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WO2018222741A1 - Bispecific antibodies for the treatment of streptococcus pneumonia - Google Patents

Bispecific antibodies for the treatment of streptococcus pneumonia Download PDF

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Publication number
WO2018222741A1
WO2018222741A1 PCT/US2018/035170 US2018035170W WO2018222741A1 WO 2018222741 A1 WO2018222741 A1 WO 2018222741A1 US 2018035170 W US2018035170 W US 2018035170W WO 2018222741 A1 WO2018222741 A1 WO 2018222741A1
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Prior art keywords
seq
amino acid
acid sequence
chain variable
variable region
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French (fr)
Inventor
Kenneth Smith
Justin Briggs
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Oklahoma Medical Research Foundation
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Oklahoma Medical Research Foundation
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    • 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
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1275—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Streptococcus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/315—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci
    • G01N2333/3156—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae [Pneumococcus]

Definitions

  • the present disclosure relates generally to antibodies, in particular bispecific antibodies, and functional fragments thereof that bind to Streptococcus pneumoniae.
  • the disclosed antibodies comprise novel complementarity determining regions (CDRs), which are combined into bispecific constructs that are capable of binding at least two serotypes of streptococcus pneumonia.
  • CDRs complementarity determining regions
  • the present disclosure also relates to methods of treating streptococcus pneumonia by administering the disclosed antibodies to a subject in need thereof.
  • CAP Community Acquired Pneumonia
  • SPN Streptococcus pneumoniae
  • CAPP severe pneumococcal CAP
  • the present disclosure provides isolated bispecific antibodies, in particular human bispecific antibodies that bind to Streptococcus pneumoniae (SPN) and exhibit desirable therapeutic properties. These properties include high affinity binding to various SPN serotypes, and each of the disclosed bispecific antibodies can bind at least two SPN serotypes.
  • SPN Streptococcus pneumoniae
  • the present disclosure provides bispecific anti -streptococcus pneumoniae antibodies or functional fragments thereof that specifically bind to at least two serotypes selected from a group consisting of serotypes 19A, 3, 22F, 6A, 6B, 6C, 19F, 15A, 23 A, 11 A, 35B, 12F, 22A and 7F.
  • the present disclosure provides bispecific antibodies or functional fragments thereof that specifically bind to at least two serotypes of streptococcus pneumoniae, wherein the bispecific antibodies or functional fragments thereof comprise: a first heavy chain variable region comprising: a complementarity determining region (CDR) HI selected from the group consisting of SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, and 43; a CDRH2 selected from the group consisting of SEQ ID NOs: 2, 8, 14, 20, 26, 32, 38, and 44; and a CDRH3 selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, and 45; a second heavy chain variable region comprising: a complementarity determining region (CDR) HI selected from the group consisting of SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, and 43; a CDRH2 selected from the group consisting of SEQ ID NOs: 2, 8, 14, 20, 26, 32, 38, and 44; and a CDRH3 selected from the group
  • the present disclosure provides bispecific antibodies or functional fragments thereof that specifically bind to at least two serotypes of streptococcus pneumoniae, wherein the bispecific antibody or functional fragment thereof comprises two different heavy chain variable regions and two different light chain variable regions, wherein the two different heavy chain variable regions and the two different light chain variable regions are encoded by four different nucleic acid sequences chosen from Table 4.
  • the bispecific antibody or functional fragment thereof can specifically bind to at least two of serotypes 19A, 3, 22F, 6A, 6B, or 6C.
  • the antibody or functional fragment thereof can specifically bind to serotypes 19A and 3, while in some embodiments, the antibody or functional fragment thereof can specifically bind to serotypes 6A and 22F.
  • a binding domain that can bind to serotype 6A can be cross-reactive with serotypes 6B and/or 6C.
  • the bispecific antibody or functional fragment thereof has a K D for the serotypes that it binds that is at least at least l .OxlO "9 M, and in some embodiments, the antibody or functional fragment thereof has an OPA titer of greater than 8.
  • the bispecific antibody or functional fragment thereof may comprise first and second heavy chain variable regions comprising an amino acid sequence comprising SEQ ID NOs: 49, 51, 53, 55, 57, 59, 61, or 63, and first and second light chain variable regions comprising an amino acid sequence comprising SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, or 64, wherein the first and second heavy chains do not comprise the same sequences and the first and second light chains do not comprise the same sequences.
  • the first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 3;
  • the second heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 25, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 26, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 27;
  • the first light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 4, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 6;
  • the second light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 28, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 29, and a CDRL3 compris
  • the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 49
  • the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 57
  • the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 50
  • the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 58.
  • the first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 7, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 9;
  • the second heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 31, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 33;
  • the first light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 10, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 12;
  • the second light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 34, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 35, and a CD
  • the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 51
  • the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 59
  • the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 52
  • the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 60.
  • the first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 7, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 9;
  • the second heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 37, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 39;
  • the first light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 10, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 12;
  • the second light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 40, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 41, and a
  • the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 51
  • the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 61
  • the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 52
  • the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 62.
  • the first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 13, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 14, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 15;
  • the second heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 43, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 45;
  • the first light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 16, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 17, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 18;
  • the second light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 46, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 47, and a
  • the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 53
  • the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 63
  • the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 54
  • the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 64.
  • the first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 19, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 21;
  • the second heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 43, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 45;
  • the first light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 22, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 23, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 24;
  • the second light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 46, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 47, and a
  • the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 55
  • the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 63
  • the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 56
  • the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 64.
  • the present disclosure provides methods of treating a Streptococcus pneumoniae infection in an individual comprising, administering to an individual with a S. pneumoniae infection, (i) a therapeutically effective amount of a bispecific antibody or a functional fragment thereof that specifically binds to at least two serotypes selected from a group consisting of serotypes 19A, 3, 22F, 6A, 6B, 6C, 19F, 15A, 23 A, 11 A, 35B, 12F, 22A and 7F; and (ii) an antibiotic.
  • the present disclosure provides methods of treating an antibiotic- resistant Streptococcus pneumoniae infection in an individual comprising, administering to an individual with an antibiotic-resistant S. pneumoniae infection, a therapeutically effective amount of a bispecific antibody or a functional fragment thereof that specifically binds to at least two serotypes selected from a group consisting of serotypes 19 A, 3, 22F, 6 A, 6B, 6C, 19F, 15A, 23 A, 11 A, 35B, 12F, 22A and 7F.
  • the present disclosure provides methods of treating a Streptococcus pneumoniae infection in an individual comprising, administering to an individual with a S. pneumoniae infection, a therapeutically effective amount of a bispecific antibody or a functional fragment thereof that specifically binds to at least two serotypes selected from a group consisting of serotypes 19A, 3, 22F, 6A, 6B, and 6C, wherein the bispecific antibody or functional fragment thereof has a K D for the serotypes that it binds that is at least at least l .OxlO "9 M.
  • the bispecific antibody or functional fragment thereof utilized in the disclosed methods can specifically bind to at least two of serotypes 19A, 3, 22F, 6A, 6B, or 6C.
  • the bispecific antibody or functional fragment thereof utilized in the disclosed methods may comprise first and second heavy chain variable regions comprising an amino acid sequence comprising SEQ ID NOs: 49, 51, 53, 55, 57, 59, 61, or 63, and first and second light chain variable regions comprising an amino acid sequence comprising SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, or 64, wherein the first and second heavy chains do not comprise the same sequences and the first and second light chains do not comprise the same sequences.
  • the bispecific antibody or functional fragment thereof utilized in the disclosed methods may comprise a first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 3; a second heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 25, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 26, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 27; a first light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 4, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 6; and a second light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 28, a CDRL2 comprising the amino acid sequence of SEQ
  • the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 49
  • the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 57
  • the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 50
  • the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 58.
  • the bispecific antibody or functional fragment thereof utilized in the disclosed methods may comprise a first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 7, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 9;
  • a second heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 31, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 33;
  • a first light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 10, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 12;
  • a second light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 34, a CDRL
  • the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 51
  • the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 59
  • the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 52
  • the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 60.
  • the bispecific antibody or functional fragment thereof utilized in the disclosed methods may comprise a first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 7, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 9;
  • a second heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 37, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 39;
  • a first light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 10, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 12;
  • a second light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 40, a CD
  • the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 51
  • the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 61
  • the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 52
  • the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 62.
  • the bispecific antibody or functional fragment thereof utilized in the disclosed methods may comprise a first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 13, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 14, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 15;
  • a second heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 43, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 45;
  • a first light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 16, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 17, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 18;
  • a second light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 46, a CD
  • the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 53
  • the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 63
  • the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 54
  • the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 64.
  • the bispecific antibody or functional fragment thereof utilized in the disclosed methods may comprise a first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 19, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 21; a second heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 43, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 45; a first light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 22, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 23, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 24; and a second light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 46, a CD
  • the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 55
  • the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 63
  • the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 56
  • the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 64.
  • the antibiotic used to treat a patient can be a ⁇ -lactam antibiotic, a tetracycline, a macrolide, a quinolone, or a fluoroquinolone, or in some embodiments, the antibiotic can be Levofloxacin or Linezolid.
  • the individual's S. pneumoniae infection comprises at least one of the serotypes specifically bound by the bispecific antibody or functional fragment thereof.
  • the bispecific antibody or functional fragment thereof is administered before, after, or concurrently with the antibiotic.
  • the individual may have previously received an antibiotic to treat the S. pneumoniae infection, and in some embodiments, the infection may be antibiotic-resistant.
  • the antibody or functional fragment thereof used in the disclosed methods may have an OPA titer of greater than 8.
  • Figure 1 shows the prevalence of S. pneumoniae serotypes in North America in 2009.
  • Figure 2 shows two parent antibodies (F405Lp4A05L and K409Rp6E03K) and their bispecific (p4A05L/p6E03K-bi) tested on ELISAs to demonstrate bispecificity with OPA titers and specificities against serotype 19A and 3.
  • Figure 3 shows in vivo efficacy of BsAb serotype 19 A/3 is comparable or better than Linezolid.
  • Panel A shows lung CFU counts taken 24 hours post intranasal infection of mice with SPN serotype 3.
  • Panel B shows a 10 day mortality study in the infected mice.
  • compositions and methods of the present disclosure employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (F. M.
  • compositions and methods include the recited elements, but not excluding others.
  • Consisting essentially of when used to define compositions and methods shall mean excluding other elements of any essential significance to the composition or method.
  • Consisting of shall mean excluding more than trace elements of other ingredients for claimed compositions and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure. Accordingly, it is intended that the methods and compositions can include additional steps and components (comprising) or alternatively including steps and compositions of no significance (consisting essentially of) or alternatively, intending only the stated method steps or compositions (consisting of).
  • the terms “individual”, “patient”, or “subject” can be an individual organism, a vertebrate, a mammal (e.g., a bovine, a canine, a feline, or an equine), or a human. In a preferred embodiment, the individual, patient, or subject is a human.
  • an "isolated antibody” is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to streptococcus pneumonia is substantially free of antibodies that do not bind to streptococcus pneumonia).
  • An isolated antibody that specifically binds to a serotype of streptococcus pneumonia may, however, have cross- reactivity to other serotypes of SPN.
  • the disclosed isolated antibodies preferably always bind to at least one specific serotype of SPN with high affinity.
  • an isolated antibody is typically substantially free of other cellular material and/or chemicals.
  • humanized antibody refers to an antibody that comprises the CDRs of antibodies derived from mammals other than human, and the framework (FR) region and the constant region of a human antibody.
  • a humanized antibody is useful as an effective component in a therapeutic agent according to the present disclosure since antigenicity of the humanized antibody in human body is lowered.
  • the term "recombinant human antibody” includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, including but not limited to (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody (e.g., from a transfectoma), (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant human antibodies have variable and constant regions derived from human germline and/or non-germline immunoglobulin sequences.
  • such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • the term "glycosylation pattern" is defined as the pattern of carbohydrate units that are covalently attached to a protein, more specifically to an immunoglobulin protein.
