503, 510, 517, 524, 531,
538, 545, 552, 559, 566, 573, 580, 587, 594, 601, 608, 615, 622, 629, 636, 643, 650, 657,
664, 671, 678, 685, 692, 699, 706, 713, 720, 727, 734, 741, 748, 755, 763, 770, 777, 784,
791, 798, 805, 812, 819, 826, 833, 840, 847, 854, 861, 868, 875, 883, 890, 897, 904, 911,
918, 925, 932, 939, 946, 953, 960, 967, 974, 981, 988, 995, 1002, 1009, 1016, 1023, 1031, 1038, 1045, 1052, 1059, 1066, 1073, 1080, 1087, 1094, 1101, 1108, 1115, 1122, 1129,
1136, 1143, 1150, 1157, 1164, 1171 , 1178, 1185, 1 192, 1199, 1206, 1213, 1220, 1227,
1234, 1241, 1248, 1494, 1496, 1498, 1500, 1502, 1506, 1508, 1510, 1512, 1514, 1516,
1518, 1522, 1526, 1528, 1530, 1532, 1534, 1536, 1538, 1540, 1542, 1544, 1546, 1548,
1550, 1552, 1554, 1556, 1558, 1560, 1564, 1566, 1568, 1570, 1572, 1574, 1576, 1578,
1580, or 1584.
[0009] In some embodiments, the human light chain variable domain (VL) CDR3 amino acid sequence is at least 85% identical to an amino acid sequence set forth in SEQ ID NO: 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 105, 112, 119, 126, 133, 140, 147, 155, 163, 170, 177, 184, 191, 198, 205, 212, 219, 226, 233, 240, 247, 254, 261, 268,
275, 282, 289, 296, 303, 310, 317, 324, 331, 338, 345, 352, 359, 366, 373, 380, 387, 394,
401, 408, 415, 422, 429, 436, 443, 450, 457, 464, 471,478, 485,492, 499, 506, 513, 520,
527, 534, 541, 548, 555, 562, 569, 576, 583, 590, 597, 604, 611, 618, 625, 632, 639, 646,
653, 660, 667, 674, 681, 688, 695, 702, 709, 716, 723, 730, 737, 744, 751, 759, 766, 773,
780, 787, 794, 801, 808, 815, 822, 829, 836, 843, 850, 857, 864, 871, 879, 886, 893, 900, 907, 914, 921, 928, 935, 942, 949, 956, 963, 970, 977, 984, 991, 998, 1005, 1012, 1019, 1027, 1034, 1041, 1048, 1055, 1062, 1069, 1076, 1083, 1090, 1097, 1104, 1111, 1118,
1125, 1132, 1139, 1146, 1153, 1160, 1167, 1174, 1 181 , 1188, 1195, 1202, 1209, 1216,
1223, 1230, 1237, 1244, 1251, 1495, 1497, 1499, 1501, 1503, 1505, 1507, 1509, 1511,
1513, 1515, 1517, 1519, 1521, 1523, 1527, 1529, 1531, 1532, 1533, 1537, 1539, 1541,
1543, 1545, 1547, 1549, 1553, 1557, 1561, 1563, 1565, 1567, 1569, 1571, 1575, 1577,
1579, 1581, 1583, or 1585. In some embodiments, the binding protein is capable of specifically binding to an epitope on Pseudomonas aeruginosa V-antigen (PcrV).
[0010] In some embodiments, the heavy chain variable domain (VH) comprises a CDR3 amino acid sequence of SEQ ID NO: 4, 11, 18, 25, 32, 39, 46, 53, 60, 67, 74, 81, 88, 95, 102, 109, 116, 123, 130, 137, 144, 151 , 159, 167, 174, 181, 188, 195, 202, 209, 216, 223, 230, 237, 244, 251, 258, 265, 272, 279, 286, 293, 300, 307, 314, 321, 328, 335, 342, 349, 356, 363, 370, 377, 385, 391, 398, 405, 412, 419, 426, 433,440, 447, 454, 461, 468, 475, 482, 489, 496, 503, 510, 517, 524, 531, 538, 545, 552, 559, 566, 573, 580, 587, 594,
601, 608, 615, 622, 629, 636, 643, 650, 657, 664, 671, 678, 685, 692, 699, 706, 713, 720,
727, 734, 741, 748, 755, 763, 770, 777, 784, 791, 798, 805, 812, 819, 826, 833, 840, 847,
854, 861, 868, 875, 883, 890, 897, 904, 911, 918, 925, 932, 939, 946, 953, 960, 967, 974,
981, 988, 995, 1002, 1009, 1016, 1023, 1031, 1038, 1045, 1052, 1059, 1066, 1073, 1080, 1087, 1094, 1101, 1108, 1115, 1122, 1129, 1136, 1143, 1150, 1157, 1164, 1171, 1178,
1185, 1192, 1199, 1206, 1213, 1220, 1227, 1234, 1241 , 1248, 1494, 1496, 1498, 1500,
1502, 1506, 1508, 1510, 1512, 1514, 1516, 1518, 1522, 1526, 1528, 1530, 1532, 1534,
1536, 1538, 1540, 1542, 1544, 1546, 1548, 1550, 1552, 1554, 1556, 1558, 1560, 1564,
1566, 1568, 1570, 1572, 1574, 1576, 1578, 1580, or 1584.
[0011] In some embodiments, the human light chain variable domain (VL) comprises a CDR3 amino acid sequence of SEQ ID NO: 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 105, 112, 119, 126, 133, 140, 147, 155, 163, 170, 177, 184, 191, 198, 205, 212, 219, 226, 233, 240, 247, 254, 261, 268, 275, 282, 289, 296, 303, 310, 317, 324,
331, 338, 345, 352, 359, 366, 373, 380, 387, 394, 401, 408, 415, 422, 429, 436, 443, 450,
457, 464, 471,478, 485,492, 499, 506, 513, 520, 527, 534, 541, 548, 555, 562, 569, 576,
583, 590, 597, 604, 611, 618, 625, 632, 639, 646, 653, 660, 667, 674, 681, 688, 695, 702,
709, 716, 723, 730, 737, 744, 751, 759, 766, 773, 780, 787, 794, 801, 808, 815, 822, 829,
836, 843, 850, 857, 864, 871, 879, 886, 893, 900, 907, 914, 921, 928, 935, 942, 949, 956,
963, 970, 977, 984, 991, 998, 1005, 1012, 1019, 1027, 1034, 1041, 1048, 1055, 1062, 1069, 1076, 1083, 1090, 1097, 1104, 1111, 1118, 1125, 1132, 1139, 1146, 1153, 1160, 1167,
1174, 1181, 1188, 1195, 1202, 1209, 1216, 1223, 1230, 1237, 1244, 1251, 1495, 1497,
1499, 1501, 1503, 1505, 1507, 1509, 1511, 1513, 1515, 1517, 1519, 1521, 1523, 1527,
1529, 1531, 1532, 1533, 1537, 1539, 1541, 1543, 1545, 1547, 1549, 1553, 1557, 1561,
1563, 1565, 1567, 1569, 1571, 1575, 1577, 1579, 1581, 1583, or 1585. In some embodiments, the binding protein is capable of specifically binding to an epitope on Pseudomonas aeruginosa V-antigen (PcrV).
[0012] In some embodiments, the VH has at least 85% sequence identity to a sequence set forth in an (a) in a pair below and the VL has at least 85% sequence identity to a sequence set forth in (b) of the same pair. In an embodiment, the pairs have the sequences set forth in:
[0013] (a) SEQ ID NO: 1 and (b) SEQ ID NO: 5; (a) SEQ ID NO: 8 and (b) SEQ ID NO: 12; (a) SEQ ID NO: 15 and (b) SEQ ID NO: 19; (a) SEQ ID NO: 22 and (b) SEQ
ID NO: 26; (a) SEQ ID NO: 29 and (b) SEQ ID NO: 33; (a) SEQ ID NO: 36 and (b) SEQ
ID NO: 40; (a) SEQ ID NO: 43 and (b) SEQ ID NO: 47; (a) SEQ ID NO: 50 and (b) SEQ
ID NO: 54; (a) SEQ ID NO: 57 and (b) SEQ ID NO: 61 ; (a) SEQ ID NO: 64 and (b) SEQ
ID NO: 68; (a) SEQ ID NO: 71 and (b) SEQ ID NO: 75; (a) SEQ ID NO: 78 and (b)
SEQ ID NO: 82; (a) SEQ ID NO: 85 and (b) SEQ ID NO: 89; (a) SEQ ID NO: 92 and (b) SEQ ID NO: 96; (a) SEQ ID NO: 99 and (b) SEQ ID NO: 103; (a) SEQ ID NO: 106 and (b) SEQ ID NO: 110; (a) SEQ ID NO: 113 and (b) SEQ ID NO: 117; (a) SEQ ID NO: 120 and (b) SEQ ID NO: 124; (a) SEQ ID NO: 127 and (b) SEQ ID NO: 131; (a) SEQ ID NO: 134 and (b) SEQ ID NO: 138; (a) SEQ ID NO: 141 and (b) SEQ ID NO: 145; (a) SEQ ID NO: 148 and (b) SEQ ID NO: 152; (a) SEQ ID NO: 156 and (b) SEQ ID NO: 160; (a) SEQ ID NO: 164 and (b) SEQ ID NO: 168; (a) SEQ ID NO: 171 and (b) SEQ ID NO: 175; (a) SEQ ID NO: 178 and (b) SEQ ID NO: 182; (a) SEQ ID NO: 185 and (b) SEQ ID NO: 189;
(a) SEQ ID NO: 192 and (b) SEQ ID NO: 196; (a) SEQ ID NO: 199 and (b) SEQ ID NO: 203; (a) SEQ ID NO: 206 and (b) SEQ ID NO: 210; (a) SEQ ID NO: 213 and (b) SEQ ID NO: 217; (a) SEQ ID NO: 220 and (b) SEQ ID NO: 224; (a) SEQ ID NO: 227 and (b) SEQ ID NO: 231 ; (a) SEQ ID NO: 234 and (b) SEQ ID NO: 238; (a) SEQ ID NO: 241 and (b) SEQ ID NO: 245; (a) SEQ ID NO: 248 and (b) SEQ ID NO: 252; SEQ ID NO: 255 and
(b) SEQ ID NO: 259 (a) SEQ ID NO: 262 and (b) SEQ ID NO: 266; (a) SEQ ID NO: 269 and (b) SEQ ID NO: 273; (a) SEQ ID NO: 276 and (b) SEQ ID NO: 280; (a) SEQ ID NO: 283 and (b) SEQ ID NO: 287; (a) SEQ ID NO: 290 and (b) SEQ ID NO: 294; (a) SEQ ID NO: 297 and (b) SEQ ID NO: 301 ; (a) SEQ ID NO: 304 and (b) SEQ ID NO: 308; (a) SEQ ID NO: 311 and (b) SEQ ID NO: 315; (a) SEQ ID NO: 318 and (b) SEQ ID NO: 322; (a) SEQ ID NO: 325 and (b) SEQ ID NO: 329; (a) SEQ ID NO: 332 and (b) SEQ ID NO: 336;
(a) SEQ ID NO: 339 and (b) SEQ ID NO: 343; (a) SEQ ID NO: 346 and (b) SEQ ID NO: 350; (a) SEQ ID NO: 353 and (b) SEQ ID NO: 357; (a) SEQ ID NO: 360 and (b) SEQ ID NO: 364; (a) SEQ ID NO: 367 and (b) SEQ ID NO: 371; (a) SEQ ID NO: 374 and (b) SEQ ID NO: 378; (a) SEQ ID NO: 381 and (b) SEQ ID NO: 385; (a) SEQ ID NO: 388 and (b) SEQ ID NO: 392; (a) SEQ ID NO: 395 and (b) SEQ ID NO: 399; (a) SEQ ID NO: 402 and
(b) SEQ ID NO: 406; (a) SEQ ID NO: 409 and (b) SEQ ID NO: 413; (a) SEQ ID NO: 416 and (b) SEQ ID NO: 420; (a) SEQ ID NO: 423 and (b) SEQ ID NO: 427; (a) SEQ ID NO: 430 and (b) SEQ ID NO: 434; (a) SEQ ID NO: 437 and (b) SEQ ID NO: 441; (a) SEQ ID NO: 444 and (b) SEQ ID NO: 448; (a) SEQ ID NO: 451 and (b) SEQ ID NO: 455; (a) SEQ ID NO: 458 and (b) SEQ ID NO: 462; (a) SEQ ID NO: 465 and (b) SEQ ID NO: 469; (a) SEQ ID NO: 472 and (b) SEQ ID NO: 476; (a) SEQ ID NO: 479 and (b) SEQ ID NO: 483;
(a) SEQ ID NO: 486 and (b) SEQ ID NO: 490; (a) SEQ ID NO: 493 and (b) SEQ ID NO: 497; (a) SEQ ID NO: 500 and (b) SEQ ID NO: 504; (a) SEQ ID NO: 507 and (b) SEQ ID NO: 511 ; (a) SEQ ID NO: 514 and (b) SEQ ID NO: 518; (a) SEQ ID NO: 521 and (b) SEQ ID NO: 525; (a) SEQ ID NO: 528 and (b) SEQ ID NO: 532; (a) SEQ ID NO: 535 and (b) SEQ ID NO: 539; (a) SEQ ID NO: 542 and (b) SEQ ID NO: 546; (a) SEQ ID NO: 549 and
(b) SEQ ID NO: 553; (a) SEQ ID NO: 556 and (b) SEQ ID NO: 560; (a) SEQ ID NO: 563 and (b) SEQ ID NO: 567; (a) SEQ ID NO: 570 and (b) SEQ ID NO: 574; (a) SEQ ID NO: 577 and (b) SEQ ID NO: 581; (a) SEQ ID NO: 584 and (b) SEQ ID NO: 588; (a) SEQ ID NO: 591 and (b) SEQ ID NO: 595; (a) SEQ ID NO: 598 and (b) SEQ ID NO: 602; (a) SEQ ID NO: 605 and (b) SEQ ID NO: 609; (a) SEQ ID NO: 626 and (b) SEQ ID NO: 630; (a) SEQ ID NO: 633 and (b) SEQ ID NO: 637; (a) SEQ ID NO: 640 and (b) SEQ ID NO: 644;
(a) SEQ ID NO: 647 and (b) SEQ ID NO: 651; (a) SEQ ID NO: 654 and (b) SEQ ID NO: 658; (a) SEQ ID NO: 661 and (b) SEQ ID NO: 665; (a) SEQ ID NO: 668 and (b) SEQ ID NO: 672; (a) SEQ ID NO: 675 and (b) SEQ ID NO: 679; (a) SEQ ID NO: 682 and (b) SEQ ID NO: 686; (a) SEQ ID NO: 689 and (b) SEQ ID NO: 693; (a) SEQ ID NO: 696 and (b) SEQ ID NO: 700; (a) SEQ ID NO: 703 and (b) SEQ ID NO: 707; (a) SEQ ID NO: 710 and
(b) SEQ ID NO: 714; (a) SEQ ID NO: 717 and (b) SEQ ID NO: 721; (a) SEQ ID NO: 724 and (b) SEQ ID NO: 728; (a) SEQ ID NO: 731 and (b) SEQ ID NO: 735; (a) SEQ ID NO: 738 and (b) SEQ ID NO: 742; (a) SEQ ID NO: 745 and (b) SEQ ID NO: 749; (a) SEQ ID NO: 752 and (b) SEQ ID NO: 756; (a) SEQ ID NO: 760 and (b) SEQ ID NO: 764; (a) SEQ ID NO: 767 and (b) SEQ ID NO: 771; (a) SEQ ID NO: 774 and (b) SEQ ID NO: 778; (a) SEQ ID NO: 781 and (b) SEQ ID NO: 785; (a) SEQ ID NO: 788 and (b) SEQ ID NO: 792;
(a) SEQ ID NO: 795 and (b) SEQ ID NO: 799; (a) SEQ ID NO: 802 and (b) SEQ ID NO: 806; (a) SEQ ID NO: 809 and (b) SEQ ID NO: 813; (a) SEQ ID NO: 816 and (b) SEQ ID NO: 820; (a) SEQ ID NO: 823 and (b) SEQ ID NO: 827; (a) SEQ ID NO: 830 and (b) SEQ ID NO: 834; (a) SEQ ID NO: 837 and (b) SEQ ID NO: 841; (a) SEQ ID NO: 844 and (b) SEQ ID NO: 848; (a) SEQ ID NO: 851 and (b) SEQ ID NO: 855; (a) SEQ ID NO: 858 and
(b) SEQ ID NO: 862; (a) SEQ ID NO: 865 and (b) SEQ ID NO: 869; (a) SEQ ID NO: 872 and (b) SEQ ID NO: 876; (a) SEQ ID NO: 880 and (b) SEQ ID NO: 884; (a) SEQ ID NO: 887 and (b) SEQ ID NO: 889; (a) SEQ ID NO: 894 and (b) SEQ ID NO: 898; (a) SEQ ID NO: 901 and (b) SEQ ID NO: 905; (a) SEQ ID NO: 908 and (b) SEQ ID NO: 912; (a) SEQ ID NO: 915 and (b) SEQ ID NO: 919; (a) SEQ ID NO: 922 and (b) SEQ ID NO: 926; (a) SEQ ID NO: 929 and (b) SEQ ID NO: 933; (a) SEQ ID NO: 936 and (b) SEQ ID NO: 940;
(a) SEQ ID NO: 943 and (b) SEQ ID NO: 947; (a) SEQ ID NO: 950 and (b) SEQ ID NO: 954; (a) SEQ ID NO: 957 and (b) SEQ ID NO: 961; (a) SEQ ID NO: 964 and (b) SEQ ID NO: 968; (a) SEQ ID NO: 971 and (b) SEQ ID NO: 975; (a) SEQ ID NO: 978 and (b) SEQ ID NO: 982; (a) SEQ ID NO: 985 and (b) SEQ ID NO: 989; (a) SEQ ID NO: 992 and (b) SEQ ID NO: 996; (a) SEQ ID NO: 999 and (b) SEQ ID NO: 1003; (a) SEQ ID NO: 1006 and (b) SEQ ID NO: 1010; (a) SEQ ID NO: 1013 and (b) SEQ ID NO: 1017; (a) SEQ ID NO: 1020 and (b) SEQ ID NO: 1024; (a) SEQ ID NO: 1028 and (b) SEQ ID NO: 1032; (a) SEQ ID NO: 1035 and (b) SEQ ID NO: 1039; (a) SEQ ID NO: 1042 and (b) SEQ ID NO: 1046; (a) SEQ ID NO: 1049 and (b) SEQ ID NO: 1053; (a) SEQ ID NO: 1056 and (b) SEQ ID NO: 1060; (a) SEQ ID NO: 1063 and (b) SEQ ID NO: 1067; (a) SEQ ID NO: 1070 and
(b) SEQ ID NO: 1074; (a) SEQ ID NO: 1077 and (b) SEQ ID NO: 1081; (a) SEQ ID NO: 1084 and (b) SEQ ID NO: 1088; (a) SEQ ID NO: 1091 and (b) SEQ ID NO: 1095; (a) SEQ ID NO: 1098 and (b) SEQ ID NO: 1102; (a) SEQ ID NO: 1105 and (b) SEQ ID NO: 1109; (a) SEQ ID NO: 1112 and (b) SEQ ID NO: 1116; (a) SEQ ID NO: 1119 and (b) SEQ ID NO: 1123; (a) SEQ ID NO: 1126 and (b) SEQ ID NO: 1130; (a) SEQ ID NO: 1133 and (b) SEQ ID NO: 1137; (a) SEQ ID NO: 1140 and (b) SEQ ID NO: 1144; (a) SEQ ID NO: 1147 and (b) SEQ ID NO: 1151; (a) SEQ ID NO: 1154 and (b) SEQ ID NO: 1158; (a) SEQ ID NO: 1161 and (b) SEQ ID NO: 1165; (a) SEQ ID NO: 1168 and (b) SEQ ID NO: 1172; (a) SEQ ID NO: 1175 and (b) SEQ ID NO: 1179; (a) SEQ ID NO: 1182 and (b) SEQ ID NO: 1186; (a) SEQ ID NO: 1189 and (b) SEQ ID NO: 1193; (a) SEQ ID NO: 1196 and (b) SEQ ID NO: 1200; (a) SEQ ID NO: 1203 and (b) SEQ ID NO: 1207; (a) SEQ ID NO: 1210 and (b) SEQ ID NO: 1214; (a) SEQ ID NO: 1217 and (b) SEQ ID NO: 1221 ;(a) SEQ ID NO: 1224 and (b) SEQ ID NO: 1228;(a) SEQ ID NO: 1231 and (b) SEQ ID NO: 1235;(a) SEQ ID NO: 1238 and (b) SEQ ID NO: 1242; (a) SEQ ID NO: 1245 and (b) SEQ ID NO: 1249;(a) SEQ ID NO: 1264 and (b) SEQ ID NO: 1265; (a) SEQ ID NO: 1264 and (b) SEQ ID NO: 1265; (a) SEQ ID NO: 1266 and (b) SEQ ID NO: 1267; (a) SEQ ID NO: 1268 and (b) SEQ ID NO: 1269; (a) SEQ ID NO: 1270 and (b) SEQ ID NO: 1271; (a) SEQ ID NO: 1272 and (b) SEQ ID NO: 1273; (a) SEQ ID NO: 12 74 and (b) SEQ ID NO: 1275; (a) SEQ ID NO: 1276 and (b) SEQ ID NO: 1277; (a) SEQ ID NO: 1278 and (b) SEQ ID NO: 1279; (a) SEQ ID NO: 1280 and (b) SEQ ID NO: 1281; (a) SEQ ID NO: 1282 and (b) SEQ ID NO: 1283; (a) SEQ ID NO: 1284 and (b) SEQ ID NO: 1285; (a) SEQ ID NO: 1286 and (b) SEQ ID NO: 1287; (a) SEQ ID NO: 1288 and (b) SEQ ID NO: 1289; (a) SEQ ID NO: 1290 and (b) SEQ ID NO: 1291 ; (a) SEQ ID NO: 1292 and (b) SEQ ID NO: 1293; (a) SEQ ID NO: 1294 and (b) SEQ ID NO: 1295; (a) SEQ ID NO: 1296 and (b) SEQ ID NO: 1297;
(a) SEQ ID NO: 1298 and (b) SEQ ID NO: 1299; (a) SEQ ID NO: 1300 and (b) SEQ ID NO: 1301 ; (a) SEQ ID NO: 1302 and (b) SEQ ID NO: 1303; (a) SEQ ID NO: 1304 and (b) SEQ ID NO: 1305; (a) SEQ ID NO: 1306 and (b) SEQ ID NO: 1307; (a) SEQ ID NO: 1308 and (b) SEQ ID NO: 1309; (a) SEQ ID NO: 1310 and (b) SEQ ID NO: 1311 ; (a) SEQ ID NO: 1312 and (b) SEQ ID NO: 1313; (a) SEQ ID NO: 1314 and (b) SEQ ID NO: 1315; (a) SEQ ID NO: 1316 and (b) SEQ ID NO: 1317; (a) SEQ ID NO: 1318 and (b) SEQ ID NO: 1319; (a) SEQ ID NO: 1320 and (b) SEQ ID NO: 1321; (a) SEQ ID NO: 1322 and (b) SEQ ID NO: 1323; (a) SEQ ID NO: 1324 and (b) SEQ ID NO: 1325; (a) SEQ ID NO: 1326 and
(b) SEQ ID NO: 1327; (a) SEQ ID NO: 1328 and (b) SEQ ID NO: 1329; (a) SEQ ID NO: 1330 and (b) SEQ ID NO: 1331 ; (a) SEQ ID NO: 1332 and (b) SEQ ID NO: 1333; (a) SEQ ID NO: 1334 and (b) SEQ ID NO: 1335; (a) SEQ ID NO: 1336 and (b) SEQ ID NO: 1337; (a) SEQ ID NO: 1338 and (b) SEQ ID NO: 1339;(a) SEQ ID NO: 1340 and (b) SEQ ID NO: 1341 ; (a) SEQ ID NO: 1342 and (b) SEQ ID NO: 1343; (a) SEQ ID NO: 1344 and (b) SEQ ID NO: 1345; (a) SEQ ID NO: 1346 and (b) SEQ ID NO: 1347; (a) SEQ ID NO: 1348 and (b) SEQ ID NO: 1349; (a) SEQ ID NO: 1350 and (b) SEQ ID NO: 1351 ; (a) SEQ ID NO: 1352 and (b) SEQ ID NO: 1353; or (a) SEQ ID NO: 1354 and (b) SEQ ID NO: 1355.
[0014] In some embodiments, the variable heavy chain (VH) has at least 90% sequence identity to a sequence as set forth above in an (a) in a pair and the variable light chain (VL) has at least 90% sequence identity to the sequence as set forth above in the (b) of the same pair. In some embodiments, the variable heavy chain (VH) has at least 95% sequence identity to a sequence as set forth above in an (a) in a pair and the variable light chain (VL) has at least 95% sequence identity to the sequence as set forth above in the (b) of the same pair. In yet another embodiment, the variable heavy chain (VH) has at least 98% sequence identity to a sequence as set forth above in an (a) in a pair and the variable light chain (VL) has at least 98% sequence identity to the sequence as set forth above in the (b) of the same pair.
[0015] In some embodiments, the binding protein comprises a set of CDRs comprising: (i) a HCDR1, HCDR2, HCDR3, comprising or contained within SEQ ID NO: 1; SEQ ID NO: 8; SEQ ID NO: 15; SEQ ID NO: 22; SEQ ID NO: 29; SEQ ID NO: 36; SEQ ID NO: 43; SEQ ID NO: 50; SEQ ID NO: 57; SEQ ID NO: 64; SEQ ID NO: 71 ; SEQ ID NO: 78; SEQ ID NO: 85; SEQ ID NO: 92; SEQ ID NO: 99; SEQ ID NO: 106; SEQ ID NO: 113; SEQ ID NO: 120; SEQ ID NO: 127; SEQ ID NO: 134; SEQ ID NO: 141; SEQ ID NO: 148; SEQ ID NO: 156; SEQ ID NO: 164; SEQ ID NO: 171; SEQ ID NO: 178; SEQ ID NO: 185; SEQ ID NO: 192; SEQ ID NO: 199; SEQ ID NO: 206; SEQ ID NO: 213; SEQ ID NO: 220; SEQ ID NO: 227; SEQ ID NO: 234; SEQ ID NO: 241; SEQ ID NO: 248; SEQ ID NO: 255; SEQ ID NO: 262; SEQ ID NO: 269; SEQ ID NO: 276; SEQ ID NO: 283; SEQ ID NO: 290; SEQ ID NO: 297; SEQ ID NO: 304; SEQ ID NO: 311; SEQ ID NO: 318; SEQ ID NO: 325; SEQ ID NO: 332; SEQ ID NO: 339; SEQ ID NO: 346; SEQ ID NO: 353; SEQ ID NO: 360; SEQ ID NO: 367; SEQ ID NO: 374; SEQ ID NO: 381; SEQ ID NO: 388; SEQ ID NO: 395; SEQ ID NO: 402; SEQ ID NO: 409;SEQ ID NO: 416; SEQ ID NO: 423; SEQ ID NO: 430; SEQ ID NO: 437; SEQ ID NO: 444; SEQ ID NO: 451; SEQ ID NO: 458; SEQ ID NO: 465; SEQ ID NO: 472; SEQ ID NO: 479; SEQ ID NO: 486; SEQ ID NO: 493; SEQ ID NO: 500; SEQ ID NO: 507; SEQ ID NO: 514; SEQ ID NO: 521 ; SEQ ID NO: 528; SEQ ID NO: 535; SEQ ID NO: 542; SEQ ID NO: 549; SEQ ID NO: 556; SEQ ID NO: 563; SEQ ID NO: 570; SEQ ID NO: 577; SEQ ID NO: 584; SEQ ID NO: 591 ; SEQ ID NO: 598; SEQ ID NO: 605; SEQ ID NO: 612; SEQ ID NO: 619; SEQ ID NO: 626; SEQ ID NO: 633; SEQ ID NO: 640; SEQ ID NO: 647; SEQ ID NO: 654; SEQ ID NO: 661 ; SEQ ID NO: 668; SEQ ID NO: 675; SEQ ID NO: 682; SEQ ID NO: 689; SEQ ID NO: 696; SEQ ID NO: 703; SEQ ID NO: 710; SEQ ID NO: 717; SEQ ID NO: 724; SEQ ID NO: 731 ; SEQ ID NO: 738; SEQ ID NO: 745; SEQ ID NO: 752; SEQ ID NO: 760; SEQ ID NO: 767; SEQ ID NO: 774; SEQ ID NO: 781; SEQ ID NO: 788; SEQ ID NO: 795; SEQ ID NO: 802; SEQ ID NO: 809; SEQ ID NO: 816; SEQ ID NO: 823; SEQ ID NO: 830; SEQ ID NO: 837; SEQ ID NO: 844; SEQ ID NO: 851; SEQ ID NO: 858; SEQ ID NO: 865; SEQ ID NO: 872; SEQ ID NO: 880; SEQ ID NO: 887; SEQ ID NO: 894; SEQ ID NO: 901 SEQ ID NO: 908 SEQ ID NO: 915; SEQ ID NO: 922; SEQ ID NO: 929; SEQ ID NO: 936; SEQ ID NO: 943; SEQ ID NO: 950; SEQ ID NO: 957; SEQ ID NO: 964; SEQ ID NO: 971; SEQ ID NO: 978; SEQ ID NO: 985; SEQ ID NO: 992; SEQ ID NO: 999; SEQ ID NO: 1006; SEQ ID NO: 1013; SEQ ID NO: 1020; SEQ ID NO: 1028; SEQ ID NO: 1035; SEQ ID NO: 1042; SEQ ID NO: 1049; SEQ ID NO: 1056; SEQ ID NO: 1063; SEQ ID NO: 1070; SEQ ID NO: 1077; SEQ ID NO: 1084; SEQ ID NO: 1091; SEQ ID NO: 1098; SEQ ID NO: 1105; SEQ ID NO: 1112; SEQ ID NO: 1119; SEQ ID NO: 1126; SEQ ID NO: 1133; SEQ ID NO: 1140; SEQ ID NO: 1147; SEQ ID NO: 1154; SEQ ID NO: 1161; SEQ ID NO: 1168; SEQ ID NO: 1175 SEQ ID NO: 1182; SEQ ID NO: 1189; SEQ ID NO: 1196; SEQ ID NO: 1203; SEQ ID NO: 1210; SEQ ID NO: 1217; SEQ ID NO: 1224; SEQ ID NO: 1231; SEQ ID NO: 1238; SEQ ID NO: 1245; SEQ ID NO: 1264; SEQ ID NO: 1266; SEQ ID: 1268; SEQ ID: 1270; SEQ ID: 1272; SEQ ID: 1274; SEQ ID NO: 1276; SEQ ID NO: 1278; SEQ ID NO: 1280; SEQ ID NO: 1282; SEQ ID NO: 1284; SEQ ID NO: 1286; SEQ ID NO: 1288; SEQ ID NO: 1290; SEQ ID NO: 1292; SEQ ID NO: 1294, SEQ ID NO: 1296; SEQ ID NO: 1298; SEQ ID NO: 1300; SEQ ID NO: 1302; SEQ ID NO: 1304, SEQ ID NO: 1306, SEQ ID NO: 1308; SEQ ID NO: 1310; SEQ ID NO: 1312; SEQ ID NO: 1314; SEQ ID NO: 1316; SEQ ID NO: 1318; SEQ ID NO: 1320; SEQ ID NO: 1322; SEQ ID NO: 1324; SEQ ID NO: 1326; SEQ ID NO: 1328; SEQ ID NO: 1330; SEQ ID NO: 1332; SEQ ID NO: 1334; SEQ ID NO: 1336, SEQ ID NO: 1338; SEQ ID NO: 1340; SEQ ID NO: 1342; SEQ ID NO: 1344; SEQ ID NO: 1346; SEQ ID NO: 1348; SEQ ID NO: 1350; SEQ ID NO: 1352; or SEQ ID NO: 1354, with one or two single amino acid substitutions in one or more of the HCDRs; and (ii) a LCDR1, LCDR2, and LCDR3 comprising or contained within SEQ ID NO: 5; SEQ ID NO: 12; SEQ ID NO: 19; SEQ ID NO: 26; SEQ ID NO: 33; SEQ ID NO: 40; SEQ ID NO: 47; SEQ ID NO: 54; SEQ ID NO: 61; SEQ ID NO: 68; SEQ ID NO: 75; SEQ ID NO: 82; SEQ ID NO: 89; SEQ ID NO: 96; SEQ ID NO: 103; SEQ ID NO: 110; SEQ ID NO: 117; SEQ ID NO: 124; SEQ ID NO: 131; SEQ ID NO: 138; SEQ ID NO: 145; SEQ ID NO: 152; SEQ ID NO: 160; SEQ ID NO: 168; SEQ ID NO: 175; SEQ ID NO: 182; SEQ ID NO: 189; SEQ ID NO: 196; SEQ ID NO: 203; SEQ ID NO: 210; SEQ ID NO: 217; SEQ ID NO: 224; SEQ ID NO: 231; SEQ ID NO: 238; SEQ ID NO: 245; SEQ ID NO: 252; SEQ ID NO: 259; SEQ ID NO: 266; SEQ ID NO: 273; SEQ ID NO: 280; SEQ ID NO: 287; SEQ ID NO: 294; SEQ ID NO: 301; SEQ ID NO: 308; SEQ ID NO: 315; SEQ ID NO: 322; SEQ ID NO: 329; SEQ ID NO: 336; SEQ ID NO: 343; SEQ ID NO: 350; SEQ ID NO: 357; SEQ ID NO: 364; SEQ ID NO: 371; SEQ ID NO: 378; SEQ ID NO: 385; SEQ ID NO: 392; SEQ ID NO: 399; SEQ ID NO: 406; SEQ ID NO: 413; SEQ ID NO: 420; SEQ ID NO: 427; SEQ ID NO: 434; SEQ ID NO: 441; SEQ ID NO: 448; SEQ ID NO: 455; SEQ ID NO: 462; SEQ ID NO: 469; SEQ ID NO: 476; SEQ ID NO: 483; SEQ ID NO: 490; SEQ ID NO: 497; SEQ ID NO: 504; SEQ ID NO: 511; SEQ ID NO: 518; SEQ ID NO: 525; SEQ ID NO: 532; SEQ ID NO: 539; SEQ ID NO: 546; SEQ ID NO: 553; SEQ ID NO: 560; SEQ ID NO: 567; SEQ ID NO: 574; SEQ ID NO: 581; SEQ ID NO: 588; SEQ ID NO: 595; SEQ ID NO: 602; SEQ ID NO: 609; SEQ ID NO: 616; SEQ ID NO: 623; SEQ ID NO: 630; SEQ ID NO: 637; SEQ ID NO: 644; SEQ ID NO: 651; SEQ ID NO: 658; SEQ ID NO: 665; SEQ ID NO: 672; SEQ ID NO: 679; SEQ ID NO: 686; SEQ ID NO: 693; SEQ ID NO: 700; SEQ ID NO: 707; SEQ ID NO: 714; SEQ ID NO: 721; SEQ ID NO: 728; SEQ ID NO: 735; SEQ ID NO: 742; SEQ ID NO: 749; SEQ ID NO: 756; SEQ ID NO: 764; SEQ ID NO: 771; SEQ ID NO: 778; SEQ ID NO: 785; SEQ ID NO: 792; SEQ ID NO: 799; SEQ ID NO: 806; SEQ ID NO: 813; SEQ ID NO: 820; SEQ ID NO: 827; SEQ ID NO: 834; SEQ ID NO: 841; SEQ ID NO: 848; SEQ ID NO: 855; SEQ ID NO: 862; SEQ ID NO: 869;S EQ ID NO: 876; SEQ ID NO: 884; SEQ ID NO: 889; SEQ ID NO: 898; SEQ ID NO: 905; SEQ ID NO: 912; SEQ ID NO: 919; SEQ ID NO: 926; SEQ ID NO: 933; SEQ ID NO: 940; SEQ ID NO: 947; SEQ ID NO: 954; SEQ ID NO: 961; SEQ ID NO: 968; SEQ ID NO: 975; SEQ ID NO: 982; SEQ ID NO: 989; SEQ ID NO: 996; SEQ ID NO: 1003; SEQ ID NO: 1010; SEQ ID NO: 1017; SEQ ID NO: 1024; SEQ ID NO: 1032; SEQ ID NO: 1039; SEQ ID NO: 1046; SEQ ID NO: 1053; SEQ ID NO: 1060; SEQ ID NO: 1067; SEQ ID NO: 1074; SEQ ID NO: 1081; SEQ ID NO: 1088; SEQ ID NO: 1095; SEQ ID NO: 1102; SEQ ID NO: 1109; SEQ ID NO: 1116; SEQ ID NO: 1123; SEQ ID NO: 1130; SEQ ID NO: 1137; SEQ ID NO: 1144; SEQ ID NO: 1151; SEQ ID NO: 1158; SEQ ID NO: 1165; SEQ ID NO: 1172; SEQ ID NO: 1179; SEQ ID NO: 1186; SEQ ID NO: 1193; SEQ ID NO: 1200; SEQ ID NO: 1207; SEQ ID NO: 1214; SEQ ID NO: 1221; SEQ ID NO: 1228; SEQ ID NO: 1235; SEQ ID NO: 1242; SEQ ID NO: 1249; SEQ ID NO: 1265; SEQ ID NO: 1267; SEQ ID NO: 1269; SEQ ID NO: 1271; SEQ ID NO: 1273; SEQ ID NO: 1275; SEQ ID NO: 1277; SEQ ID NO: 1279; SEQ ID NO: 1281; SEQ ID NO: 1283; SEQ ID NO: 1285; SEQ ID NO: 1287; SEQ ID NO: 1289; SEQ ID NO: 1291; SEQ ID NO: 1293; SEQ ID NO: 1295; SEQ ID NO: 1297; SEQ ID NO: 1299; SEQ ID NO: 1301 ; SEQ ID NO: 1303; SEQ ID NO: 1305; SEQ ID NO: 1307; SEQ ID NO: 1309; SEQ ID NO: 1311; SEQ ID NO: 1313; SEQ ID NO: 1315; SEQ ID NO: 1317; SEQ ID NO: 1319; SEQ ID NO: 1321; SEQ ID NO: 1323; SEQ ID NO: 1325; SEQ ID NO: 1327; SEQ ID NO: 1329; SEQ ID NO: 1331; SEQ ID NO: 1333; SEQ ID NO: 1335; SEQ ID NO: 1337; SEQ ID NO: 1339; SEQ ID NO: 1341 ; SEQ ID NO: 1343; SEQ ID NO: 1345; SEQ ID NO: 1347; SEQ ID NO: 1349; SEQ ID NO: 1351; SEQ ID NO: 1353; or SEQ ID NO: 1355, with one or two single amino acid substitutions in one or more of the HCDRs.