  • the phrases "therapeutically effective amount” and “therapeutic level” mean that drug dosage or plasma concentration in a subject, respectively, that provides the specific pharmacological effect for which the drug is administered in a subject in need of such treatment, i.e. to reduce, ameliorate, or eliminate the symptoms or effects of streptococcus pneumonia. It is emphasized that a therapeutically effective amount or therapeutic amount of an antibody will not always be effective in treating the conditions/diseases described herein, even though such dosage is deemed to be a therapeutically effective amount by those of skill in the art. The therapeutically effective amount may vary based on the route of administration and dosage form, the age and weight of the subject, and/or the subject's condition, including the serotype and stage of the streptococcus pneumonia infection, among other factors.
  • treatment or “treating” as used herein with reference to streptococcus pneumonia refer to reducing, ameliorating or eliminating one or more symptoms or effects of streptococcus pneumonia, including but not limited to, bacterial load ⁇ i.e., CFU count) within the subject, difficulty breathing, chest pains, fever, fatigue, chills, bloody mucus, increased heart rate, nausea, diarrhea, coughing and/or sneezing, etc.
  • bacterial load ⁇ i.e., CFU count
  • binding fragment refers to one or more fragments of a bispecific anti-SPN antibody, as disclosed herein, that retains the ability to bind to at least two serotypes of SPN.
  • binding fragments include (i) Fab fragments (monovalent fragments consisting of the VL, VH, CL and CHI domains); (ii) F(ab')2 fragments (bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region); (iii) Fd fragments (comprising the VH and CHI domains); (iv) Fv fragments (comprising the VL and VH domains of a single arm of an antibody), (v) dAb fragments (comprising a VH domain); and (vi) isolated complementarity determining regions (CDR), e.g., VH CDR3.
  • CDR complementarity determining regions
  • scFv single chain Fv constructs. See e.g., Bird et al., Science, 242:423-26 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-83 (1988).
  • PD-L1 -binding-domain immunoglobulin fusion proteins comprising (i) a PD-L1 -binding domain polypeptide (such as a heavy chain variable region, a light chain variable region, or a heavy chain variable region fused to a light chain variable region via a linker peptide) fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region.
  • a PD-L1 -binding domain polypeptide such as a heavy chain variable region, a light chain variable region, or a heavy chain variable region fused to a light chain variable region via a linker peptide
  • Streptococcus pneumoniae an often drug-resistant pathogen, is the leading cause of community-acquired pneumonia, and a key contributor to morbidity/mortality worldwide.
  • SPN Streptococcus pneumoniae
  • community-acquired pneumococcal pneumonia infects more than 5 million/yr (despite the availability of several vaccines), causes 1.4 million hospital visits, 1.1 million hospitalizations, and even with modern antibacterial intervention, results in 68,000 deaths annually. Hospitalized pneumonia patients stay an average of 5.6 days. Pediatric need is even greater, as pneumonia causes more than 156 million infections and kills an estimated 2 million children under 5 every year— more than AIDS, malaria and tuberculosis combined. Of these pneumonia-related deaths, over 50% are caused by SPN.
  • antibody (Ab) therapies are generally more expensive compared to antibiotics, their ability to expedite microbial clearance and disease resolution reduces total days in the hospital and total cost of CAP. Indeed, a 1.7-day reduction in hospital stays for CAP resulted in an estimated cost saving of $1,700 per patient. Thus, a non-antibiotic treatment for CAP could address multiple issues: Growing antibiotic resistance, risk of potentially fatal CDI, and significant patient morbidity/mortality.
  • bispecific antibodies that specifically bind to at least two serotypes of SPN.
  • Polysaccharide type-specific Abs mediating opsonin-dependent phagocytosis are considered the main mechanism of host protection against Streptococcus pneumoniae, and levels of opsonic Abs correlate with protection in preclinical models. Abs to the non-opsonogenic targets of pneumococcal surface protein A (PspA), pneumolysin (Ply), and adhesins have had mixed results as therapeutic interventions and have not shown efficacy in preventing/treating pneumococcal infection in several cases.
  • PspA pneumococcal surface protein A
  • Ply pneumolysin
  • adhesins adhesins
  • anti- polysaccharide Abs are the ideal therapeutic to clear SPN infection, but there are over 90 known SPN serotypes, and developing so many monospecific antibodies would be expensive and therapeutically challenging in an acute-care setting.
  • the present disclosure provides a therapeutic that contains multi-specific antibodies targeting the most predominant, disease-causing serotypes would overcome the challenges of serotype-specific treatment.
  • the disclosed bispecific antibodies can opsonize at least two different SPN serotypes, which provides at least three beneficial clinical results: 1) an increased number of patients will be treatable with the BsAbs compared to monospecific Abs; 2) the need for Ab mixtures of multiple, individual Abs will be obviated; and 3) the total protein load administered to a given patient will be reduced, thereby improving the safety of the Ab therapy.
  • an antibody consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two copies of a light (L) chain polypeptide.
  • each heavy chain contains one N-terminal variable (VH) region and three C-terminal constant (CHI, CH2 and CH3) regions
  • each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region.
  • the variable regions of each pair of light and heavy chains form the antigen binding site of an antibody.
  • Bispecific antibodies typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen (i.e., a different SPN serotype).
  • Each antigen-binding domain of a bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR).
  • HCVR heavy chain variable domain
  • LCVR light chain variable domain
  • each antigen binding domain comprises at least one CDR that alone, or in combination with one or more additional CDRs and/or FRs, specifically binds to a particular antigen (i.e., serotype).
  • the first antigen-binding domain and the second antigen-binding domain may be directly or indirectly connected to one another to form a bispecific antigen-binding molecule (i.e., bispecific ScFv) further bound to an Fc domain.
  • the first antigen-binding domain and the second antigen-binding domain may each be connected to a separate Fc domain.
  • Bispecific antigen-binding molecules of the present disclosure may comprise two Fc domains that are each individually part of a separate antibody heavy chain.
  • the first and second Fc domains may be of the same sequence, or the Fc domains may have a mutation in the CH3 domain intended for the facilitation or ease of purification of heterodimeric (i.e., bispecific) molecules.
  • the disclosed bispecific antibodies may be produced by any means known in the art for producing bispecific antibodies, so long as the resulting bispecific antibody retains the functional characteristic of being able to specifically bind at least two serotypes of SPN.
  • the disclosed BsAbs will comprise the CDR sequences or variable region sequences disclosed herein.
  • the BsAbs may be created using the methods described in Labrijin et al., Proc. Natl. Acad. Sci. USA, 110(13):5145-50 (2013). Briefly, the two parental Abs— each containing single matched point mutations in the CH3 domains— are separately expressed and then mixed under reducing conditions in vitro.
  • bispecific antibody formats or technologies may be used to make the bispecific antigen-binding molecules of the present disclosure.
  • an antibody or fragment thereof having a first antigen binding specificity can be functionally linked ⁇ e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity to produce a bispecific antigen-binding molecule.
  • bispecific formats that can be used in the context of the present invention include, without limitation, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chain ⁇ e.g., common light chain with knobs-into- holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgGl/IgG2, dual acting Fab (DAF)-IgG, and Mab 2 bispecific formats ⁇ see, e.g., Klein et al. 2012, mAbs 4:6, 1-1 1 , and references cited therein, for a review of the foregoing formats).
  • the disclosed bispecific antibodies can be made from or incorporate the CDRs or variable regions from polyclonal, monoclonal, chimeric, human, partially or fully humanized, and/or recombinant antibodies.
  • the "parent" antibodies for the disclosed BsAbs are not particularly limited; however, they are preferably fully human.
  • the parent antibody can be a polyclonal antibody.
  • the parent antibody can be a monoclonal.
  • the parent antibody can be a human antibody.
  • Standard means of making antibodies are known in the art.
  • polyclonal antibodies may be obtained by methods known in the art, such as by immunizing a selected animal with a SPN serotype-specific antigen, collecting serum from the animal, and isolating and/or purifying antibodies from the serum.
  • Monoclonal antibodies (mAbs) may be obtained by methods known in the art, for example, by fusing antibody-producing cells with immortalized cells to obtain a hybridoma, and/or by generating mAbs from mRNA extracted from bone marrow and spleen cells of immunized animals using combinatorial antibody library technology.
  • Recombinant antibodies may be obtained by methods known in the art, for example, using phage or yeast display technologies and/or expressing or co- expressing antibody polypeptides. Other techniques for making antibodies are known in the art, and can be used to obtain antibodies used in the methods described herein.
  • the parent anti-SPN antibodies may be fully human antibodies isolated from human subjects that were vaccinated with a pneumococcal vaccine (e.g., P EUMOVAX®23 or PREVINAR®13) containing a large number of SPN serotypes, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F.
  • the serotype-specific antibodies can be isolated from these subject and cloned.
  • This platform leverages the natural "memory" response to isolate a broad range of high-affinity Abs to the target pathogen following vaccination or infection and provides cross-sectional characterization of the anti-polysaccharide response.
  • the serotype- specificity of these antibodies can then be characterized using routine methods known in the art (see, e.g., U.S. Patent No. 9,279,815, which is hereby incorporated by reference).
  • Pneumococcal vaccine serotypes represent nearly 75% of CAPP infections in the U.S. and worldwide, but a handful of serotypes predominate (Figure 1). In fact, just 10 serotypes account for over half of pneumococcal infections worldwide. These serotypes include 19A, 3, 22F, 6A, 6B, 6C, 19F, 15A, 23 A, 11 A, 35B, 12F, 22A and 7F.
  • the present inventors have developed fully human Abs that specifically bind at least one of each of the 24 SPN vaccine serotypes, and demonstrated that these full-length, fully human Abs have high binding affinity and potent opsonophagocytotic assay (OP A) activity (>8 titers) against SPN serotypes that account for almost three-quarters of invasive pneumococcal disease in hospitalized patients. While elderly with dampened immune responses and immunocompromised individuals account for a significant fraction of CAPP -infected individuals, less than 5% were found to be leukopenic.
  • the disclosed BsAbs which incorporate the variable sequences of the disclosed fully human Abs, along with patient neutrophils, will be successful in clearing the SPN infection via opsonophagocytosis.
  • the disclosed BsAbs are capable of providing a broad spectrum of protection without the need for multiple, distinct Abs.
  • BsAbs are emerging as a novel approach for immunotherapies. Utilizing immuno-engineering techniques, it is possible to combine 2 antigen-recognizing elements into a single construct that simultaneously binds to 2 distinct targets (see, e.g., Spasevska et al., J. Blood Disord. Transfus., 6:243. doi: 10.4172/2155-9864.1000243 (2015)).
  • hmAbs high- affinity, high OPA titer human monoclonal antibodies
  • hmAbs high- affinity, high OPA titer human monoclonal antibodies
  • the OPA activity for these bispecific antibodies can be significantly higher than those of the monospecific antibodies on which they are based. See Table 1 below.
  • the disclosed anti-SPN BsAbs can bind at least two serotypes of S. pneumoniae. In some embodiments, the disclosed anti-SPN BsAbs may possess cross- reactivity, allowing them to bind to more than two serotypes. For examples, in some embodiments, the disclosed anti-SPN BsAbs may bind two, three, four, five, or more serotypes of S. pneumoniae.
  • the cross-reactivity of the disclosed antibodies can significantly increase the therapeutic coverage of the disclosed anti-SPN BsAbs. For instance, an anti-6A mAb may bind serotypes 6A/B/C, or an anti-19A mAb may bind both 19A and 19F
  • the disclosed BsAbs are specific for at least two of serotypes 19A, 3, 22F, or 6A.
  • some BsAbs may be specific for serotypes 19A and 3; 22F and 6A; 19A and 22F, 19A and 6A; 3 and 22F; or 3 and 6A.
  • These serotypes are the four most prevalent among hospitalized patients over 65 in the North America and often possess penicillin and erythromycin resistance.
  • a first BsAb that specifically binds to serotypes 19A and 3 would address 25-30% of SPN infections in the U.S. and worldwide, and a second BsAb that specifically binds to serotypes 22F and 6 A would address another 15-20% of SPN infections.
  • Further adding additional BsAbs to other serotypes, particularly those with known antibiotic resistance would substantially increase therapeutic coverage without having to administer numerous monospecific antibodies to an individual.