[0016] In another aspect, the present disclosure provides a composition comprising an isolated human monoclonal antibody or derivative thereof which binds to a specific epitope of Pseudomonas aeruginosa V-antigen (PcrV). In some embodiments, the composition comprises: a heavy chain variable domain (VH); a light chain variable domain (VL); and a pharmaceutically acceptable carrier.
[0017] In some embodiments, the antibody or derivative thereof is selected from: an antibody or derivative thereof wherein at least one variable region has at least 85% sequence identity to a sequence set forth in and selected from the group consisting of: SEQ ID NO: 1 ; SEQ ID NO: 8; SEQ ID NO: 15; SEQ ID NO: 22; SEQ ID NO: 29; SEQ ID NO: 36; SEQ ID NO: 43; SEQ ID NO: 50; SEQ ID NO: 57; SEQ ID NO: 64; SEQ ID NO: 71 ; SEQ ID NO: 78; SEQ ID NO: 85; SEQ ID NO: 92; SEQ ID NO: 99; SEQ ID NO: 106; SEQ ID NO: 113; SEQ ID NO: 120; SEQ ID NO: 127; SEQ ID NO: 134; SEQ ID NO: 141; SEQ ID NO: 148; SEQ ID NO: 156; SEQ ID NO: 164; SEQ ID NO: 171; SEQ ID NO: 178; SEQ ID NO: 185; SEQ ID NO: 192; SEQ ID NO: 199; SEQ ID NO: 206; SEQ ID NO: 213; SEQ ID NO: 220; SEQ ID NO: 227; SEQ ID NO: 234; SEQ ID NO: 241; SEQ ID NO: 248; SEQ ID NO: 255; SEQ ID NO: 262; SEQ ID NO: 269; SEQ ID NO: 276; SEQ ID NO: 283; SEQ ID NO: 290; SEQ ID NO: 297; SEQ ID NO: 304; SEQ ID NO: 311; SEQ ID NO: 318; SEQ ID NO: 325; SEQ ID NO: 332; SEQ ID NO: 339; SEQ ID NO: 346; SEQ ID NO: 353; SEQ ID NO: 360; SEQ ID NO: 367; SEQ ID NO: 374; SEQ ID NO: 381; SEQ ID NO: 388; SEQ ID NO: 395; SEQ ID NO: 402; SEQ ID NO: 409;SEQ ID NO: 416; SEQ ID NO: 423; SEQ ID NO: 430; SEQ ID NO: 437; SEQ ID NO: 444; SEQ ID NO: 451 ; SEQ ID NO: 458; SEQ ID NO: 465; SEQ ID NO: 472; SEQ ID NO: 479; SEQ ID NO: 486; SEQ ID NO: 493; SEQ ID NO: 500; SEQ ID NO: 507; SEQ ID NO: 514; SEQ ID NO: 521; SEQ ID NO: 528; SEQ ID NO: 535; SEQ ID NO: 542; SEQ ID NO: 549; SEQ ID NO: 556; SEQ ID NO: 563; SEQ ID NO: 570; SEQ ID NO: 577; SEQ ID NO: 584; SEQ ID NO: 591; SEQ ID NO: 598; SEQ ID NO: 605; SEQ ID NO: 612; SEQ ID NO: 619; SEQ ID NO: 626; SEQ ID NO: 633; SEQ ID NO: 640; SEQ ID NO: 647; SEQ ID NO: 654; SEQ ID NO: 661; SEQ ID NO: 668; SEQ ID NO: 675; SEQ ID NO: 682; SEQ ID NO: 689; SEQ ID NO: 696; SEQ ID NO: 703; SEQ ID NO: 710; SEQ ID NO: 717; SEQ ID NO: 724; SEQ ID NO: 731; SEQ ID NO: 738; SEQ ID NO: 745; SEQ ID NO: 752; SEQ ID NO: 760; SEQ ID NO: 767; SEQ ID NO: 774; SEQ ID NO: 781; SEQ ID NO: 788; SEQ ID NO: 795; SEQ ID NO: 802; SEQ ID NO: 809; SEQ ID NO: 816; SEQ ID NO: 823; SEQ ID NO: 830; SEQ ID NO: 837; SEQ ID NO: 844; SEQ ID NO: 851; SEQ ID NO: 858; SEQ ID NO: 865; SEQ ID NO: 872; SEQ ID NO: 880; SEQ ID NO: 887; SEQ ID NO: 894; SEQ ID NO: 901 SEQ ID NO: 908 SEQ ID NO: 915; SEQ ID NO: 922; SEQ ID NO: 929; SEQ ID NO: 936; SEQ ID NO: 943; SEQ ID NO: 950; SEQ ID NO: 957; SEQ ID NO: 964; SEQ ID NO: 971; SEQ ID NO: 978; SEQ ID NO: 985; SEQ ID NO: 992; SEQ ID NO: 999; SEQ ID NO: 1006; SEQ ID NO: 1013; SEQ ID NO: 1020; SEQ ID NO: 1028; SEQ ID NO: 1035; SEQ ID NO: 1042; SEQ ID NO: 1049; SEQ ID NO: 1056; SEQ ID NO: 1063; SEQ ID NO: 1070; SEQ ID NO: 1077; SEQ ID NO: 1084; SEQ ID NO: 1091; SEQ ID NO: 1098; SEQ ID NO: 1105; SEQ ID NO: 1112; SEQ ID NO: 1119; SEQ ID NO: 1126; SEQ ID NO: 1133; SEQ ID NO: 1140; SEQ ID NO: 1147; SEQ ID NO: 1154; SEQ ID NO: 1161; SEQ ID NO: 1168; SEQ ID NO: 1175 SEQ ID NO: 1182; SEQ ID NO: 1189; SEQ ID NO: 1196; SEQ ID NO: 1203; SEQ ID NO: 1210; SEQ ID NO: 1217; SEQ ID NO: 1224; SEQ ID NO: 1231; SEQ ID NO: 1238; SEQ ID NO: 1245; SEQ ID NO: 1264; SEQ ID NO: 1266; SEQ ID: 1268; SEQ ID: 1270; SEQ ID: 1272; SEQ ID: 1274; SEQ ID NO: 1276; SEQ ID NO: 1278; SEQ ID NO: 1280; SEQ ID NO: 1282; SEQ ID NO: 1284; SEQ ID NO: 1286; SEQ ID NO: 1288; SEQ ID NO: 1290; SEQ ID NO: 1292; SEQ ID NO: 1294, SEQ ID NO: 1296; SEQ ID NO: 1298; SEQ ID NO: 1300; SEQ ID NO: 1302; SEQ ID NO: 1304, SEQ ID NO: 1306, SEQ ID NO: 1308; SEQ ID NO: 1310; SEQ ID NO: 1312; SEQ ID NO: 1314; SEQ ID NO: 1316; SEQ ID NO: 1318; SEQ ID NO: 1320; SEQ ID NO: 1322; SEQ ID NO: 1324; SEQ ID NO: 1326; SEQ ID NO: 1328; SEQ ID NO: 1330; SEQ ID NO: 1332; SEQ ID NO: 1334; SEQ ID NO: 1336, SEQ ID NO: 1338; SEQ ID NO: 1340; SEQ ID NO: 1342; SEQ ID NO: 1344; SEQ ID NO: 1346; SEQ ID NO: 1348; SEQ ID NO: 1350; SEQ ID NO: 1352; SEQ ID NO: 1354; SEQ ID NO: 5; SEQ ID NO: 12; SEQ ID NO: 19; SEQ ID NO: 26; SEQ ID NO: 33; SEQ ID NO: 40; SEQ ID NO: 47; SEQ ID NO: 54; SEQ ID NO: 61 ; SEQ ID NO: 68; SEQ ID NO: 75; SEQ ID NO: 82; SEQ ID NO: 89; SEQ ID NO: 96; SEQ ID NO: 103; SEQ ID NO: 110; SEQ ID NO: 117; SEQ ID NO: 124; SEQ ID NO: 131; SEQ ID NO: 138; SEQ ID NO: 145; SEQ ID NO: 152; SEQ ID NO: 160; SEQ ID NO: 168; SEQ ID NO: 175; SEQ ID NO: 182; SEQ ID NO: 189; SEQ ID NO: 196; SEQ ID NO: 203; SEQ ID NO: 210; SEQ ID NO: 217; SEQ ID NO: 224; SEQ ID NO: 231; SEQ ID NO: 238; SEQ ID NO: 245; SEQ ID NO: 252; SEQ ID NO: 259; SEQ ID NO: 266; SEQ ID NO: 273; SEQ ID NO: 280; SEQ ID NO: 287; SEQ ID NO: 294; SEQ ID NO: 301; SEQ ID NO: 308; SEQ ID NO: 315; SEQ ID NO: 322; SEQ ID NO: 329; SEQ ID NO: 336; SEQ ID NO: 343; SEQ ID NO: 350; SEQ ID NO: 357; SEQ ID NO: 364; SEQ ID NO: 371; SEQ ID NO: 378; SEQ ID NO: 385; SEQ ID NO: 392; SEQ ID NO: 399; SEQ ID NO: 406; SEQ ID NO: 413; SEQ ID NO: 420; SEQ ID NO: 427; SEQ ID NO: 434; SEQ ID NO: 441; SEQ ID NO: 448; SEQ ID NO: 455; SEQ ID NO: 462; SEQ ID NO: 469; SEQ ID NO: 476; SEQ ID NO: 483; SEQ ID NO: 490; SEQ ID NO: 497; SEQ ID NO: 504; SEQ ID NO: 511; SEQ ID NO: 518; SEQ ID NO: 525; SEQ ID NO: 532; SEQ ID NO: 539; SEQ ID NO: 546; SEQ ID NO: 553; SEQ ID NO: 560; SEQ ID NO: 567; SEQ ID NO: 574; SEQ ID NO: 581; SEQ ID NO: 588; SEQ ID NO: 595; SEQ ID NO: 602; SEQ ID NO: 609; SEQ ID NO: 616; SEQ ID NO: 623; SEQ ID NO: 630; SEQ ID NO: 637; SEQ ID NO: 644; SEQ ID NO: 651; SEQ ID NO: 658; SEQ ID NO: 665; SEQ ID NO: 672; SEQ ID NO: 679; SEQ ID NO: 686; SEQ ID NO: 693; SEQ ID NO: 700; SEQ ID NO: 707; SEQ ID NO: 714; SEQ ID NO: 721; SEQ ID NO: 728; SEQ ID NO: 735; SEQ ID NO: 742; SEQ ID NO: 749; SEQ ID NO: 756; SEQ ID NO: 764; SEQ ID NO: 771; SEQ ID NO: 778; SEQ ID NO: 785; SEQ ID NO: 792; SEQ ID NO: 799; SEQ ID NO: 806; SEQ ID NO: 813; SEQ ID NO: 820; SEQ ID NO: 827; SEQ ID NO: 834; SEQ ID NO: 841; SEQ ID NO: 848; SEQ ID NO: 855; SEQ ID NO: 862; SEQ ID NO: 869;S EQ ID NO: 876; SEQ ID NO: 884; SEQ ID NO: 889; SEQ ID NO: 898; SEQ ID NO: 905; SEQ ID NO: 912; SEQ ID NO: 919; SEQ ID NO: 926; SEQ ID NO: 933; SEQ ID NO: 940; SEQ ID NO: 947; SEQ ID NO: 954; SEQ ID NO: 961; SEQ ID NO: 968; SEQ ID NO: 975; SEQ ID NO: 982; SEQ ID NO: 989; SEQ ID NO: 996; SEQ ID NO: 1003; SEQ ID NO: 1010; SEQ ID NO: 1017; SEQ ID NO: 1024; SEQ ID NO: 1032; SEQ ID NO: 1039; SEQ ID NO: 1046; SEQ ID NO: 1053; SEQ ID NO: 1060; SEQ ID NO: 1067; SEQ ID NO: 1074; SEQ ID NO: 1081; SEQ ID NO: 1088; SEQ ID NO: 1095; SEQ ID NO: 1102; SEQ ID NO: 1109; SEQ ID NO: 1116; SEQ ID NO: 1123; SEQ ID NO: 1130; SEQ ID NO: 1137; SEQ ID NO: 1144; SEQ ID NO: 1151; SEQ ID NO: 1158; SEQ ID NO: 1165; SEQ ID NO: 1172; SEQ ID NO: 1179; SEQ ID NO: 1186; SEQ ID NO: 1193; SEQ ID NO: 1200; SEQ ID NO: 1207; SEQ ID NO: 1214; SEQ ID NO: 1221 ; SEQ ID NO: 1228; SEQ ID NO: 1235; SEQ ID NO: 1242; SEQ ID NO: 1249; SEQ ID NO: 1265; SEQ ID NO: 1267; SEQ ID NO: 1269; SEQ ID NO: 1271; SEQ ID NO: 1273; SEQ ID NO: 1275; SEQ ID NO: 1277; SEQ ID NO: 1279; SEQ ID NO: 1281 ; SEQ ID NO: 1283; SEQ ID NO: 1285; SEQ ID NO: 1287; SEQ ID NO: 1289; SEQ ID NO: 1291 ; SEQ ID NO: 1293; SEQ ID NO: 1295; SEQ ID NO: 1297; SEQ ID NO: 1299; SEQ ID NO: 1301; SEQ ID NO: 1303; SEQ ID NO: 1305; SEQ ID NO: 1307; SEQ ID NO: 1309; SEQ ID NO: 1311; SEQ ID NO: 1313; SEQ ID NO: 1315; SEQ ID NO: 1317; SEQ ID NO: 1319; SEQ ID NO: 1321; SEQ ID NO: 1323; SEQ ID NO: 1325; SEQ ID NO: 1327; SEQ ID NO: 1329; SEQ ID NO: 1331; SEQ ID NO: 1333; SEQ ID NO: 1335; SEQ ID NO: 1337; SEQ ID NO: 1339; SEQ ID NO: 1341 ; SEQ ID NO: 1343; SEQ ID NO: 1345; SEQ ID NO: 1347; SEQ ID NO: 1349; SEQ ID NO: 1351; SEQ ID NO: 1353; and SEQ ID NO: 1355.
[0018] In some embodiments, the antibody or derivative thereof is selected from: an antibody or derivative thereof where at least one variable region is a sequence set forth in and selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 8; SEQ ID NO: 15; SEQ ID NO: 22; SEQ ID NO: 29; SEQ ID NO: 36; SEQ ID NO: 43; SEQ ID NO: 50; SEQ ID NO: 57; SEQ ID NO: 64; SEQ ID NO: 71 ; SEQ ID NO: 78; SEQ ID NO: 85; SEQ ID NO: 92; SEQ ID NO: 99; SEQ ID NO: 106; SEQ ID NO: 113; SEQ ID NO: 120; SEQ ID NO: 127; SEQ ID NO: 134; SEQ ID NO: 141; SEQ ID NO: 148; SEQ ID NO: 156; SEQ ID NO: 164; SEQ ID NO: 171 ; SEQ ID NO: 178; SEQ ID NO: 185; SEQ ID NO: 192; SEQ ID NO: 199; SEQ ID NO: 206; SEQ ID NO: 213; SEQ ID NO: 220; SEQ ID NO: 227; SEQ ID NO: 234; SEQ ID NO: 241 ; SEQ ID NO: 248;SEQ ID NO: 255; SEQ ID NO: 262; SEQ ID NO: 269; SEQ ID NO: 276; SEQ ID NO: 283; SEQ ID NO: 290; SEQ ID NO: 297; SEQ ID NO: 304; SEQ ID NO: 311 ; SEQ ID NO: 318; SEQ ID NO: 325; SEQ ID NO: 332; SEQ ID NO: 339; SEQ ID NO: 346; SEQ ID NO: 353; SEQ ID NO: 360; SEQ ID NO: 367; SEQ ID NO: 374; SEQ ID NO: 381; SEQ ID NO: 388; SEQ ID NO: 395; SEQ ID NO: 402; SEQ ID NO: 409;SEQ ID NO: 416; SEQ ID NO: 423; SEQ ID NO: 430; SEQ ID NO: 437; SEQ ID NO: 444; SEQ ID NO: 451; SEQ ID NO: 458; SEQ ID NO: 465; SEQ ID NO: 472; SEQ ID NO: 479; SEQ ID NO: 486; SEQ ID NO: 493; SEQ ID NO: 500;SEQ ID NO: 507; SEQ ID NO: 514; SEQ ID NO: 521 ; SEQ ID NO: 528; SEQ ID NO: 535; SEQ ID NO: 542; SEQ ID NO: 549; SEQ ID NO: 556; SEQ ID NO: 563;SEQ ID NO: 570; SEQ ID NO: 577; SEQ ID NO: 584; SEQ ID NO: 591; SEQ ID NO: 598; SEQ ID NO: 605; SEQ ID NO: 612; SEQ ID NO: 619; SEQ ID NO: 626; SEQ ID NO: 633; SEQ ID NO: 640; SEQ ID NO: 647; SEQ ID NO: 654; SEQ ID NO: 661; SEQ ID NO: 668; SEQ ID NO: 675; SEQ ID NO: 682; SEQ ID NO: 689; SEQ ID NO: 696; SEQ ID NO: 703; SEQ ID NO: 710; SEQ ID NO: 717; SEQ ID NO: 724; SEQ ID NO: 731; SEQ ID NO: 738; SEQ ID NO: 745; SEQ ID NO: 752; SEQ ID NO: 760; SEQ ID NO: 767; SEQ ID NO: 774; SEQ ID NO: 781; SEQ ID NO: 788; SEQ ID NO: 795; SEQ ID NO: 802; SEQ ID NO: 809; SEQ ID NO: 816; SEQ ID NO: 823; SEQ ID NO: 830; SEQ ID NO: 837; SEQ ID NO: 844; SEQ ID NO: 851; SEQ ID NO: 858; SEQ ID NO: 865; SEQ ID NO: 872; SEQ ID NO: 880; SEQ ID NO: 887; SEQ ID NO: 894; SEQ ID NO: 901 SEQ ID NO: 908 SEQ ID NO: 915; SEQ ID NO: 922; SEQ ID NO: 929; SEQ ID NO: 936; SEQ ID NO: 943; SEQ ID NO: 950; SEQ ID NO: 957; SEQ ID NO: 964; SEQ ID NO: 971; SEQ ID NO: 978; SEQ ID NO: 985; SEQ ID NO: 992; SEQ ID NO: 999; SEQ ID NO: 1006; SEQ ID NO: 1013; SEQ ID NO: 1020; SEQ ID NO: 1028; SEQ ID NO: 1035; SEQ ID NO: 1042; SEQ ID NO: 1049; SEQ ID NO: 1056; SEQ ID NO: 1063; SEQ ID NO: 1070; SEQ ID NO: 1077; SEQ ID NO: 1084; SEQ ID NO: 1091; SEQ ID NO: 1098; SEQ ID NO: 1105; SEQ ID NO: 1112; SEQ ID NO: 1119; SEQ ID NO: 1126; SEQ ID NO: 1133; SEQ ID NO: 1140; SEQ ID NO: 1147; SEQ ID NO: 1154; SEQ ID NO: 1161; SEQ ID NO: 1168; SEQ ID NO: 1175 SEQ ID NO: 1182; SEQ ID NO: 1189; SEQ ID NO: 1196; SEQ ID NO: 1203; SEQ ID NO: 1210; SEQ ID NO: 1217; SEQ ID NO: 1224; SEQ ID NO: 1231; SEQ ID NO: 1238; SEQ ID NO: 1245; SEQ ID NO: 1264; SEQ ID NO: 1266; SEQ ID: 1268; SEQ ID: 1270; SEQ ID: 1272; SEQ ID: 1274; SEQ ID NO: 1276; SEQ ID NO: 1278; SEQ ID NO: 1280; SEQ ID NO: 1282; SEQ ID NO: 1284; SEQ ID NO: 1286; SEQ ID NO: 1288; SEQ ID NO: 1290; SEQ ID NO: 1292; SEQ ID NO: 1294, SEQ ID NO: 1296; SEQ ID NO: 1298; SEQ ID NO: 1300; SEQ ID NO: 1302; SEQ ID NO: 1304, SEQ ID NO: 1306, SEQ ID NO: 1308; SEQ ID NO: 1310; SEQ ID NO: 1312; SEQ ID NO: 1314; SEQ ID NO: 1316; SEQ ID NO: 1318; SEQ ID NO: 1320; SEQ ID NO: 1322; SEQ ID NO: 1324; SEQ ID NO: 1326; SEQ ID NO: 1328; SEQ ID NO: 1330; SEQ ID NO: 1332; SEQ ID NO: 1334; SEQ ID NO: 1336, SEQ ID NO: 1338; SEQ ID NO: 1340; SEQ ID NO: 1342; SEQ ID NO: 1344; SEQ ID NO: 1346; SEQ ID NO: 1348; SEQ ID NO: 1350; SEQ ID NO: 1352; SEQ ID NO: 1354; SEQ ID NO: 5; SEQ ID NO: 12; SEQ ID NO: 19; SEQ ID NO: 26; SEQ ID NO: 33; SEQ ID NO: 40; SEQ ID NO: 47; SEQ ID NO: 54; SEQ ID NO: 61; SEQ ID NO: 68; SEQ ID NO: 75; SEQ ID NO: 82; SEQ ID NO: 89; SEQ ID NO: 96; SEQ ID NO: 103; SEQ ID NO: 110; SEQ ID NO: 117; SEQ ID NO: 124; SEQ ID NO: 131; SEQ ID NO: 138; SEQ ID NO: 145; SEQ ID NO: 152; SEQ ID NO: 160; SEQ ID NO: 168; SEQ ID NO: 175; SEQ ID NO: 182; SEQ ID NO: 189; SEQ ID NO: 196; SEQ ID NO: 203; SEQ ID NO: 210; SEQ ID NO: 217; SEQ ID NO: 224; SEQ ID NO: 231; SEQ ID NO: 238; SEQ ID NO: 245; SEQ ID NO: 252; SEQ ID NO: 259; SEQ ID NO: 266; SEQ ID NO: 273; SEQ ID NO: 280; SEQ ID NO: 287; SEQ ID NO: 294; SEQ ID NO: 301; SEQ ID NO: 308; SEQ ID NO: 315; SEQ ID NO: 322; SEQ ID NO: 329; SEQ ID NO: 336; SEQ ID NO: 343; SEQ ID NO: 350; SEQ ID NO: 357; SEQ ID NO: 364; SEQ ID NO: 371; SEQ ID NO: 378; SEQ ID NO: 385; SEQ ID NO: 392; SEQ ID NO: 399; SEQ ID NO: 406; SEQ ID NO: 413; SEQ ID NO: 420; SEQ ID NO: 427; SEQ ID NO: 434; SEQ ID NO: 441; SEQ ID NO: 448; SEQ ID NO: 455; SEQ ID NO: 462; SEQ ID NO: 469; SEQ ID NO: 476; SEQ ID NO: 483; SEQ ID NO: 490; SEQ ID NO: 497; SEQ ID NO: 504; SEQ ID NO: 511; SEQ ID NO: 518; SEQ ID NO: 525; SEQ ID NO: 532; SEQ ID NO: 539; SEQ ID NO: 546; SEQ ID NO: 553; SEQ ID NO: 560; SEQ ID NO: 567; SEQ ID NO: 574; SEQ ID NO: 581; SEQ ID NO: 588; SEQ ID NO: 595; SEQ ID NO: 602; SEQ ID NO: 609; SEQ ID NO: 616; SEQ ID NO: 623; SEQ ID NO: 630; SEQ ID NO: 637; SEQ ID NO: 644; SEQ ID NO: 651; SEQ ID NO: 658; SEQ ID NO: 665; SEQ ID NO: 672; SEQ ID NO: 679; SEQ ID NO: 686; SEQ ID NO: 693; SEQ ID NO: 700; SEQ ID NO: 707; SEQ ID NO: 714; SEQ ID NO: 721; SEQ ID NO: 728; SEQ ID NO: 735; SEQ ID NO: 742; SEQ ID NO: 749; SEQ ID NO: 756; SEQ ID NO: 764; SEQ ID NO: 771; SEQ ID NO: 778; SEQ ID NO: 785; SEQ ID NO: 792; SEQ ID NO: 799; SEQ ID NO: 806; SEQ ID NO: 813; SEQ ID NO: 820; SEQ ID NO: 827; SEQ ID NO: 834; SEQ ID NO: 841; SEQ ID NO: 848; SEQ ID NO: 855; SEQ ID NO: 862; SEQ ID NO: 869;S EQ ID NO: 876; SEQ ID NO: 884; SEQ ID NO: 889; SEQ ID NO: 898; SEQ ID NO: 905; SEQ ID NO: 912; SEQ ID NO: 919; SEQ ID NO: 926; SEQ ID NO: 933; SEQ ID NO: 940; SEQ ID NO: 947; SEQ ID NO: 954; SEQ ID NO: 961; SEQ ID NO: 968; SEQ ID NO: 975; SEQ ID NO: 982; SEQ ID NO: 989; SEQ ID NO: 996; SEQ ID NO: 1003; SEQ ID NO: 1010; SEQ ID NO: 1017; SEQ ID NO: 1024; SEQ ID NO: 1032; SEQ ID NO: 1039; SEQ ID NO: 1046; SEQ ID NO: 1053; SEQ ID NO: 1060; SEQ ID NO: 1067; SEQ ID NO: 1074; SEQ ID NO: 1081; SEQ ID NO: 1088; SEQ ID NO: 1095; SEQ ID NO: 1102; SEQ ID NO: 1109; SEQ ID NO: 1116; SEQ ID NO: 1123; SEQ ID NO: 1130; SEQ ID NO: 1137; SEQ ID NO: 1144; SEQ ID NO: 1151; SEQ ID NO: 1158; SEQ ID NO: 1165; SEQ ID NO: 1172; SEQ ID NO: 1179; SEQ ID NO: 1186; SEQ ID NO: 1193; SEQ ID NO: 1200; SEQ ID NO: 1207; SEQ ID NO: 1214; SEQ ID NO: 1221 ; SEQ ID NO: 1228; SEQ ID NO: 1235; SEQ ID NO: 1242; SEQ ID NO: 1249; SEQ ID NO: 1265; SEQ ID NO: 1267; SEQ ID NO: 1269; SEQ ID NO: 1271 ; SEQ ID NO: 1273; SEQ ID NO: 1275; SEQ ID NO: 1277; SEQ ID NO: 1279; SEQ ID NO: 1281; SEQ ID NO: 1283; SEQ ID NO: 1285; SEQ ID NO: 1287; SEQ ID NO: 1289; SEQ ID NO: 1291 ; SEQ ID NO: 1293; SEQ ID NO: 1295; SEQ ID NO: 1297; SEQ ID NO: 1299; SEQ ID NO: 1301 ; SEQ ID NO: 1303; SEQ ID NO: 1305; SEQ ID NO: 1307; SEQ ID NO: 1309; SEQ ID NO: 1311; SEQ ID NO: 1313; SEQ ID NO: 1315; SEQ ID NO: 1317; SEQ ID NO: 1319; SEQ ID NO: 1321 ; SEQ ID NO: 1323; SEQ ID NO: 1325; SEQ ID NO: 1327; SEQ ID NO: 1329; SEQ ID NO: 1331 ; SEQ ID NO: 1333; SEQ ID NO: 1335; SEQ ID NO: 1337; SEQ ID NO: 1339; SEQ ID NO: 1341; SEQ ID NO: 1343; SEQ ID NO: 1345; SEQ ID NO: 1347; SEQ ID NO: 1349; SEQ ID NO: 1351 ; SEQ ID NO: 1353; and SEQ ID NO: 1355.