  • the disclosed anti-SPN BsAbs may comprise any combination of the disclosed variable regions or CDRs so long as the BsAb is able to bind to at least two serotypes of S. pneumonia. Accordingly, in some embodiments, the disclosed BsAb can comprise a combination of an anti-19A, anti-3, anti-22F, anti-6A, anti-6B, anti-6C, anti-19F, anti-15A, anti-23A, anti-l lA, anti-35B, anti-12F, anti-22A or anti-7F variable regions or CDRs.
  • the BsAbs may be defined according to their amino acid sequence or according to the nucleic acid sequence that encodes the amino acids. Exemplary antibody sequences are shown in the following Tables 2-4.
  • TLVFGGGTKLTVL (SEQ ID NO: 52)
  • TFGPGTKVDIK (SEQ ID NO: 54)
  • CARGQSYPGIWGQGTMVTVSS (SEQ ID NO: 57)
  • GQGTKVEIK (SEQ ID NO: 60)
  • PV4p6C02H caggtgcagctggtgcagtctggggccgaggtgaagaagcctggggcctcagtcaaggtttcctgcaagg / Heavy* cttctggatacatgttcactagctatactatgcattgggtgcgccaggcccccggacaaaggcttgagtggat gggatggatcggcgcggcgatggtaacataaaatattcacagaagttccagggcagagccaccaccattacc agggacacatccgcgagcaccgcgtacatggagctgagcagcctgagatctgaagacacggctatgt actgtgcgagagagggtacagaagtcggttggacattgactactggggccagggaaccctggtcacc
  • PV4p6C02L cagtctgccctgactcagccaccctccgtgtccgggtctcctggacagtcagtcaccatctcctgcactgga / Light* agtagcagtgacgttggaagttataacggtgtctcctggtacaagcagcccccaggcacagtccccaaact catgatttatgaggtcactaatcggcccctcaggggtccctgatcgcttctctggggccaagtctggcaacacg gcctcctgaccatctctgggctcctgctgaggacgaggctgattattactgcagctcatatacaagcagca acacttatgtcttcggaactgggaccacggtcaccgtcctag (
  • the anti-SPN BsAbs or functional fragments will share about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%), or about 99% sequence identity with the disclosed sequences.
  • the nucleic acid encoding the anti-SPN BsAbs or functional fragments thereof will share about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the disclosed sequences.
  • the disclosed bispecific antibodies and functional fragments thereof may be defined by sequence, as above, or by functional characteristics.
  • the present disclosure provides anti-SPN high-affinity Abs that have K D values better than nanomolar range and opsonophagocytosis assays (OP A) titers greater than 8.
  • OP A measures the ability of an Ab to kill the pneumococcus and has been used as a surrogate marker for vaccine efficacy.
  • OPA titers over 8 are considered protective.
  • the disclosed BsAbs have high-binding affinity and greater than 8 multiplexed OPA titers (MOPA-measures Ab killing of multiple distinct serotypes in one test) against specific SPN serotypes (see , e.g., Table 1 and Figure 2).
  • the disclosed bispecific antibodies and functional fragments thereof can have a K D of at least 3.0xl0 "8 , at least 2.5xl0 "8 , at least 2.0xl0 “8 , at least 1.5x10 " 8 , at least l .OxlO "8 , at least 0.5xl0 "8 , at least 9.95xl0 "9 , at least 9.90xl0 "9 , at least 9.85xl0 "9 , at least 9.80xl0 “9 , at least 9.75xl0 “9 , at least 9.70xl0 "9 , at least 9.65xl0 "9 , at least 9.60xl0 “9 , at least 9.55xl0 "9 , at least 9.5xl0 “9 , at least 9.45xl0 "9 , at least 9.40xl0 “9 , at least 9.35xl0 "9 , at least 9.30xl0 "9 , at least 9.25xl0 "9
  • the disclosed bispecific antibodies and functional fragments thereof can have an OPA or MOPA titer of at least 8, at least 9, at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, at least 5500, at least 6000, at least 6500, ate least 7000, at least 7500, at least 8000, at least 8500, at least 9000, at least 9500, at least 10000, at least 10500, at least 11000, at least 11500, at least 12000, at least 12500, or at least 13000.
  • OPA or MOPA titer of at least 8, at least 9, at least
  • the hinge region of the disclosed antibodies may be modified by replacing one or more cysteine residues with, for example, serine residues, to prevent dimerization. See, e.g., U.S. Patent Application Publication 2003/0118592; U.S. Patent Application Publication U.S. 2003/0133939. Additionally, in some embodiments, the disclosed antibodies may comprise other mutations, including but not limited to a variant Fc portion of an IgGl having the point mutations S239D/I332E, S239D, or I332E, or any combination thereof, or a variant Fc portion of an IgG4 having the point mutation S228P.
  • Such modifications may alter the binding of the disclosed antibodies and functional fragments to Fc receptors (FcRs), and in some embodiments, the antibody may be modified to be more stable, while in some embodiments, the antibody may be modified to enhance ADCC function.
  • the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.
  • the glycosylation patterns of the disclosed antibodies may be modified or altered.
  • the disclosed antibodies and functional fragments thereof may be low fucose antibodies or they may be defucosylated or the antibodies may be expressed or produced in such a way that they are lacking fucose altogether (i.e. afucosylated).
  • Modifying the fucose content of the antibody or functional fragment may be accomplished through various means known in the art, for instance, expressing the antibody or functional fragment in a cell that is FUT8 deficient or that has a mutated version of FUT8.
  • Low fucose or defucosylated antibodies and functional fragment have increased ADCC activity.
  • the disclosed antibodies and functional fragments may comprise other functional modifications to their glycosylation patterns.
  • modifications at position 297 e.g. N297A and N297Q
  • modifications at position 297 can prevent glycosylation of the Fc region altogether, thus eliminating Fc function, ADCC, and CDC.
  • the anti-SPN BsAbs or functional fragments thereof can be formulated in a pharmaceutical composition suitable for administration to the target subject by the intended route of administration, as discussed in more detail below.
  • compositions suitable for use in the methods described herein can include the therapeutically active agent (i.e., the disclosed anti-SPN BsAbs or functional fragments thereof) and a pharmaceutically acceptable carrier or diluent.
  • the composition may be formulated for intravenous, subcutaneous, intraperitoneal, intramuscular, oral, nasal, pulmonary, ocular, vaginal, or rectal administration.
  • the anti-SPN BsAbs or functional fragments thereof are formulated for intravenous, subcutaneous, intraperitoneal, or intramuscular administration, such as in a solution, suspension, emulsion, liposome formulation, etc.
  • the pharmaceutical composition can be formulated to be an immediate-release composition, sustained-release composition, delayed-release composition, etc., using techniques known in the art.
  • Pharmacologically acceptable carriers for various dosage forms are known in the art.
  • excipients, lubricants, binders, and disintegrants for solid preparations are known; solvents, solubilizing agents, suspending agents, isotonicity agents, buffers, and soothing agents for liquid preparations are known.
  • the pharmaceutical compositions include one or more additional components, such as one or more preservatives, antioxidants, stabilizing agents and the like.
  • the disclosed pharmaceutical compositions comprising at least one anti-SPN BsAbs can be formulated as a solution, microemulsion, nanoparticle, liposome, or other ordered structure suitable to high drug concentration.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (i.e. the disclosed anti-SPN BsAbs or functional fragments thereof) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • compositions of the disclosure can be administered in combination with other therapeutics, such as antibiotics.
  • the combination therapy can include a pharmaceutical composition comprising at least one of the disclosed anti-SPN BsAbs or functional fragments thereof with at least one or more additional therapeutic agents, including but not limited to, antibiotics commonly used to treat SPN infections (discussed in more detail below).
  • kits for treating streptococcus pneumonia with the disclosed bispecific anti-SPN antibodies More specifically, the disclosure provides for methods of enhancing a subject's immunity or ability to fight a SPN infection comprising administering a therapeutically effective amount of any of the above described anti-SPN BsAbs or pharmaceutical compositions comprising the same.
  • administration of the disclosed BsAbs can serve as a standalone treatment.
  • patient outcome may be improved by administering the disclosed BsAbs in conjunction with antibiotic therapy.
  • the disclosed BsAbs may be administered before, concurrently with, or after administration of at least one antibiotic.
  • antibiotics that may be used in conjunction with the disclosed BsAbs include, but are not limited to, ⁇ -lactam antibiotics (e.g., penicillin, ampicillin, methicillin, amoxicillin, etc.), tetracyclines (e.g., tetracycline, minocycline, methacycline, doxycycline, etc.), macrolides (e.g., erythromycin, azithromycin, clarithromycin, etc.), and quinolones or fluoroquinolones (e.g., ciprofloxacin, garenoxacin, gatifloxacin, gemifloxacin, levofloxacin, moxifloxacin, linezolid (ot other oxazolidinones), etc.).
  • ⁇ -lactam antibiotics e.g., penicillin, ampicillin, methicillin, amoxicillin, etc.
  • tetracyclines e.g., tetracycline
  • the method of treatment may comprise administering more than one of the disclosed BsAbs.
  • a patient may be administered a first BsAb that is specific for serotypes 19 A/3 and a second BsAb that is specific for serotypes 22F/6A.
  • the first and second BsAbs may be administered at the same time or at separate times, for instance, one after the other.
  • the disclosed BsAb can be administered to a patient with SPN or CAPP that is resistant to antibiotics.
  • the disclosed BsAb can be administered to a patient with SPN having a serotype of 19A, 3, 22F, 6A, 6B, 6C, 19F, 15A, 23 A, 1 1 A, 35B, 12F, 22A or 7F.
  • the serotype of the infection may be determined prior to administration of the disclosed BsAb using, for example, a lateral-flow point-of-care diagnostic, like those disclosed herein, or another diagnostic capable of distinguishing SPN serotypes.
  • the mode of administration is not particularly limited and may be based on the needs of an individual patient.
  • the disclosed BsAbs are administered via injection, but other modes of administration may be applicable.
  • the BsAbs may be formulated for intravenous, intraperitoneal, intra-arterial, intramuscular, or subcutaneous administration.
  • the dosage regimens are likewise not particularly limited. Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response like a decrease in CFU count or reduction in symptoms of infection). For example, in some embodiments, a single bolus of BsAb may be administered, while in some embodiments, several divided doses may be administered over time. In some embodiments, the dose may be proportionally reduced or increased as indicated by the situation. For example, in some embodiments the disclosed antibodies or functional fragments may be administered once, twice, three, four, five, six, or seven times a week by subcutaneous or intravenous injection. In some embodiments, the disclosed antibodies or functional fragments may be administered once, twice, three, or four times a month by subcutaneous injection.
  • the disclosed antibodies or functional fragments may be administered once a day, once every other day, once every week, once every other week, once every three weeks, once every four weeks, once every other month, once every three months, once every four months, once every five months, or once every six months.
  • Exemplary doses can vary according to the size and health of the individual being treated, as well as the condition being treated.
  • the disclosed antibodies or functional fragments may be administered in a dose of 0.1-150 mg/kg, or any range in between.
  • an applicable dose range may be 1-100 mg/kg, 5-50 mg/kg, 10-25 mg/kg, or any ranges therein.
  • the disclosed antibodies and functional fragments may be administered in a dose of about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, or about 150 mg/kg.
  • Particular treatment regimens may be evaluated according to whether they will improve a given patient's outcome, meaning it will reduce the risk of recurrence of infection or increase the likelihood of survival of the patient with a SPN infection.
  • beneficial or desired clinical results include, but are not limited to, one or more of the following: decreasing one or more symptoms resulting from the infection, increasing the quality of life of those suffering from an infection, decreasing the dose of other medications required to treat the infection, delaying the progression of the infection, reducing the bacterial load (i.e., CFU count) of a patient with an infection and/or curing the patient of the SPN infection.
  • the subject of the methods is generally a patient with a SPN infection
  • the age and health of the patient is not limited.
  • the disclosed methods are useful for treating immunocompromised patients or patients with allergies to antibiotic.
  • the patient may be a pediatric patient, while in other embodiments, the subject may be an adult patient or an elderly patient (i.e. a patient that is 70 years of age or older).