[0019] In some embodiments, the antibody or derivative thereof is selected from: an antibody or derivative thereof comprising from one to three heavy chain CDR sequences and/or from one to three light chain CDR sequences selected from the group consisting of sequences as set forth in SEQ ID NO: 2, 3, 4, 6, 7, 9, 10, 11, 13, 14, 16, 17, 18, 20, 21, 23, 24, 25, 27, 28, 30, 31, 32, 34, 35, 37, 38, 39, 41, 42, 44, 45, 46, 48, 49, 51, 52, 53, 55, 56, 58, 59, 60, 62, 63, 65, 66, 67, 69, 70, 72, 73, 74, 76, 77, 79. 80, 81, 83, 84, 86, 87, 88, 90, 91, 93, 94, 95, 97, 98, 100, 101,102,104, 105,107,108,109, 111, 112, 114, 115, 116, 118, 119, 121, 122, 123, 125, 126, 128, 129, 130, 132, 133, 135, 136, 137, 139, 140, 142, 143,
144, 146, 147, 149, 150, 151, 153, 154, 155, 157, 158, 159, 161, 162, 163, 165, 166, 167,
169, 170, 172, 173, 174, 176, 177, 179, 180, 181, 183, 184, 186, 187, 188, 190, 191, 193,
194, 195, 197, 198, 200, 201, 202, 204, 205, 207, 208, 209, 211, 212, 214, 215, 216, 218,
219, 221, 222, 223, 225, 226, 228, 229, 230, 232, 233, 235, 236, 237, 239, 240, 242, 243,
244, 246, 247, 249, 250, 251, 253, 254, 256, 257, 258, 260, 261, 263, 264, 265, 267, 268,
270, 271, 272, 274, 275, 277, 278, 279, 281, 282, 284, 285, 286, 288, 289, 291, 292, 293,
295, 296, 298, 299, 300, 302, 303, 305, 306, 307, 309, 310, 312, 313, 314, 316, 317, 319,
320, 321, 323, 324, 326, 327, 328, 330, 331, 333, 334, 335, 337, 338, 340, 341, 342, 344,
345, 347, 348, 349, 351, 352, 354, 355, 356, 358, 359, 361, 362, 363, 365, 366, 368, 369,
370, 372, 373, 375, 376, 377, 379, 380, 382, 383, 384, 386, 387, 389, 390, 391, 393, 394,
396, 397, 398, 400, 402, 403, 404, 405, 407, 408, 410, 411, 412, 414, 415, 417, 418, 419,
421, 422, 424, 425, 426, 428, 429, 431, 432, 433, 435, 436, 438, 439, 440, 442, 443, 445, 446, 447, 449, 450, 452, 453, 454, 456, 457, 459, 460, 461, 463, 464, 466, 467, 468, 470, 471, 473, 474, 475, 477, 478, 480, 481, 482, 484, 485, 487, 488, 489, 491, 492, 494, 495, 496, 498, 499, 501, 502, 503, 505, 506, 508, 509, 510, 512, 513, 515, 516, 517, 519, 520, 522, 523, 524, 526, 527, 529, 530, 531, 533, 534, 536, 537, 538, 540, 541, 543, 544, 545, 547, 548, 550, 551, 552, 554, 555, 557, 558, 559, 561, 562, 564, 565, 566, 568, 569, 571, 572, 573, 575, 576, 578, 579, 580, 582, 583, 585, 586, 587, 589, 590, 592, 593, 594, 596, 597, 599, 600, 601, 603, 604, 606, 607, 608, 610, 611, 613, 614, 615, 617, 618, 620, 621, 622, 624, 625, 627, 628, 629, 631, 632, 634, 635, 636, 638, 639, 641, 642, 643, 645, 646, 648, 649, 650, 652, 653, 655, 656, 657, 659, 660, 662, 663, 664, 666, 667, 669, 670, 671, 673, 674, 676, 677, 678, 680, 681, 683, 684, 685, 687, 688, 690, 691, 692, 694, 695, 697, 698, 699, 701, 702, 704, 705, 706, 708, 709, 711, 712, 713, 715, 716, 718, 719, 720, 722, 723, 725, 726, 727, 729, 730, 732, 733, 734, 736, 737, 739, 740, 741, 743, 744, 746, 747, 748, 750, 751, 753, 754, 755, 757, 758, 759, 761, 762, 763, 765, 766, 768, 769, 770, 772, 773, 775, 776, 777, 779, 780, 782, 783, 784, 786, 787, 789, 790, 791, 793, 794, 796, 797, 798, 800, 801, 803, 804, 805, 807, 808, 810, 811, 812, 814, 815, 817, 818, 819, 821, 822, 824, 825, 826, 828, 829, 831, 832, 833, 835, 836, 838, 839, 840, 842, 843, 845, 846, 847, 849, 850, 852, 853, 854, 856, 857, 859, 860, 861, 863, 864, 866, 867, 868, 870, 871, 873, 874, 875, 877, 878, 879, 881, 882, 883, 885, 886, 888, 889, 890, 892, 893, 895, 896, 897, 899, 900, 902, 903, 904, 906, 907, 909, 910, 911, 913, 914, 916, 917, 918, 920, 921, 923, 924, 925, 927, 928, 930, 931, 932, 934, 935, 937, 938, 939, 941, 942, 944, 945, 946, 948, 949, 951, 952, 953, 955, 956, 958, 959, 960, 962, 963, 965, 966, 967, 969, 970, 972, 973, 974, 976, 977, 979, 980, 981, 983, 984, 986, 987, 988, 990, 991, 993, 994, 995, 997, 998, 1000, 1001, 1002, 1004, 1005, 1007, 1008, 1009, 1011, 1012, 1014, 1015, 1016, 1018, 1019, 1021, 1022, 1023, 1025, 1026, 1027, 1029, 1030, 1031, 1033, 1034, 1036, 1037, 1038, 1040, 1041, 1043, 1044, 1045, 1047, 1048, 1050, 1051, 1052, 1054, 1055, 1057, 1058, 1059, 1061, 1062, 1064, 1065, 1066, 1068, 1069, 1071, 1072, 1073, 1075, 1076, 1078, 1079, 1080, 1082, 1083, 1085, 1086, 1087, 1089, 1090, 1092, 1093, 1094, 1096, 1097, 1099, 1100, 1101, 1103, 1104, 1106, 1107, 1 108, 11 10, 111 1, 1 113, 11 14, 1115, 1117, 1118, 1120, 1121, 1122, 1124, 1125, 1127, 1 128, 1129, 1131, 1132, 1134, 1135, 1136, 1138, 1139, 1141, 1142, 1143, 1145, 1146, 1148, 1149, 1150, 1152, 1153, 1155, 1156, 1157, 1159, 1160, 1162, 1163, 1164, 1166, 1167, 1169, 1170, 1171, 1173, 1174, 1176, 1177, 1178, 1180, 1181, 1183, 1184, 1185, 1187, 1188, 1190, 1191, 1192, 1194, 1195, 1197, 1198, 1199, 1201, 1202, 1204, 1205, 1206, 1208, 1209, 1211, 1212, 1213, 1215, 1216, 1218, 1219, 1220, 1222, 1223, 1225, 1226, 1227, 1229, 1230, 1232, 1233, 1234, 1236, 1237, 1239, 1240, 1241, 1243, 1244, 1246, 1247, 1248, 1250, 1251, 1356, 1357, 1358, 1359, 1360, 1361, 1362, 1364, 1365, 1367, 1368, 1369, 1370, 1371, 1372,
1374, 1375, 1376, 1378, 1380, 1381, 1384, 1389, 1392, 1394, 1396, 1400, 1407, 1408,
1410, 1411, 1415, 1418, 1419, 1420, 1423, 1426, 1427, 1433, 1434, 1442, 1448, 1449,
1450, 1451, 1452, 1454, 1455, 1456, 1457, 1458, 1459, 1460, 1461, 1462, 1466, 1470,
1471, 1474, 1475, 1478, 1479, 1480, 1487, 1488, 1491 , 1494, 1495, 1496, 1497, 1498,
1499, 1500, 1501, 1502, 1503, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513,
1514, 1515, 1516, 1517, 1518, 1519, 1521, 1522, 1523, 1526, 1527, 1528, 1529, 1530,
1531, 1532, 1533, 1534, 1536, 1537, 1538, 1539, 1540, 1541, 1542, 1543, 1544, 1545,
1546, 1547, 1548, 1549, 1550, 1552, 1553, 1554, 1556, 1557, 1558, 1560, 1561, 1563,
1564, 1565, 1566, 1567, 1568, 1569, 1570, 1571, 1572, 1574, 1575, 1576, 1577, 1578,
1579, 1580, 1581, 1583, 1584, and 1585.
[0020] In some embodiments, the antibody or derivative thereof is selected from: an antibody comprising a means for binding the Pseudomonas aeruginosa V-antigen (PcrV) antigen. In some such embodiments, the antibody comprises (a) means for binding the PcrV antigen and (b) an immunoglobulin Fc region. Exemplified structures for binding the PcrV antigen include the heavy and light chain variable region pairs and corresponding CDR sets shown in Table 1 and Table 2 of the present disclosure.
[0021] In some embodiments, the binding protein comprises a set of CDRs having the sequences as set forth in Table 1 and Table 2 of the present disclosure.
[0022] In some embodiments, the CDRs are grafted into a human IgG acceptor antibody framework or an IgM acceptor antibody framework. In some embodiments, the acceptor framework comprises an Fc region of the IgG or the IgM linked to the variable heavy chain. In an embodiment, the Fc region is a variant Fc region, wherein the variant Fc region comprises one or more amino acid substitutions, insertions, or deletions that alter an effector function relative to a naturally occurring Fc region.
[0023] In some embodiments, the binding protein is a genetically engineered intact antibody comprising a variant Fc region. In an embodiment, the variant Fc region comprises one or more amino acid substitutions that alter an effector function relative to a naturally-occurring Fc region. In some embodiments, the binding protein is a genetically engineered intact antibody or antibody fragment or a derivative thereof. In an embodiment, the binding protein is formatted into a multimer, a single chain variable fragment (scFv), or an antibody conjugate.
[0024] In yet another aspect, provided herein is composition comprising a binding protein of the present disclosure and a pharmaceutically acceptable carrier, diluent, or excipient.
[0025] In an aspect, the present disclosure also provides a polynucleotide encoding a binding protein of the present disclosure. In some embodiments, the polynucleotide is codon optimized. In an embodiment, the present disclosure provides an expression vector comprising the polynucleotide encoding a binding protein of the present disclosure. In some embodiments, the polynucleotide is operably linked to an expression control sequence. In some embodiments, the expression vector is capable of delivering the polynucleotide to a host cell. Further, provided herein is a recombinant host cell comprising the polynucleotide encoding a binding protein of the present disclosure. In some embodiments, provided herein is a recombinant host cell comprising the expression vector comprising the polynucleotide encoding a binding protein of the present disclosure. In an embodiment, the host cell is capable of expressing the encoded binding protein.
[0026] Provided herein is also a composition comprising: (a) means for binding a specific epitope of Pseudomonas aeruginosa V-antigen (PcrV); and (b) a pharmaceutically acceptable carrier.
[0027] In yet another aspect, the present disclosure provides a method of treating and/or preventing a disease. In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a binding protein, or a composition disclosed herein. In some embodiments, the subject is a human. In an embodiment, the disease is an infectious disease. In some embodiments, the disease is an infectious disease caused by Pseudomonas aeruginosa. In an embodiment, the Pseudomonas aeruginosa infection is associated with an inflammatory lung disease. In some embodiments, the inflammatory lung disease is cystic fibrosis (CF). In an embodiment, the administration comprises intravenous administration. In some embodiments, the method further comprises administering another therapeutic agent and/or therapy to the subject.
[0028] In yet another aspect, the present disclosure provides a method of immunizing a subject against a pathogen. In some embodiments, the method comprises administering to the subject in need thereof an effective amount of a binding protein, a composition, a polynucleotide, an expression vector, or a recombinant host cell, disclosed herein.
[0029] In another aspect, provided herein is a method of treating and/or preventing a disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of a composition disclosed herein.
[0030] In another aspect, provided herein is a method of immunizing a subject against Pseudomonas aeruginosa. In an embodiment, the method comprises administering to the subject in need thereof an effective amount of a composition discloses herein.
[0031] In another aspect, the present disclosure provides a method of detecting the presence of Pseudomonas aeruginosa V-antigen (PcrV), or a cell expressing PcrV, in a sample. In some embodiments, the sample is a biological sample. In some embodiments, the method comprises contacting the sample with a binding protein or an isolated human monoclonal antibody disclosed herein. In some embodiments, the sample is a biological sample.
DESCRIPTION OF THE DRAWINGS
[0032] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:
[0033] FIGS. 1A-1C show PcrV-specific B cells in subjects with cystic fibrosis (CF). Schematic of primary human B cells binding to PcrV tetramer reagent and decoy (FIG. 1A, Left). Representative flow plot for B cells after enrichment (FIG. 1A, Right). Cells binding only to the PcrV tetramer are indicated with the box. Percentage (FIG. IB) and frequency (FIG. 1C) of PcrV-specific B cells in 14 human subjects with cystic fibrosis (CF) vs. control, blood bank donors (non-CF).
[0034] FIG. 2 shows tetramer-specific class-switched B cells in mice immunized with PcrV. Flow cytometry plots from lymphoid tissue in representative PcrV-immunized or control (naive) mice sacrificed on day 7 post-immunization by intraperitoneal injection. Cells were analyzed after the magnetic enrichment of tetramerbound cells. Class-switched B cells are highlighted within the boxes.
[0035] FIGS. 3A-3D show generation of anti-PcrV mAbs derived from B cells isolated from a chronically PA infected CF donor. Percentage of somatic hypermutation (SHM) detected in B cell receptor (BCR) sequences from cells of the indicated phenotype for CF donor 1 (FIG. 3A). ELIS As showing PcrV binding using supernatants derived from 293T cells transfected with IgM expression plasmids containing the indicated BCR sequences (FIG. 3B) Area under the curve (AUC) for representative ELISAs of purified antibodies 408 and 411 expressed alternatively as IgM vs. IgG (FIG. 3C). AUC for representative ELISA of purified mAbs V2L2 and 411 expressed, alternatively as, IgG vs. IgM; and for the commercially sourced, clinical, bi-specific mAb, gremubamab. mAbs derived from CF subject B cells are labeled with the CF prefix for clarity (FIG. 3D).
[0036] FIGS. 4A-4C show CF-subject derived, germline, anti-PcrV-specific mAbs exhibit robust anti-PA activity in an in vivo mouse pneumonia model. Schematic illustrating the experimental PA infection and mAb delivery protocol (FIG. 4A). Bacterial load in mouse lungs at 48 h post-infection for mice that received a 60 pg (FIG. 4B) or 20 pg (FIG. 4C) intranasal dose of off-target, control IgGl mAb (ctl), indicated anti-PcrV mAbs, or diluent alone (none).
[0037] FIGS. 5A-5C show high affinity anti-PcrV mAbs derived from memory B cells isolated from CF Donor 2. Somatic hypermutation (%SHM) rates in BCR sequences from individual B cells with the indicated surface phenotype isolated from CF donor 2. Each circle represents a singly sorted cell (FIG. 5A). Heatmap showing paired heavy (x-axis) and light (y-axis) V gene families for 31 MBCs in donor 3 (FIG. 5B). The grey scale depicts SHM for the heavy chains in each pairing. Clone numbers for BCRs to be expressed as mAbs are included to the left of their corresponding box. PcrV binding by ELISA for purified mAbs generated from five MBC BCRs (FIG. 5C). The two CF subject 1-derived mAbs with in vivo protective activity (CF 408 and CF 411) are used as benchmarks for relative binding activity (dashed lines). The off-target control (anti-SARS- CoV-2 RBD) line appears hidden because it overlaps with the line for (CF 432).
[0038] FIG. 6 shows lung bacterial load for mice treated intranasally with 20 pg of the indicated anti-PcrV mAb, or vehicle only (PBS). Data from two independent experiments is shown, each with n = 5 mice per condition.
[0039] FIGS. 7A-7C show B cell receptor (BCR) sequencing of PcrV-tetramer- specific B cells derived from 3 CF donors. Percentage of somatic hypermutation (SHM) detected in BCR sequences from cells of the indicated phenotype in chronically PA infected, CF donor 3 (FIG. 7A). Each circle represents a singly sorted cell. Histograms show the number of unique BCR sequences obtained for each V gene family (y-axis) for heavy and light (kappa or lambda) chains (FIG. 7B). Data from each CF donor is shown in a separate panel of graphs (with Donor number indicated at top). The bars for the V gene families used by in vivo-tested mAbs are colored as: blue-green (CF 411) and orange (CF 408). Heatmap showing pairings of heavy- (x-axis) and light (y-axis) chain V gene families where full-length, high quality V region sequence was attained (FIG. 7C). For each heavy/light chain pair, the percentage of heavy chain sequence which differs from the germline sequence is depicted by the grey scale .
[0040] FIG. 8 shows transfectant supernatant screen of 12 MBC-derived mAbs. ELISA assessing PcrV binding for supernatants from 293T cells transfected with IgG expression plasmids. Numbers (430-442) indicate the BCR sequences identified from individual, PcrV- specific, MBCs from CF donor 2. Supernatant for mAb 411 IgG (dotted line; isolated from CF Donor 1) is included as a benchmark (positive control).
DETAILED DESCRIPTION
[0041] Definitions
[0042] It is to be noted that the term “a” or “an” entity refers to one or more of that entity, for example, “a binding protein or a binding molecule,” is understood to represent one or more binding proteins or binding molecules. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
[0043] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.
[0044] As used herein, “about” or “comprising essentially of’ mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” or “comprising essentially of’ can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, “about” or “comprising essentially of” can mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values are provided in the application and claims, unless otherwise stated, the meaning of “about” or “comprising essentially of’ should be assumed to be within an acceptable error range for that particular value.
[0045] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
[0046] As used herein, the term “binding protein” refers in its broadest sense to a molecule that specifically binds to a receptor, e.g., an epitope or an antigenic determinant. As described further herein, a binding protein can comprise one of more “antigen binding domains” described herein. A non-limiting example of a binding protein is an antibody, a derivative, or fragment thereof that retains antigen- specific binding.
[0047] The term “epitope” includes any molecular determinant capable of specific binding to a binding protein, e.g., an antibody. In certain aspects, an epitope can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain aspects, can have three-dimensional structural characteristics, and or specific charge characteristics. An epitope is a region of a target that is bound by a binding protein.
[0048] As used herein, the terms “binding domain” or “binding fragment,” or “antigen binding domain” or “antigen binding fragment” may be used interchangeably and refer to a region of a binding protein that is necessary and sufficient to specifically bind to an epitope of the antigen. For example, in an antibody, an “Fv,” e.g., a variable heavy chain (VH) and variable light chain (VL) of an antibody, either as two separate polypeptide subunits or as a single chain, is considered to be a “binding domain.” Other binding domains include, without limitation, the variable heavy chain (VH) of an antibody derived from a camelid species, or six immunoglobulin complementarity determining regions (CDRs) expressed in a fibronectin scaffold. A “binding protein” as described herein can include one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more “antigen binding domains.”
[0049] As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of “polypeptide,” and the term “polypeptide” can be used instead of, or interchangeably with any of these terms. The term “polypeptide” is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, and derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide can be derived from a biological source or produced by recombinant technology but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.
[0050] By an “isolated” polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated as disclosed herein, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.
[0051] A “conservative amino acid substitution” is one in which one amino acid is replaced with another amino acid having a similar side chain. Families of amino acids having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g. , aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. In certain embodiments, conservative substitutions in the sequences of the polypeptides and antibodies of the present disclosure do not abrogate the binding of the polypeptide or antibody containing the amino acid sequence, to the antigen to which the binding molecule binds. Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen-binding are well-known in the art (see, e.g., Brummell et al , Biochem. 32: 1 180-1 187 (1993); Kobayashi et al , Protein Eng. 12(10): 879-884 (1999); and Burks et al, Proc. Natl. Acad. Sci. USA 94:.412-417 (1997)).
[0052] The term “polynucleotide” is intended to encompass a singular nucleic acid as well as plural nucleic acids and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA), cDNA, or plasmid DNA (pDNA). A polynucleotide can comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)). The terms “nucleic acid” or “nucleic acid sequence” refer to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. By an “isolated” nucleic acid or polynucleotide is intended any form of the nucleic acid or polynucleotide that is separated from its native environment. For example, gel-purified polynucleotide, or a recombinant polynucleotide encoding a polypeptide contained in a vector would be considered to be “isolated.” Also, a polynucleotide segment, e.g., a PCR product, which has been engineered to have restriction sites for cloning is considered to be “isolated.”
[0053] Disclosed herein are certain binding proteins, or antigen-binding fragments, variants, or derivatives thereof that specifically bind P. aeruginosa V-antigen (PcrV) and exhibit robust bacterial neutralization activity. Unless specifically referring to full-sized antibodies, the term “binding protein” encompasses full-sized antibodies as well as antigenbinding subunits, fragments, variants, analogs, or derivatives thereof, e.g., isolated monoclonal antibody molecules, engineered antibody molecules or fragments that bind antigen in a manner similar to antibody molecules, but which use a different scaffold.
[0054] The terms “antibody” and “immunoglobulin” can be used interchangeably herein. The term “antibody” as referred to herein includes whole antibodies and any antigen-binding fragment (i.e., “antigen-binding portion”) or single chains thereof. A “whole antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three or four domains, depending on antibody isotype (specifically, CHI, CH2, and CH3 in the case of IgG, IgA, and IgD, or CHI, CH2, CH3, and CH4 in the case of IgM and IgE). Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. 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 regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may 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. An antibody (or a fragment, variant, or derivative thereof as disclosed herein) includes at least the variable domain of a heavy chain or at least the variable domains of a heavy chain and a light chain. Basic immunoglobulin structures in vertebrate systems are relatively well understood. See, e.g., Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988). Unless otherwise stated, the term “antibody” encompasses anything ranging from a small antigen- binding fragment of an antibody to a full sized antibody, e.g., an IgG antibody that includes two complete heavy chains and two complete light chains, an IgA antibody that includes four complete heavy chains and four complete light chains and optionally includes a J chain and/or a secretory component, or an IgM antibody that includes ten or twelve complete heavy chains and ten or twelve complete light chains and optionally includes a J chain.
[0055] The term “immunoglobulin” comprises various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (y, p, a, 5, s) with some subclasses among them (e.g., yl-y4 or 1- a.2). It is the nature of this chain that determines the “isotype” of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (subtypes) e.g., IgGi, IgG2, IgG3, IgG4, IgAi, IgA?, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these immunoglobulins are readily discernible to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of this disclosure.
[0056] Light chains are classified as either kappa or lambda (K, X). Each heavy chain class can be bound with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the “tail” portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are expressed, e.g., by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N- terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain. The basic structure of certain antibodies, e.g., IgG antibodies, includes two heavy chain subunits and two light chain subunits covalently connected via disulfide bonds to form a “Y” structure.”
[0057] As indicated above, variable region(s) allows a binding molecule/protein to selectively recognize and specifically bind epitopes on antigens. For example, in context of the binding protein of the present disclosure comprising an antibody, the VL domain and VH domain, or subset of the complementarity determining regions (CDRs), of the antibody, combine to form the antigen binding domain or antigen-binding fragment. More specifically, an antigen binding domain or antigen-binding fragment can be defined by three CDRs on each of the VH and VL chains.
[0058] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigenbinding fragment,” as used herein.
[0059] The six “complementarity determining regions” or “CDRs” present in an antigen-binding domain or antigen-binding fragment are short, non-contiguous sequences of amino acids that are specifically positioned to form the binding domain as the antibody assumes its three-dimensional configuration in an aqueous environment. The remainder of the amino acids in the binding domain, referred to as “framework” regions, show less inter- molecular variability. The framework regions largely adopt a P-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the P-sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the protein/molecule to its cognate epitope.
[0060] The amino acids that make up the CDRs and the framework regions, respectively, can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been defined in various different ways (see, “Sequences of Proteins of Immunological Interest,” Kabat, E., et al, U.S. Department of Health and Human Services, (1983); IMGT (Lefranc M P et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 January; 27(1 ):55-77 (“IMGT” numbering scheme)); and Chothia and Lesk, J. Mol. Biol., 796:901-917 (1987), which are incorporated herein by reference in their entireties).
[0061] In the case where there are two or more definitions of a term which are used and/or accepted within the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated to the contrary. A specific example is the use of the term “complementarity determining region” (“CDR”) to describe the noncontiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described, for example, by Kabat et al, U.S. Dept, of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983), IMGT (Lefranc M P et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 January; 27(l):55-77 (“IMGT” numbering scheme)), and by Chothia et al, J. Mol. Biol. 196:901-917 (1987), which are incorporated herein by reference. Nevertheless, application of either definition (or other definitions known to those of ordinary skill in the art) to refer to a CDR of an antibody or variant thereof is intended to be within the scope of the term as defined and used herein, unless otherwise indicated. Numbering of all CDR definitions in Table 1 and Table 2 is according to the numbering conventions set forth by IMGT.
[0062] A binding protein comprising an antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH — VH, VH — VL or VL — VL dimers. Alternatively, the antigen-binding fragment may contain a monomeric VH or VL domain.
[0063] In certain embodiments, an antigen-binding fragment of a binding protein, for example an antibody, may contain at least one variable domain covalently linked to at least one constant domain. Non- limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of the present disclosure include: (i) VH— CHI ; (ii) VH— CH2; (iii) VH— CH3; (iv) VH— CH1-CH2; (v) VH— CH1-CH2-C H3; (vi) VH— CH2-CH3; (vii) VH— CL; (viii) VL— CHI ; (ix) VL— CH2; (x) VL— CH3; (xi) VL— CH1-CH2; (xii) VL— CH1-CH2-CH3; (xiii) VL— CH2-CH3; and (xiv) VL — CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of the present disclosure may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).
[0064] As with full antibody molecules, antigen-binding fragments of the present disclosure may be monospecific or multispecific (e.g., bispecific). A multispecific antigenbinding fragment will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of the present disclosure using routine techniques available in the art.
[0065] By “specifically binds,” it is generally meant that a binding protein, e.g., an antibody or fragment, variant, or derivative thereof binds to an epitope via its antigenbinding domain or antigen-binding fragment, and that the binding entails some complementarity between the antigen binding- fragment and the epitope. According to this definition, a binding protein is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen binding domain more readily than it would bind to a random, unrelated epitope. The term “specificity” is used herein to qualify the relative affinity by which certain binding proteins bind to a certain epitope. For example, binding protein “A” can be deemed to have a higher specificity for a given epitope than binding protein “B,” or binding protein “A” can be said to bind to epitope “C” with a higher specificity than it has for related epitope “D.”
[0066] As used herein, the term “affinity” refers to a measure of the strength of the binding of an individual epitope with one or more binding domains, e.g., of an immunoglobulin molecule. See, e.g., Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) at pages 27-28.
[0067] A binding protein, e.g., an antibody or fragment, variant, or derivative thereof of the present disclosure can be said to bind a target antigen with an off rate (k(off)) of less than or equal to 5 X 10"2 sec-1, 10"2 sec-1, 5 X 10"3 sec-1, 10'3 sec-1, 5 X 10"4 sec-1, 10"
4 sec-1, 5 X 10'5 sec-1, or 10'5 sec-1 5 X 10"6 sec-1, 10"6 sec-1, 5 X 10"7 sec-1 or 10'7 sec-1. A binding protein, e.g., an antibody or antigen-binding fragment, variant, or derivative disclosed herein can be said to bind a target antigen with an on rate (k(on)) of greater than or equal to 103 M-1 sec-1, 5 X 103 M-1 sec-1, 104 M-1 sec-1, 5 X 104 M-1 sec-1, 105 M-1 sec-1,
5 X 1CP M-1 sec-1, 106 M’1 sec'1, or 5 X 106 M-1 sec-1 or 107 M-1 sec-1.
[0068] A binding protein of the present disclosure can also be described or specified in terms of their binding affinity to an antigen. For example, a binding protein can bind to an antigen with a dissociation constant or KD no greater than 5 x 10"2 M, 10" 2 M, 5 x IO’3 M, IO’3 M, 5 x 10’4 M, IO’4 M, 5 x 10’5 M, 10’5 M, 5 x IO’6 M, IO’6 M, 5 x 10’ 7 M, IO’7 M, 5 x 10’8 M, 10’s M, 5 x IO’9 M, IO 9 M, 5 x IO'10 M, IO’10 M, 5 x 10’1 1 M, 10’ 11 M, 5 x IO 12 M, 10‘12 M, 5 x 10’13 M, 10‘13 M, 5 x 10 14 M, 10 14 M, 5 x 10‘15 M, or W 15 M.
[0069] A binding protein of the present disclosure, e.g., an antibody or fragment, variant, or derivative thereof including single-chain antibodies or other antigen-binding fragments can exist alone or in combination with one or more of the following: hinge region, CHI, CH2, CH3, or CH4 domains, J chain, or secretory component. Also included are antigen- binding fragments that can include any combination of variable region(s) with one or more of a hinge region, CHI, CH2, CH3, or CH4 domains, a J chain, or a secretory component.
[0070] In certain embodiments, the binding protein comprises an anti-PA PcrV antibody. In some embodiments, the antibody is a human antibody. The term “human antibody,” as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “human antibody,” as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
[0071] The binding proteins of the disclosure may, in some embodiments, comprise anti-PA PcrV recombinant human antibodies. The term “recombinant human antibody,” as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis and thus the amino acid sequences of the Vn and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
[0072] As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and can in some instances express endogenous immunoglobulins and some not, as described, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.
[0073] As used herein, the term “heavy chain subunit” includes amino acid sequences derived from an immunoglobulin heavy chain, A binding protein, e.g., an antibody comprising a heavy chain subunit can include at least one of: a heavy chain variable (VH) domain, a CHI domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, or a variant or fragment thereof. For example, a binding protein, e.g., an antibody or fragment, variant, or derivative thereof can include without limitation, in addition to a VH domain:, a CHI domain; a CHI domain, a hinge, and a CH2 domain; a CHI domain and a CH3 domain; a CHI domain, a hinge, and a CH3 domain; or a CHI domain, a hinge domain, a CH2 domain, and a CH3 domain. In certain aspects a binding protein, e.g., an antibody or fragment, variant, or derivative thereof can include, in addition to a VH domain, a CH3 domain and a CH4 domain; or a CH3 domain, a CH4 domain, and a J chain. Further, a binding protein for use in the disclosure can lack certain constant region portions, e.g., all or part of a CH2 domain. It will be understood by one of ordinary skill in the art that these domains (e.g., the heavy chain subunit) can be modified such that they vary in amino acid sequence from the original immunoglobulin molecule.