  • the disclosed anti-SPN BsAbs can be incorporated into any antibody- based diagnostic known in the art in order to identify the specific serotype or serotypes present in a given patient's infection.
  • the disclosed anti-SPN BsAbs may be affixed to a bead, a dipstick, a chip, or a plate that can be contacted with a sample from an individual suspected of having a SPN infection.
  • Further explanation of exemplary diagnostics can be found in U.S. Patent No. 9,279,815, which is hereby incorporated by reference.
  • the diagnostic may comprise a lateral-flow test or kit.
  • Lateral-flow tests are simple devices intended to detect the presence (or absence) of a specific serotype in sample (e.g., blood, plasma, saliva, urine, etc.) without the need for specialized and costly equipment.
  • the test may comprise a at least one or a series of capillary beds, such as pieces of porous paper, microstructured polymer, or sintered polymer that are capable of transporting fluid.
  • the disclosed anti-SPN BsAbs can be immobilized on these capillary beds such that they will bind the SPN serotype for which they possess specificity, thus indicating that a given serotype is present in the sample.
  • the lateral flow test may be a competitive test, while in some embodiments, the lateral flow test may be a sandwich assay test.
  • the diagnostic may be a point-of-care (POC) diagnostic, while in some embodiments, the diagnostic may require operation or interpretation by a specialty laboratory.
  • POC point-of-care
  • the disclosed devices and kits may be used in methods of diagnosing an individual as having SPN or, more specifically, a given serotype of SPN.
  • mice receiving 10 mg/kg mAb 3 and BsAb 19 A/3 had reduced bacterial burden in their lungs 24 hr post-infection (Figure 3A) and improved survival rates (Figure 3B).
  • Success will be measured as BsAb-mediated 3-log kill, comparable to untreated SPN-infected mice, and that the CFU reduction is similar to or better than that of antibiotic- treated SPN-infected mice. Success will also be measured as prolonged survival of the BsAb treated mice compared to untreated mice. Survival rates of BsAb treated mice will be compared to that of mAb treated and antibiotic treated mice.
  • the anti-19A/3 BsAb (p4A05L/p6E03K-bi), which has good OP A activity for 19A and 3 and in vivo activity against serotype 3 infection, may not prove efficacious in vivo against serotype 19A infection.
  • the treatment plan can be modified to include repeated Ab dosing or another of the disclosed 19A-specific variable regions can be substituted in.
  • the disclosed Ab library allows for the potential development of at least one additional 19A/3 BsAb and 3 22F/6A BsAbs.
  • the susceptibility of the SPN strains to antibiotics will be determined.
  • the MIC for Levofloxacin and Linezolid against SPN 19A will be determined using the broth microdilution method described by the Clinical and Laboratory Standards Institute. Resistance will be defined as MIC values > than the breakpoints. If the 19A SPN strain is resistant to both antibiotics, a different strain of 19A serotype will be obtained (readily available from JMI) and tested for susceptibility.
  • mice will be challenged with SPN 19A at the Appropriate Challenge Dose (ACD) intranasally or intravenously (determined previously).
  • ACD Appropriate Challenge Dose
  • BsAb 19 A/3 and mAb 19A will be given intravenously at 10 mg/kg (dose selected based on previous mAb and BsAb studies).
  • Controls will be SPN infection with no treatment, treatment with antibiotics, and treatment with parental mAb.
  • Lung tissue will be harvested at 24 hr after SPN challenge and bacterial burden per gram of lung tissue will be determined. Success will be Ab-mediated reduction in bacterial load by 3 loglO CFU ⁇ see Table 5 for example of a BsAb in vivo study outline).
  • the 3 logio CFU reduction metric is based on a similar reduction seen with antibiotics resulting in optimal bactericidal action.
  • mice CBA/J
  • 19A SPN Appropriate Challenge Dose intranasally.
  • control will be treated with Levaquin or Linezolid (antibiotic dose will be selected based on literature and MIC results).
  • Mice will be treated with lOmg/kg of the parental mAb 19A or with a dose range of BsAb 19 A/3. After 10 days, percent survival will be calculated. Success is BsAb-mediated survival comparable to antibiotics and mAb.
  • Example 3 Testing an Anti-6A/22F BsAb in Mice [0107] This study will be performed similar to those described in Examples 1 and 2, but utilizing SPN 6A and SPN 22F.
  • Success will be measured as BsAb-mediated 3-log kill, comparable to untreated SPN-infected mice, and that the CFU reduction is similar to that of antibiotic-treated SPN- infected mice. Success will also be measured as prolonged survival of the BsAb treated mice compared to untreated mice. Survival rates of BsAb treated mice will be compared to that of mAb treated and antibiotic treated mice.
  • SPN serotypes 6A and 3 can cause pulmonary infection in ICR mice and SPN serotype 19A in CBA/J mice; SPN serotype 22F is not able to cause a pulmonary infection in ICR mice (data not shown).
  • SPN serotype 19A can cause a pulmonary infection in CBA/J mice, it is likely that SPN serotype 22F will also able to cause a pulmonary infection in CBA/J mice.
  • it may be determined that the SPN serotype 22F is not able to cause a pulmonary infection, in which case the study will be performed in an intravenous bacteremia murine model in a 10-day mortality study.
  • mice will be challenged with SPN 22F at approximately 3, 4, 5, or 6 loglO CFU via intranasal administration. Success Metric: Finding a challenge dose resulting in 5-7 loglO CFUs in the lungs after 24 h post-infection will represent the ACD. The effectiveness of Levofloxacin and Linezolid against SPN serotype 6A and 22F will also be determined. This will be done as described in Example 2 above, but for SPN serotype 6A and SPN serotype 22F.
  • Example 2 if the anti-6A/22F BsAb is ineffective, the treatment plan can be modified to include additional or alternative doses, or the BsAb can be redesigned to include alternative 6A- or 22F-specific variable regions. [0113] These exemplary experimental methods could be used to test any other bispecific antibody disclosed herein with any combination of serotype specificities. The use of further antibodies alone and in combination with standard-of-care antibiotics is planned for subsequent studies.

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Abstract

The present disclosure relates generally to bispecific antibodies and functional fragments thereof that bind to at least two serotypes of streptococcus pneumonia. In particular, the disclosed bispecific antibodies and fragments can bind to serotypes 19A, 3, 22F, 6A, 6B, 6C, 19F, 15A, 23 A, 11 A, 35B, 12F, 22A and 7F of streptococcus pneumonia, and comprise novel complementarity determining regions (CDRs) also disclosed herein. The present disclosure further relates to treating streptococcus pneumonia infection by administering the disclosed bispecific antibodies to an individual with such an infection.

Description

BISPECIFIC ANTIBODIES FOR THE TREATMENT OF STREPTOCOCCUS
PNEUMONIA
Field of Invention
[0001] The present disclosure relates generally to antibodies, in particular bispecific antibodies, and functional fragments thereof that bind to Streptococcus pneumoniae. The disclosed antibodies comprise novel complementarity determining regions (CDRs), which are combined into bispecific constructs that are capable of binding at least two serotypes of streptococcus pneumonia. Finally, the present disclosure also relates to methods of treating streptococcus pneumonia by administering the disclosed antibodies to a subject in need thereof.
Background
[0002] Community Acquired Pneumonia (CAP) affects approximately 5 million patients, and is associated with over 1 million hospitalizations and 68,000 deaths annually in the United States. Streptococcus pneumoniae (SPN) causes up to 50% of these pneumonia cases in the U.S. and throughout the developed world. Pneumonia is the second leading cause of hospitalization in the U.S. and costs the economy $40 billion per year, of which $9.9 billion are direct hospitalization costs. The greatest unmet clinical need among SPN infections is to improve survival among patients with severe pneumococcal CAP (CAPP) that results in hospitalization. The mortality rate among patients who are hospitalized in the ICU with severe pneumonia (Pneumonia Severity Index grade IV- V) is 20-40% and has remained relatively unchanged since the introduction of antibiotics. Development of organism- specific immunotherapeutics administered as a supplement to current treatments is often listed as one needed new approach to address poor patient outcomes that are due, in part, to the growing crisis of antibiotic drug resistance.
[0003] The present disclosure satisfies this pressing need.
Summary
[0004] The present disclosure provides isolated bispecific antibodies, in particular human bispecific antibodies that bind to Streptococcus pneumoniae (SPN) and exhibit desirable therapeutic properties. These properties include high affinity binding to various SPN serotypes, and each of the disclosed bispecific antibodies can bind at least two SPN serotypes. [0005] In one aspect, the present disclosure provides bispecific anti -streptococcus pneumoniae antibodies or functional fragments thereof that specifically bind to at least two serotypes selected from a group consisting of serotypes 19A, 3, 22F, 6A, 6B, 6C, 19F, 15A, 23 A, 11 A, 35B, 12F, 22A and 7F.
[0006] In another aspect, the present disclosure provides bispecific antibodies or functional fragments thereof that specifically bind to at least two serotypes of streptococcus pneumoniae, wherein the bispecific antibodies or functional fragments thereof comprise: a first heavy chain variable region comprising: a complementarity determining region (CDR) HI selected from the group consisting of SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, and 43; a CDRH2 selected from the group consisting of SEQ ID NOs: 2, 8, 14, 20, 26, 32, 38, and 44; and a CDRH3 selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, and 45;a second heavy chain variable region comprising: a complementarity determining region (CDR) HI selected from the group consisting of SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, and 43; a CDRH2 selected from the group consisting of SEQ ID NOs: 2, 8, 14, 20, 26, 32, 38, and 44; and a CDRH3 selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, and 45; a first light chain variable region comprising: a CDRL1 selected from the group consisting of SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, and 46; a CDRL2 selected from the group consisting of SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, and 47; and a CDRL3 selected from the group consisting of SEQ ID NOs: 6, 12, 18, 24, 30, 36, 42, and 48; and a second light chain variable region comprising: a CDRL1 selected from the group consisting of SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, and 46; a CDRL2 selected from the group consisting of SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, and 47; and a CDRL3 selected from the group consisting of SEQ ID NOs: 6, 12, 18, 24, 30, 36, 42, and 48; wherein the first and second heavy chain do not comprise the same CDR sequences, and wherein the first and second light chain do not comprise the same CDR sequences.
[0007] In another aspect, the present disclosure provides bispecific antibodies or functional fragments thereof that specifically bind to at least two serotypes of streptococcus pneumoniae, wherein the bispecific antibody or functional fragment thereof comprises two different heavy chain variable regions and two different light chain variable regions, wherein the two different heavy chain variable regions and the two different light chain variable regions are encoded by four different nucleic acid sequences chosen from Table 4. [0008] In some embodiments, the bispecific antibody or functional fragment thereof can specifically bind to at least two of serotypes 19A, 3, 22F, 6A, 6B, or 6C. In some embodiments, the antibody or functional fragment thereof can specifically bind to serotypes 19A and 3, while in some embodiments, the antibody or functional fragment thereof can specifically bind to serotypes 6A and 22F. In some embodiments, a binding domain that can bind to serotype 6A can be cross-reactive with serotypes 6B and/or 6C.
[0009] In some embodiments, the bispecific antibody or functional fragment thereof has a KD for the serotypes that it binds that is at least at least l .OxlO"9 M, and in some embodiments, the antibody or functional fragment thereof has an OPA titer of greater than 8.
[0010] In some embodiments, the bispecific antibody or functional fragment thereof may comprise first and second heavy chain variable regions comprising an amino acid sequence comprising SEQ ID NOs: 49, 51, 53, 55, 57, 59, 61, or 63, and first and second light chain variable regions comprising an amino acid sequence comprising SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, or 64, wherein the first and second heavy chains do not comprise the same sequences and the first and second light chains do not comprise the same sequences.
[0011] In some embodiments, the first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 3; the second heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 25, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 26, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 27; the first light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 4, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 6; and the second light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 28, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 29, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 30. For example, in some embodiments, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 49, the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 57, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 50, and the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 58.
[0012] In some embodiments, the first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 7, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 9; the second heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 31, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 33; the first light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 10, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 12; and the second light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 34, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 36. For example, in some embodiments, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 51, the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 59, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 52, and the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 60.