[0074] As used herein, the term “light chain subunit” includes amino acid sequences derived from an immunoglobulin light chain. The light chain subunit includes at least a light chain variable domain (VL), and can further include a CL (e.g., CK or C) domain.
[0075] The term “expression” as used herein refers to a process by which a gene produces a biochemical, for example, a polypeptide. The process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient expression and stable expression. It includes without limitation transcription of the gene into RNA, e.g., messenger RNA (mRNA), and the translation of such mRNA into polypeptide(s). If the final desired product is a biochemical, expression includes the creation of that biochemical and any precursors. Expression of a gene produces a “gene product.” As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide that is translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.
[0076] The present disclosure also provides polynucleotides encoding the binding proteins, fragments, and derivatives thereof, disclosed herein, as well as expression vectors comprising such polynucleotides, and host cells comprising such expression vectors. Additionally, provided are methods of production of at least one binding protein, a fragment, or a derivative, thereof of the present disclosure by recombinant techniques, as is well known in the art. See, e.g., Sambrook, et al., supra; Ausubel, et al., supra, each entirely incorporated herein by reference.
[0077] For example, to express the binding protein, or a fragment thereof, DNAs encoding partial or full-length light and heavy chains, can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma that expresses the antibody of interest) and the DNAs can be inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. In this context, the term “operatively linked” is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. Additionally, separate expression vectors can be utilized. For example, the antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors or, more typically, both genes are inserted into the same expression vector. The polynucleotide encoding the binding protein is inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites, or blunt end ligation if no restriction sites are present).
[0078] Additionally, or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the binding protein from a host cell. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein). In addition to the polynucleotide encoding the binding protein, the expression vectors of the present disclosure carry regulatory sequences that control the expression of the binding protein in a host cell. The term “regulatory sequence” is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. [0079] Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter (AdIVILP) and polyoma. Alternatively, nonviral regulatory sequences may be used, such as the ubiquitin promoter or 13-globin promoter. Still further, regulatory elements composed of sequences from different sources, such as the SRa, promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8:466-472).
[0080] The expression vectors of the disclosure may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
[0081] Preferred mammalian host cells for expressing the binding proteins of the present disclosure include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mot Biol. 1.59:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular, for use with NSO myeloma cells, another preferred expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036, and EP 338,841.
[0082] 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 or slow the progression of an existing diagnosed pathologic condition or disorder. Terms such as “prevent,” “prevention,” “avoid,” “deterrence” and the like refer to prophylactic or preventative measures that prevent the development of an undiagnosed targeted pathologic condition or disorder. Thus, “those in need of treatment” can include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. [0083] By “subject” or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, swine, cows, bears, and so on.
[0084] A “therapeutically effective amount” is a dose or an amount sufficient to provide a useful therapeutic or prophylactic effect against a particular disease or disease condition. For example, for a subject infected with a harmful bacteria or virus, an effective amount is sufficient to achieve one or more of the following therapeutic effects: reduce the ability of the bacteria or virus to propagate in the patient or reduce the amount of bacteria or viral load in the patient. With respect to anti -infective applications for an uninfected patient, an effective amount is sufficient to achieve one or more of the following prophylactic effects: a reduced susceptibility to a bacterial or viral infection or a reduced ability of an infecting bacterium or virus to establish persistent infection. In another example, a therapeutically effective amount or an effective amount of the compositions disclosed herein includes an amount sufficient to generate an immune response useful to provide a therapeutic or prophylactic effect against a particular disease or disease condition.
[0085] The dose or amount of a composition of the present disclosure administered to a subject may vary depending upon the age and the size of the subject, conditions, route of administration, and the like. The preferred dose is typically calculated according to body weight or body surface area. When an antibody of the present disclosure is used for treating a subject, it may be advantageous to intravenously administer the antibody of the present disclosure normally at a single dose of about 0.01 to about 20 mg/kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. Effective dosages and schedules for administering of the compositions of the present disclosure may be determined empirically; for example, progress can be monitored by periodic assessment, and the dose adjusted accordingly. Moreover, interspecies scaling of dosages can be performed using well-known methods in the art (e.g., Mordenti et al., Pharmaceut Res 8: 1351 (1991)).
[0086] Various delivery systems are known and can be used to administer the composition of the disclosure, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing an antibody or other therapeutic protein of the invention, receptor mediated endocytosis (see, e.g., Wu et al., J Biol Chem 262:4429-4432 (1987)). The binding proteins, antibodies and other therapeutically active compositions of the present disclosure may also be delivered by gene therapy techniques. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
[0087] A pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.
[0088] Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition as discussed herein. Examples include, but are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but are not limited to the SOLOSTAR™ pen (sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), and the HUMIRA™ Pen (Abbott Labs, Abbott Park IL), to name only a few.
[0089] In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987)). In another embodiment, polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed in proximity of the composition’s target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, Science 249:1527-1533 (1990).
[0090] The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending, or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents.
[0091] In some cases, the binding proteins, antibodies, and other therapeutically active compositions of the present disclosure can be administered with a further therapeutic agent. The further therapeutic agent can be administered prior to, concurrently, or after the administration of the binding proteins, antibodies, and other therapeutically active compositions of the present disclosure.
[0092] In the face of rising antibiotic resistance by pathogenic bacteria, the development of new antimicrobial treatment modalities is becoming increasingly desirable. Because mechanisms of intrinsic resistance already limit therapeutic options, multidrugresistant Pseudomonas aeruginosa (PA) can be especially difficult to treat. Unfortunately, relative to the projected need, few novel antimicrobials are under active pharmaceutical development. Antimicrobial monoclonal antibodies (mAbs) may fill a widening gap as alternatives, or adjuncts, to traditional antibiotics. [0093] Traditional methods of monoclonal antibody (mAb) development require extensive labor in the early stages, from immunization of mice with a recombinant protein to screening hundreds of hybridomas. Further, to address the immunogenicity of the murine constant region, mouse-derived antibodies must be iteratively re-humanized in vitro and/or modified into antibody fragments (Fab) which lack effector functions and are subject to rapid renal clearance. Although fully in vitro strategies like phage display can enable the use of human variable domains, the high-throughput screening processes are vulnerable to bottleneck effects and drift, do not select against autoreactivity or immunogenicity, and do not screen paired VH/VL sequences within the context of full-length paired chains. An alternative approach that has also sought to maximize throughput in screening for high affinity anti-PcrV binders from a library of llama-derived nanobodies shares similar advantages and drawbacks.
[0094] Monoclonal antibodies (mAbs) that bind to key PA virulence factors have shown promise in animal models. One such PA virulence factor, P. aeruginosa V-antigen (PcrV), a homolog of the Yersinia V-antigen LcrV, is a 28kDa surface protein that forms the distal tip of the type III secretion system (T3S) required for the toxin injection into host cells. PcrV facilitates the integration of pore-forming proteins into the eukaryotic cell membrane and is required for translocation of cytotoxins into the host cell. Efforts to develop mAbs for PA antibodies targeting PcrV, a critical component of the toxin injection apparatus, are bolstered by strong evidence for antibody-mediated protection in animal models. Unfortunately, two engineered antibody-like drug candidates (a Fab fragment and a bi-specific) derived from mice failed to achieve positive end points in Phase II trials. The humanized anti-PcrV antibody fragment (Fab) and a bi-specific containing an anti-PcrV binding moiety raised in transgenic mice demonstrated safety in ventilated patients at high risk for PA pneumonia, but each failed to achieve the targeted efficacy outcomes in Phase 2 trials. Based on the evidence for protection by species-concordant mAbs in animal models of PA pneumonia and safety and suggestions for partial efficacy of early candidate mAbs in clinical trials, pursuit of improved anti-PcrV therapies for use in humans remains attractive.
[0095] In prior studies of Covid- 19 and malaria, potent anti-pathogen mAbs have been generated by sequencing the variable regions of the proto- antibody B cell receptor (BCR) in antigen- specific memory B cells (MBCs) that arise following natural infection in humans. Utilizing single cell sequencing strategy, the present disclosure identifies and provides protective anti-PcrV monoclonal antibodies derived from B cells in individuals previously infected with PA.
[0096] Persons living with cystic fibrosis (CF) experience frequent airway PA infections. Intermittent infections early in childhood eventually progress to a state of persistent, chronic airway colonization. CF is a multi-organ disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an ion channel. Several mechanisms related to the biology of the CF airway have been proposed to explain susceptibility to PA. Importantly, mutations in CFTR do not produce intrinsic deficits in adaptive immunity. Class-switched anti-PcrV antibodies are present in CF serum and sputum, at higher concentrations than the general population. However, the PcrV- specific B cells that give rise to antibody- secreting cells have not been previously studied. The present disclosure shows that individuals with CFhave more PcrV- specific circulating B cells. Building on this demonstration, the present disclosure utilized single-cell sequencing to identify PcrV-specific paired-chain BCR sequences in CF subjects. From BCR sequences derived from CF plasmablasts and MBCs, novel anti-PcrV mAbs, including several that confer robust protection against a virulent strain of PA in an in vivo pneumonia challenge model, were generated.
[0097] In this disclosure, multiple high-affinity, PcrV-specific mAbs that exhibit robust bacterial neutralization activity are described. In addition to using IgG monomers, IgM, or IgG multimers or IgA isotypes are also included. mAbs derived from IgM memory B cells including IgM antibodies that exhibit neutralizing activity are particularly described.
[0098] Thus, the present disclosure addresses the unmet need in the art by generating multiple anti-PcrV mAbs directly from the BCR variable regions of antigenspecific B cells derived from CF donors. The present disclosure provides human anti-PcrV mAbs that improve immunogenicity risk and pharmacokinetics, while maintaining robust protective activity.
[0099] Overall, the present disclosure demonstrates that, while lower throughput than library screens, antibody discovery was extremely efficient, requiring expression of very few (< 12) BCRs to yield protective anti-pseudomonal mAbs from each of 2 donors. Interestingly, sequences encoding high-affinity IgG mAbs were obtained from a phenotypically diverse set of B cells, including class-switched and IgM MBCs and IgM- expressing cells that lacked canonical MBC markers and employed fully germline BCRs. Strikingly, 4 of 5 tested mAbs directly cloned from human B cells achieved control of PA infection that matched V2L2MD IgG, the PcrV-specific binding component of gremubamab. Notably, promising results in animal models may not predict clinical success for novel anti-PA therapeutics. However, mAbs derived from human B cells have theoretical advantages versus previously trialed KB001 and gremubamab that would not be reflected in murine models, including reduced immunogenicity and fully human effector functions.
[0100] The antibodies are derived from a broad range of memory B cell subsets including IgG, IgM, and IgA memory cells. Notably, it is shown that these antibodies exhibit protective, neutralizing activity against P. aeruginosa. They exhibit high affinity to a range of PcrV epitopes and limit bacterial infection.
[0101] While the goal of the present disclosure was to generate novel protective mAbs for therapeutic use, the findings herein have an additional benefit of insight into the biology of the adaptive humoral response to PA in CF, which has thus far been limited to comparing titrations of secreted antibodies. As hypothesized given the uniquely high frequency of PA infections in the CF population, circulating PcrV-specific B cells in CF individuals were expanded compared to non-CF individuals, a finding that raises interesting new questions about barriers to clearance of infection. While the ability to generate protective mAbs ex vivo from single cell CF BCRs demonstrates that B cells with the technical capacity to produce functional anti-PA antibodies, while rare, are present in the peripheral blood, multiple in vivo challenges may limit PA clearance in individuals with CF.
[0102] Besides eliminating the humanization requirement and concern for residual immunogenicity, human MBC-derived mAbs are the product of evolved B cell development and maturation processes that encompass tremendous diversity and include selection against auto-reactivity and, for cells that have exited GCs, pre-optimized binding to antigen via affinity maturation. When combined with the results of our present study, human antigen- specific B cells may be broadly considered as an underutilized high-yield resource in the critical endeavor to discover new treatments for infectious disease.
[0103] Naturally occurring antibody structural units disclosed herein include a tetramer. Each tetramer includes two pairs of polypeptide chains, each pair having one light chain and one heavy chain. The amino-terminal portion of each chain includes a variable region that is responsible for antigen recognition and epitope binding. The variable regions exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions (CDRs). The CDRs from the two chains of each pair are aligned by the framework regions, which enables binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chain variable regions include the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is typically in accordance with the definitions of IMGT (Manso T, Folch G, Giudicelli V, Jabado-Michaloud J, Kushwaha A, Nguefack Ngoune V, Georga M, Papadaki A, Debbagh C, Pegorier P, Bertignac M, Hadi- Saljoqi S, Chentli I, Cherouali K, Aouinti S, El Hamwi A, Albani A, Elazami Elhassani M, Viart B, Goret A, Tran A, Sanou G, Rollin M, Duroux P, Kossida S., IMGT® databases, related tools and web resources through three main axes of research and development. Nucleic Acids Res. 2022 Jan 7;50(Dl):D1262-D1272.), as described in more detail below.
[0104] The carboxy-terminal portion of each chain defines a constant region, which can be responsible for effector function particularly in the heavy chain (the Fc). Examples of effector functions include: Clq binding and complement dependent cytotoxicity (CDC); antibody dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B-cell receptors); and B-cell activation.
[0105] Within full-length light and heavy chains, the variable and constant regions are joined by a “J” region of amino acids, with the heavy chain also including a “D” region of amino acids. See, e.g., Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)).
[0106] Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody’s isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including IgGl , lgG2, lgG3, and lgG4. IgM has subclasses including IgMl and lgM2. IgA is similarly subdivided into subclasses including IgAl and lgA2. IgG causes opsonization and cellular cytotoxicity and crosses the placenta, IgA functions on the mucosal surface, IgM is most effective in complement fixation, and IgE mediates degranulation of mast cells and basophils. The function of IgD is still not well understood. Resting B cells, which are immunocompetent but not yet activated, express IgM and IgD. Once activated and committed to secrete antibodies these B cells can express any of the five isotypes. The heavy chain isotypes of IgG, IgA, IgM, IgD and IgE are respectively designated the y, a, p, 8, and 8 chains. [0107] Antibodies and antibody-like molecules that can multimerize, such as IgA and IgM antibodies, have emerged as promising drug candidates in the fields of, e.g., immuno-oncology and infectious diseases allowing for improved specificity, improved avidity, and the ability to bind to multiple binding targets. See, e.g., U.S. Patent Nos. 9,951 ,134, 10,400,038, and 9,938,347, U.S. Patent Application Publication Nos. US20190100597A1, US20180118814A1, US20180118816A1, US20190185570A1 , and US20180265596A1, and PCT Publication Nos. WO 2018/017888, WO 2018/017763, WO 2018/017889, WO 2018/017761, and WO 2019/165340. Multimers based on IgA and IgM (among other multimerization strategies) are discussed in more detail below.
[0108] As indicated above, antibodies bind epitopes on antigens. The term antigen refers to a molecule or a portion of a molecule capable of being bound by an antibody. An epitope is a region of an antigen that is bound by the variable region of an antibody. Epitope determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups, and can have specific three- dimensional structural characteristics, and/or specific charge characteristics. When the antigen is a protein or peptide, the epitope includes specific amino acids within that protein or peptide that contact the variable region of an antibody.
[0109] In particular embodiments, an epitope denotes the binding site on the PcrV protein bound by a corresponding variable region of an antibody. The variable region either binds to a linear epitope, (e.g., an epitope including a stretch of 5 to 12 consecutive amino acids), or the variable region binds to a three-dimensional structure formed by the spatial arrangement of several short stretches of the protein target. Three-dimensional epitopes recognized by a variable region, e.g., by the epitope recognition site or paratope of an antibody or antibody fragment, can be thought of as three-dimensional surface features of an epitope molecule. These features fit precisely (in) to the corresponding binding site of the variable region and thereby binding between the variable region and its target protein (more generally, antigen) is facilitated. In particular embodiments, an epitope can be considered to have two levels: (i) the “covered patch” which can be thought of as the shadow an antibody variable region would cast on the antigen to which it binds; and (ii) the individual participating side chains and backbone residues that facilitate binding. Binding is then due to the aggregate of ionic interactions, hydrogen bonds, and hydrophobic interactions. [0110] The term “binding protein” and/or “antibody” includes (in addition to antibodies having two full-length heavy chains and two full-length light chains as described above) variants, derivatives, and fragments thereof, examples of which are described below. Furthermore, unless explicitly excluded, antibodies or binding proteins can include monoclonal antibodies, human antibodies, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, multi- specific antibodies, polyclonal antibodies, linear antibodies, minibodies, domain antibodies, synthetic antibodies, chimeric antibodies, antibody fusions, and fragments thereof, respectively. In particular embodiments, antibodies can include oligomers or multiplexed versions of antibodies.
[0111] A monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies including the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which include different antibodies directed against different epitopes, each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be made by a variety of techniques, including the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci.
[0112] A chimeric antibody is a monoclonal antibody constructed from variable regions derived from a different organism from the constant regions. For example, a chimeric antibody can contain variable regions from a murine source and constant regions derived from the intended host source (e.g., human; for a review, see Morrison and Oi 1989, Advances in Immunology 44: 65-92).
[0113] A “human antibody” is one which includes an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibodyencoding sequences. The strategy of “humanizing” involves sequence comparison between the non-human and human antibody variable domain sequences to determine whether specific amino acid substitutions from a non-human to human consensus is appropriate (Jones et al., 1986, Nature 321 : 522-526). This technology is well known in the art and is represented by numerous strategies for its improvement, including but not limited to, “reshaping” (see Verhoeyen, et al., 1988, Science 239:1534-1536), “hyperchimerization” (see Queen, et al., 1991, Proc. Natl. Acad. Sci. 88:2869-2873), “CDR grafting” (Winter and Harris, 1993, Immunol. Today 14:243-246), “veneering” (Mark, et al., 1994, Derivation of Therapeutically Active Humanized and Veneered anti-CD18 Antibodies. In: Metcalf and Dalton, eds. Cellular Adhesion: Molecular Definition to Therapeutic Potential. New York: Plenum Press, 291-312), and “SDR grafting” (Kashmiri et al., 2005, Methods 36:25-34).
[0114] Fully human mAbs can be produced using genetically engineered mouse strains which possess an immune system whereby the mouse antibody genes have been inactivated and in turn replaced with a repertoire of functional human antibody genes, leaving other components of the mouse immune system unchanged. Such genetically engineered mice allow for the natural in vivo immune response and affinity maturation process, resulting in high affinity, fully human monoclonal antibodies. This technology is well known in the art and is fully detailed in various publications, including but not limited to U.S. Pat. Nos. 5,939,598; 6,075,181 ; 6,114,598; 6,150,584 and related family members; as well as U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429. See also a review from Kellerman and Green, 2002, Curr. Opinion in Biotechnology 13: 593-597. Human antibodies can also be produced starting with a human phage display library.
[0115] A “human consensus framework” is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. The subgroup of sequences can be a subgroup as in IMGT (supra).
[0116] The antibodies and/or binding proteins disclosed herein include the following sequences, and functional variants thereof as described below:
[0117] Table 1: Antibody Variable Chains & Representative CDR Sequences
Figure imgf000047_0001
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[0119] The CDR sequences provided above are based on IMGT numbering (Lefranc M P et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 January; 27(l):55-77 (“IMGT” numbering scheme)). However, other CDRs based on the provided CDRs can be determined by other methods known in the art.
[0120] Definitive delineation of a CDR and identification of residues including the binding site of an antibody can be accomplished by solving the structure of the antibody and/or solving the structure of the antibody-epitope complex. In particular embodiments, this can be accomplished by methods such as X-ray crystallography. Alternatively, CDRs are determined by comparison to known antibodies (linear sequence) and without resorting to solving a crystal structure. [0121] In addition to IMGT, CDR sets can be based on Kabat numbering (Kabat et al. (1991), “Sequences of Proteins of Immunological Interest.'’ 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme)), Chothia (Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme)), Martin (Abinandan et al., Mol Immunol. 45:3832- 3839 (2008), “Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains”), Gelfand, Contact (MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745.” (Contact numbering scheme)), AHo (Honegger A and Pluckthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun. 8; 309(3):657-70, (AHo numbering scheme)), North (North et al., J Mol Biol. 406(2) :228-256 (2011), “A new clustering of antibody CDR loop conformations”), or other numbering schemes.
[0122] To determine CDR residues involved in binding, a co-crystal structure of the Fab (antibody fragment) bound to the target can be determined. Software programs and bioinformatical tools, such as ABodyBuilder, Paratome, and LlamaMagic (Fridy et al. Nature Methods 2014;l 1 (12):1253-1260) can also be used to determine CDR and FR sequences.
[0123] The Kabat numbering scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. Similar to the IMGT scheme, the AHo scheme can skip numbers in the sequential residue numbering.
[0124] In certain examples, antibodies disclosed herein are expressed as IgA, IgG, or IgM isotypes.
[0125] Antibodies and/or binding proteins disclosed herein can be utilized to prepare various forms of relevant binding domain molecules. For example, particular embodiments can include binding fragments of an antibody, e.g., Fv, Fab, Fab’, F(ab’)2, and single chain Fv fragments (scFvs) or any biologically effective fragments of an immunoglobulin that bind specifically to an epitope described herein.
[0126] In particular embodiments, an antibody fragment is used. An “antibody fragment” denotes a portion of a complete or full-length antibody that retains the ability to bind to an epitope. Antibody fragments can be made by various techniques, including proteolytic digestion of an intact antibody as well as production by recombinant host-cells (e.g., mammalian suspension cell lines, E. coli or phage). Antibody fragments can be screened for their binding properties in the same manner as intact antibodies. Examples of antibody fragments include Fv, scFv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; and linear antibodies.
[0127] A single chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy and light chains of immunoglobulins connected with a short linker peptide. Fv fragments include the VL and VH domains of a single arm of an antibody but lack the constant regions. Although the two domains of the Fv fragment, VL and VH, are coded by separate genes, they can be joined, using, for example, recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (single chain Fv (scFv)). For additional information regarding Fv and scFv, see e.g., Bird, el al., Science 242:423-426, 1988; Huston, et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; Plueckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore (eds.), Springer- Verlag, New York), (1994) 269-315; WO 1993/16185; U.S. Pat. No. 5,571 ,894; and U.S. Pat. No. 5,587,458.
[0128] Linker sequences that are used to connect the VL and VH of an scFv are generally five to 35 amino acids in length. In particular embodiments, a VL-VH linker includes from five to 35, ten to 30 amino acids or from 15 to 25 amino acids. Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies. Linker sequences of scFv are commonly Gly-Ser linkers.
[0129] A linker is an amino acid sequence which can provide flexibility and room for conformational movement between the binding domains of a LAM. Any appropriate linker may be used. [0108] Examples of linkers can be found in Chen et al., Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357-1369. Linkers can be flexible, rigid, or semi-rigid, depending on the desired functional domain presentation to a target. [0130] Commonly used flexible linkers include linker sequence with the amino acids glycine and serine (Gly-Ser linkers). In particular embodiments, the linker sequence includes sets of glycine and serine repeats such as from one to ten repeats of (GlyxSery)n, wherein x and y are independently an integer from 0 to 10 provided that x and y are not both 0 and wherein n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) (SEQ ID NO: 1586).
[0131] Linkers that include one or more antibody hinge regions and/or immunoglobulin heavy chain constant regions, such as CH3 alone or a CH2CH3 sequence can also be used.
[0132] Additional examples of antibody -based binding domain formats include scFv-based grababodies and soluble VH domain antibodies. These antibodies form binding regions using only heavy chain variable regions. See, for example, Jespers et al., Nat. Biotechnol. 22: 1161, 2004; Cortez-Retamozo et al., Cancer Res. 64:2853, 2004; Baral et al., Nature Med. 12:580, 2006; and Barthelemy et al., J. Biol. Chem. 283:3639, 2008.
[0133] A Fab fragment is a monovalent antibody fragment including VL, VH, CL and CHI domains. A F(ab’)2 fragment is a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region. For discussion of Fab and F(ab’)2 fragments having increased in vivo half-life, see U.S. Patent 5,869,046. Diabodies include two epitope-binding sites that may be bivalent. See, for example, EP 0404097; WO1993/01161; and Holliger, et al., Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993. Dual affinity retargeting antibodies (DART™; based on the diabody format but featuring a C- terminal disulfide bridge for additional stabilization (Moore et al., Blood 117:4542-51, 2011)) can also be used. Antibody fragments can also include isolated CDRs. For a review of antibody fragments, see Hudson, et al., Nat. Med. 9: 129-134, 2003.
[0134] As indicated, variants of antibodies and/or binding proteins described herein are also included. Variants can include those having one or more conservative amino acid substitutions or one or more non-conservative substitutions that do not adversely affect the binding of the protein.
[0135] In particular embodiments, a conservative amino acid substitution may not substantially change the structural characteristics of the reference sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the reference sequence or disrupt other types of secondary structure that characterizes the reference sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden & J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al., Nature, 354: 105 (1991). For more information regarding conservative amino acid substitutions, see the Closing Paragraphs section of this disclosure.
[0136] In particular embodiments, a VL region can be derived from or based on a disclosed VL and can include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the VL regions disclosed herein. An insertion, deletion or substitution may be anywhere in the VL region, including at the amino- or carboxy-terminus or both ends of this region, provided that each CDR includes zero changes or at most one, two, or three changes, and provided an antibody including the modified VL region can still specifically bind its target epitope with an affinity similar to the wild type binding domain.
[0137] In particular embodiments, a VH region can be derived from or based on a disclosed VH and can include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the VH regions disclosed herein. An insertion, deletion or substitution may be anywhere in the VH region, including at the amino- or carboxy-terminus or both ends of this region, provided that each CDR includes zero changes or at most one, two, or three changes and provided an antibody including the modified VH region can still specifically bind its target epitope with an affinity similar to the wild type binding domain.
[0138] In particular embodiments, a variant includes or is a sequence that has at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to an antibody sequence disclosed herein. In particular embodiments, a variant includes or is a sequence that has at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to a light chain variable region (VL) and/or to a heavy chain variable region ( VH), or both, wherein each CDR includes zero changes or at most one, two, or three changes, from the reference antibody disclosed herein or fragment or derivative thereof that specifically binds a PcrV epitope as described herein.
[0139] In particular embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody, thereby generating an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g. , a human IgG 1 , lgG2, lgG3 or lgG4 Fc region) including an amino acid modification (e.g., a substitution) at one or more amino acid positions. Numerous Fc variants are described below that provide administration benefits.
[0140] In particular embodiments, variants have been modified from a reference sequence to produce an administration benefit. Exemplary administration benefits can include (1) reduced susceptibility to proteolysis, (2) reduced susceptibility to oxidation, (3) altered binding affinity for forming protein complexes, (4) altered binding affinities, (5) reduced immunogenicity; and/or (6) extended half-live. While the disclosure below describes these modifications in terms of their application to antibodies and/or binding proteins disclosed herein, when applicable to another particular PcrV binding domain format (e.g., an scFv, bispecific antibodies), the modifications can also be applied to these other formats.
[0141] In particular embodiments, the antibodies can be mutated to increase their affinity for Fc receptors. Exemplary mutations that increase the affinity for Fc receptors include: G236A/S239D/A330L/I332E (GASDALIE). Smith et al., Proceedings of the National Academy of Sciences of the United States of America, 109(16), 6181-6186, 2012. In particular embodiments, an antibody variant includes an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues). In particular embodiments, alterations are made in the Fc region that result in altered Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551 , WO 99/51642, and Idusogie et al., J. Immunol. 164: 4178-4184, 2000.
[0142] In particular embodiments, it may be desirable to create cysteine engineered antibodies, e.g., “thioMAbs,” in which one or more residues of an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further below. In particular embodiments, residue 5400 (EU numbering) of the heavy chain Fc region is selected. Cysteine engineered antibodies may be generated as described, e.g., in U.S. Pat. No. 7,521 ,541.
[0143] Antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1 % to 80%, from 1 % to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI- TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., WG2000/61739; WO 2001/29246; W02002/031140; US2002/0164328; W02003/085119; W02003/084570; US2003/0115614; US2003/0157108; US2004/0093621 ;
US2004/0110704; US2004/0132140; US2004/0110282; US2004/0109865;
W02005/035586; W02005/035778; W02005/053742; Okazaki el al. J. Mol. Biol. 336: 1239-1249 (2004); and Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Led 3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545, 1986, and knockout cell lines, such as alpha- 1 ,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614, 2004; Kanda et al., Biotechnol. Bioeng., 94(4):680-688, 2006; and W02003/085107).
[0144] In particular embodiments, modified antibodies include those wherein one or more amino acids have been replaced with a non- amino acid component, or where the amino acid has been conjugated to a functional group or a functional group has been otherwise associated with an amino acid. The modified amino acid may be, e.g., a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, or an amino acid conjugated to an organic derivatizing agent. Amino acid(s) can be modified, for example, co-translationally or post-translationally during recombinant production (e.g., N-linked glycosylation at N-X-S/T motifs during expression in mammalian cells) or modified by synthetic means. The modified amino acid can be within the sequence or at the terminal end of a sequence. Modifications also include nitrited constructs.
[0145] In particular embodiments, variants include glycosylation variants wherein the number and/or type of glycosylation site has been altered compared to the amino acid sequences of a reference sequence. In particular embodiments, glycosylation variants include a greater or a lesser number of N-linked glycosylation sites than the reference sequence. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X can be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions which eliminate this sequence will remove an existing N-linked carbohydrate chain. Also provided is a rearrangement of N-linked carbohydrate chains wherein one or more N-linked glycosylation sites (e.g., those that are naturally occurring) are eliminated and one or more new N-linked sites are created. Additional antibody variants include cysteine variants wherein one or more cysteine residues are deleted from or substituted for another amino acid (e.g., serine) as compared to the reference sequence. These cysteine variants can be useful when antibodies must be refolded into a biologically active conformation such as after the isolation of insoluble inclusion bodies. These cysteine variants generally have fewer cysteine residues than the reference sequence, and typically have an even number to minimize interactions resulting from unpaired cysteines.
[0146] PEGylation particularly is a process by which polyethylene glycol (PEG) polymer chains are covalently conjugated to other molecules such as proteins. Several methods of PEGylating proteins have been reported in the literature. For example, N- hydroxy succinimide (NHS)-PEG was used to PEGylate the free amine groups of lysine residues and N-terminus of proteins; PEGs bearing aldehyde groups have been used to PEGylate the amino-termini of proteins in the presence of a reducing reagent; PEGs with maleimide functional groups have been used for selectively PEGylating the free thiol groups of cysteine residues in proteins; and site-specific PEGylation of acetylphenylalanine residues can be performed.
[0147] Covalent attachment of proteins to PEG has proven to be a useful method to increase the half-lives of proteins in the body (Abuchowski, A. et al., Cancer Biochem. Biophys., 1984, 7: 175- 186; Hershfield, M. S. et al., N. Engl. J. Medicine, 1987, 316:589- 596; and Meyers, F. J. et al., Clin. Pharmacol. Then, 49:307-313, 1991). The attachment of PEG to proteins not only protects the molecules against enzymatic degradation, but also reduces their clearance rate from the body. The size of PEG attached to a protein has significant impact on the half-life of the protein. The ability of PEGylation to decrease clearance is generally not a function of how many PEG groups are attached to the protein, but the overall molecular weight of the altered protein. Usually the larger the PEG is, the longer the in vivo half-life of the attached protein. In addition, PEGylation can also decrease protein aggregation (Suzuki et al., Biochem. Bioph. Acta 788:248, 1984), alter protein immunogenicity (Abuchowski et al., J. Biol. Chem. 252: 3582, 1977), and increase protein solubility as described, for example, in PCT Publication No. WO 92/16221).
[0148] Several sizes of PEGs are commercially available (Nektar Advanced PEGylation Catalog 2005-2006; and NOF DDS Catalogue Ver 7. 1), which are suitable for producing proteins with targeted circulating half-lives. A variety of active PEGs have been used including mPEG succinimidyl succinate, mPEG succinimidyl carbonate, and PEG aldehydes, such as mPEG- propionaldehyde.