[0013] In some embodiments, the first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 7, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 9; the second heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 37, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 39; the first light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 10, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 12; and the second light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 40, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 42. For example, in some embodiments, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 51, the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 61, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 52, and the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 62.
[0014] In some embodiments, the first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 13, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 14, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 15; the second heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 43, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 45; the first light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 16, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 17, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 18; and the second light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 46, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 48. For example, in some embodiments, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 53, the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 63, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 54, and the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 64.
[0015] In some embodiments, the first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 19, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 21; the second heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 43, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 45; the first light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 22, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 23, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 24; and the second light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 46, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 48. For example, in some embodiments, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 55, the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 63, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 56, and the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 64.
[0016] In another aspect, the present disclosure provides methods of treating a Streptococcus pneumoniae infection in an individual comprising, administering to an individual with a S. pneumoniae infection, (i) a therapeutically effective amount of a bispecific antibody or a functional fragment thereof that specifically binds to at least two serotypes selected from a group consisting of serotypes 19A, 3, 22F, 6A, 6B, 6C, 19F, 15A, 23 A, 11 A, 35B, 12F, 22A and 7F; and (ii) an antibiotic.
[0017] In another aspect, the present disclosure provides methods of treating an antibiotic- resistant Streptococcus pneumoniae infection in an individual comprising, administering to an individual with an antibiotic-resistant S. pneumoniae infection, a therapeutically effective amount of a bispecific antibody or a functional fragment thereof that specifically binds to at least two serotypes selected from a group consisting of serotypes 19 A, 3, 22F, 6 A, 6B, 6C, 19F, 15A, 23 A, 11 A, 35B, 12F, 22A and 7F.
[0018] In another aspect, the present disclosure provides methods of treating a Streptococcus pneumoniae infection in an individual comprising, administering to an individual with a S. pneumoniae infection, a therapeutically effective amount of a bispecific antibody or a functional fragment thereof that specifically binds to at least two serotypes selected from a group consisting of serotypes 19A, 3, 22F, 6A, 6B, and 6C, wherein the bispecific antibody or functional fragment thereof has a KD for the serotypes that it binds that is at least at least l .OxlO"9 M.
[0019] In some embodiments, the bispecific antibody or functional fragment thereof utilized in the disclosed methods can specifically bind to at least two of serotypes 19A, 3, 22F, 6A, 6B, or 6C.
[0020] In some embodiments, the bispecific antibody or functional fragment thereof utilized in the disclosed methods may comprise first and second heavy chain variable regions comprising an amino acid sequence comprising SEQ ID NOs: 49, 51, 53, 55, 57, 59, 61, or 63, and first and second light chain variable regions comprising an amino acid sequence comprising SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, or 64, wherein the first and second heavy chains do not comprise the same sequences and the first and second light chains do not comprise the same sequences.
[0021] In some embodiments, the bispecific antibody or functional fragment thereof utilized in the disclosed methods may comprise a first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 3; a second heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 25, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 26, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 27; a first light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 4, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 6; and a second light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 28, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 29, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 30. For example, in some embodiments, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 49, the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 57, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 50, and the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 58.
[0022] In some embodiments, the bispecific antibody or functional fragment thereof utilized in the disclosed methods may comprise a first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 7, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 9; a second heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 31, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 33; a first light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 10, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 12; and a second light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 34, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 36. For example, in some embodiments, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 51, the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 59, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 52, and the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 60.
[0023] In some embodiments, the bispecific antibody or functional fragment thereof utilized in the disclosed methods may comprise a first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 7, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 9; a second heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 37, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 39; a first light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 10, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 12; and a second light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 40, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 42. For example, in some embodiments, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 51, the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 61, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 52, and the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 62.
[0024] In some embodiments, the bispecific antibody or functional fragment thereof utilized in the disclosed methods may comprise a first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 13, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 14, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 15; a second heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 43, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 45; a first light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 16, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 17, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 18; and a second light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 46, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 48. For example, in some embodiments, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 53, the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 63, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 54, and the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 64.
[0025] In some embodiments, the bispecific antibody or functional fragment thereof utilized in the disclosed methods may comprise a first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 19, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 21; a second heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 43, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 45; a first light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 22, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 23, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 24; and a second light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 46, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 48. For example, in some embodiments, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 55, the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 63, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 56, and the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 64.
[0026] In some embodiments, the antibiotic used to treat a patient can be a β-lactam antibiotic, a tetracycline, a macrolide, a quinolone, or a fluoroquinolone, or in some embodiments, the antibiotic can be Levofloxacin or Linezolid.
[0027] In some embodiments, it was determined prior to administration of the bispecific antibody or functional fragment thereof that the individual's S. pneumoniae infection comprises at least one of the serotypes specifically bound by the bispecific antibody or functional fragment thereof.
[0028] In some embodiments when the disclosed bispecific antibodies are administered in conjunction with an antibiotic, the bispecific antibody or functional fragment thereof is administered before, after, or concurrently with the antibiotic.
[0029] In some embodiments, the individual may have previously received an antibiotic to treat the S. pneumoniae infection, and in some embodiments, the infection may be antibiotic-resistant.
[0030] In some embodiments, the antibody or functional fragment thereof used in the disclosed methods may have an OPA titer of greater than 8.
[0031] The foregoing general description and following detailed description are exemplary and explanatory and are intended to provide further explanation of the disclosure as claimed. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following brief description of the drawings and detailed description of the disclosure.
Brief Description of the Drawings
[0032] Figure 1 shows the prevalence of S. pneumoniae serotypes in North America in 2009.
[0033] Figure 2 shows two parent antibodies (F405Lp4A05L and K409Rp6E03K) and their bispecific (p4A05L/p6E03K-bi) tested on ELISAs to demonstrate bispecificity with OPA titers and specificities against serotype 19A and 3.
[0034] Figure 3 shows in vivo efficacy of BsAb serotype 19 A/3 is comparable or better than Linezolid. Panel A shows lung CFU counts taken 24 hours post intranasal infection of mice with SPN serotype 3. Panel B shows a 10 day mortality study in the infected mice.
Detailed Description
[0035] The compositions and methods of the present disclosure employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds 1987, and periodic updates); PCR: The Polymerase Chain Reaction, (Mullis et al., ed., 1994); A Practical Guide to Molecular Cloning (Perbal Bernard V., 1988); Phage Display: A Laboratory Manual (Barbas et al., 2001).
I. Definitions
[0036] It is to be understood that methods are not limited to the particular embodiments described, and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. The scope of the present technology will be limited only by the appended claims.
[0037] As used herein, certain terms may have the following defined meanings. As used in the specification and claims, the singular form "a," "an" and "the" include singular and plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a single cell as well as a plurality of cells, including mixtures thereof.
[0038] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but not excluding others. "Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the composition or method. "Consisting of shall mean excluding more than trace elements of other ingredients for claimed compositions and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure. Accordingly, it is intended that the methods and compositions can include additional steps and components (comprising) or alternatively including steps and compositions of no significance (consisting essentially of) or alternatively, intending only the stated method steps or compositions (consisting of).
[0039] As used herein, "about" means plus or minus 10%.
[0040] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. [0041] As used herein, the terms "individual", "patient", or "subject" can be an individual organism, a vertebrate, a mammal (e.g., a bovine, a canine, a feline, or an equine), or a human. In a preferred embodiment, the individual, patient, or subject is a human.
[0042] As used herein, the term an "isolated antibody" is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to streptococcus pneumonia is substantially free of antibodies that do not bind to streptococcus pneumonia). An isolated antibody that specifically binds to a serotype of streptococcus pneumonia may, however, have cross- reactivity to other serotypes of SPN. However, the disclosed isolated antibodies preferably always bind to at least one specific serotype of SPN with high affinity. In addition, an isolated antibody is typically substantially free of other cellular material and/or chemicals.
[0043] As used herein, the term "humanized antibody" refers to an antibody that comprises the CDRs of antibodies derived from mammals other than human, and the framework (FR) region and the constant region of a human antibody. A humanized antibody is useful as an effective component in a therapeutic agent according to the present disclosure since antigenicity of the humanized antibody in human body is lowered.
[0044] As used herein, the term "recombinant human antibody" includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, including but not limited to (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody (e.g., from a transfectoma), (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline and/or non-germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo. [0045] As used herein, the term "glycosylation pattern" is defined as the pattern of carbohydrate units that are covalently attached to a protein, more specifically to an immunoglobulin protein.
[0046] As used herein, the phrases "therapeutically effective amount" and "therapeutic level" mean that drug dosage or plasma concentration in a subject, respectively, that provides the specific pharmacological effect for which the drug is administered in a subject in need of such treatment, i.e. to reduce, ameliorate, or eliminate the symptoms or effects of streptococcus pneumonia. It is emphasized that a therapeutically effective amount or therapeutic amount of an antibody will not always be effective in treating the conditions/diseases described herein, even though such dosage is deemed to be a therapeutically effective amount by those of skill in the art. The therapeutically effective amount may vary based on the route of administration and dosage form, the age and weight of the subject, and/or the subject's condition, including the serotype and stage of the streptococcus pneumonia infection, among other factors.
[0047] The terms "treatment" or "treating" as used herein with reference to streptococcus pneumonia refer to reducing, ameliorating or eliminating one or more symptoms or effects of streptococcus pneumonia, including but not limited to, bacterial load {i.e., CFU count) within the subject, difficulty breathing, chest pains, fever, fatigue, chills, bloody mucus, increased heart rate, nausea, diarrhea, coughing and/or sneezing, etc.
[0048] The term "functional fragment," as used herein, refers to one or more fragments of a bispecific anti-SPN antibody, as disclosed herein, that retains the ability to bind to at least two serotypes of SPN. Examples of binding fragments include (i) Fab fragments (monovalent fragments consisting of the VL, VH, CL and CHI domains); (ii) F(ab')2 fragments (bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region); (iii) Fd fragments (comprising the VH and CHI domains); (iv) Fv fragments (comprising the VL and VH domains of a single arm of an antibody), (v) dAb fragments (comprising a VH domain); and (vi) isolated complementarity determining regions (CDR), e.g., VH CDR3. Other examples include single chain Fv (scFv) constructs. See e.g., Bird et al., Science, 242:423-26 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-83 (1988). Other examples include PD-L1 -binding-domain immunoglobulin fusion proteins comprising (i) a PD-L1 -binding domain polypeptide (such as a heavy chain variable region, a light chain variable region, or a heavy chain variable region fused to a light chain variable region via a linker peptide) fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region.
II. Community Acquired Pneumonia (CAP) and Streptococcus pneumoniae (SPN)
[0049] Streptococcus pneumoniae (SPN), an often drug-resistant pathogen, is the leading cause of community-acquired pneumonia, and a key contributor to morbidity/mortality worldwide. In the U.S. alone, community-acquired pneumococcal pneumonia infects more than 5 million/yr (despite the availability of several vaccines), causes 1.4 million hospital visits, 1.1 million hospitalizations, and even with modern antibacterial intervention, results in 68,000 deaths annually. Hospitalized pneumonia patients stay an average of 5.6 days. Pediatric need is even greater, as pneumonia causes more than 156 million infections and kills an estimated 2 million children under 5 every year— more than AIDS, malaria and tuberculosis combined. Of these pneumonia-related deaths, over 50% are caused by SPN. Nearly 40% of patients with severe CAPP, and up to 80% of those with pneumococcal bacteremia, will die. The greatest unmet clinical need is to improve survival against severe pneumococcal infections. Standard of care is primarily antibiotics and supportive care. However, the morbidity/mortality of ICU patients with CAPP unfortunately has remained unchanged since the introduction of antibiotics due to increased antibiotic resistance. Combined with an increased risk of antibiotic-associated disease, low vaccination rates, and poor vaccine efficacy, there is a clear need to significantly improve poor patient outcomes related to this disease.