[0149] In particular embodiments, the antibody can include an Fc polypeptide that includes amino acid alterations that extend the in vivo half-life of the antibody that contains the altered Fc polypeptide as compared to the half-life of a similar antibody containing the same Fc polypeptide without the amino acid alterations. In particular embodiments, Fc polypeptide amino acid alterations can include M252Y, T252L, T253S, S254T, T254F, T256E, T256N, N286/D/E/Q, E294delta, T307P, Q31 IV, A379V, S383N, M428L, N434S, N434A, N434Y, and/or R435H. J chain mutations that extend half-life include Y102A and R106A/E. These mutations are described in Braathen et al., Journal of Immunology, (2007) 178(3) 1589-1597. The R435H mutation is described in more detail in Stapleton et al., Nat. Comm. (2011)2:599. The N434A mutation is described in more detail in Shields et al., J. Biol. Chem. (2001) 276: 6591-604. The E294delta mutation is described in more detail in Monnet et al., Mabs (2014) 6:422-36 and Bas et al., J. Immunol. (2019) 202:1582094. M428L/N434S is a pair of mutations that increase the half-life of antibodies in serum, as described in Zalevsky et al., Nature Biotechnology 28, 157-159, 2010. M252Y/S254T/T256E are a trio of mutations described in Dall’acqua et al., J. Immunol. (2002) 169: 5171-80. T252L/T253S/T254F is a trio described in Ghetie et al., Nat. Biotechnol. (1997) 15:637-40. Additional combinations include E294delta/T307P/N434Y (Monnet et al., Mabs (2014) 6:422-36) and T256N/A379V/S383N/N434Y (Monnet et al., Mabs (2014) 6:422-36). The N286/D/E/Q and Q31 IV mutations are described in Booth et al., mAbs, (2018) 10(7) 1098-1110.
[0150] Other alterations that can be helpful are described in US Patent No. 7,083,784, US Patent No. 7,670,600, US Publication No. 2010/0234575, PCT/US2012/070146, and Zwolak, Scientific Reports 7: 15521, 2017. In particular embodiments, any substitution at one of the following amino acid positions in an Fc polypeptide can be considered an Fc alteration that extends half-life: 250, 251, 252, 253, 254, 256, 286, 294, 259, 307, 308, 311 , 332, 378, 379, 380, 383,428, 430, 434, 435, 436. Each of these alterations or combinations of these alterations can be used to extend the halflife of an antibody as described herein.
[0151] For additional information regarding Fc mutations that create administration benefits, see Saunders, Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life, Frontiers in Immunology (2019) Vol. 10, Article 1296.
[0152] In particular embodiments, PcrV-specific antibodies of the present disclosure can be multimers. As used herein, multimers include at least two binding domains. Examples of multimers include dimers, trimers, tetramers, pentamers, hexamers, and higher order binding molecules. In particular embodiments, the binding domains of a multimer can all bind the same epitope. In other embodiments, the binding domains of a multimer can bind the same antigen, but distinct epitopes on the antigen. In still other embodiments, the binding domains of a multimer can bind different antigens.
[0153] Multi- specific antibodies are examples of multimers and include bispecific, trispecific, or tetraspecific antibodies. PcrV bispecific antibodies bind at least two epitopes wherein at least one of the epitopes is located on PcrV antigen. PcrV trispecific antibodies bind at least 3 epitopes, wherein at least one of the epitopes is located on PcrV antigen, and so on. In particular embodiments, a dimer includes a bispecific antibody. In particular embodiments, a trimer includes a trispecific antibody.
[0154] Dimers can be prepared as full-length antibodies or antibody fragments (for example, F(ab’)2 bispecific antibodies). For example, WO 1996/016673 describes a bispecific ErbB2/ Fc gamma RIH antibody; US Pat. No. 5,837,234 describes a bispecific ErbB2/ Fc gamma R1 antibody; WO 1998/002463 describes a bispecific ErbB2/Fc alpha antibody; and US 5,821 ,337 describes a bispecific ErbB2/ CD3 antibody. [0155] Some additional exemplary dimers or bispecific antibodies have two heavy chains (each having three heavy chain CDRs, followed by (N-terminal to C-terminal) a CHI domain, a hinge, a CH2 domain, and a CH3 domain), and two immunoglobulin light chains that confer antigen-binding specificity through association with each heavy chain. However, as indicated, additional architectures are envisioned, including dimers or bispecific antibodies in which the light chain(s) associate with each heavy chain but do not (or minimally) contribute to antigen-binding specificity, or that can bind one or more of the epitopes bound by the heavy chain antigen-binding regions, or that can associate with each heavy chain and enable binding of one or both of the heavy chains to one or both epitopes.
[0156] As indicated previously, IgA and IgM antibodies have the ability to multimerize. IgA, as the major class of antibody present in the mucosal secretions of most mammals, represents a key first line of defense against invasion by inhaled and ingested pathogens. IgA is also found at significant concentrations in the serum of many species, where it functions as a second line of defense mediating elimination of pathogens that have breached the mucosal surface. Receptors specific for the Fc region of IgA, FcaR, are key mediators of IgA effector function. Native IgA is a tetrameric protein including two identical light chains (K or A) and two identical heavy chains. IgA, similarly, to IgG, contains three constant domains (CA1-CA3), with a hinge region between the CAI and CA2 domains. The main difference between IgA I and lgA2 resides in the hinge region that lies between the two Fab arms and the Fc region. IgAl has an extended hinge region due to the insertion of a duplicated stretch of amino acids, which is absent in lgA2. Both forms of IgA have the capacity to form dimers, in which two monomer units, each including two heavy chains and light chains, are arranged in an end-to-end configuration stabilized by disulfide bridges and incorporation of a J-chain. J-chains are also part of IgM pentamers and are discussed in more detail below. Dimeric IgA, produced locally at mucosal sites, is transported across the epithelial cell boundary and out into the secretions by interaction with the polymeric immunoglobulin receptor (plgR). During this process the plgR is cleaved and the major fragment, termed secretory component (SC), becomes covalently attached to the IgA dimer.
[0157] ] Both IgA and IgM (discussed further below in relation to pentamers and hexamers) possess an 18-amino acid extension in the C terminus called the “tail piece” (tp). The IgA and IgM tail piece is highly conserved among various animal species. The conserved penultimate cysteine residue in the IgA and IgM tail pieces has been demonstrated to be involved in multimerization by forming a disulfide bond between heavy chains to permit formation of a multimer. Both tail pieces contain an N-linked carbohydrate addition site, the presence of which is required for dimer formation in IgA and J-chain incorporation and pentamer formation in IgM. However, the structure and composition of the N-linked carbohydrates in the tail pieces differ, suggesting differences in the accessibility of the glycans to processing by glycosyltransferases. Particularly, the IgA (atp) and IgM (ptp) tail pieces differ at seven amino acid positions.
[0158] The human IgAl constant region typically includes the amino acid sequence: ASPTSPKVFPLSLCSTQPDGNWIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQ DASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPS TPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKS AVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGN TFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTW ASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAG KPTHVNVSVVMAEVDGTCY (SEQ ID NO: 1252). Referring to this SEQ ID NO: 1252, the human CAI domain extends from amino acid 6 to amino acid 98; the human IgAl hinge region extends from amino acid 102 to amino acid 124, the human CA2 domain extends from amino acid 125 to amino acid 219, the human CA3 domain extends from amino acid 228 to amino acid 330, and the tailpiece extends from amino acid 331 to amino acid 352.
[0159] The human lgA2 constant region typically includes the amino acid sequence ASPTSPKVFPLSLDSTPQDGNWVACLVQGFFPQEPLSVTWSESGQNVTARNFPPS QDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCH PRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDL CGCYSVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLP PPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGT TTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSV VMAEVDGTCY(SEQ ID NO: 1253). Referring to this SEQ ID NO: 1253, the human CAI domain extends from amino acid 6 to amino acid 98, the human lgA2 hinge region extends from amino acid 102 to amino acid 111 , the human CA2 domain extends from amino acid 113 to amino acid 206, the human CA3 domain extends from amino acid 215 to amino acid 317, and the tailpiece extends from amino acid 318 to amino acid 340. [0160] As indicated, two IgA binding units can form a complex with two additional polypeptide chains, the J-chain (e.g., the mature human J chain) and the secretory component to form a bivalent secretory IgA (slgA)-derived binding molecule. While not wishing to be bound by theory, the assembly of two IgA binding units into a dimeric IgA- derived binding molecule is thought to involve the CA3 and tailpiece domains. See, e.g., Braathen, R., et al., J. Biol. Chem. 277:42755-42762 (2002). Accordingly, a multimerizing dimeric IgA-derived binding molecule provided in this disclosure typically includes IgA constant regions that include at least the CA3 and tailpiece domains.
[0161] An engineered IgA heavy chain constant region can additionally include a CA2 domain or a fragment thereof, an IgA hinge region or fragment thereof, a CAI domain or a fragment thereof, and/or other IgA (or other immunoglobulin, e.g., IgG) heavy chain domains, including, e.g., an IgG hinge region. In certain embodiments, a binding molecule as provided herein can include a complete IgA heavy chain constant domain (e.g., SEQ ID NO: 1252 or SEQ ID NO: 1253), or a variant, derivative, or analog thereof.
[0162] scFv dimers or diabodies may also be used, rather than whole antibodies. Diabodies and scFv can be constructed without an Fc region, using only variable domains (usually including the variable domain components from both light and heavy chains of the source antibody), potentially reducing the effects of anti-idiotypic reaction. Other forms of bispecific antibodies include the single chain “Janusins” described in Traunecker et al. (Embo lournal, 10, 3655-3659, 1991).
[0163] Dimers (e.g., bispecific antibodies) with extended half-lives are described in, for example, US Patent No. 8,921 ,528 and US Patent Publication No. 2014/0308285.
[0164] Other methods for making dimers are similarly well-known in the art. For example, traditional production of full-length antibodies, such as IgA antibodies, is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have the same specificities. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (see, for example, Millstein et al. Nature 305:37-39, 1983). Similar procedures are disclosed in, for example, WO 1993/008829, Traunecker et al., EMBO I. 10:3655- 3659, 1991 and Holliger & Winter, Current Opinion Biotechnol. 4, 446-449 (1993).
[0165] In particular embodiments, dimers can be prepared using chemical linkage. For example, Brennan et al. (Science 229: 81, 1985) describes a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab’)2 fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize vicinal di thiols and prevent intermolecular disulfide formation. The Fab’ fragments generated then are converted to thionitrobenzoate (TNB) derivatives. One of the Fab’-TNB derivatives then is reconverted to the Fab’ -thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab’-TNB derivative to form the dimer.
[0166] Particular embodiments include IgM immunoglobulin constant region domains that allow the binding portion of molecules provided herein to readily multimerize into pentamers or hexamers.
[0167] Particular embodiments include IgM constant regions (or variants thereof). These embodiments have the ability to form hexamers, or in association with a J-chain, form pentamers. Embodiments with an IgM constant region typically include at least the Cp4-tp domains of the IgM constant region but can include heavy chain constant region domains from other antibody isotypes, e.g., IgG, from the same species or from a different species. In particular embodiments, one or more constant region domains can be deleted so long as the IgM antibody is capable of forming hexamers and/or pentamers. Thus, an IgM antibody can be, e.g., a hybrid IgM/IgG antibody or can be a “multimerizing fragment” of an IgM-derived binding molecule.
[0168] The assembly of five or six IgM binding units into a pentameric or hexameric IgM antibody is thought to involve the Cp4 and tail piece domains. See, e.g., Braathen, R., et al., J Biol. Chem. 277:42755-42762 (2002). Accordingly, a pentameric or hexameric IgM antibody described in this disclosure typically includes at least the Cp4 and/or tailpiece domains (also referred to herein collectively as Cp4-tp). A “multimerizing fragment” of an IgM heavy chain constant region thus includes at least the Cp4-tp domains. An IgM heavy chain constant region can additionally include a Cp3 domain or a fragment thereof, a Cp2 domain or a fragment thereof, a Cpl domain or a fragment thereof, and/or other IgM heavy chain domains.
[0169] Five IgM monomers form a complex with a J-chain to form a native IgM molecule. The J-chain is considered to facilitate polymerization of chains before IgM is secreted from antibody producing cells. Sequences for the human IGJ gene are known in the art, for example, (IGMT Accession: J00256, X86355, M25625, AJ879487). The J chain establishes the disulfide bridges between IgM antibodies to form multimeric structures such as pentamers. See, for example, Sorensen et al. International Immunology, (2000), pages 19-27. While crystallization of IgM has proved to be notoriously challenging, Czajkowsky and Shao (PNAS 106(35): 14960-14965, 2009) published a homology-based structural model of IgM, based on the structure of the IgE Fc domain and the known disulfide pairings. The authors report that the human IgM pentamer is a mushroom- shaped molecule with a flexural bias. The IgM heavy (p) chain contains five N-linked glycosylation sites: Asn-171 , Asn-332, Asn-395, Asn-402 and Asn-563. In an IgM antibody where each binding unit is bivalent, the binding molecule itself can have 10 or 12 valencies.
[0170] The Kabat numbering system for the human IgM constant domain can be found in Kabat, et. al. “Tabulation and Analysis of Amino acid and nucleic acid Sequences of Precursors, V- Regions, C-Regions, J-Chain, T-Cell Receptors for Antigen, T-Cell Surface Antigens, b-2 Microglobulins, Major Histocompatibility Antigens, Thy-I, Complement, C-Reactive Protein, Thymopoietin, Integrins, Post-gamma Globulin, a-2 Macroglobulins, and Other Related Proteins,” U.S. Dept of Health and Human Services (1991). IgM constant regions can be numbered sequentially (i.e., amino acid #1 starting with the first amino acid of the constant region) or by using the Kabat numbering scheme.
[0171] A “full length IgM antibody heavy chain” is a polypeptide that includes, in N- terminal to C- terminal direction, an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CMl or Cpl), an antibody heavy chain constant domain 2 (CM2 or Cp2), an antibody heavy chain constant domain 3 (CM3 or Cp3), and an antibody heavy chain constant domain 4 (CM4 or Cp4) that can include a tailpiece, as indicated above.
[0172] In particular embodiments, each binding unit of a multimeric binding molecule as provided herein includes two IgM heavy chain constant regions or multimerizing fragments or variants thereof, each including at least an IgM Cp4 domain and an IgM tail piece domain. In certain embodiments the IgM heavy chain constant regions can each further include an IgM Cp3 domain situated N-terminal to the IgM Cp4 and IgM tail piece domains.
[0173] In particular embodiments, the IgM heavy chain constant regions can each further include an IgM Cp2 domain situated N-terminal to the IgM Cp3 domain. Exemplary multimeric binding molecules provided herein include human IgM constant regions that include the wild-type human Cp2, Cp3, and Cp4-TP domains as follows: VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVT TDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMC VPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHT NISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALH RPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKP TLYNVSLVMSDTAGTCY (SEQ ID NO: 1254).
[0174] In certain IgM-derived multimeric binding molecules as provided herein each IgM constant region can include, instead of, or in addition to an IgM Cp2 domain, an IgG hinge region or functional variant thereof situated N-terminal to the IgM Cp3 domain. An exemplary variant human IgGl hinge region amino acid sequence in which the cysteine at position 6 is substituted with serine is VEPKSSDKTHTCPPCPAP (SEQ ID NO: 1255). An exemplary IgM constant region of this type includes the variant human IgGl hinge region fused to a multimerizing fragment of the human IgM constant region including the Cp3, Cp4, and TP domains, and includes the amino acid sequence: VEPKSSDKTHTCPPCPAPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSV TISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDL PSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQW MQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEA LPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 1256).
[0175] The human IgM constant region typically includes the amino acid sequence GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFP SVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPP KVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQA EAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDT AIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHP NATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYL LPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQA PGRYFAHSILTVSEEEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYNVSL VM SDTAGTCY (SEQ ID NO: 1257; identical to, e.g., GenBank Accession Nos. pirllS37768, CAA47708.1 , and CAA47714.1). Referring to this SEQ ID NO: 1257, the human Cpl region ranges from amino acid 5 to amino acid 102; the human Cp2 region ranges from amino acid 114 to amino acid 205, the human Cp3 region ranges from amino acid 224 to amino acid 319, the Cp4 region ranges from amino acid 329 to amino acid 430, and the tailpiece ranges from amino acid 431 to amino acid 453. [0176] In particular embodiments, an IgM heavy chain constant region includes the sequence:GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDI SSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPL PVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGV TTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSM CVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKT HTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVA LHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTS APMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTG KPTLYNVSLVMSDTAGTCY (SEQ ID NO: 1258; (UniProt ID P01871)— allele IGHM*04). This sequence differs from SEQ ID NO: 1257 by one amino acid at position 191.
[0177] Other forms of the human IgM constant region with minor sequence variations exist, including GenBank Accession Nos. P01871.4, CAB37838.1, and pirllMHHU. The amino acid substitutions, insertions, and/or deletions at positions corresponding to SEQ ID NO: 1257 described herein can likewise be incorporated into alternate human IgM sequences, as well as into IgM constant region amino acid sequences of other species.
[0178] As indicated, five IgM binding units can form a complex with a J-chain to form a pentameric IgM antibody.
[0179] The precursor form of the human J-chain includes: MKNHLLFWGVLAVFIKAVHVKAQEDERIVLVDNKCKCARITSRIIRSSEDPNEDI VERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSN ICDEDSATETCYTYDRNKC YTAVVPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 1259). The signal peptide extends from amino acid 1 to amino acid 22 of SEQ ID NO: 1259 and the mature human J-chain extends from amino acid 23 to amino acid 159 of SEQ ID NO: 1259.
[0180] The mature human J-chain includes the amino acid sequence QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRT RFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAV VPLVYGGETKMVETALTPD ACYPD (SEQ ID NO: 1260).
[0181] The term “J-chain” as used herein refers to the J-chain of native sequence IgM or IgA antibodies of any animal species. When specified, it can also refer to any functional fragment thereof, derivative thereof, and/or variant thereof. Functional fragments, derivatives and variants retain the multimerizing functionality of the reference sequence and also include at least 90% or 95% sequence identity to the reference sequence.
[0182] An antibody binding domain can be introduced into the J-chain at any location that allows the binding of the binding domain to its binding target without interfering with J-chain function or the function of an associated IgA, IgM, or hybrid IgG antibody. Insertion locations include at or near the C- terminus, at or near the N-terminus or at an internal location that, based on the three- dimensional structure of the J-chain, is accessible.
[0183] The human lgG2 Fc region includes the amino acid sequence: PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTFRWSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PMLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1261). Referring to SEQ ID NO: 1261, the human CH2 region extends from amino acid 10 to amino acid 107 and the human CH3 region extends from amino acid 116 to amino acid 212.
[0184] The human lgG3 Fc region includes the amino acid sequence: PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVQFKWYVDGVEV HNAKTKPREEQFNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKT KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTT PPMLDSDGSFFLYSKLTVDKSRWQ QGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 1262). Referring to SEQ ID NO: 1262, the human CH2 region extends from amino acid 1 1 to amino acid 108 and the human CH3 region extends from amino acid 117 to amino acid 212.
[0185] The human lgG4 Fc region includes the amino acid sequence: PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 1263). Referring to SEQ ID NO: 1263, the human CH2 region extends from amino acid 1 to amino acid 111 and the human CH3 region extends from amino acid 112 to amino acid 218. [0186] Particular embodiments include an IgG Fc region with an IgA or IgM tailpiece inserted into the IgG Fc region or fused to the C-terminus. J-chains can also be inserted or fused.
[0187] In particular embodiments, the PcrV-specific antibodies and/or binding proteins disclosed herein may be conjugated to an additional molecule. Exemplary molecules may include immunotoxins, drugs, radioisotopes, and detectable labels.
[0188] Any of the antibodies and/or binding proteins described herein in any exemplary format can be formulated alone or in combination into compositions for administration to subjects. Salts and/or pro-drugs of the antibodies can also be used.
[0189] A pharmaceutically acceptable salt includes any salt that retains the activity of the antibody and is acceptable for pharmaceutical use. A pharmaceutically acceptable salt also refers to any salt which may form in vivo as a result of administration of an acid, another salt, or a prodrug which is converted into an acid or salt. A prodrug includes an active ingredient which is converted to a therapeutically active compound after administration, such as by cleavage or by hydrolysis of a biologically labile group.
[0190] In particular embodiments, the compositions include antibodies and/or binding proteins of at least 0.1 % w/v or w/w of the composition; at least 1 % w/v or w/w of composition; at least 10% w/v or w/w of composition; at least 20% w/v or w/w of composition; at least 30% w/v or w/w of composition; at least 40% w/v or w/w of composition; at least 50% w/v or w/w of composition; at least 60% w/v or w/w of composition; at least 70% w/v or w/w of composition; at least 80% w/v or w/w of composition; at least 90% w/v or w/w of composition; at least 95% w/v or w/w of composition; or at least 99% w/v or w/w of composition.
[0191] Exemplary generally used pharmaceutically acceptable carriers include any and all absorption delaying agents, antioxidants, binders, buffering agents, bulking agents or fillers, chelating agents, coatings, disintegration agents, dispersion media, gels, isotonic agents, lubricants, preservatives, salts, solvents or co-solvents, stabilizers, surfactants, and/or delivery vehicles.
[0192] Exemplary antioxidants include ascorbic acid, methionine, and vitamin E.
[0193] Exemplary buffering agents include citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and/or trimethylamine salts. [0194] An exemplary chelating agent is EDTA (ethylene-diamine-tetra-acetic acid).
[0195] Exemplary isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.
[0196] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, and benzalkonium halides.
[0197] Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the antibodies or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can include polyhydric sugar alcohols, amino acids, organic sugars or sugar alcohols, PEG, amino acid polymers, sulfur-containing reducing agents, low molecular weight polypeptides (i.e., <10 residues), proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins, hydrophilic polymers, monosaccharides, disaccharides, trisaccharides, and polysaccharides.
[0198] Cell-based compositions (e.g., cells genetically modified to express a binding domain of an antibody disclosed herein, are also contemplated.
[0199] The compositions disclosed herein can be formulated for administration by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion. The compositions disclosed herein can further be formulated for intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral, sublingual, and/or subcutaneous administration. For injection, compositions can be formulated as aqueous solutions, such as in buffers including Hanks’ solution, Ringer’s solution, or physiological saline. The aqueous solutions can include formulatory agents such as suspending, stabilizing, and/or dispersing agents. Alternatively, the formulation can be in lyophilized and/or powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0200] For oral administration, the compositions can be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like. Compositions can be formulated as an aerosol. In particular embodiments, the aerosol is provided as part of an anhydrous, liquid, or dry powder inhaler. Aerosol sprays from pressurized packs or nebulizers can also be used with a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
[0201] Compositions can also be formulated as depot preparations. Depot preparations can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
[0202] Additionally, compositions can be formulated as sustained-release systems utilizing semipermeable matrices of solid polymers including at least one type of antibody. Various sustained-release materials have been established and are well known by those of ordinary skill in the art.
[0203] Any composition disclosed herein can advantageously include any other pharmaceutically acceptable carriers which include those that do not produce significantly adverse, allergic, or other untoward reactions that outweigh the benefit of administration. Exemplary pharmaceutically acceptable carriers and formulations are disclosed in Remington’ s Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Moreover, formulations can be prepared to meet sterility, pyrogenicity, general safety, and purity standards as required by U.S. FDA Office of Biological Standards and/or other relevant foreign regulatory agencies.
[0204] Methods disclosed herein include treating subjects (e.g., humans, veterinary animals (dogs, cats, reptiles, birds) livestock (e.g., horses, cattle, goats, pigs, chickens) and research animals (e.g., monkeys, rats, mice, fish) with compositions disclosed herein. Treating subjects includes delivering therapeutically effective amounts. Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments, and/or therapeutic treatments.
[0205] An “effective amount” is the amount of a composition necessary to result in a desired physiological change in the subject. Effective amounts are often administered for research purposes. Effective amounts disclosed herein can cause a statistically- significant effect in an animal model or in vitro assay relevant to the assessment of an infection’s development, progression, and/or resolution.
[0206] ’’prophylactic treatment” includes a treatment administered to a subject who does not display signs or symptoms of an infection or displays only early signs or symptoms of an infection such that treatment is administered for the purpose of diminishing or decreasing the risk of developing the infection further. Thus, a prophylactic treatment functions as a preventative treatment against an infection. In particular embodiments, prophylactic treatments reduce, delay, or prevent the worsening of an infection.
[0207] A “therapeutic treatment” includes a treatment administered to a subject who displays symptoms or signs of an infection and is administered to the subject for the purpose of diminishing or eliminating those signs or symptoms of the infection. The therapeutic treatment can reduce, control, or eliminate the presence or activity of the infection and/or reduce control or eliminate side effects of the infection.
[0208] Function as an effective amount, prophylactic treatment or therapeutic treatment are not mutually exclusive, and in particular embodiments, administered dosages may accomplish more than one treatment type.
[0209] For administration, therapeutically effective amounts (also referred to herein as doses) can be initially estimated based on results from in vitro assays and/or animal model studies. Such information can be used to more accurately determine useful doses in subjects of interest. The actual dose amount administered to a particular subject can be determined by a physician, veterinarian or researcher taking into account parameters such as physical and physiological factors including target, body weight, severity of condition, type of infection, stage of infection, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.
[0210] Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly).
[0211] Biological samples can include any biological sample obtained from a subject from which B cells or B cell progenitor cells can be isolated and include bone marrow, spleen, lymph node, blood, e.g., peripheral blood, tissue biopsies or samples, surgical specimens, fine needle aspirates, autopsy material, and the like. In particular embodiments, a biological sample refers to a sample isolated from a subject, such as a peripheral blood sample, which is then further processed, for example, by cell sorting (e.g., magnetic sorting and/or FACS), to obtain a population of antigen-specific IgM, IgG, or IgA memory B cells. In particular embodiments, a biological sample including IgM, IgG, or IgA memory B cells refers to an in vitro or ex vivo culture of expanded antigen- specific IgM, IgG, or IgA memory B cells. Such a sample is enriched for antigen- specific IgM, IgG, or IgA memory B cells relative to the proportion of such cells that might occur in, e.g., a blood sample from a subject exposed to the antigen.
[0212] In particular embodiments, methods disclosed herein include a method of making an antibody construct including a PcrV antigen-binding domain from a memory B cell that specifically binds to a PcrV antigen of interest. In particular embodiments, the method includes: (a) isolating a population of IgM, IgG, or IgA expressing memory B cells from a biological sample; (b) amplifying heavy chain and light chain variable domain sequences from the memory B cell population of step (a); (c) ligating heavy chain variable domain sequences amplified in step (b) into a heavy chain expression vector sequence and ligating light chain variable domain sequences amplified in step (b) into a light chain expression vector sequence; (d) introducing one or more vectors encoding heavy chain and light chain expression vector sequences of step (c) to a cell and culturing the cell under conditions that permit expression of antibody polypeptides from the expression vector sequences; (e) contacting antibody polypeptides expressed by the cell with an antigen of interest; and (f) isolating antibodies that bind to the PcrV antigen of interest, thereby making an antibody construct including a PcrV antigen-binding domain from a memory B cell and that specifically binds to an antigen of interest.
[0213] In particular embodiments, the method further includes, prior to step (a), a step of immunizing a subject and obtaining a biological sample.
[0214] In particular embodiments, the method further includes, prior to step (e), collecting cell culture medium from cells of step (d).
[0215] In particular embodiments, step (e) includes contacting antibody polypeptides with the PcrV antigen of interest immobilized on a solid support.
[0216] In particular embodiments, methods include sorting PcrV-specific IgM, IgG, or IgA memory B cells (MBCs), including: contacting a biological sample obtained from a subject infected with Pseudomonas aeruginosa with a tetramer (or other multimer) including a PcrV antigen; and sorting a cell population including PcrV-specific IgM, IgG, or IgA MBCs based on binding to the tetramer (or other multimer). In particular embodiments, the method further includes generating tetramers or other multimers including PcrV antigens prior to the contacting step. In particular embodiments, the methods further include a step of sequencing one or more BCRs, or at least the PcrV antigen-binding domains thereof, expressed by the cell population including IgM, IgG, or IgA MBCs. Methods of sequencing are known in the art. See, for example, Schwartz et al. (2014). J Immunol 193, 3492-3502. In particular embodiments, the methods further include a step of cloning the one or more BCRs and expressing the one or more BCRs as recombinant PcrV-specific antibodies or antigen-binding fragments thereof. See, for example, Tiller et al. (2009) J Immunol Methods 350, 183-193.
[0217] A multimer of a PcrV antigen of interest can be formed by labeling the PcrV antigen with a labeling system that allows multimerization and detection by flow cytometry. For example, a phycoerythrin (PE)-conjugated tetramer containing the PcrV antigen can be generated. In particular embodiments, tagged (e.g., His tagged) recombinant PcrV antigen can be produced and purified using the tag. The purified recombinant antigen can be biotinylated and tetramerized with streptavidin-PE (e.g., from Prozyme, Agilent Technologies, Santa Clara, CA) as described in Taylor et al. (2012). J Exp Med 209, 2065- 2077). B cells binding PcrV antigen in the tetramers can be enriched using anti-PE magnetic microbeads. A decoy reagent to gate out non-antigen-specific B cells (e.g., B cells binding to non-antigen tetramer components such as PE, streptavidin, and biotin epitopes) can be made by conjugating streptavidin-PE to Alexa Fluor 647 (AF647) using an AF647 protein labeling kit (ThermoFisher Scientific, Waltham, MA), washing and removing any unbound AF647, and incubating with an excess of an irrelevant biotinylated His-tagged protein, similar to what has been previously described (Taylor et al. (2012). J Exp Med 209, 2065-2077).
[0218] In particular embodiments, splenocytes can first be stained with a decoy reagent to exclude cells binding other components of the tetramer and then with an antigen PE tetramer (Taylor et al. (2012). J Exp Med 209, 2065-2077). Anti-PE coated magnetic beads can be used to enrich both decoy-specific and PcrV antigen-specific B cells, which can subsequently be stained with antibodies for analysis by multiparameter flow cytometry. In particular embodiments, antibody panels can be used that allow visualization of all stages of mature B2 B cell differentiation.
[0219] Control experiments can be conducted using B cells with BCRs that do not bind the PcrV antigen tetramer and to assess that they are not activated non-specifically by PcrV antigen.
[0220] Single antigen- specific MBCs can be sorted by flow cytometry into 96-well plates. BCRs can be amplified and sequenced from the cDNA of single cells as previously described (Schwartz et al. (2014). J Immunol 193, 3492-3502), with additional IgH primers described in Tiller et al. (2009) J Immunol Methods 350, 183-193. Amplified products can be cloned and mAbs generated using previously described methods (Schwartz et al., 2014; Tiller et al., 2009).
[0221] Provided herein, in particular embodiments, are compositions including a population of PcrV antigen-specific IgM, IgG, or IgA memory B cells bound via their B cell receptors to PcrV antigen immobilized on a solid support. In particular embodiments, the PcrV antigen immobilized on the solid support includes a multimer construct including the PcrV antigen.
[0222] In particular embodiments, the compositions described herein selectively bind the PcrV antigen of interest. Methods of measuring binding of a polypeptide to an antigen are known in the art (e.g., enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immune absorbent spot (ELISPOT) assay).
[0223] Another aspect of the present disclosure describes a method of detecting the presence of a PA PcrV antigen in a solution or on a cell. The method involves providing a binding protein or an antibody described herein to the solution or cell and measuring the ability of the protein to bind to the PA PcrV antigen in the solution or cell. Measurements can be quantitative or qualitative.
[0224] Another aspect of the present disclosure describes a cell line producing a binding protein disclosed herein.
[0225] While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.
[0226] The Experimental Examples below are included to demonstrate particular, non-limiting embodiments of the disclosure. Those of ordinary skill in the art will recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure.
EXAMPLE 1
PBMC and serum collection
[0227] Under protocols approved by the Seattle Children’s Institutional Review Board (SCH#10325), CF donors were recruited from patients receiving care that day at Seattle Children’s Cystic Fibrosis Clinic in March-lune 2019 (for initial B cell phenotyping and serum ELIS As) and April- July 2021 (serum ELIS As and BCR sequencing experiments). Individuals who declined to participate or were assessed by the clinical team to be unable to safely donate at least 30 mL of blood were excluded. Blood was transported at room temperature to the University of Washington Department of Immunology and processed within 4 hours of collection. Serum was collected by centrifugation at 1500 g for 10 min, and then frozen at -80 C. Peripheral mononuclear blood cells (PBMCs) were collected using SepMate-50 PBMC Isolation Tubes (STEMCELL Technologies) and frozen slowly at -80 C before transfer to liquid nitrogen for long-term storage. Non-CF donor samples were provided by BloodWorks Northwest from regular blood donors or from non-mobilized healthy donors through the Fred Hutch Hematopoietic Cell Procurement and Processing core.