[0050] Increasingly, SPN isolates are resistant to many antibiotics and are frequently resistant to not just one, but multiple drugs. Results from national studies show that -30% of pneumococci are resistant to penicillin, and -25% of pneumococcal strains are resistant to macrolides. Although fluoroquinolones are effective against SPN, they are typically reserved for more difficult-to-treat infections due to their side-effect profiles. Also, antibiotic treatment can alter gut flora, allowing for more pathogenic organisms such as Clostridium difficile to flourish. Clostridium difficile infection (CDI) is common in CAPP patients, and antibiotic use, particularly Levaquin, for CAPP contributes to CDI. Patients who contract CDI in the hospital must remain in the hospital 3.6 days longer (on average), adding hospital costs that average $3,669. [0051] Two types of vaccines against SPN are available for subjects under 5 or over 50: pneumococcal polysaccharide vaccine (PPV) and pneumococcal conjugate vaccine (PCV). Both are designed to generate antibodies against capsular polysaccharide (CPS). Unfortunately, vaccination rates of adult patients are under 60%, leaving approximately 70 million U.S. adults unvaccinated. In addition to poor vaccination rates, non-conjugate vaccine (PPV) efficacy rates are low. In a retrospective cohort analysis study based on the CDC pneumococcal surveillance system, 23-valent PNEUMOVAX® (Merck), demonstrated only a 57% overall protective effectiveness against invasive infections caused by the serotypes in the vaccine in persons >6 years of age.
III. Anti-SPN Bispecific Antibodies (BsAbs)
[0052] Although antibody (Ab) therapies are generally more expensive compared to antibiotics, their ability to expedite microbial clearance and disease resolution reduces total days in the hospital and total cost of CAP. Indeed, a 1.7-day reduction in hospital stays for CAP resulted in an estimated cost saving of $1,700 per patient. Thus, a non-antibiotic treatment for CAP could address multiple issues: Growing antibiotic resistance, risk of potentially fatal CDI, and significant patient morbidity/mortality.
[0053] Accordingly, disclosed herein are bispecific antibodies that specifically bind to at least two serotypes of SPN. Polysaccharide type-specific Abs mediating opsonin-dependent phagocytosis are considered the main mechanism of host protection against Streptococcus pneumoniae, and levels of opsonic Abs correlate with protection in preclinical models. Abs to the non-opsonogenic targets of pneumococcal surface protein A (PspA), pneumolysin (Ply), and adhesins have had mixed results as therapeutic interventions and have not shown efficacy in preventing/treating pneumococcal infection in several cases. Thus, anti- polysaccharide Abs are the ideal therapeutic to clear SPN infection, but there are over 90 known SPN serotypes, and developing so many monospecific antibodies would be expensive and therapeutically challenging in an acute-care setting.
[0054] The present disclosure provides a therapeutic that contains multi-specific antibodies targeting the most predominant, disease-causing serotypes would overcome the challenges of serotype-specific treatment. The disclosed bispecific antibodies (BsAbs) can opsonize at least two different SPN serotypes, which provides at least three beneficial clinical results: 1) an increased number of patients will be treatable with the BsAbs compared to monospecific Abs; 2) the need for Ab mixtures of multiple, individual Abs will be obviated; and 3) the total protein load administered to a given patient will be reduced, thereby improving the safety of the Ab therapy.
[0055] Typically, an antibody consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two copies of a light (L) chain polypeptide. Typically, each heavy chain contains one N-terminal variable (VH) region and three C-terminal constant (CHI, CH2 and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen binding site of an antibody. Bispecific antibodies typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen (i.e., a different SPN serotype). Each antigen-binding domain of a bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In the context of a bispecific antigen- binding molecule comprising a first and a second antigen-binding domain (e.g., a bispecific antibody), each antigen binding domain comprises at least one CDR that alone, or in combination with one or more additional CDRs and/or FRs, specifically binds to a particular antigen (i.e., serotype).
[0056] The first antigen-binding domain and the second antigen-binding domain may be directly or indirectly connected to one another to form a bispecific antigen-binding molecule (i.e., bispecific ScFv) further bound to an Fc domain. Alternatively, the first antigen-binding domain and the second antigen-binding domain may each be connected to a separate Fc domain. Bispecific antigen-binding molecules of the present disclosure may comprise two Fc domains that are each individually part of a separate antibody heavy chain. The first and second Fc domains may be of the same sequence, or the Fc domains may have a mutation in the CH3 domain intended for the facilitation or ease of purification of heterodimeric (i.e., bispecific) molecules.
[0057] The disclosed bispecific antibodies may be produced by any means known in the art for producing bispecific antibodies, so long as the resulting bispecific antibody retains the functional characteristic of being able to specifically bind at least two serotypes of SPN. In some embodiments, the disclosed BsAbs will comprise the CDR sequences or variable region sequences disclosed herein. In some embodiments, the BsAbs may be created using the methods described in Labrijin et al., Proc. Natl. Acad. Sci. USA, 110(13):5145-50 (2013). Briefly, the two parental Abs— each containing single matched point mutations in the CH3 domains— are separately expressed and then mixed under reducing conditions in vitro. This separates the Abs into HL half-molecules, followed by reassembly, to form BsAbs, and is compatible with large-scale manufacturing of BsAbs. However, this is simply one exemplary method for making a BsAb. Those of skill in the art will be aware that other methods of producing BsAbs are available, and the present disclosure is not intended to be limited solely to the methods of making and type of bi specific antibodies disclosed herein.
[0058] Other bispecific antibody formats or technologies may be used to make the bispecific antigen-binding molecules of the present disclosure. For example, an antibody or fragment thereof having a first antigen binding specificity can be functionally linked {e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity to produce a bispecific antigen-binding molecule. Specific exemplary bispecific formats that can be used in the context of the present invention include, without limitation, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chain {e.g., common light chain with knobs-into- holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgGl/IgG2, dual acting Fab (DAF)-IgG, and Mab2 bispecific formats {see, e.g., Klein et al. 2012, mAbs 4:6, 1-1 1 , and references cited therein, for a review of the foregoing formats).
[0059] The disclosed bispecific antibodies can be made from or incorporate the CDRs or variable regions from polyclonal, monoclonal, chimeric, human, partially or fully humanized, and/or recombinant antibodies. Thus, the "parent" antibodies for the disclosed BsAbs are not particularly limited; however, they are preferably fully human. In some embodiments, the parent antibody can be a polyclonal antibody. In some embodiments, the parent antibody can be a monoclonal. In some embodiments, the parent antibody can be a human antibody.
[0060] Standard means of making antibodies are known in the art. For instance, polyclonal antibodies may be obtained by methods known in the art, such as by immunizing a selected animal with a SPN serotype-specific antigen, collecting serum from the animal, and isolating and/or purifying antibodies from the serum. Monoclonal antibodies (mAbs) may be obtained by methods known in the art, for example, by fusing antibody-producing cells with immortalized cells to obtain a hybridoma, and/or by generating mAbs from mRNA extracted from bone marrow and spleen cells of immunized animals using combinatorial antibody library technology. Recombinant antibodies may be obtained by methods known in the art, for example, using phage or yeast display technologies and/or expressing or co- expressing antibody polypeptides. Other techniques for making antibodies are known in the art, and can be used to obtain antibodies used in the methods described herein.
[0061] In some embodiments, the parent anti-SPN antibodies may be fully human antibodies isolated from human subjects that were vaccinated with a pneumococcal vaccine (e.g., P EUMOVAX®23 or PREVINAR®13) containing a large number of SPN serotypes, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. The serotype-specific antibodies can be isolated from these subject and cloned. This platform leverages the natural "memory" response to isolate a broad range of high-affinity Abs to the target pathogen following vaccination or infection and provides cross-sectional characterization of the anti-polysaccharide response. The serotype- specificity of these antibodies can then be characterized using routine methods known in the art (see, e.g., U.S. Patent No. 9,279,815, which is hereby incorporated by reference).
A. SPN Serotypes
[0062] Pneumococcal vaccine serotypes represent nearly 75% of CAPP infections in the U.S. and worldwide, but a handful of serotypes predominate (Figure 1). In fact, just 10 serotypes account for over half of pneumococcal infections worldwide. These serotypes include 19A, 3, 22F, 6A, 6B, 6C, 19F, 15A, 23 A, 11 A, 35B, 12F, 22A and 7F. The present inventors have developed fully human Abs that specifically bind at least one of each of the 24 SPN vaccine serotypes, and demonstrated that these full-length, fully human Abs have high binding affinity and potent opsonophagocytotic assay (OP A) activity (>8 titers) against SPN serotypes that account for almost three-quarters of invasive pneumococcal disease in hospitalized patients. While elderly with dampened immune responses and immunocompromised individuals account for a significant fraction of CAPP -infected individuals, less than 5% were found to be leukopenic. Thus, the disclosed BsAbs, which incorporate the variable sequences of the disclosed fully human Abs, along with patient neutrophils, will be successful in clearing the SPN infection via opsonophagocytosis.
[0063] Thus, rather than treating a patient with a therapeutic cocktail of several different individual, monospecific Abs, the disclosed BsAbs are capable of providing a broad spectrum of protection without the need for multiple, distinct Abs. BsAbs are emerging as a novel approach for immunotherapies. Utilizing immuno-engineering techniques, it is possible to combine 2 antigen-recognizing elements into a single construct that simultaneously binds to 2 distinct targets (see, e.g., Spasevska et al., J. Blood Disord. Transfus., 6:243. doi: 10.4172/2155-9864.1000243 (2015)). Disclosed herein are high- affinity, high OPA titer human monoclonal antibodies (hmAbs) that can be combined in various ways to produce bispecific antibodies capable of binding at least two serotypes of SPN. Moreover, the OPA activity for these bispecific antibodies can be significantly higher than those of the monospecific antibodies on which they are based. See Table 1 below.
Binding affinity and OPA titers for selected S. pneumoniae monoclonal antibodies
Figure imgf000020_0001
[0064] In some embodiments, the disclosed anti-SPN BsAbs can bind at least two serotypes of S. pneumoniae. In some embodiments, the disclosed anti-SPN BsAbs may possess cross- reactivity, allowing them to bind to more than two serotypes. For examples, in some embodiments, the disclosed anti-SPN BsAbs may bind two, three, four, five, or more serotypes of S. pneumoniae. The cross-reactivity of the disclosed antibodies can significantly increase the therapeutic coverage of the disclosed anti-SPN BsAbs. For instance, an anti-6A mAb may bind serotypes 6A/B/C, or an anti-19A mAb may bind both 19A and 19F
[0065] In some embodiments, the disclosed BsAbs are specific for at least two of serotypes 19A, 3, 22F, or 6A. For instance, some BsAbs may be specific for serotypes 19A and 3; 22F and 6A; 19A and 22F, 19A and 6A; 3 and 22F; or 3 and 6A. These serotypes are the four most prevalent among hospitalized patients over 65 in the North America and often possess penicillin and erythromycin resistance. A first BsAb that specifically binds to serotypes 19A and 3 would address 25-30% of SPN infections in the U.S. and worldwide, and a second BsAb that specifically binds to serotypes 22F and 6 A would address another 15-20% of SPN infections. Further adding additional BsAbs to other serotypes, particularly those with known antibiotic resistance, would substantially increase therapeutic coverage without having to administer numerous monospecific antibodies to an individual.
B. Anti-SPN BsAb Sequences
[0066] Disclosed herein are various anti-SPN antibody variable regions and complementarity determining regions (CDRs). The disclosed anti-SPN BsAbs may comprise any combination of the disclosed variable regions or CDRs so long as the BsAb is able to bind to at least two serotypes of S. pneumonia. Accordingly, in some embodiments, the disclosed BsAb can comprise a combination of an anti-19A, anti-3, anti-22F, anti-6A, anti-6B, anti-6C, anti-19F, anti-15A, anti-23A, anti-l lA, anti-35B, anti-12F, anti-22A or anti-7F variable regions or CDRs.
[0067] For the purposes of this disclosure, the BsAbs may be defined according to their amino acid sequence or according to the nucleic acid sequence that encodes the amino acids. Exemplary antibody sequences are shown in the following Tables 2-4.
Table 2 - Amino Acid Sequences of Complementarity Determining Regions (CDRs) of
Exemplary Serotype-Specific Abs
Figure imgf000021_0001
Figure imgf000022_0001
*Only binds to 6A/B, not C
Table 3 - Amino Acid Sequences of Variable Regions of Exemplary Serotype-Specific Abs
Figure imgf000022_0002
FGGGTKLTVL (SEQ ID NO: 50)
Sei ol> pc 22 l-
Con3p5B01H EVQLVESGGGLVQPGGSLRLSCVASGLTFSSHWMTWVRQAPGKGPEW / Heavy VANIKEDGSEKNYADSVKGRFTISRDNAENSLYLQLNSLSADDTAVYY
CASRRCSTGVCYRYSHYYMDDWGKGTTVTVSS (SEQ ID NO: 51)
Con3p5B01L QSALTQPRSVSGSPGQSVAISCTGTSGDVGGYNFVSWYQQHPGKAPKL / Light MIFDVNRRPSGVPDRFSGSKSGNTASMTISGLQHEDEADYYCCSYAGIY
TLVFGGGTKLTVL (SEQ ID NO: 52)
Conlp3C03H EVQLVESGGGLVQPGGSLRLSCAASGFAISGNYMSWVRQAPGKGLEW / Heavy VSLIYWTDDTVYADSVKGRFTISRDVSKNMVHLQMSSLRVEDTAVYYC
ARELGVFHSGGDQWLGPLDCWGQGTLVTVSS (SEQ ID NO: 53)
Conlp3C03K EIVMTQSPATLSVSPGETATLSCRASQSVNSFLAWYQQKPGQAPRLLIY / Light AASTRATGVPARFSGSGSGTEFTLTISSLQSEDFAVYYCHQYKNWPPMG
TFGPGTKVDIK (SEQ ID NO: 54)
Con2p6G04H QVQLVQSGAEVKKPGASVKLSCKTSGYSFTSNYLHWVRQAPGQGLEW / Heavy MGMVYPNDGTTTYAQKFQGRVTMTSETSTTTIYMDLSGLTSEDTAIYY
CARDDSAFDYWGQGTLVTVSS (SEQ ID NO: 55)
Con2p6G04K DIVMTQTPLSLPVTLGQPASISCISSQSLVHSDGNTYLSWLQQRPGQPPR / Light LLI YKI SNRF SGVPDRF SGS GAGTDF TLKI SRVE AED VGV Y YCMQ AS Q S T
WTLGQGTKVEIK (SEQ ID NO: 56)
Scrol> |)0
Conlp6E03H EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEW / Heavy VGKIKEDGSEKYYVDSVKGRFAISRDNAKNSLSLQMNSLRAEDTAVYY
CARGQSYPGIWGQGTMVTVSS (SEQ ID NO: 57)
Conlp6E03K EIVMTQSPATLSVSPGERATLSCRASQSVSGDLVWYQQKPGQAPRLLIY / Light GATTRASGVPARFSGSGSGTEFTLTISSLQSEDFAIYYCQQYNNWPRTFG
QGTKVEIK (SEQ ID NO: 58)
Scrolypc 6 (Λ B ( )
SLE2plD04H E VQL VES GGGL VKPGE SLRL S CAT S GVNFNI AWMTW VRQ APGKGLEW / Heavy VGRIKSKIGGGTTDYAAPVKGRFTMSIDDSKNTLYLQMNSLKTEDTAV
YYCTTVRNMADLSLNHWGQGTLVTVSS (SEQ ID NO: 59)
SLE2plD04K EIVLTQSPGTLSLSPGERATLSCRASQSVSSTYLNWYQQKPGQAPRLLIY / Light GASTRATGIPDRFSGSGSGADFTLTISRLEPEDFAVYYCQQYDDSRWTF
GQGTKVEIK (SEQ ID NO: 60)
Con3p6C02H EVQLLESGGDLVQPGGSLRLSCAASGFIFSRSDMTWVRQAPGKGLEWV / Heavy SSIRAAGVETFYADSVKGRFTISRDNFKNTLHLQMNSLRAEDTAVFYCT
RGIRDISYWGQGTLVTVSS (SEQ ID NO: 61)
Con3p6C02K DVVMTQSPLSLPVTLGQPASISCSSSQSLVYSDGNTYLNWFQQRPGQSP / Light RRLI YR VSNRD S GVPDRF S GS GS GT YFTLKI SRVE AED VGI YYC AQGAH
WPLTFGGGTKVED (SEQ ID NO: 62)
PV4p6C02H / QVQLVQSGAEVKKPGASVKVSCKASGYMFTSYTMHWVRQAPGQRLE Heavy* WMGWIGAGDGNIKYSQKFQGRATITRDTSASTAYMELSSLRSEDTAMY
YC AREGTE VGLDID YWGQGTL VT VS S (SEQ ID NO: 63)
PV4p6C02L / QSALTQPPSVSGSPGQSVTISCTGSSSDVGSYNGVSWYKQPPGTVPKLMI Light* YEVTNRPSGVPDRF SGAKSGNT ASLTISGLLPEDEAD YYC S S YTS SNTYV
FGTGTTVTVL (SEQ ID NO: 64)
^Only binds to 6A/B, not C Table 4 - Nucleic Acid Sequences Encoding Variable Regions of Exemplary Serotype-
Specific Abs
Figure imgf000024_0001
caagactcctgatttataagatttctaaccggttctctggggtcccagacagattcagtggcagtggggcagg gacagatttcacactgaaaatcagcagggtggaagctgaggatgtcggggtttattactgcatgcaagcttc acaatctacgtggacgctcggccaagggaccaaggtggagatcaaac (SEQ ID NO: 72)
Serotype 3
Conlp6E03H Gaggtgcagctggtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgca / Heavy gcctctggattcacctttagtagctattggatgagctgggtccgccaggctccagggaagggactggagtg ggtgggcaaaataaaggaagacggaagtgagaaatactatgtggactctgtgaagggccgattcgccatct ccagagacaacgccaagaactccctgtctctgcaaatgaacagcctgagagccgaggacacggccgtgt attactgtgcgagaggtcaatcatatccgggaatttggggccaagggacaatggtcaccgtctcttcag
(SEQ ID NO: 73)
Conlp6E03K Gaaatagtgatgacgcagtctccagccaccctgtctgtgtctccaggggaaagagccaccctctcctgcag / Light ggccagtcagagtgttagcggcgacttagtctggtaccagcagaaacctggccaggctcccaggctcctca tctatggtgccaccaccagggcctctggtgtcccagccaggttcagtggcagtgggtctgggacagagttc actctcaccatcagcagcctgcagtctgaagattttgcaatttattactgtcagcagtataataactggccccg gacgttcggccaagggaccaaggtggaaatcaaac (SEQ ID NO: 74)
Serot> pe 6 \ B (
SLE2plD04 gaggtgcagctggtggagtccgggggaggcttggtaaagccgggggagtcccttagactctcgtgtgcaa H / Heavy cctctggagtcaacttcaacatcgcctggatgacctgggtccgccaggctccagggaagggactggagtg ggttggccgtattaaaagcaaaattggtggtgggacaacagactatgctgcacccgtgaaaggcagattca ccatgtcaatagatgattcaaaaaataccctatatctgcaaatgaacagcctgaaaaccgaggacacagccg tgtattattgtaccacagtccgcaatatggccgacttgtcccttaatcactggggccagggaaccctggtcac cgtctcctcag (SEQ ID NO: 75)
SLE2plD04 gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagg K / Light gccagtcagagtgtcagcagcacctacttaaactggtaccagcagaagcctggccaggctcccaggctcct catctatggtgcgtccaccagggccactggcatcccagacaggttcagtggcagtgggtctggggcagact tcactctaaccatcagcagactggagcctgaagactttgcagtgtactactgtcagcaatatgatgactcacg gtggacgttcggccaagggaccaaggtggaaatcaaac (SEQ ID NO: 76)
Con3p6C02H gaggtgcagctgttggagtctgggggagacttggtacagccgggggggtccctgagactctcctgtgcag / Heavy cctctggattcatctttagcaggtcagacatgacctgggtccgccaggctccagggaaggggctggagtgg gtctcaagtattagagctgctggtgttgagacattctacgcagactccgtgaagggccggttcaccatatcca gagacaatttcaagaatacgttgcatcttcaaatgaatagcctgagagccgaggacacggccgtcttttactg cacgaggggtatccgtgacattagctactggggccagggaaccctggtcaccgtctcctcag (SEQ ID
NO: 77)
Con3p6C02K gatgttgtgatgactcagtctccactctccctgcccgtcacccttggacagccggcctccatctcctgcagctc / Light tagtcaaagcctcgtatacagtgatggaaacacctacttgaattggtttcaacagaggccaggccaatctcca aggcgcctaatttatagggtttctaaccgggactctggggtcccagacagattcagcggcagtgggtcagg cacttatttcacactgaaaatcagcagggtggaggctgaggatgttgggatttattactgcgcgcaaggtgca cactggcctctcactttcggcggagggaccaaggtggaaatcaaac (SEQ ID NO: 78)
PV4p6C02H caggtgcagctggtgcagtctggggccgaggtgaagaagcctggggcctcagtcaaggtttcctgcaagg / Heavy* cttctggatacatgttcactagctatactatgcattgggtgcgccaggcccccggacaaaggcttgagtggat gggatggatcggcgccggcgatggtaacataaaatattcacagaagttccagggcagagccaccattacc agggacacatccgcgagcaccgcgtacatggagctgagcagcctgagatctgaagacacggctatgtatt actgtgcgagagagggtacagaagtcggtttggacattgactactggggccagggaaccctggtcaccgt ctcctcag (SEQ ID NO: 79)
PV4p6C02L cagtctgccctgactcagccaccctccgtgtccgggtctcctggacagtcagtcaccatctcctgcactgga / Light* agtagcagtgacgttggaagttataacggtgtctcctggtacaagcagcccccaggcacagtccccaaact catgatttatgaggtcactaatcggccctcaggggtccctgatcgcttctctggggccaagtctggcaacacg gcctccctgaccatctctgggctcctgcctgaggacgaggctgattattactgcagctcatatacaagcagca acacttatgtcttcggaactgggaccacggtcaccgtcctag (SEQ ID NO: 80) *Only binds to 6A/B, not C
[0068] One of ordinary skill in the art will understand that certain changes can be made to the disclosed sequences without compromising the binding affinity or function of the disclosed anti-SPN BsAbs and functional fragments. According, in some embodiments, the anti-SPN BsAbs or functional fragments will share about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%), or about 99% sequence identity with the disclosed sequences. In some embodiments, the nucleic acid encoding the anti-SPN BsAbs or functional fragments thereof will share about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the disclosed sequences.
C. Anti-SPN BsAb Functional Characteristics
[0069] The disclosed bispecific antibodies and functional fragments thereof may be defined by sequence, as above, or by functional characteristics. The present disclosure provides anti-SPN high-affinity Abs that have KD values better than nanomolar range and opsonophagocytosis assays (OP A) titers greater than 8. OP A measures the ability of an Ab to kill the pneumococcus and has been used as a surrogate marker for vaccine efficacy. For Abs produced following vaccine administration, OPA titers over 8 are considered protective. The disclosed BsAbs have high-binding affinity and greater than 8 multiplexed OPA titers (MOPA-measures Ab killing of multiple distinct serotypes in one test) against specific SPN serotypes (see , e.g., Table 1 and Figure 2).