EXAMPLE 2
Tetramer production
[0228] Recombinant PcrV was expressed using E. coli transformed with low copy plasmid containing the PcrV CDS (GenBank: AF010149.1) and a 10 X N terminal His tag and purified from filtered bacterial supernatant on a His affinity column. To enable tetramerization, PcrV was biotinylated using EZ-Link Sulfo-NHS-LC Biotinylation Kit (ThermoFisher). The production of antigen-specific B cell tetramer reagents has been previously described in some detail. Briefly, biotinylated PcrV was co-incubated with streptavidin-fluorophore (SA-PE or SA-APC; Agilent). Decoy tetramers are produced by tetramerization of an irrelevant protein with a matched conjugated fluorophore (e.g., PE- Cy7 for use in experiments requiring the PE-conjugated PcrV tetramer).
EXAMPLE 3
Identification of PcrV-specific cells
[0229] Methods for isolating antigen- specific B cells using tetramer reagents are described in Taylor, Justin J et al. “A germinal center-independent pathway generates unswitched memory B cells early in the primary response.” The Journal of experimental medicine vol. 209,3 (2012): 597-606. doi: 10. 1084/jem.20111696, incorporated herein by reference in its entirety. Flow cytometry was performed on a BD LSR II (Becton Dickinson). For BCR sequencing experiments, single cells were sorted into a 96 wellmicroplate using a BDFACS Aria II that was contained within a biosafety cabinet. EXAMPLE 4
BCR sequencing
[0230] cDNA was amplified from singly sorted B cells using SMART-Seq v4 (Takara Bio) at half reaction volumes. Initial BCR sequencing for donor 1 followed protocols previously described in detail in Hale, Malika et al. “IgM antibodies derived from memory B cells are potent cross-variant neutralizers of SARS-CoV-2.” The Journal of experimental medicine vol. 219,9 (2022): e20220849. doi:10. 1084/jem.20220849 and Thouvenel, Christopher D et al. “Multimeric antibodies from antigen-specific human IgM-i- memory B cells restrict Plasmodium parasites.” The Journal of experimental medicine vol. 218,4 (2021): e20200942. doi: 10.1084/jem.20200942, incorporated herein by reference in their entirety. Briefly, a single, multiplex PCR was performed for each B cell using a universal primer for the template switch region and pooled constant region primers for the p, y, a, K, and /. constant regions. Amplicons were then purified and sequenced by Sanger sequencing. For donors 2 and 3, a custom protocol that enables high-quality, single cell BCR sequencing by MiSeq was employed (manuscript in preparation as Thouvenel et al). Alignment of all trimmed sequences was performed using both TRUST4 and IGMT/HighV-QUEST. Rare conflicts (e.g., differences in reported %SHM) were resolved by review of the raw sequence data and individual analysis in IgBlast.
EXAMPLE 5
BCR cloning
[0231] Our methods for cloning BCR variable region sequences into antibody expression plasmids were described previously in detail in Hale, Malika et al. “IgM antibodies derived from memory B cells are potent cross-variant neutralizers of SARS- CoV-2.” The Journal of experimental medicine vol. 219,9 (2022): e20220849. doi: 10.1084/jem.20220849 and Thouvenel, Christopher D et al. “Multimeric antibodies from antigen- specific human IgM-i- memory B cells restrict Plasmodium parasites.” The Journal of experimental medicine vol. 218,4 (2021): e20200942. doi: 10. 1084/jem.20200942, incorporated herein by reference in their entirety. Briefly, each light chain was cloned into vectors of its isotype, K or X, following the manufacturer’s protocol for in-fusion cloning (Takara Bio). All heavy chains were similarly cloned into IgGl and IgM plasmids in parallel. Concordance with the parental cDNA was confirmed by Sanger sequencing of the cloned plasmids. [0232] For the V2L2MD IgG and IgM mAbs, heavy and light chain sequences were synthesized as a gBlock (IDT) by introducing mutations to match the amino acid sequence for the anti-PcrV VH/VL in gremubamab (INN 10909; 17) in the closest human VH and VL nucleotide sequences. The resultant V(D)J sequences were then synthesized as a gBlock (IDT) and then cloned into expression plasmids using the same methods described for generation of our human B cell BCR sequences.
[0233] Whole-plasmid sequences were obtained from a subset of plasmids as a quality control measure (Primordium Labs).
EXAMPLE 6
Production of mAbs
[0234] IgG mAbs were produced in HEK-293T cells (ATCC) by co-transfection of heavy- and light-chains in polyethylenimine as previously described in Thouvenel, Christopher D et al. “Multimeric antibodies from antigen- specific human IgM-i- memory B cells restrict Plasmodium parasites.” The Journal of experimental medicine vol. 218,4 (2021): e20200942. doi:10.1084/jem.20200942, incorporated herein by reference. Production of IgM mAbs was carried out as described in Hale et al., using the same human J chain plasmid. For initial screens, supernatant was harvested at 4 days post-transfection and concentrated and buffer-exchanged into PBS using 50,000 MWCO Millipore Amicon Ultra- 15 Centrifugal Filter units (Thermo Fisher Scientific). Purification was carried out following manufacturer’s instructions on HiTrap Protein G HP purification column for IgG mAbs (GE Healthcare), and a POROS CaptureSelect IgM-XL Affinity Matrix Column (Thermo Fisher Scientific) for IgM. Antibodies were concentrated and buffer-exchanged into phosphate buffered saline to at least 1 mg/mL, and then stored at -80C in 120-200 pL aliquots.
EXAMPLE 7
ELISAs
[0235] To prepare for antigen-specific antibody ELISAs, recombinant PcrV was prepared as described above and diluted to 2 pg/ml in PBS and incubated on high-binding 96 well plates (Coming) overnight at 4° C. Plates were then washed thoroughly with PBS and 0.05% Tween 20 (PBS-T). Next, non-specific interactions were blocked using 200 pl/well of PBS-T with 3% bovine serum albumin (3%) at room temperature for 1 - 3 hours. Samples of interest were serially diluted in PBS-T immediately prior to use. Dilution series for each sample were incubated on washed plates for 2 hours at room temperature. After thorough washing with PBS-T, bound antibodies were detected by incubation for 1 hour at room temperature with HRP-conjugated goat anti-human IgG (diluted 1:3000 in PBS-T; SouthemBiotech), IgM (1 :1500; SouthemBiotech), or IgA (1 :1500; ThermoFisher), washed again, and developed using lx 3,39,5,50-tetramethylbenzidine (Invitrogen) and 1 M H2SO4. OD was measured on a SpectroMax i3X (Molecular Devices, San Jose, CA) at 450 and 570 nm, and OD450-750 was analyzed in Prism (v9.5; GraphPad). When un-purified transfectant supernatants were used, ELIS As for total IgM or IgG were also performed in parallel using human uncoated IgM or IgG ELISA kits (Invitrogen), following manufacturer’s instructions.
EXAMPLE 8
Murine pneumonia challenge
[0236] All animal procedures were conducted according to the guidelines of the Emory University Institutional Animal Care and Use Committee (IACUC), under approved protocol number PROTO 201700441. The study was carried out in strict accordance with established guidelines and policies at Emory University School of Medicine, and recommendations in the Guide for Care and Use of Laboratory Animals, as well as local, state, and federal laws. Eight- to ten- week-old female BALB/c mice (Jackson Laboratories, Bar Harbor, ME) were anesthetized by intraperitoneal injection of 0.2 ml of a cocktail of ketamine (100 mg/ml) and xylazine (5 mg/ml) and intranasally instilled with approximately 105 CFU P. aeruginosa PA103 (in 10-20 pL of PBS). At 15 minutes post-infection, monoclonal antibodies or PBS were delivered via the same route in a 20 pL volume. Mice were euthanized at 24 hours post-infection, and whole lungs were collected aseptically, weighed, and homogenized for 20 seconds in 1 ml of PBS, followed by serial dilution onto Difco™ Pseudomonas isolation agar, and plated for CFU enumeration.
EXAMPLE 9
PcrV-specific B cells are enriched in CF individuals
[0237] To prepare to isolate PcrV-specific B cells, antigen- specific tetramer reagent consisting of four recombinant PcrV proteins linked to a fluorophore were first generated. In parallel, a decoy tetramer consisting of irrelevant proteins linked to a tandem fluorophore was also generated (FIG. 1A). To test the tetramer reagent, immune competent mice were immunized with recombinant PcrV. Immunization induced class-switched B cells that bound to the tetramer. These findings are consistent with successful detection of cells derived from a PcrV- specific germinal center response and imply that this reagent should function similarly for detection of antigen- specific human B cells (FIG. 2).
[0238] Peripheral blood samples were obtained from a cohort of 14 young adults with CF who had received a swab test for PA as part of routine outpatient care. As B cells specific for any single antigen are rare, tetramer-bound cells were enriched via an anti- fluorophore magnetic column prior to analysis. After enrichment, PcrV-specific cells made up > 5% of B cells in 7 of 14 (50%) of CF donors, while 0 of 14 (0%) samples obtained from non-CF donors (isolated from a local blood bank) exhibited binding (FIG. IB). Similar differences between CF vs. non-CF donors were obtained when the number of PcrV-specific B cells in each sample was normalized to lymphocyte count (FIG. 1C).
EXAMPLE 10
Generation of mAbs from IgM* B cells derived from a CF donor with chronic PA pulmonary infection
[0239] To initially test the ability to isolate PcrV-specific cells, studies were performed using a blood sample from an additional CF donor (Donor 1), who was known to be chronically PA-infected. Following single cell sorting of PcrV-specific cells, paired chain sequencing of the heavy and light chain variable regions was performed. FACS sorting methods used herein allow single-cell surface phenotyping data to be linked to the BCR sequence for individual cells. Interestingly, PcrV-specific B cells were rare, and only a very small number of PcrV-specific B cells expressed the canonical MBC surface markers (CD21+CD27+). Consistent with these observations, BCR sequencing of PcrV-specific B cells revealed predominantly germline sequences with little or no evidence for somatic hypermutation (FIG. 3A).
[0240] To directly confirm the specificity of newly acquired variable region sequences, mAbs that employed the BCR variable regions from 10 of the tetramer-bound B cells isolated in this donor were generated. As the surface-expressed isotype of all these PcrV-specific B cells was IgM, the BCR sequences were first expressed as pentameric IgM antibodies. When tested for binding to recombinant PcrV in a plate-bound ELISA assay, supernatants collected from cells co-transfected with heavy, light, and J chain plasmids (to make pentameric IgM) exhibited strong binding to PcrV for 3 of 10 CF-derived BCRs (FIG. 3B) The light chain for BCR 421 contains several mutations in CDR1. A strong binding by germline antibody sequences 408 and 411 was observed. The source cells for 411 and 421, but not 408, expressed a surface marker suggestive of a plasmablast/plasma cell phenotype (CD38+).
[0241] As clinical application was likely to require large-scale production in a similar system, the best binders that also were most efficiently expressed in the 293T cell line were selected: 408 and 411.
[0242] The inventors have previously found that for mAbs that target divergent pathogens, including SARS-CoV-2 and Plasmodium falciparum, multimerized antibodies (e.g., pentameric IgM) have enhanced binding and protection properties in vitro. However, nearly all current monoclonal antibody therapeutics employ the IgGl isotype. Therefore, the activity of these mAbs in both the IgM and IgGl formats, was next compared and expression plasmids containing the heavy chain variable regions upstream of the gammal constant region to enable expression as IgGl mAbs were generated. After co-transfection with the paired light chain (+ J chain for IgM) and antibody purification, binding to recombinant PcrV for BCR sequences 408 and 411 when expressed as IgG vs. IgM mAbs (FIG. 3C) was compared. CF BCRs 408 and 411 exhibited binding to PcrV as both IgM and IgG mAbs; with higher binding as IgM, presumably reflecting the avidity of IgM multimers.
[0243] One challenge in studying new candidate human PcrV mAbs is the lack of readily available comparators. V2L2MD is a heavily engineered anti-PcrV mAb that was generated by phage display binding optimization from a pre-cursor candidate identified by hybridoma screening from PcrV-immunized, human-variable region transgenic mice. The clinical candidate bi-specific, gremubamab, consists of the paired heavy-and light chain variable regions of V2L2MD fused to an anti-polys accharide (Psi) single chain variable fragment (scFv) and the human IgGl constant region. To prepare for in vivo protection assays, gremubamab’ s anti-PcrV variable regions (V2L2MD) was introduced into expression plasmids using the same strategies employed for expressing CF BCRs. This approach enabled production and purification of a positive control human V2L2MD IgG and IgM mAbs in parallel with the candidate CF BCR-derived mAbs.
[0244] To confirm specificity, binding to recombinant PcrV for purified IgG and IgM mAbs produced in parallel from CF BCR 411 and the newly cloned V2L2MD, was measured together with commercially sourced gremubamab (FIG. 3D). As IgM mAbs, CF BCR 411 and V2L2MD exhibited similar binding to PcrV, on par with the commercial gremubamab. EXAMPLE 11
Anti-PcrV mAbs derived from CF B cells protect mice from PA pneumonia
[0245] Next, a challenge model of pneumonia was utilized to test the anti-PA activity of candidate mAbs (FIG. 4A). Mice treated with a single, 60 pg intranasal dose of anti-PcrV IgG exhibited an ~ 2 log reduction in burden of bacteria in the lungs at 48 h in comparison with an off-target control mAb or vehicle (PBS) only (FIG. 4B). A similarly dramatic reduction in lung bacteria load was also achieved when the experiment was repeated with a 3 -fold lower dose of anti-PcrV IgG (FIG. 4C). The CF-derived mAbs performed equivalently to the positive control V2L2MD mAb at both doses. While assessment of lung bacterial burden requires sacrifice of the treated mice, doses achieving similar reductions in lung bacterial burdens have resulted in 100% survival in cohorts of mice in prior studies of V2L2MD.
EXAMPLE 12
Anti-PcrV mAbs derived from MBCs of individuals with CF
[0246] The above findings demonstrate that isolation of PcrV-specific B cells chronically infected CF donor enabled the rapid discovery of 2 protective mAbs. Notably, these mAb were derived from B cells lacking an MBC phenotype. In earlier studies of other pathogens, antigen- specific MBCs had been excellent sources for high-affinity, protective BCR sequences. Therefore, whether additional, and perhaps, higher affinity, anti-PcrV mAb could be isolated from antigen-experienced, somatically hypermutated, PcrV-specific MBCs was investigated. Unfortunately, PcrV-specific MBCs were extremely rare in the subject who was initially selected for BCR sequencing (FIG. 3A). Therefore, peripheral blood samples from 2 additional donors with CF were obtained: Donor 2, had previously tested positive for PA but was negative on most recent testing, while Donor 3, like Donor 1, was chronically infected. Using improved methods that enhanced the efficiency and depth of sequencing, single cell BCR sequencing of PcrV-specific B cells from each donor was performed. Strikingly, Donor 2, had abundant PcrV-specific MBCs after tetramer enrichment. Of the ~ 40 sequences from PcrV-specific cells identified by surface phenotype as likely MBCs (CD21+CD27+), many were somatically hypermutated (FIG. 5A). In contrast, Donor 3 had fewer MBCs and fewer somatically hypermutated cells (FIG. 7A).
[0247] There was significant breadth in V family usage within and between donors (FIG. 7B). The small number of cells analyzed, compared to B cell repertoire studies that employ bulk sorted populations, limits the interpretation of our data. However, a strength of singly sorted B cell sequencing is the ability to obtain paired chain information. The heavy chain IGHV3-23 is used, with mutations, in the V2L2MD antibody, and is a very common VH gene segment in most human B cell repertoire studies. Accordingly, it was not surprising that IGHV3-23 was also well represented in this dataset. Notably, none of the PcrV-specific B cells sequenced for the studies described herein paired VH3-23 heavy chains with IGKV1-6, the V2L2MD light chain variable gene (FIG. 7C) and IGKV1-6 was infrequent in the dataset. Heavy- and light-chain pairings for CD21+CD27+ cells are shown in FIG. 5B. These data represent the first attempt to sequence human PcrV-specific BCRs.
[0248] To pursue the goal of identifying potential protective mAbs among MBCs, 12 of these BCRs were randomly selected for cloning into expression plasmids. Supernatants from transfected cells were screened for binding to PcrV (FIG. 8). BCRs 435 and 442 were the most striking binders in the transfection screen. Because the initial screen did not adequately control for antibody concentration in the supernatant, antibodies that are less efficiently expressed by 293T cells might appear to be poor binders. Based on performance in the screen or for reasons of special interest (e.g., BCR clone 439 was derived from an IgA MBC, the predominant isotype in respiratory secretions), 5 MBC- derived BCRs were chosen for production as purified IgG mAbs. When this panel of purified mAbs was evaluated at matched concentrations (FIG. 5C), 2 of 5 of MBC-derived mAbs matched or exceeded PcrV binding exhibited by the protective, donor 1 (plasmablast derived) mAb, 411. Further, 4 of 5 (including the IgA-derived mAb, 439) matched or exceeded the other protective donor- 1 derived protective mAb, 408 IgG. Noting that 408 IgG exhibited reduced binding to PcrV in ELISA vs. 411 (FIG. 3B) but conferred equivalent protection (FIG. 4B-4C), a moderate- affinity MBC-derived mAb (439) was elected for testing in vivo, in addition to the two best performing MBC-derived mAbs (437 and 442). At a low, 20 pg dose, CF MBC-derived mAbs 437 (originally derived from an IgG MBC), 439 (derived from an IgA MBC), and 442 (derived from an IgM MBC) achieved significant reductions in bacterial burden at 48 h when compared to an off-target control mAb or saline alone (FIG. 6).
[0249] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of’ excludes any element, step, ingredient, or component not specified. The transition phrase “consisting essentially of’ limits the scope of the embodiment to the specified elements, steps, ingredients, or components and to those that do not materially affect the embodiment. A material effect would cause a statistically significant reduction in the ability to obtain a claimed effect according to a relevant experimental method described in the current disclosure.
[0250] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.
[0251] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
[0252] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0253] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
[0254] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
[0255] Furthermore, numerous references have been made to patents, printed publications, journal articles and other written text throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching. [0338] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.
[0256] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and/or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.
[0257] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster’s Dictionary, 3rd Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford, 2006).

Claims

CLAIMS The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
1. A binding protein comprising: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR), CDR3 amino acid sequence that is at least 85% identical to an amino acid sequence set forth in SEQ ID NO: 4, 11, 18, 25, 32, 39, 46, 53, 60, 67, 74, 81, 88, 95, 102, 109, 116, 123, 130, 137, 144, 151, 159, 167, 174, 181, 188, 195, 202, 209, 216, 223, 230,
237, 244, 251, 258, 265, 272, 279, 286, 293, 300, 307, 314, 321, 328, 335, 342, 349, 356,
363, 370, 377, 385, 391, 398, 405, 412, 419, 426, 433,440, 447, 454, 461, 468, 475, 482, 489, 496, 503, 510, 517, 524, 531, 538, 545, 552, 559, 566, 573, 580, 587, 594, 601, 608,
615, 622, 629, 636, 643, 650, 657, 664, 671, 678, 685, 692, 699, 706, 713, 720, 727, 734,
741, 748, 755, 763, 770, 777, 784, 791, 798, 805, 812, 819, 826, 833, 840, 847, 854, 861,
868, 875, 883, 890, 897, 904, 911, 918, 925, 932, 939, 946, 953, 960, 967, 974, 981, 988,
995, 1002, 1009, 1016, 1023, 1031, 1038, 1045, 1052, 1059, 1066, 1073, 1080, 1087, 1094, 1101, 1108, 1115, 1122, 1129, 1136, 1143, 1150, 1157, 1164, 1171, 1178, 1185, 1192,
1 199, 1206, 1213, 1220, 1227, 1234, 1241, 1248, 1494, 1496, 1498, 1500, 1502, 1506,
1508, 1510, 1512, 1514, 1516, 1518, 1522, 1526, 1528, 1530, 1532, 1534, 1536, 1538,
1540, 1542, 1544, 1546, 1548, 1550, 1552, 1554, 1556, 1558, 1560, 1564, 1566, 1568,
1570, 1572, 1574, 1576, 1578, 1580, or 1584; and a human light chain variable domain (VL) comprising a CDR3 amino acid sequence that is at least 85% identical to an amino acid sequence set forth in SEQ ID NO: 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 105, 112, 119, 126, 133, 140, 147, 155, 163, 170, 177, 184, 191, 198, 205, 212, 219, 226, 233, 240, 247, 254, 261, 268, 275, 282, 289, 296,
303, 310, 317, 324, 331, 338, 345, 352, 359, 366, 373, 380, 387, 394, 401 , 408, 415, 422,
429, 436, 443, 450, 457, 464, 471,478, 485,492, 499, 506, 513, 520, 527, 534, 541, 548,
555, 562, 569, 576, 583, 590, 597, 604, 611, 618, 625, 632, 639, 646, 653, 660, 667, 674,
681, 688, 695, 702, 709, 716, 723, 730, 737, 744, 751, 759, 766, 773, 780, 787, 794, 801,
808, 815, 822, 829, 836, 843, 850, 857, 864, 871 , 879, 886, 893, 900, 907, 914, 921, 928,
935, 942, 949, 956, 963, 970, 977, 984, 991, 998, 1005, 1012, 1019, 1027, 1034, 1041, 1048, 1055, 1062, 1069, 1076, 1083, 1090, 1097, 1104, 1111, 1118, 1125, 1132, 1139, 1146, 1153, 1160, 1167, 1174, 1181, 1188, 1195, 1202, 1209, 1216, 1223, 1230, 1237,
1244, 1251, 1495, 1497, 1499, 1501, 1503, 1505, 1507, 1509, 1511, 1513, 1515, 1517,
1519, 1521, 1523, 1527, 1529, 1531, 1532, 1533, 1537, 1539, 1541, 1543, 1545, 1547,
1549, 1553, 1557, 1561, 1563, 1565, 1567, 1569, 1571, 1575, 1577, 1579, 1581, 1583, or 1585, wherein the binding protein is capable of specifically binding to an epitope on Pseudomonas aeruginosa V-antigen (PcrV).
2. A binding protein comprising: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR), CDR3 amino acid sequence of SEQ ID NO: 4, 11, 18, 25, 32, 39, 46, 53, 60, 67, 74, 81, 88, 95, 102, 109, 116, 123, 130, 137, 144, 151, 159, 167, 174, 181, 188, 195, 202, 209, 216, 223, 230, 237, 244, 251, 258, 265, 272, 279, 286, 293, 300, 307, 314, 321, 328, 335, 342, 349, 356, 363, 370, 377, 385, 391, 398, 405, 412, 419, 426, 433,440, 447, 454, 461, 468, 475, 482, 489, 496, 503, 510, 517, 524, 531, 538, 545, 552, 559, 566,
573, 580, 587, 594, 601, 608, 615, 622, 629, 636, 643, 650, 657, 664, 671, 678, 685, 692,
699, 706, 713, 720, 727, 734, 741, 748, 755, 763, 770, 777, 784, 791, 798, 805, 812, 819,
826, 833, 840, 847, 854, 861, 868, 875, 883, 890, 897, 904, 911, 918, 925, 932, 939, 946,
953, 960, 967, 974, 981, 988, 995, 1002, 1009, 1016, 1023, 1031, 1038, 1045, 1052, 1059, 1066, 1073, 1080, 1087, 1094, 1101, 1108, 1115, 1122, 1129, 1136, 1143, 1150, 1157,
1164, 1171, 1178, 1185, 1192, 1199, 1206, 1213, 1220, 1227, 1234, 1241, 1248, 1494,
1496, 1498, 1500, 1502, 1506, 1508, 1510, 1512, 1514, 1516, 1518, 1522, 1526, 1528,
1530, 1532, 1534, 1536, 1538, 1540, 1542, 1544, 1546, 1548, 1550, 1552, 1554, 1556,
1558, 1560, 1564, 1566, 1568, 1570, 1572, 1574, 1576, 1578, 1580, or 1584; and a human light chain variable domain (VL) comprising a CDR3 amino acid sequence of SEQ ID NO: 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 105, 112, 119, 126, 133, 140, 147, 155, 163, 170, 177, 184, 191, 198, 205, 212, 219, 226, 233, 240, 247, 254, 261,
268, 275, 282, 289, 296, 303, 310, 317, 324, 331, 338, 345, 352, 359, 366, 373, 380, 387,
394, 401, 408, 415, 422, 429, 436, 443, 450, 457, 464, 471,478, 485,492, 499, 506, 513,
520, 527, 534, 541, 548, 555, 562, 569, 576, 583, 590, 597, 604, 611, 618, 625, 632, 639,
646, 653, 660, 667, 674, 681, 688, 695, 702, 709, 716, 723, 730, 737, 744, 751, 759, 766,
773, 780, 787, 794, 801, 808, 815, 822, 829, 836, 843, 850, 857, 864, 871, 879, 886, 893, 900, 907, 914, 921, 928, 935, 942, 949, 956, 963, 970, 977, 984, 991, 998, 1005, 1012,
1019, 1027, 1034, 1041, 1048, 1055, 1062, 1069, 1076, 1083, 1090, 1097, 1104, 1111, 1118, 1125, 1132, 1139, 1146, 1153, 1160, 1167, 1 174, 1181, 1188, 1 195, 1202, 1209, 1216, 1223, 1230, 1237, 1244, 1251, 1495, 1497, 1499, 1501, 1503, 1505, 1507, 1509, 1511, 1513, 1515, 1517, 1519, 1521, 1523, 1527, 1529, 1531, 1532, 1533, 1537, 1539, 1541, 1543, 1545, 1547, 1549, 1553, 1557, 1561, 1563, 1565, 1567, 1569, 1571, 1575, 1577, 1579, 1581, 1583, or 1585, wherein the binding protein is capable of specifically binding to an epitope on Pseudomonas aeruginosa V-antigen (PcrV).
3. The binding protein of claim 1 or claim 2, wherein the VH has at least 85% sequence identity to a sequence set forth in an (a) in a pair below and the VL has at least 85% sequence identity to a sequence set forth in (b) of the same pair, wherein the pairs have the sequences set forth in:
(a) SEQ ID NO: 1 and (b) SEQ ID NO: 5;
(a) SEQ ID NO: 8 and (b) SEQ ID NO: 12;
(a) SEQ ID NO: 15 and (b) SEQ ID NO: 19;
(a) SEQ ID NO: 22 and (b) SEQ ID NO: 26;
(a) SEQ ID NO: 29 and (b) SEQ ID NO: 33;
(a) SEQ ID NO: 36 and (b) SEQ ID NO: 40;
(a) SEQ ID NO: 43 and (b) SEQ ID NO: 47;
(a) SEQ ID NO: 50 and (b) SEQ ID NO: 54;
(a) SEQ ID NO: 57 and (b) SEQ ID NO: 61;
(a) SEQ ID NO: 64 and (b) SEQ ID NO: 68;
(a) SEQ ID NO: 71 and (b) SEQ ID NO: 75; (a) SEQ ID NO: 78 and (b) SEQ ID NO: 82;
(a) SEQ ID NO: 85 and (b) SEQ ID NO: 89;
(a) SEQ ID NO: 92 and (b) SEQ ID NO: 96;
(a) SEQ ID NO: 99 and (b) SEQ ID NO: 103;
(a) SEQ ID NO: 106 and (b) SEQ ID NO: 110;
(a) SEQ ID NO: 113 and (b) SEQ ID NO: 117;
(a) SEQ ID NO: 120 and (b) SEQ ID NO: 124;
(a) SEQ ID NO: 127 and (b) SEQ ID NO: 131;
(a) SEQ ID NO: 134 and (b) SEQ ID NO: 138;
(a) SEQ ID NO: 141 and (b) SEQ ID NO: 145;
(a) SEQ ID NO: 148 and (b) SEQ ID NO: 152;
(a) SEQ ID NO: 156 and (b) SEQ ID NO: 160;
(a) SEQ ID NO: 164 and (b) SEQ ID NO: 168;
(a) SEQ ID NO: 171 and (b) SEQ ID NO: 175;
(a) SEQ ID NO: 178 and (b) SEQ ID NO: 182;
(a) SEQ ID NO: 185 and (b) SEQ ID NO: 189;
(a) SEQ ID NO: 192 and (b) SEQ ID NO: 196;
(a) SEQ ID NO: 199 and (b) SEQ ID NO: 203;
(a) SEQ ID NO: 206 and (b) SEQ ID NO: 210;
(a) SEQ ID NO: 213 and (b) SEQ ID NO: 217;
(a) SEQ ID NO: 220 and (b) SEQ ID NO: 224; (a) SEQ ID NO: 227 and (b) SEQ ID NO: 231
(a) SEQ ID NO: 234 and (b) SEQ ID NO: 238
(a) SEQ ID NO: 241 and (b) SEQ ID NO: 245
(a) SEQ ID NO: 248 and (b) SEQ ID NO: 252
(a) SEQ ID NO: 255 and (b) SEQ ID NO: 259
(a) SEQ ID NO: 262 and (b) SEQ ID NO: 266
(a) SEQ ID NO: 269 and (b) SEQ ID NO: 273
(a) SEQ ID NO: 276 and (b) SEQ ID NO: 280
(a) SEQ ID NO: 283 and (b) SEQ ID NO: 287
(a) SEQ ID NO: 290 and (b) SEQ ID NO: 294;
(a) SEQ ID NO: 297 and (b) SEQ ID NO: 301 ;
(a) SEQ ID NO: 304 and (b) SEQ ID NO: 308;
(a) SEQ ID NO: 311 and (b) SEQ ID NO: 315
(a) SEQ ID NO: 318 and (b) SEQ ID NO: 322
(a) SEQ ID NO: 325 and (b) SEQ ID NO: 329
(a) SEQ ID NO: 332 and (b) SEQ ID NO: 336
(a) SEQ ID NO: 339 and (b) SEQ ID NO: 343:
(a) SEQ ID NO: 346 and (b) SEQ ID NO: 350
(a) SEQ ID NO: 353 and (b) SEQ ID NO: 357:
(a) SEQ ID NO: 360 and (b) SEQ ID NO: 364:
(a) SEQ ID NO: 367 and (b) SEQ ID NO: 371 (a) SEQ ID NO: 374 and (b) SEQ ID NO: 378;
(a) SEQ ID NO: 381 and (b) SEQ ID NO: 385;
(a) SEQ ID NO: 388 and (b) SEQ ID NO: 392;
(a) SEQ ID NO: 395 and (b) SEQ ID NO: 399;
(a) SEQ ID NO: 402 and (b) SEQ ID NO: 406;
(a) SEQ ID NO: 409 and (b) SEQ ID NO: 413;
(a) SEQ ID NO: 416 and (b) SEQ ID NO: 420;
(a) SEQ ID NO: 423 and (b) SEQ ID NO: 427;
(a) SEQ ID NO: 430 and (b) SEQ ID NO: 434;
(a) SEQ ID NO: 437 and (b) SEQ ID NO: 441;
(a) SEQ ID NO: 444 and (b) SEQ ID NO: 448;
(a) SEQ ID NO: 451 and (b) SEQ ID NO: 455;
(a) SEQ ID NO: 458 and (b) SEQ ID NO: 462;
(a) SEQ ID NO: 465 and (b) SEQ ID NO: 469;
(a) SEQ ID NO: 472 and (b) SEQ ID NO: 476;
(a) SEQ ID NO: 479 and (b) SEQ ID NO: 483;
(a) SEQ ID NO: 486 and (b) SEQ ID NO: 490;
(a) SEQ ID NO: 493 and (b) SEQ ID NO: 497;
(a) SEQ ID NO: 500 and (b) SEQ ID NO: 504;
(a) SEQ ID NO: 507 and (b) SEQ ID NO: 511;
(a) SEQ ID NO: 514 and (b) SEQ ID NO: 518; (a) SEQ ID NO: 521 and (b) SEQ ID NO: 525;
(a) SEQ ID NO: 528 and (b) SEQ ID NO: 532;
(a) SEQ ID NO: 535 and (b) SEQ ID NO: 539;
(a) SEQ ID NO: 542 and (b) SEQ ID NO: 546;
(a) SEQ ID NO: 549 and (b) SEQ ID NO: 553;
(a) SEQ ID NO: 556 and (b) SEQ ID NO: 560;
(a) SEQ ID NO: 563 and (b) SEQ ID NO: 567;
(a) SEQ ID NO: 570 and (b) SEQ ID NO: 574;
(a) SEQ ID NO: 577 and (b) SEQ ID NO: 581
(a) SEQ ID NO: 584 and (b) SEQ ID NO: 588
(a) SEQ ID NO: 591 and (b) SEQ ID NO: 595
(a) SEQ ID NO: 598 and (b) SEQ ID NO: 602
(a) SEQ ID NO: 605 and (b) SEQ ID NO: 609
(a) SEQ ID NO: 626 and (b) SEQ ID NO: 630
(a) SEQ ID NO: 633 and (b) SEQ ID NO: 637
(a) SEQ ID NO: 640 and (b) SEQ ID NO: 644
(a) SEQ ID NO: 647 and (b) SEQ ID NO: 651
(a) SEQ ID NO: 654 and (b) SEQ ID NO: 658
(a) SEQ ID NO: 661 and (b) SEQ ID NO: 665
(a) SEQ ID NO: 668 and (b) SEQ ID NO: 672:
(a) SEQ ID NO: 675 and (b) SEQ ID NO: 679 (a) SEQ ID NO: 682 and (b) SEQ ID NO: 686;
(a) SEQ ID NO: 689 and (b) SEQ ID NO: 693;
(a) SEQ ID NO: 696 and (b) SEQ ID NO: 700;
(a) SEQ ID NO: 703 and (b) SEQ ID NO: 707;
(a) SEQ ID NO: 710 and (b) SEQ ID NO: 714;
(a) SEQ ID NO: 717 and (b) SEQ ID NO: 721;
(a) SEQ ID NO: 724 and (b) SEQ ID NO: 728;
(a) SEQ ID NO: 731 and (b) SEQ ID NO: 735;
(a) SEQ ID NO: 738 and (b) SEQ ID NO: 742;
(a) SEQ ID NO: 745 and (b) SEQ ID NO: 749;
(a) SEQ ID NO: 752 and (b) SEQ ID NO: 756;
(a) SEQ ID NO: 760 and (b) SEQ ID NO: 764;
(a) SEQ ID NO: 767 and (b) SEQ ID NO: 771;
(a) SEQ ID NO: 774 and (b) SEQ ID NO: 778;
(a) SEQ ID NO: 781 and (b) SEQ ID NO: 785;
(a) SEQ ID NO: 788 and (b) SEQ ID NO: 792;
(a) SEQ ID NO: 795 and (b) SEQ ID NO: 799;
(a) SEQ ID NO: 802 and (b) SEQ ID NO: 806;
(a) SEQ ID NO: 809 and (b) SEQ ID NO: 813;
(a) SEQ ID NO: 816 and (b) SEQ ID NO: 820;
(a) SEQ ID NO: 823 and (b) SEQ ID NO: 827; (a) SEQ ID NO: 830 and (b) SEQ ID NO: 834;
(a) SEQ ID NO: 837 and (b) SEQ ID NO: 841;
(a) SEQ ID NO: 844 and (b) SEQ ID NO: 848;
(a) SEQ ID NO: 851 and (b) SEQ ID NO: 855;
(a) SEQ ID NO: 858 and (b) SEQ ID NO: 862;
(a) SEQ ID NO: 865 and (b) SEQ ID NO: 869;
(a) SEQ ID NO: 872 and (b) SEQ ID NO: 876;
(a) SEQ ID NO: 880 and (b) SEQ ID NO: 884;
(a) SEQ ID NO: 887 and (b) SEQ ID NO: 889;
(a) SEQ ID NO: 894 and (b) SEQ ID NO: 898;
(a) SEQ ID NO: 901 and (b) SEQ ID NO: 905;
(a) SEQ ID NO: 908 and (b) SEQ ID NO: 912;
(a) SEQ ID NO: 915 and (b) SEQ ID NO: 919;
(a) SEQ ID NO: 922 and (b) SEQ ID NO: 926;
(a) SEQ ID NO: 929 and (b) SEQ ID NO: 933;
(a) SEQ ID NO: 936 and (b) SEQ ID NO: 940;
(a) SEQ ID NO: 943 and (b) SEQ ID NO: 947;
(a) SEQ ID NO: 950 and (b) SEQ ID NO: 954;
(a) SEQ ID NO: 957 and (b) SEQ ID NO: 961;
(a) SEQ ID NO: 964 and (b) SEQ ID NO: 968;
(a) SEQ ID NO: 971 and (b) SEQ ID NO: 975; (a) SEQ ID NO: 978 and (b) SEQ ID NO: 982;
(a) SEQ ID NO: 985 and (b) SEQ ID NO: 989;
(a) SEQ ID NO: 992 and (b) SEQ ID NO: 996;
(a) SEQ ID NO: 999 and (b) SEQ ID NO: 1003;
(a) SEQ ID NO: 1006 and (b) SEQ ID NO: 1010;
(a) SEQ ID NO: 1013 and (b) SEQ ID NO: 1017;
(a) SEQ ID NO: 1020 and (b) SEQ ID NO: 1024;
(a) SEQ ID NO: 1028 and (b) SEQ ID NO: 1032;
(a) SEQ ID NO: 1035 and (b) SEQ ID NO: 1039;
(a) SEQ ID NO: 1042 and (b) SEQ ID NO: 1046;
(a) SEQ ID NO: 1049 and (b) SEQ ID NO: 1053;
(a) SEQ ID NO: 1056 and (b) SEQ ID NO: 1060;
(a) SEQ ID NO: 1063 and (b) SEQ ID NO: 1067;
(a) SEQ ID NO: 1070 and (b) SEQ ID NO: 1074;
(a) SEQ ID NO: 1077 and (b) SEQ ID NO: 1081;
(a) SEQ ID NO: 1084 and (b) SEQ ID NO: 1088;
(a) SEQ ID NO: 1091 and (b) SEQ ID NO: 1095;
(a) SEQ ID NO: 1098 and (b) SEQ ID NO: 1102;
(a) SEQ ID NO: 1105 and (b) SEQ ID NO: 1109;
(a) SEQ ID NO: 1112 and (b) SEQ ID NO: 1116;
(a) SEQ ID NO: 1119 and (b) SEQ ID NO: 1123; (a) SEQ ID NO: 1126 and (b) SEQ ID NO: 1130;
(a) SEQ ID NO: 1133 and (b) SEQ ID NO: 1137;
(a) SEQ ID NO: 1140 and (b) SEQ ID NO: 1144;
(a) SEQ ID NO: 1147 and (b) SEQ ID NO: 1151;
(a) SEQ ID NO: 1154 and (b) SEQ ID NO: 1158;
(a) SEQ ID NO: 1161 and (b) SEQ ID NO: 1165;
(a) SEQ ID NO: 1168 and (b) SEQ ID NO: 1172;
(a) SEQ ID NO: 1175 and (b) SEQ ID NO: 1179;
(a) SEQ ID NO: 1182 and (b) SEQ ID NO: 1186;
(a) SEQ ID NO: 1189 and (b) SEQ ID NO: 1193;
(a) SEQ ID NO: 1196 and (b) SEQ ID NO: 1200;
(a) SEQ ID NO: 1203 and (b) SEQ ID NO: 1207;
(a) SEQ ID NO: 1210 and (b) SEQ ID NO: 1214;
(a) SEQ ID NO: 1217 and (b) SEQ ID NO: 1221;
(a) SEQ ID NO: 1224 and (b) SEQ ID NO: 1228;
(a) SEQ ID NO: 1231 and (b) SEQ ID NO: 1235;
(a) SEQ ID NO: 1238 and (b) SEQ ID NO: 1242;
(a) SEQ ID NO: 1245 and (b) SEQ ID NO: 1249;
(a) SEQ ID NO: 1264 and (b) SEQ ID NO: 1265;
(a) SEQ ID NO: 1264 and (b) SEQ ID NO: 1265;
(a) SEQ ID NO: 1266 and (b) SEQ ID NO: 1267; (a) SEQ ID NO: 1268 and (b) SEQ ID NO: 1269;
(a) SEQ ID NO: 1270 and (b) SEQ ID NO: 1271;
(a) SEQ ID NO: 1272 and (b) SEQ ID NO: 1273;
(a) SEQ ID NO: 1274 and (b) SEQ ID NO: 1275;
(a) SEQ ID NO: 1276 and (b) SEQ ID NO: 1277;
(a) SEQ ID NO: 1278 and (b) SEQ ID NO: 1279;
(a) SEQ ID NO: 1280 and (b) SEQ ID NO: 1281;
(a) SEQ ID NO: 1282 and (b) SEQ ID NO: 1283;
(a) SEQ ID NO: 1284 and (b) SEQ ID NO: 1285;
(a) SEQ ID NO: 1286 and (b) SEQ ID NO: 1287;
(a) SEQ ID NO: 1288 and (b) SEQ ID NO: 1289;
(a) SEQ ID NO: 1290 and (b) SEQ ID NO: 1291;
(a) SEQ ID NO: 1292 and (b) SEQ ID NO: 1293;
(a) SEQ ID NO: 1294 and (b) SEQ ID NO: 1295;
(a) SEQ ID NO: 1296 and (b) SEQ ID NO: 1297;
(a) SEQ ID NO: 1298 and (b) SEQ ID NO: 1299;
(a) SEQ ID NO: 1300 and (b) SEQ ID NO: 1301 ;
(a) SEQ ID NO: 1302 and (b) SEQ ID NO: 1303;
(a) SEQ ID NO: 1304 and (b) SEQ ID NO: 1305;
(a) SEQ ID NO: 1306 and (b) SEQ ID NO: 1307;
(a) SEQ ID NO: 1308 and (b) SEQ ID NO: 1309; (a) SEQ ID NO: 1310 and (b) SEQ ID NO: 1311;
(a) SEQ ID NO: 1312 and (b) SEQ ID NO: 1313;
(a) SEQ ID NO: 1314 and (b) SEQ ID NO: 1315;
(a) SEQ ID NO: 1316 and (b) SEQ ID NO: 1317;
(a) SEQ ID NO: 1318 and (b) SEQ ID NO: 1319;
(a) SEQ ID NO: 1320 and (b) SEQ ID NO: 1321;
(a) SEQ ID NO: 1322 and (b) SEQ ID NO: 1323;
(a) SEQ ID NO: 1324 and (b) SEQ ID NO: 1325;
(a) SEQ ID NO: 1326 and (b) SEQ ID NO: 1327;
(a) SEQ ID NO: 1328 and (b) SEQ ID NO: 1329;
(a) SEQ ID NO: 1330 and (b) SEQ ID NO: 1331;
(a) SEQ ID NO: 1332 and (b) SEQ ID NO: 1333;
(a) SEQ ID NO: 1334 and (b) SEQ ID NO: 1335;
(a) SEQ ID NO: 1336 and (b) SEQ ID NO: 1337;
(a) SEQ ID NO: 1338 and (b) SEQ ID NO: 1339;
(a) SEQ ID NO: 1340 and (b) SEQ ID NO: 1341;
(a) SEQ ID NO: 1342 and (b) SEQ ID NO: 1343;
(a) SEQ ID NO: 1344 and (b) SEQ ID NO: 1345;
(a) SEQ ID NO: 1346 and (b) SEQ ID NO: 1347;
(a) SEQ ID NO: 1348 and (b) SEQ ID NO: 1349;
(a) SEQ ID NO: 1350 and (b) SEQ ID NO: 1351; (a) SEQ ID NO: 1352 and (b) SEQ ID NO: 1353; or
(a) SEQ ID NO: 1354 and (b) SEQ ID NO: 1355.
4. The binding protein of claim 3, wherein the variable heavy chain has at least 90% sequence identity to a sequence as set forth in an (a) in a pair and the variable light chain has at least 90% sequence identity to the sequence as set forth in the (b) of the same pair.
5. The binding protein of claim 3, wherein the variable heavy chain has at least 95% sequence identity to a sequence as set forth in an (a) in a pair and the variable light chain has at least 95% sequence identity to the sequence as set forth in the (b) of the same pair.
6. The binding protein of claim 3, wherein the variable heavy chain has at least 98% sequence identity to a sequence as set forth in an (a) in a pair and the variable light chain has at least 98% sequence identity to the sequence as set forth in the (b) of the same pair.
7. The binding protein of claim 3, having a set of CDRs comprising:
(i) a HCDR1, HCDR2, HCDR3, comprising or contained within SEQ ID NO: 1; SEQ ID NO: 8; SEQ ID NO: 15; SEQ ID NO: 22; SEQ ID NO: 29; SEQ ID NO: 36; SEQ ID NO: 43; SEQ ID NO: 50; SEQ ID NO: 57; SEQ ID NO: 64; SEQ ID NO: 71; SEQ ID NO: 78; SEQ ID NO: 85; SEQ ID NO: 92; SEQ ID NO: 99; SEQ ID NO: 106; SEQ ID NO: 113; SEQ ID NO: 120; SEQ ID NO: 127; SEQ ID NO: 134; SEQ ID NO: 141 ; SEQ ID NO: 148; SEQ ID NO: 156; SEQ ID NO: 164; SEQ ID NO: 171; SEQ ID NO: 178; SEQ ID NO: 185; SEQ ID NO: 192; SEQ ID NO: 199; SEQ ID NO: 206; SEQ ID NO: 213; SEQ ID NO: 220; SEQ ID NO: 227; SEQ ID NO: 234; SEQ ID NO: 241; SEQ ID NO: 248; SEQ ID NO: 255; SEQ ID NO: 262; SEQ ID NO: 269; SEQ ID NO: 276; SEQ ID NO: 283; SEQ ID NO: 290; SEQ ID NO: 297; SEQ ID NO: 304; SEQ ID NO: 311; SEQ ID NO: 318; SEQ ID NO: 325; SEQ ID NO: 332; SEQ ID NO: 339; SEQ ID NO: 346; SEQ ID NO: 353; SEQ ID NO: 360; SEQ ID NO: 367; SEQ ID NO: 374; SEQ ID NO: 381; SEQ ID NO: 388; SEQ ID NO: 395; SEQ ID NO: 402; SEQ ID NO: 409;SEQ ID NO: 416; SEQ ID NO: 423; SEQ ID NO: 430; SEQ ID NO: 437; SEQ ID NO: 444; SEQ ID NO: 451; SEQ ID NO: 458; SEQ ID NO: 465; SEQ ID NO: 472; SEQ ID NO: 479; SEQ ID NO: 486; SEQ ID NO: 493; SEQ ID NO: 500; SEQ ID NO: 507; SEQ ID NO: 514; SEQ ID NO: 521 ; SEQ ID NO: 528; SEQ ID NO: 535; SEQ ID NO: 542; SEQ ID NO: 549; SEQ ID NO: 556; SEQ ID NO: 563; SEQ ID NO: 570; SEQ ID NO: 577; SEQ ID NO: 584; SEQ ID NO: 591; SEQ ID NO: 598; SEQ ID NO: 605; SEQ ID NO: 612; SEQ ID NO: 619; SEQ ID NO: 626; SEQ ID NO: 633; SEQ ID NO: 640; SEQ ID NO: 647; SEQ ID NO: 654; SEQ ID NO: 661; SEQ ID NO: 668; SEQ ID NO: 675; SEQ ID NO: 682; SEQ ID NO: 689; SEQ ID NO: 696; SEQ ID NO: 703; SEQ ID NO: 710; SEQ ID NO: 717; SEQ ID NO: 724; SEQ ID NO: 731; SEQ ID NO: 738; SEQ ID NO: 745; SEQ ID NO: 752; SEQ ID NO: 760; SEQ ID NO: 767; SEQ ID NO: 774; SEQ ID NO: 781; SEQ ID NO: 788; SEQ ID NO: 795; SEQ ID NO: 802; SEQ ID NO: 809; SEQ ID NO: 816; SEQ ID NO: 823; SEQ ID NO: 830; SEQ ID NO: 837; SEQ ID NO: 844; SEQ ID NO: 851; SEQ ID NO: 858; SEQ ID NO: 865; SEQ ID NO: 872; SEQ ID NO: 880; SEQ ID NO: 887; SEQ ID NO: 894; SEQ ID NO: 901 SEQ ID NO: 908 SEQ ID NO: 915; SEQ ID NO: 922; SEQ ID NO: 929; SEQ ID NO: 936; SEQ ID NO: 943; SEQ ID NO: 950; SEQ ID NO: 957; SEQ ID NO: 964; SEQ ID NO: 971; SEQ ID NO: 978; SEQ ID NO: 985; SEQ ID NO: 992; SEQ ID NO: 999; SEQ ID NO: 1006; SEQ ID NO: 1013; SEQ ID NO: 1020; SEQ ID NO: 1028; SEQ ID NO: 1035; SEQ ID NO: 1042; SEQ ID NO: 1049; SEQ ID NO: 1056; SEQ ID NO: 1063; SEQ ID NO: 1070; SEQ ID NO: 1077; SEQ ID NO: 1084; SEQ ID NO: 1091; SEQ ID NO: 1098; SEQ ID NO: 1105; SEQ ID NO: 1112; SEQ ID NO: 1119; SEQ ID NO: 1126; SEQ ID NO: 1133; SEQ ID NO: 1140; SEQ ID NO: 1147; SEQ ID NO: 1154; SEQ ID NO: 1161; SEQ ID NO: 1168; SEQ ID NO: 1175 SEQ ID NO: 1182; SEQ ID NO: 1189; SEQ ID NO: 1196; SEQ ID NO: 1203; SEQ ID NO: 1210; SEQ ID NO: 1217; SEQ ID NO: 1224; SEQ ID NO: 1231; SEQ ID NO: 1238; SEQ ID NO: 1245; SEQ ID NO: 1264; SEQ ID NO: 1266; SEQ ID: 1268; SEQ ID: 1270; SEQ ID: 1272; SEQ ID: 1274; SEQ ID NO: 1276; SEQ ID NO: 1278; SEQ ID NO: 1280; SEQ ID NO: 1282; SEQ ID NO: 1284; SEQ ID NO: 1286; SEQ ID NO: 1288; SEQ ID NO: 1290; SEQ ID NO: 1292; SEQ ID NO: 1294, SEQ ID NO: 1296; SEQ ID NO: 1298; SEQ ID NO: 1300; SEQ ID NO: 1302; SEQ ID NO: 1304, SEQ ID NO: 1306, SEQ ID NO: 1308; SEQ ID NO: 1310; SEQ ID NO: 1312; SEQ ID NO: 1314; SEQ ID NO: 1316; SEQ ID NO: 1318; SEQ ID NO: 1320; SEQ ID NO: 1322; SEQ ID NO: 1324; SEQ ID NO: 1326; SEQ ID NO: 1328; SEQ ID NO: 1330; SEQ ID NO: 1332; SEQ ID NO: 1334; SEQ ID NO: 1336, SEQ ID NO: 1338; SEQ ID NO: 1340; SEQ ID NO: 1342; SEQ ID NO: 1344; SEQ ID NO: 1346; SEQ ID NO: 1348; SEQ ID NO: 1350; SEQ ID NO: 1352; or SEQ ID NO: 1354, with one or two single amino acid substitutions in one or more of the HCDRs; and (ii) a LCDR1, LCDR2, and LCDR3 comprising or contained within SEQ ID NO: 5; SEQ ID NO: 12; SEQ ID NO: 19; SEQ ID NO: 26; SEQ ID NO: 33; SEQ ID NO: 40; SEQ ID NO: 47; SEQ ID NO: 54; SEQ ID NO: 61 ; SEQ ID NO: 68; SEQ ID NO: 75; SEQ ID NO: 82; SEQ ID NO: 89; SEQ ID NO: 96; SEQ ID NO: 103; SEQ ID NO: 110; SEQ ID NO: 117; SEQ ID NO: 124; SEQ ID NO: 131; SEQ ID NO: 138; SEQ ID NO: 145; SEQ ID NO: 152; SEQ ID NO: 160; SEQ ID NO: 168; SEQ ID NO: 175; SEQ ID NO: 182; SEQ ID NO: 189; SEQ ID NO: 196; SEQ ID NO: 203; SEQ ID NO: 210; SEQ ID NO: 217; SEQ ID NO: 224; SEQ ID NO: 231; SEQ ID NO: 238; SEQ ID NO: 245; SEQ ID NO: 252; SEQ ID NO: 259; SEQ ID NO: 266; SEQ ID NO: 273; SEQ ID NO: 280; SEQ ID NO: 287; SEQ ID NO: 294; SEQ ID NO: 301; SEQ ID NO: 308; SEQ ID NO: 315; SEQ ID NO: 322; SEQ ID NO: 329; SEQ ID NO: 336; SEQ ID NO: 343; SEQ ID NO: 350; SEQ ID NO: 357; SEQ ID NO: 364; SEQ ID NO: 371; SEQ ID NO: 378; SEQ ID NO: 385; SEQ ID NO: 392; SEQ ID NO: 399; SEQ ID NO: 406; SEQ ID NO: 413; SEQ ID NO: 420; SEQ ID NO: 427; SEQ ID NO: 434; SEQ ID NO: 441; SEQ ID NO: 448; SEQ ID NO: 455; SEQ ID NO: 462; SEQ ID NO: 469; SEQ ID NO: 476; SEQ ID NO: 483; SEQ ID NO: 490; SEQ ID NO: 497; SEQ ID NO: 504; SEQ ID NO: 511; SEQ ID NO: 518; SEQ ID NO: 525; SEQ ID NO: 532; SEQ ID NO: 539; SEQ ID NO: 546; SEQ ID NO: 553; SEQ ID NO: 560; SEQ ID NO: 567; SEQ ID NO: 574; SEQ ID NO: 581; SEQ ID NO: 588; SEQ ID NO: 595; SEQ ID NO: 602; SEQ ID NO: 609; SEQ ID NO: 616; SEQ ID NO: 623; SEQ ID NO: 630; SEQ ID NO: 637; SEQ ID NO: 644; SEQ ID NO: 651; SEQ ID NO: 658; SEQ ID NO: 665; SEQ ID NO: 672; SEQ ID NO: 679; SEQ ID NO: 686; SEQ ID NO: 693; SEQ ID NO: 700; SEQ ID NO: 707; SEQ ID NO: 714; SEQ ID NO: 721; SEQ ID NO: 728; SEQ ID NO: 735; SEQ ID NO: 742; SEQ ID NO: 749; SEQ ID NO: 756; SEQ ID NO: 764; SEQ ID NO: 771; SEQ ID NO: 778; SEQ ID NO: 785; SEQ ID NO: 792; SEQ ID NO: 799; SEQ ID NO: 806; SEQ ID NO: 813; SEQ ID NO: 820; SEQ ID NO: 827; SEQ ID NO: 834; SEQ ID NO: 841; SEQ ID NO: 848; SEQ ID NO: 855; SEQ ID NO: 862; SEQ ID NO: 869;S EQ ID NO: 876; SEQ ID NO: 884; SEQ ID NO: 889; SEQ ID NO: 898; SEQ ID NO: 905; SEQ ID NO: 912; SEQ ID NO: 919; SEQ ID NO: 926; SEQ ID NO: 933; SEQ ID NO: 940; SEQ ID NO: 947; SEQ ID NO: 954; SEQ ID NO: 961; SEQ ID NO: 968; SEQ ID NO: 975; SEQ ID NO: 982; SEQ ID NO: 989; SEQ ID NO: 996; SEQ ID NO: 1003; SEQ ID NO: 1010; SEQ ID NO: 1017; SEQ ID NO: 1024; SEQ ID NO: 1032; SEQ ID NO: 1039; SEQ ID NO: 1046; SEQ ID NO: 1053; SEQ ID NO: 1060; SEQ ID NO: 1067; SEQ ID NO: 1074; SEQ ID NO: 1081 ; SEQ ID NO: 1088; SEQ ID NO: 1095; SEQ ID NO: 1102; SEQ ID NO: 1109; SEQ ID NO: 1116; SEQ ID NO: 1123; SEQ ID NO: 1130; SEQ ID NO: 1 137; SEQ ID NO: 1144; SEQ ID NO: 1151 ; SEQ ID NO: 1158; SEQ ID NO: 1165; SEQ ID NO: 1172; SEQ ID NO: 1179; SEQ ID NO: 1186; SEQ ID NO: 1193; SEQ ID NO: 1200; SEQ ID NO: 1207; SEQ ID NO: 1214; SEQ ID NO: 1221; SEQ ID NO: 1228; SEQ ID NO: 1235; SEQ ID NO: 1242; SEQ ID NO: 1249; SEQ ID NO: 1265; SEQ ID NO: 1267; SEQ ID NO: 1269; SEQ ID NO: 1271 ; SEQ ID NO: 1273; SEQ ID NO: 1275; SEQ ID NO: 1277; SEQ ID NO: 1279; SEQ ID NO: 1281 ; SEQ ID NO: 1283; SEQ ID NO: 1285; SEQ ID NO: 1287; SEQ ID NO: 1289; SEQ ID NO: 1291; SEQ ID NO: 1293; SEQ ID NO: 1295; SEQ ID NO: 1297; SEQ ID NO: 1299; SEQ ID NO: 1301; SEQ ID NO: 1303; SEQ ID NO: 1305; SEQ ID NO: 1307; SEQ ID NO: 1309; SEQ ID NO: 1311; SEQ ID NO: 1313; SEQ ID NO: 1315; SEQ ID NO: 1317; SEQ ID NO: 1319; SEQ ID NO: 1321; SEQ ID NO: 1323; SEQ ID NO: 1325; SEQ ID NO: 1327; SEQ ID NO: 1329; SEQ ID NO: 1331; SEQ ID NO: 1333; SEQ ID NO: 1335; SEQ ID NO: 1337; SEQ ID NO: 1339; SEQ ID NO: 1341 ; SEQ ID NO: 1343; SEQ ID NO: 1345; SEQ ID NO: 1347; SEQ ID NO: 1349; SEQ ID NO: 1351 ; SEQ ID NO: 1353; or SEQ ID NO: 1355, with one or two single amino acid substitutions in one or more of the HCDRs.
8. The binding protein of any one of claims 1-7, wherein the CDRs are grafted into a human IgG acceptor antibody framework or an IgM acceptor antibody framework.
9. The binding protein of claim 8, wherein the acceptor framework comprises an Fc region of the IgG or the IgM linked to the variable heavy chain.
10. The binding protein of claim 9, wherein the Fc region is a variant Fc region, wherein the variant Fc region comprises one or more amino acid substitutions, insertions, or deletions that alter an effector function relative to a naturally occurring Fc region.
11. The binding protein of any one of claims 1-10, wherein the binding protein is a genetically engineered intact antibody comprising a variant Fc region, wherein the variant Fc region comprises one or more amino acid substitutions that alter an effector function relative to a naturally-occurring Fc region.
12. The binding protein of any one of claims 1-11, wherein the binding protein is a genetically engineered intact antibody or antibody fragment or a derivative thereof.
13. The binding protein of claim 12, wherein the binding protein is formatted into a multimer, a single chain variable fragment (scFv), or an antibody conjugate.
14. A composition comprising the binding protein of any one of claims 1-13 and a pharmaceutically acceptable carrier, diluent, or excipient.
15. A polynucleotide encoding the binding protein of any one of claims 1-13.
16. The polynucleotide of claim 15, wherein the polynucleotide is codon optimized.
17. An expression vector comprising the polynucleotide of claim 15 or claim 16 operably linked to an expression control sequence.
18. The expression vector of claim 17, wherein the expression vector is capable of delivering the polynucleotide to a host cell.
19. A recombinant host cell comprising the polynucleotide of claim 15, or the expression vector of claim 17, wherein the host cell is capable of expressing the encoded binding protein.
20. A composition comprising: (a) means for binding a specific epitope of Pseudomonas aeruginosa V-antigen (PcrV); and (b) a pharmaceutically acceptable carrier.
21. A method of treating and/or preventing a disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of the binding protein of any one of claims 1-13, or the composition of claim 14 or claim 20.
22. The method of claim 21, wherein the subject is a human.
23. The method of claim 21 or claim 22, wherein the disease is an infectious disease.
24. The method of any one of claims 21-23, wherein the disease is an infectious disease caused by Pseudomonas aeruginosa.
25. The method of claim 24, wherein the Pseudomonas aeruginosa infection is associated with an inflammatory lung disease.
26. The method of claim 25, wherein the inflammatory lung disease is cystic fibrosis (CF).
27. The method of any one of claims 21-26, wherein the administration comprises intravenous administration.
28. The method of any one of claims 21-27, wherein the method further comprises administering another therapeutic agent and/or therapy to the subject.
29. A method of immunizing a subject against a pathogen, the method comprising: administering an effective amount of a binding protein of any one of claims 1-13, the composition of claim 14, the polynucleotide of claim 15 or 16, the expression vector of claim 17 or claim 18, the recombinant host cell of claim 19, or the composition of claim 20 to the subject in need thereof.
30. A composition comprising an isolated human monoclonal antibody or derivative thereof which binds to a specific epitope of Pseudomonas aeruginosa V-antigen (PcrV), comprising: a heavy chain variable domain and a light chain variable domain; and a a pharmaceutically acceptable carrier, wherein the antibody or derivative thereof is selected from the group consisting of:
(i) an antibody or derivative thereof wherein at least one variable region has at least 85% sequence identity to a sequence set forth in and selected from the group consisting of: SEQ ID NO: 1 ; SEQ ID NO: 8; SEQ ID NO: 15; SEQ ID NO: 22; SEQ ID NO: 29; SEQ ID NO: 36; SEQ ID NO: 43; SEQ ID NO: 50; SEQ ID NO: 57; SEQ ID NO: 64; SEQ ID NO: 71 ; SEQ ID NO: 78; SEQ ID NO: 85; SEQ ID NO: 92; SEQ ID NO: 99; SEQ ID NO: 106; SEQ ID NO: 113; SEQ ID NO: 120; SEQ ID NO: 127; SEQ ID NO:
134; SEQ ID NO: 141 ; SEQ ID NO: 148; SEQ ID NO: 156; SEQ ID NO:
164; SEQ ID NO: 171 ; SEQ ID NO: 178; SEQ ID NO: 185; SEQ ID NO:
192; SEQ ID NO: 199; SEQ ID NO: 206; SEQ ID NO: 213; SEQ ID NO:
220; SEQ ID NO: 227; SEQ ID NO: 234; SEQ ID NO: 241 ; SEQ ID NO:
248; SEQ ID NO: 255; SEQ ID NO: 262; SEQ ID NO: 269; SEQ ID NO:
276; SEQ ID NO: 283; SEQ ID NO: 290; SEQ ID NO: 297; SEQ ID NO: 304; SEQ ID NO 311; SEQ ID NO 318; SEQ ID NO 325; SEQ ID NO: 332; SEQ ID NO 339; SEQ ID NO 346; SEQ ID NO 353; SEQ ID NO: 360; SEQ ID NO 367; SEQ ID NO 374; SEQ ID NO 381; SEQ ID NO: 388; SEQ ID NO 395; SEQ ID NO 402; SEQ ID NO 409;SEQ ID NO: 416; SEQ ID NO 423; SEQ ID NO 430; SEQ ID NO 437; SEQ ID NO: 444; SEQ ID NO 451; SEQ ID NO 458; SEQ ID NO 465; SEQ ID NO: 472; SEQ ID NO 479; SEQ ID NO 486; SEQ ID NO 493; SEQ ID NO: 500; SEQ ID NO 507; SEQ ID NO 514; SEQ ID NO 521; SEQ ID NO: 528; SEQ ID NO 535; SEQ ID NO 542; SEQ ID NO 549; SEQ ID NO: 556; SEQ ID NO 563; SEQ ID NO 570; SEQ ID NO 577; SEQ ID NO: 584; SEQ ID NO 591; SEQ ID NO 598; SEQ ID NO 605; SEQ ID NO: 612; SEQ ID NO 619; SEQ ID NO 626; SEQ ID NO 633; SEQ ID NO: 640; SEQ ID NO 647; SEQ ID NO 654; SEQ ID NO 661; SEQ ID NO: 668; SEQ ID NO 675; SEQ ID NO 682; SEQ ID NO 689; SEQ ID NO: 696; SEQ ID NO 703; SEQ ID NO 710; SEQ ID NO 717; SEQ ID NO: 724; SEQ ID NO 731; SEQ ID NO 738; SEQ ID NO 745; SEQ ID NO: 752; SEQ ID NO 760; SEQ ID NO 767; SEQ ID NO 774; SEQ ID NO: 781; SEQ ID NO 788; SEQ ID NO 795; SEQ ID NO 802; SEQ ID NO: 809; SEQ ID NO 816; SEQ ID NO 823; SEQ ID NO 830; SEQ ID NO: 837; SEQ ID NO 844; SEQ ID NO: 851; SEQ ID NO: 858; SEQ ID NO: 865; SEQ ID NO 872; SEQ ID NO: 880; SEQ ID NO: 887; SEQ ID NO: 894; SEQ ID NO 901 SEQ ID NO: 508 SEQ ID NO: S 15; SEQ ID NO:
922; SEQ ID NO 929; SEQ ID NO: 936; SEQ ID NO: 943; SEQ ID NO:
950; SEQ ID NO 957; SEQ ID NO: 964; SEQ ID NO: 971; SEQ ID NO:
978; SEQ ID NO 985; SEQ ID NO: 992; SEQ ID NO: 999; SEQ ID NO:
1006; SEQ ID NO: 1013; SEQ ID NO: 1020; SEQ ID NO: 1028; SEQ ID NO: 1035; SEQ ID NO: 1042; SEQ ID NO: 1049; SEQ ID NO: 1056;
SEQ ID NO: 1063; SEQ ID NO: 1070; SEQ ID NO: 1077; SEQ ID NO: 1084; SEQ ID NO: 1091 ; SEQ ID NO: 1098; SEQ ID NO: 1105; SEQ ID NO: 1112; SEQ ID NO: 1119; SEQ ID NO: 1126; SEQ ID NO: 1133;
SEQ ID NO: 1140; SEQ ID NO: 1147; SEQ ID NO: 1154; SEQ ID NO: 1161; SEQ ID NO: 1168; SEQ ID NO: 1175 SEQ ID NO: 1182; SEQ ID NO: 1189; SEQ ID NO: 1196; SEQ ID NO: 1203; SEQ ID NO: 1210; SEQ ID NO: 1217; SEQ ID NO: 1224; SEQ ID NO: 1231; SEQ ID NO:
1238; SEQ ID NO: 1245; SEQ ID NO: 1264; SEQ ID NO: 1266; SEQ ID:
1268; SEQ ID: 1270; SEQ ID: 1272; SEQ ID: 1274; SEQ ID NO: 1276;
SEQ ID NO: 1278; SEQ ID NO: 1280; SEQ ID NO: 1282; SEQ ID NO: 1284; SEQ ID NO: 1286; SEQ ID NO: 1288; SEQ ID NO: 1290; SEQ ID
NO: 1292; SEQ ID NO: 1294, SEQ ID NO: 1296; SEQ ID NO: 1298;
SEQ ID NO: 1300; SEQ ID NO: 1302; SEQ ID NO: 1304, SEQ ID NO: 1306, SEQ ID NO: 1308; SEQ ID NO: 1310; SEQ ID NO: 1312; SEQ ID
NO: 1314; SEQ ID NO: 1316; SEQ ID NO: 1318; SEQ ID NO: 1320;
SEQ ID NO: 1322; SEQ ID NO: 1324; SEQ ID NO: 1326; SEQ ID NO: 1328; SEQ ID NO: 1330; SEQ ID NO: 1332; SEQ ID NO: 1334; SEQ ID NO: 1336, SEQ ID NO: 1338; SEQ ID NO: 1340; SEQ ID NO: 1342;
SEQ ID NO: 1344; SEQ ID NO: 1346; SEQ ID NO: 1348; SEQ ID NO: 1350; SEQ ID NO: 1352; SEQ ID NO: 1354; SEQ ID NO: 5; SEQ ID NO 12; SEQ ID NO: 19; SEQ ID NO: 26; SEQ ID NO: 33; SEQ ID NO: 40;
SEQ ID NO: 47; SEQ ID NO: 54; SEQ ID NO: 61; SEQ ID NO: 68; SEQ ID NO: 75; SEQ ID NO: 82; SEQ ID NO: 89; SEQ ID NO: 96; SEQ ID
NO: 103; SEQ ID NO: 110; SEQ ID NO: 117; SEQ ID NO: 124; SEQ ID NO: 131; SEQ ID NO: 138; SEQ ID NO: 145; SEQ ID NO: 152; SEQ ID NO: 160; SEQ ID NO: 168; SEQ ID NO: 175; SEQ ID NO: 182; SEQ ID NO: 189; SEQ ID NO: 196; SEQ ID NO: 203; SEQ ID NO: 210; SEQ ID NO: 217; SEQ ID NO: 224; SEQ ID NO: 231; SEQ ID NO: 238; SEQ ID NO: 245; SEQ ID NO: 252; SEQ ID NO: 259; SEQ ID NO: 266; SEQ ID NO: 273; SEQ ID NO: 280; SEQ ID NO: 287; SEQ ID NO: 294; SEQ ID NO: 301; SEQ ID NO: 308; SEQ ID NO: 315; SEQ ID NO: 322; SEQ ID NO: 329; SEQ ID NO: 336; SEQ ID NO: 343; SEQ ID NO: 350; SEQ ID NO: 357; SEQ ID NO: 364; SEQ ID NO: 371; SEQ ID NO: 378; SEQ ID NO: 385; SEQ ID NO: 392; SEQ ID NO: 399; SEQ ID NO: 406; SEQ ID NO: 413; SEQ ID NO: 420; SEQ ID NO: 427; SEQ ID NO: 434; SEQ ID NO: 441; SEQ ID NO: 448; SEQ ID NO: 455; SEQ ID NO: 462; SEQ ID NO: 469; SEQ ID NO: 476; SEQ ID NO: 483; SEQ ID NO: 490; SEQ ID NO: 497; SEQ ID NO: 504; SEQ ID NO: 511; SEQ ID NO: 518; SEQ ID NO: 525; SEQ ID NO: 532; SEQ ID NO: 539; SEQ ID NO: 546; SEQ ID NO: 553; SEQ ID NO: 560; SEQ ID NO: 567; SEQ ID NO: 574; SEQ ID NO: 581; SEQ ID NO: 588; SEQ ID NO: 595; SEQ ID NO: 602; SEQ ID NO: 609; SEQ ID NO: 616; SEQ ID NO: 623; SEQ ID NO: 630; SEQ ID NO: 637; SEQ ID NO: 644; SEQ ID NO: 651; SEQ ID NO: 658; SEQ ID NO: 665; SEQ ID NO: 672; SEQ ID NO: 679; SEQ ID NO: 686; SEQ ID NO: 693; SEQ ID NO: 700; SEQ ID NO: 707; SEQ ID NO: 714; SEQ ID NO: 721; SEQ ID NO: 728; SEQ ID NO: 735; SEQ ID NO: 742; SEQ ID NO: 749; SEQ ID NO: 756; SEQ ID NO: 764; SEQ ID NO: 771; SEQ ID NO: 778; SEQ ID NO: 785; SEQ ID NO: 792; SEQ ID NO: 799; SEQ ID NO: 806; SEQ ID NO: 813; SEQ ID NO: 820; SEQ ID NO: 827; SEQ ID NO: 834; SEQ ID NO: 841; SEQ ID NO: 848; SEQ ID NO: 855; SEQ ID NO: 862; SEQ ID NO: 869;S EQ ID NO: 876; SEQ ID NO: 884; SEQ ID NO: 889; SEQ ID NO: 898; SEQ ID NO: 905; SEQ ID NO: 912; SEQ ID NO: 919; SEQ ID NO: 926; SEQ ID NO: 933; SEQ ID NO: 940; SEQ ID NO: 947; SEQ ID NO: 954; SEQ ID NO: 961; SEQ ID NO: 968; SEQ ID NO: 975; SEQ ID NO: 982; SEQ ID NO: 989; SEQ ID NO: 996; SEQ ID
NO: 1003; SEQ ID NO: 1010; SEQ ID NO: 1017; SEQ ID NO: 1024;
SEQ ID NO: 1032; SEQ ID NO: 1039; SEQ ID NO: 1046; SEQ ID NO: 1053; SEQ ID NO: 1060; SEQ ID NO: 1067; SEQ ID NO: 1074; SEQ ID NO: 1081; SEQ ID NO: 1088; SEQ ID NO: 1095; SEQ ID NO: 1102;
SEQ ID NO: 1109; SEQ ID NO: 1116; SEQ ID NO: 1123; SEQ ID NO: 1130; SEQ ID NO: 1137; SEQ ID NO: 1144; SEQ ID NO: 1151; SEQ ID NO: 1158; SEQ ID NO: 1165; SEQ ID NO: 1172; SEQ ID NO: 1179;
SEQ ID NO: 1186; SEQ ID NO: 1193; SEQ ID NO: 1200; SEQ ID NO: 1207; SEQ ID NO: 1214; SEQ ID NO: 1221 ; SEQ ID NO: 1228; SEQ ID NO: 1235; SEQ ID NO: 1242; SEQ ID NO: 1249; SEQ ID NO: 1265;
SEQ ID NO: 1267; SEQ ID NO: 1269; SEQ ID NO: 1271; SEQ ID NO: 1273; SEQ ID NO: 1275; SEQ ID NO: 1277; SEQ ID NO: 1279; SEQ ID NO: 1281; SEQ ID NO: 1283; SEQ ID NO: 1285; SEQ ID NO: 1287;
SEQ ID NO: 1289; SEQ ID NO: 1291; SEQ ID NO: 1293; SEQ ID NO: 1295; SEQ ID NO: 1297; SEQ ID NO: 1299; SEQ ID NO: 1301; SEQ ID NO: 1303; SEQ ID NO: 1305; SEQ ID NO: 1307; SEQ ID NO: 1309;
SEQ ID NO: 1311; SEQ ID NO: 1313; SEQ ID NO: 1315; SEQ ID NO: 1317; SEQ ID NO: 1319; SEQ ID NO: 1321; SEQ ID NO: 1323; SEQ ID
NO: 1325; SEQ ID NO: 1327; SEQ ID NO: 1329; SEQ ID NO: 1331;
SEQ ID NO: 1333; SEQ ID NO: 1335; SEQ ID NO: 1337; SEQ ID NO:
1339; SEQ ID NO: 1341; SEQ ID NO: 1343; SEQ ID NO: 1345; SEQ ID
NO: 1347; SEQ ID NO: 1349; SEQ ID NO: 1351; SEQ ID NO: 1353; and
SEQ ID NO: 1355;
(ii) an antibody or derivative thereof wherein at least one variable region is a sequence set forth in and selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 8; SEQ ID NO: 15; SEQ ID NO: 22; SEQ ID NO: 29;
SEQ ID NO: 36; SEQ ID NO: 43; SEQ ID NO: 50; SEQ ID NO: 57; SEQ
ID NO: 64; SEQ ID NO: 71; SEQ ID NO: 78; SEQ ID NO: 85; SEQ ID
NO 92; SEQ ID NO: 99; SEQ ID NO: 106; SEQ ID NO: 1 13; SEQ ID
NO 120; SEQ ID NO 127; SEQ ID NO: 134; SEQ ID NO: 141; SEQ ID
NO 148; SEQ ID NO 156; SEQ ID NO: 164; SEQ ID NO: 171; SEQ ID
NO 178; SEQ ID NO 185; SEQ ID NO: 192; SEQ ID NO: 199; SEQ ID
NO 206; SEQ ID NO 213; SEQ ID NO: 220; SEQ ID NO: 227; SEQ ID
NO 234; SEQ ID NO 241; SEQ ID NO: 248;SEQ ID NO: 255; SEQ ID
NO 262; SEQ ID NO 269; SEQ ID NO: 276; SEQ ID NO: 283; SEQ ID
NO 290; SEQ ID NO 297; SEQ ID NO: 304; SEQ ID NO: 311; SEQ ID
NO 318; SEQ ID NO 325; SEQ ID NO: 332; SEQ ID NO: 339; SEQ ID
NO 346; SEQ ID NO 353; SEQ ID NO: 360; SEQ ID NO: 367; SEQ ID
NO 374; SEQ ID NO 381 ; SEQ ID NO: 388; SEQ ID NO: 395; SEQ ID
NO 402; SEQ ID NO 409;SEQ ID NO: 416; SEQ ID NO: 423; SEQ ID
NO 430; SEQ ID NO 437; SEQ ID NO: 444; SEQ ID NO: 451; SEQ ID
NO 458; SEQ ID NO 465; SEQ ID NO: 472; SEQ ID NO: 479; SEQ ID
NO 486; SEQ ID NO 493; SEQ ID NO: 500;SEQ ID NO: 507; SEQ ID
NO 514; SEQ ID NO 521 ; SEQ ID NO: 528; SEQ ID NO: 535; SEQ ID
NO 542; SEQ ID NO 549; SEQ ID NO: 556; SEQ ID NO: 563;SEQ ID
NO 570; SEQ ID NO 577; SEQ ID NO: 584; SEQ ID NO: 591; SEQ ID
NO 598; SEQ ID NO 605; SEQ ID NO: 612; SEQ ID NO: 619; SEQ ID
NO 626; SEQ ID NO 633; SEQ ID NO: 640; SEQ ID NO: 647; SEQ ID
NO 654; SEQ ID NO 661; SEQ ID NO: 668; SEQ ID NO: 675; SEQ ID NO: 682; SEQ ID NO: 689; SEQ ID NO: 696; SEQ ID NO: 703; SEQ ID NO: 710; SEQ ID NO: 717; SEQ ID NO: 724; SEQ ID NO: 731; SEQ ID NO: 738; SEQ ID NO: 745; SEQ ID NO: 752; SEQ ID NO: 760; SEQ ID NO: 767; SEQ ID NO: 774; SEQ ID NO: 781; SEQ ID NO: 788; SEQ ID NO: 795; SEQ ID NO: 802; SEQ ID NO: 809; SEQ ID NO: 816; SEQ ID NO: 823; SEQ ID NO: 830; SEQ ID NO: 837; SEQ ID NO: 844; SEQ ID NO: 851; SEQ ID NO: 858; SEQ ID NO: 865; SEQ ID NO: 872; SEQ ID NO: 880; SEQ ID NO: 887; SEQ ID NO: 894; SEQ ID NO: 901 SEQ ID NO: 908 SEQ ID NO: 915; SEQ ID NO: 922; SEQ ID NO: 929; SEQ ID NO: 936; SEQ ID NO: 943; SEQ ID NO: 950; SEQ ID NO: 957; SEQ ID NO: 964; SEQ ID NO: 971; SEQ ID NO: 978; SEQ ID NO: 985; SEQ ID NO: 992; SEQ ID NO: 999; SEQ ID NO: 1006; SEQ ID NO: 1013; SEQ ID NO: 1020; SEQ ID NO: 1028; SEQ ID NO: 1035; SEQ ID NO: 1042; SEQ ID NO: 1049; SEQ ID NO: 1056; SEQ ID NO: 1063; SEQ ID NO: 1070; SEQ ID NO: 1077; SEQ ID NO: 1084; SEQ ID NO: 1091; SEQ ID NO: 1098; SEQ ID NO: 1105; SEQ ID NO: 1112; SEQ ID NO: 1119;
SEQ ID NO: 1126; SEQ ID NO: 1133; SEQ ID NO: 1140; SEQ ID NO: 1147; SEQ ID NO: 1154; SEQ ID NO: 1161; SEQ ID NO: 1168; SEQ ID NO: 1175 SEQ ID NO: 1182; SEQ ID NO: 1189; SEQ ID NO: 1196; SEQ ID NO: 1203; SEQ ID NO: 1210; SEQ ID NO: 1217; SEQ ID NO: 1224; SEQ ID NO: 1231; SEQ ID NO: 1238; SEQ ID NO: 1245; SEQ ID NO: 1264; SEQ ID NO: 1266; SEQ ID: 1268; SEQ ID: 1270; SEQ ID: 1272; SEQ ID: 1274; SEQ ID NO: 1276; SEQ ID NO: 1278; SEQ ID NO: 1280; SEQ ID NO: 1282; SEQ ID NO: 1284; SEQ ID NO: 1286; SEQ ID NO: 1288; SEQ ID NO: 1290; SEQ ID NO: 1292; SEQ ID NO: 1294, SEQ ID NO: 1296; SEQ ID NO: 1298; SEQ ID NO: 1300; SEQ ID NO: 1302;
SEQ ID NO: 1304, SEQ ID NO: 1306, SEQ ID NO: 1308; SEQ ID NO: 1310; SEQ ID NO: 1312; SEQ ID NO: 1314; SEQ ID NO: 1316; SEQ ID NO: 1318; SEQ ID NO: 1320; SEQ ID NO: 1322; SEQ ID NO: 1324;
SEQ ID NO: 1326; SEQ ID NO: 1328; SEQ ID NO: 1330; SEQ ID NO: 1332; SEQ ID NO: 1334; SEQ ID NO: 1336, SEQ ID NO: 1338; SEQ ID NO: 1340; SEQ ID NO: 1342; SEQ ID NO: 1344; SEQ ID NO: 1346;
SEQ ID NO: 1348; SEQ ID NO: 1350; SEQ ID NO: 1352; SEQ ID NO: 1354; SEQ ID NO: 5; SEQ ID NO: 12; SEQ ID NO: 19; SEQ ID NO: 26;
SEQ ID NO: 33; SEQ ID NO: 40; SEQ ID NO: 47; SEQ ID NO: 54; SEQ ID NO: 61; SEQ ID NO: 68; SEQ ID NO: 75; SEQ ID NO: 82; SEQ ID
NO 89; SEQ ID NO: 96; SEQ ID NO: 103; SEQ ID NO: 110; SEQ ID
NO 117; SEQ ID NO 124; SEQ ID NO 131; SEQ ID NO 138; SEQ ID
NO 145; SEQ ID NO 152; SEQ ID NO 160; SEQ ID NO 168; SEQ ID
NO 175; SEQ ID NO 182; SEQ ID NO 189; SEQ ID NO 196; SEQ ID
NO 203; SEQ ID NO 210; SEQ ID NO 217; SEQ ID NO 224; SEQ ID
NO 231; SEQ ID NO 238; SEQ ID NO 245; SEQ ID NO 252; SEQ ID
NO 259; SEQ ID NO 266; SEQ ID NO 273; SEQ ID NO 280; SEQ ID
NO 287; SEQ ID NO 294; SEQ ID NO 301 ; SEQ ID NO 308; SEQ ID
NO 315; SEQ ID NO 322; SEQ ID NO 329; SEQ ID NO 336; SEQ ID
NO 343; SEQ ID NO 350; SEQ ID NO 357; SEQ ID NO 364; SEQ ID
NO 371; SEQ ID NO 378; SEQ ID NO 385; SEQ ID NO 392; SEQ ID
NO 399; SEQ ID NO 406; SEQ ID NO 413; SEQ ID NO 420; SEQ ID
NO 427; SEQ ID NO 434; SEQ ID NO 441 ; SEQ ID NO 448; SEQ ID
NO 455; SEQ ID NO 462; SEQ ID NO 469; SEQ ID NO 476; SEQ ID
NO 483; SEQ ID NO 490; SEQ ID NO 497; SEQ ID NO 504; SEQ ID
NO 511; SEQ ID NO 518; SEQ ID NO 525; SEQ ID NO 532; SEQ ID
NO 539; SEQ ID NO 546; SEQ ID NO 553; SEQ ID NO 560; SEQ ID
NO 567; SEQ ID NO 574; SEQ ID NO 581; SEQ ID NO 588; SEQ ID
NO 595; SEQ ID NO 602; SEQ ID NO 609; SEQ ID NO 616; SEQ ID
NO 623; SEQ ID NO 630; SEQ ID NO 637; SEQ ID NO 644; SEQ ID
NO 651; SEQ ID NO 658; SEQ ID NO 665; SEQ ID NO 672; SEQ ID
NO 679; SEQ ID NO 686; SEQ ID NO 693; SEQ ID NO 700; SEQ ID
NO 707; SEQ ID NO 714; SEQ ID NO 721; SEQ ID NO 728; SEQ ID
NO 735; SEQ ID NO 742; SEQ ID NO 749; SEQ ID NO 756; SEQ ID
NO 764; SEQ ID NO 771; SEQ ID NO 778; SEQ ID NO 785; SEQ ID
NO 792; SEQ ID NO 799; SEQ ID NO 806; SEQ ID NO 813; SEQ ID
NO 820; SEQ ID NO 827; SEQ ID NO 834; SEQ ID NO 841; SEQ ID
NO 848; SEQ ID NO 855; SEQ ID NO 862; SEQ ID NO 869;S EQ ID
NO 876; SEQ ID NO 884; SEQ ID NO 889; SEQ ID NO 898; SEQ ID
NO 905; SEQ ID NO 912; SEQ ID NO 919; SEQ ID NO 926; SEQ ID NO: 933; SEQ ID NO: 940; SEQ ID NO: 947; SEQ ID NO: 954; SEQ ID NO: 961; SEQ ID NO: 968; SEQ ID NO: 975; SEQ ID NO: 982; SEQ ID NO: 989; SEQ ID NO: 996; SEQ ID NO: 1003; SEQ ID NO: 1010; SEQ ID NO: 1017; SEQ ID NO: 1024; SEQ ID NO: 1032; SEQ ID NO: 1039; SEQ ID NO: 1046; SEQ ID NO: 1053; SEQ ID NO: 1060; SEQ ID NO: 1067; SEQ ID NO: 1074; SEQ ID NO: 1081 ; SEQ ID NO: 1088; SEQ ID NO: 1095; SEQ ID NO: 1102; SEQ ID NO: 1 109; SEQ ID NO: 1116;
SEQ ID NO: 1123; SEQ ID NO: 1130; SEQ ID NO: 1137; SEQ ID NO: 1144; SEQ ID NO: 1151; SEQ ID NO: 1158; SEQ ID NO: 1165; SEQ ID NO: 1172; SEQ ID NO: 1179; SEQ ID NO: 1186; SEQ ID NO: 1193;
SEQ ID NO: 1200; SEQ ID NO: 1207; SEQ ID NO: 1214; SEQ ID NO: 1221; SEQ ID NO: 1228; SEQ ID NO: 1235; SEQ ID NO: 1242; SEQ ID NO: 1249; SEQ ID NO: 1265; SEQ ID NO: 1267; SEQ ID NO: 1269;
SEQ ID NO: 1271; SEQ ID NO: 1273; SEQ ID NO: 1275; SEQ ID NO: 1277; SEQ ID NO: 1279; SEQ ID NO: 1281 ; SEQ ID NO: 1283; SEQ ID NO: 1285; SEQ ID NO: 1287; SEQ ID NO: 1289; SEQ ID NO: 1291;
SEQ ID NO: 1293; SEQ ID NO: 1295; SEQ ID NO: 1297; SEQ ID NO: 1299; SEQ ID NO: 1301; SEQ ID NO: 1303; SEQ ID NO: 1305; SEQ ID NO: 1307; SEQ ID NO: 1309; SEQ ID NO: 1311; SEQ ID NO: 1313;
SEQ ID NO: 1315; SEQ ID NO: 1317; SEQ ID NO: 1319; SEQ ID NO: 1321; SEQ ID NO: 1323; SEQ ID NO: 1325; SEQ ID NO: 1327; SEQ ID NO: 1329; SEQ ID NO: 1331 ; SEQ ID NO: 1333; SEQ ID NO: 1335;
SEQ ID NO: 1337; SEQ ID NO: 1339; SEQ ID NO: 1341; SEQ ID NO: 1343; SEQ ID NO: 1345; SEQ ID NO: 1347; SEQ ID NO: 1349; SEQ ID NO: 1351 ; SEQ ID NO: 1353; and SEQ ID NO: 1355;
(iii) an antibody or derivative thereof comprising from one to three heavy chain CDR sequences and/or from one to three light chain CDR sequences selected from the group consisting of sequences as set forth in SEQ ID NO: 2, 3, 4, 6, 7, 9, 10, 11, 13, 14, 16, 17, 18, 20, 21, 23, 24, 25, 27, 28, 30, 31,
32, 34, 35, 37, 38, 39, 41, 42, 44, 45, 46, 48, 49, 51, 52, 53, 55, 56, 58, 59,
60, 62, 63, 65, 66, 67, 69, 70, 72, 73, 74, 76, 77, 79. 80, 81, 83, 84, 86, 87,
88, 90, 91, 93, 94, 95, 97, 98, 100, 101,102,104, 105,107,108,109, 111, 112, , 115, 116 , 118, 119, 121, 122, 123, 125, 126, 128, 129, 130, 132, 133,, 136, 137 , 139, 140, 142, 143, 144, 146, 147, 149, 150, 151, 153, 154,, 157, 158 , 159, 161, 162, 163, 165, 166, 167, 169, 170, 172, 173, 174,, 177, 179 , 180, 181, 183, 184, 186, 187, 188, 190, 191, 193, 194, 195,, 198, 200 , 201, 202, 204, 205, 207, 208, 209, 211, 212, 214, 215, 216,, 219, 221 , 222, 223, 225, 226, 228, 229, 230, 232, 233, 235, 236, 237,, 240, 242 , 243, 244, 246, 247, 249, 250, 251, 253, 254, 256, 257, 258,, 261, 263 , 264, 265, 267, 268, 270, 271, 272, 274, 275, 277, 278, 279,, 282, 284 , 285, 286, 288, 289, 291, 292, 293, 295, 296, 298, 299, 300,, 303, 305 , 306, 307, 309, 310, 312, 313, 314, 316, 317, 319, 320, 321,, 324, 326 , 327, 328, 330, 331, 333, 334, 335, 337, 338, 340, 341, 342,, 345, 347 , 348, 349, 351, 352, 354, 355, 356, 358, 359, 361, 362, 363,, 366, 368 , 369, 370, 372, 373, 375, 376, 377, 379, 380, 382, 383, 384,, 387, 389 , 390, 391, 393, 394, 396, 397, 398, 400, 402, 403, 404, 405,, 408, 410 , 411, 412, 414, 415, 417, 418, 419, 421, 422, 424, 425, 426,, 429, 431 , 432, 433, 435, 436, 438, 439, 440, 442, 443, 445, 446, 447,, 450, 452 , 453, 454, 456, 457, 459, 460, 461, 463, 464, 466, 467, 468,, 471, 473 , 474, 475, 477, 478, 480, 481, 482, 484, 485, 487, 488, 489,, 492, 494 , 495, 496, 498, 499, 501, 502, 503, 505, 506, 508, 509, 510,, 513, 515 , 516, 517, 519, 520, 522, 523, 524, 526, 527, 529, 530, 531,, 534, 536 , 537, 538, 540, 541, 543, 544, 545, 547, 548, 550, 551, 552,, 555, 557 , 558, 559, 561, 562, 564, 565, 566, 568, 569, 571, 572, 573,, 576, 578 , 579, 580, 582, 583, 585, 586, 587, 589, 590, 592, 593, 594,, 597, 599 , 600, 601, 603, 604, 606, 607, 608, 610, 611, 613, 614, 615,, 618, 620 , 621, 622, 624, 625, 627, 628, 629, 631, 632, 634, 635, 636,, 639, 641 , 642, 643, 645, 646, 648, 649, 650, 652, 653, 655, 656, 657,, 660, 662 , 663, 664, 666, 667, 669, 670, 671, 673, 674, 676, 677, 678,, 681, 683 , 684, 685, 687, 688, 690, 691, 692, 694, 695, 697, 698, 699,, 702, 704 , 705, 706, 708, 709, 711, 712, 713, 715, 716, 718, 719, 720,, 723, 725 , 726, 727, 729, 730, 732, 733, 734, 736, 737, 739, 740, 741,, 744, 746 , 747, 748, 750, 751, 753, 754, 755, 757, 758, 759, 761, 762,, 765, 766 , 768, 769, 770, 772, 773, 775, 776, 777, 779, 780, 782, 783,, 786, 787 , 789, 790, 791, 793, 794, 796, 797, 798, 800, 801, 803, 804, 805, 807, 808, 810, 811, 812, 814, 815, 817, 818, 819, 821, 822, 824, 825
826, 828, 829, 831, 832, 833, 835, 836, 838, 839, 840, 842, 843, 845, 846
847, 849, 850, 852, 853, 854, 856, 857, 859, 860, 861, 863, 864, 866, 867
868, 870, 871, 873, 874, 875, 877, 878, 879, 881, 882, 883, 885, 886, 888
889, 890, 892, 893, 895, 896, 897, 899, 900, 902, 903, 904, 906, 907, 909
910, 911, 913, 914, 916, 917, 918, 920, 921, 923, 924, 925, 927, 928, 930
931, 932, 934, 935, 937, 938, 939, 941, 942, 944, 945, 946, 948, 949, 951
952, 953, 955, 956, 958, 959, 960, 962, 963, 965, 966, 967, 969, 970, 972
973, 974, 976, 977, 979, 980, 981, 983, 984, 986, 987, 988, 990, 991, 993
994, 995, 997, 998, 1000, 1001, 1002 1004, 1005, 1007, 1008, 1009, 1011, 1012, 1014, 1015, 1016, 1018, 1019, 1021, 1022, 1023, 1025, 1026, 1027, 1029, 1030, 1031, 1033, 1034, 1036, 1037, 1038, 1040, 1041, 1043, 1044, 1045, 1047, 1048, 1050, 1051, 1052, 1054, 1055, 1057, 1058, 1059, 1061, 1062, 1064, 1065, 1066, 1068, 1069, 1071, 1072, 1073, 1075, 1076, 1078, 1079, 1080, 1082, 1083, 1085, 1086, 1087, 1089, 1090, 1092, 1093, 1094, 1096, 1097, 1099, 1100, 1101, 1103, 1104, 1106, 1 107, 1108, 1110, 1111, 1113, 1114, 1115, 1117, 1118, 1120, 1121, 1122, 1124, 1125, 1127, 1128, 1129, 1131, 1132, 1134, 1135, 1136, 1138, 1139, 1141, 1142, 1143, 1145, 1146, 1148, 1149, 1150, 1152, 1153, 1155, 1156, 1157, 1159, 1160, 1162, 1163, 1164, 1166, 1167, 1169, 1170, 1171, 1173, 1 174, 1176, 1177, 1178, 1180, 1181, 1183, 1184, 1185, 1187, 1188, 1190, 1191, 1192, 1194, 1195, 1197, 1198, 1199, 1201, 1202, 1204, 1205, 1206, 1208, 1209, 1211, 1212, 1213, 1215, 1216, 1218, 1219, 1220, 1222, 1223, 1225, 1226, 1227, 1229, 1230, 1232, 1233, 1234, 1236, 1237, 1239, 1240, 1241 , 1243, 1244, 1246, 1247, 1248, 1250, 1251, 1356, 1357, 1358, 1359, 1360, 1361, 1362, 1364, 1365, 1367, 1368, 1369, 1370, 1371, 1372, 1374, 1375, 1376, 1378, 1380, 1381, 1384, 1389, 1392, 1394, 1396, 1400, 1407, 1408, 1410, 1411, 1415, 1418, 1419, 1420, 1423, 1426, 1427, 1433, 1434, 1442, 1448, 1449, 1450, 1451, 1452, 1454, 1455, 1456, 1457, 1458, 1459, 1460, 1461, 1462, 1466, 1470, 1471, 1474, 1475, 1478, 1479, 1480, 1487, 1488, 1491, 1494, 1495, 1496, 1497, 1498, 1499, 1500, 1501, 1502, 1503, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1518, 1519, 1521, 1522, 1523, 1526, 1527, 1528, 1529, 1530, 1531, 1532, 1533, 1534, 1536, 1537, 1538, 1539, 1540, 1541, 1542, 1543, 1544, 1545, 1546, 1547, 1548,
1549, 1550, 1552, 1553, 1554, 1556, 1557, 1558, 1560, 1561, 1563, 1564, 1565, 1566, 1567, 1568, 1569, 1570, 1571, 1572, 1574, 1575, 1576, 1577, 1578, 1579, 1580, 1581, 1583, 1584, and 1585; or
(iv) an antibody comprising a means for binding PcrV antigen.
31. A method of treating and/or preventing a disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of the composition of claim 30.
32. A method of immunizing a subject against Pseudomonas aeruginosa, the method comprising: administering to the subject in need thereof an effective amount of the composition of claim 30.
33. A method of detecting the presence of Pseudomonas aeruginosa V-antigen (PcrV), or a cell expressing PcrV, in a sample comprising: contacting the sample with an isolated human monoclonal antibody of claim 30.
PCT/US2024/057782 2023-12-01 2024-11-27 Protective monoclonal antibodies to pseudomonas aeruginosa Pending WO2025117765A1 (en)

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Citations (5)

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