[0070] In some embodiments, the disclosed bispecific antibodies and functional fragments thereof, can have a KD of at least 3.0xl0"8, at least 2.5xl0"8, at least 2.0xl0"8, at least 1.5x10" 8, at least l .OxlO"8, at least 0.5xl0"8, at least 9.95xl0"9, at least 9.90xl0"9, at least 9.85xl0"9, at least 9.80xl0"9, at least 9.75xl0"9, at least 9.70xl0"9, at least 9.65xl0"9, at least 9.60xl0"9, at least 9.55xl0"9, at least 9.5xl0"9, at least 9.45xl0"9, at least 9.40xl0"9, at least 9.35xl0"9, at least 9.30xl0"9, at least 9.25xl0"9, at least 9.20xl0"9, at least 9.15xl0"9, at least 9.10xl0"9, at least 9.05xl0"9, at least 9.0xl0"9, at least 8.95xl0"9, at least 8.90xl0"9, at least 8.85xl0"9, at least 8.80xl0"9, at least 8.75xl0"9, at least 8.70xl0"9, at least 8.65xl0"9, at least 8.60xl0"9, at least 8 55xlO"9, at least 8.5xlO"9, at least 8.45xl0"9, at least 8.40xl0"9, at least 8 35xlO"9, at least 8 30xlO"9, at least 8.25xl0"9 at least 8.20xl0"9 at least 8.15xlO"9, at least 8 lOxlO"9, at least 8 05xlO"9, at least 8.0xl0"9, at least 7.95xl0"9, at least 7.90xl0"9, at least 7 85xlO"9, at least 7 80xl0"9, at least 7.75xl0"9 at least 7.70xl0"9 at least 7.65xl0"9, at least 7 60x10"9, at least 7 55xlO"9, at least 7.5xl0"9, at least 7.45xl0"9, at least 7.40xl0"9, at least 7 35xlO"9, at least 7 30xlO"9, at least 7.25xl0"9 at least 7.20xl0"9 at least 7.15xl0"9, at least 7 lOxlO"9, at least 7 05xlO"9, at least 7.0x10"9, at least 6.95xl0"9, at least 6.90xl0"9, at least 6 85xlO"9, at least 6 80xl0"9, at least 6.75xl0"9 at least 6.70xl0"9 at least 6.65xl0"9, at least 6 60x10"9, at least 6 55xlO"9, at least 6.5xl0"9, at least 6.45xl0"9, at least 6.40xl0"9, at least 6 35xlO"9, at least 6 30xlO"9, at least 6.25xl0"9 at least 6.20xl0"9 at least 6.15xl0"9, at least 6 lOxlO"9, at least 6 05xlO"9, at least 6.0x10"9, at least 5.95xl0"9, at least 5.90xl0"9, at least 5 85xlO"9, at least 5 80xl0"9, at least 5.75xl0"9 at least 5.70xl0"9 at least 5.65xl0"9, at least 5 60x10"9, at least 5 55xlO"9, at least 5.5xlO"9, at least 5.45xl0"9, at least 5.40xl0"9, at least 5 35xlO"9, at least 5 30xlO"9, at least 5.25xl0"9 at least 5.20xl0"9 at least 5.15xlO"9, at least 5 lOxlO"9, at least 5 05xlO"9, at least 5.0xlO"9, at least 4.95xl0"9, at least 4.90xl0"9, at least 4 85xlO"9, at least 4 80xl0"9, at least 4.75xl0"9 at least 4.70xl0"9 at least 4.65xl0"9, at least 4 60x10"9, at least 4 55xlO"9, at least 4.5xl0"9, at least 4.45xl0"9, at least 4.40xl0"9, at least 4 35xlO"9, at least 4 30xlO"9, at least 4.25xl0"9 at least 4.20xl0"9 at least 4.15xl0"9, at least 4 lOxlO"9, at least 4 05xlO"9, at least 4.0x10"9, at least 3.95xl0"9, at least 3.90xl0"9, at least 3 85xlO"9, at least 3 80xl0"9, at least 3.75xl0"9 at least 3.70xl0"9 at least 3.65xl0"9, at least 3 60x10"9, at least 3 55xlO"9, at least 3.5xlO"9, at least 3.45xl0"9, at least 3.40xl0"9, at least 3 35xlO"9, at least 3 30xlO"9, at least 3.25xl0"9 at least 3.20xl0"9 at least 3.15xlO"9, at least 3 lOxlO"9, at least 3 05xlO"9, at least 3.0xlO"9, at least 2.95xl0"9, at least 2.90xl0"9, at least 2 85xlO"9, at least 2 80xl0"9, at least 2.75xl0"9 at least 2.70xl0"9 at least 2.65xl0"9, at least 2 60x10"9, at least 2 55xlO"9, at least 2.5xl0"9, at least 2.45xl0"9, at least 2.40xl0"9, at least 2 35xlO"9, at least 2 30xlO"9, at least 2.25xl0"9 at least 2.20xl0"9 at least 2.15xl0"9, at least 2 lOxlO"9, at least 2 05xlO"9, at least 2.0x10"9, at least 1.95xl0"9, at least 1.90xl0"9, at least 1 85xlO"9, at least 1 80xl0"9, at least 1.75xl0"9 at least 1.70xl0"9 at least 1.65xl0"9, at least 1 60x10"9, at least 1 55xlO"9, at least 1.5xlO"9, at least 1.45xl0"9, at least 1.40xl0"9, at least 1 35xlO"9, at least 1 30xlO"9, at least 1.25xl0"9 at least 1.20xl0"9 at least 1.15xlO"9, at least 1 lOxlO"9, at least 1 05xlO"9, at least l .OxlO"9, at least 0.95xl0"9, at least 0.90xl0"9, at least 0 85xlO"9, at least 0 80xl0"9, at least 0.75xl0"9 at least 0.70xl0"9 at least 0.65xl0"9, at least 0 60x10"9, at least 0 55xlO"9, at least 0.5xlO"9, at least 0.45xl0"9, at least 0.40xl0"9, at least 0 35xlO"9, at least 0.30xl0"9, at least 0.25xl0"9, at least 0.20xl0"9, at least 0.15xlO"9, at least O. lOxlO"9, at least 0.05xlO"9, at least 9.5xl0"10, at least 9.0xl0"10, at least 8.5xlO"10, at least 8.0xlO"10 M, or any value in between.
[0071] In some embodiments, the disclosed bispecific antibodies and functional fragments thereof, can have an OPA or MOPA titer of at least 8, at least 9, at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, at least 5500, at least 6000, at least 6500, ate least 7000, at least 7500, at least 8000, at least 8500, at least 9000, at least 9500, at least 10000, at least 10500, at least 11000, at least 11500, at least 12000, at least 12500, or at least 13000.
D. Additional Components of the disclosed BsAbs
[0072] The hinge region of the disclosed antibodies may be modified by replacing one or more cysteine residues with, for example, serine residues, to prevent dimerization. See, e.g., U.S. Patent Application Publication 2003/0118592; U.S. Patent Application Publication U.S. 2003/0133939. Additionally, in some embodiments, the disclosed antibodies may comprise other mutations, including but not limited to a variant Fc portion of an IgGl having the point mutations S239D/I332E, S239D, or I332E, or any combination thereof, or a variant Fc portion of an IgG4 having the point mutation S228P. Such modifications may alter the binding of the disclosed antibodies and functional fragments to Fc receptors (FcRs), and in some embodiments, the antibody may be modified to be more stable, while in some embodiments, the antibody may be modified to enhance ADCC function. When determining the number of the residue, the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.
[0073] In some embodiments, the glycosylation patterns of the disclosed antibodies may be modified or altered. For instance, in some embodiments, the disclosed antibodies and functional fragments thereof may be low fucose antibodies or they may be defucosylated or the antibodies may be expressed or produced in such a way that they are lacking fucose altogether (i.e. afucosylated). Modifying the fucose content of the antibody or functional fragment may be accomplished through various means known in the art, for instance, expressing the antibody or functional fragment in a cell that is FUT8 deficient or that has a mutated version of FUT8. Low fucose or defucosylated antibodies and functional fragment have increased ADCC activity. In addition to alterations in fucose, the disclosed antibodies and functional fragments may comprise other functional modifications to their glycosylation patterns. For instance, modifications at position 297 (e.g. N297A and N297Q) can prevent glycosylation of the Fc region altogether, thus eliminating Fc function, ADCC, and CDC.
[0074] The anti-SPN BsAbs or functional fragments thereof can be formulated in a pharmaceutical composition suitable for administration to the target subject by the intended route of administration, as discussed in more detail below.
IV. Pharmaceutical Formulations
[0075] Pharmaceutical compositions suitable for use in the methods described herein can include the therapeutically active agent (i.e., the disclosed anti-SPN BsAbs or functional fragments thereof) and a pharmaceutically acceptable carrier or diluent.
[0076] The composition may be formulated for intravenous, subcutaneous, intraperitoneal, intramuscular, oral, nasal, pulmonary, ocular, vaginal, or rectal administration. In some embodiments, the anti-SPN BsAbs or functional fragments thereof are formulated for intravenous, subcutaneous, intraperitoneal, or intramuscular administration, such as in a solution, suspension, emulsion, liposome formulation, etc. The pharmaceutical composition can be formulated to be an immediate-release composition, sustained-release composition, delayed-release composition, etc., using techniques known in the art.
[0077] Pharmacologically acceptable carriers for various dosage forms are known in the art. For example, excipients, lubricants, binders, and disintegrants for solid preparations are known; solvents, solubilizing agents, suspending agents, isotonicity agents, buffers, and soothing agents for liquid preparations are known. In some embodiments, the pharmaceutical compositions include one or more additional components, such as one or more preservatives, antioxidants, stabilizing agents and the like.
[0078] Additionally, the disclosed pharmaceutical compositions comprising at least one anti-SPN BsAbs can be formulated as a solution, microemulsion, nanoparticle, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In some embodiment, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
[0079] Sterile injectable solutions can be prepared by incorporating the active compound (i.e. the disclosed anti-SPN BsAbs or functional fragments thereof) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
[0080] Pharmaceutical compositions of the disclosure can be administered in combination with other therapeutics, such as antibiotics. For example, the combination therapy can include a pharmaceutical composition comprising at least one of the disclosed anti-SPN BsAbs or functional fragments thereof with at least one or more additional therapeutic agents, including but not limited to, antibiotics commonly used to treat SPN infections (discussed in more detail below).
V. Methods of Treating Streptococcus Pneumoniae
[0081] Provided herein are methods of treating streptococcus pneumonia with the disclosed bispecific anti-SPN antibodies. More specifically, the disclosure provides for methods of enhancing a subject's immunity or ability to fight a SPN infection comprising administering a therapeutically effective amount of any of the above described anti-SPN BsAbs or pharmaceutical compositions comprising the same.
[0082] In some embodiments, administration of the disclosed BsAbs can serve as a standalone treatment. In some embodiments, patient outcome may be improved by administering the disclosed BsAbs in conjunction with antibiotic therapy. The disclosed BsAbs may be administered before, concurrently with, or after administration of at least one antibiotic.
[0083] Exemplary antibiotics that may be used in conjunction with the disclosed BsAbs include, but are not limited to, β-lactam antibiotics (e.g., penicillin, ampicillin, methicillin, amoxicillin, etc.), tetracyclines (e.g., tetracycline, minocycline, methacycline, doxycycline, etc.), macrolides (e.g., erythromycin, azithromycin, clarithromycin, etc.), and quinolones or fluoroquinolones (e.g., ciprofloxacin, garenoxacin, gatifloxacin, gemifloxacin, levofloxacin, moxifloxacin, linezolid (ot other oxazolidinones), etc.).
[0084] In some embodiments, the method of treatment may comprise administering more than one of the disclosed BsAbs. For instance, in some embodiments, a patient may be administered a first BsAb that is specific for serotypes 19 A/3 and a second BsAb that is specific for serotypes 22F/6A. When more than one type of BsAb is used, the first and second BsAbs may be administered at the same time or at separate times, for instance, one after the other.
[0085] In some embodiments, the disclosed BsAb can be administered to a patient with SPN or CAPP that is resistant to antibiotics. In some embodiments, the disclosed BsAb can be administered to a patient with SPN having a serotype of 19A, 3, 22F, 6A, 6B, 6C, 19F, 15A, 23 A, 1 1 A, 35B, 12F, 22A or 7F. In some embodiments, the serotype of the infection may be determined prior to administration of the disclosed BsAb using, for example, a lateral-flow point-of-care diagnostic, like those disclosed herein, or another diagnostic capable of distinguishing SPN serotypes.
[0086] The mode of administration is not particularly limited and may be based on the needs of an individual patient. In general, the disclosed BsAbs are administered via injection, but other modes of administration may be applicable. In some embodiments, the BsAbs may be formulated for intravenous, intraperitoneal, intra-arterial, intramuscular, or subcutaneous administration.
[0087] The dosage regimens are likewise not particularly limited. Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response like a decrease in CFU count or reduction in symptoms of infection). For example, in some embodiments, a single bolus of BsAb may be administered, while in some embodiments, several divided doses may be administered over time. In some embodiments, the dose may be proportionally reduced or increased as indicated by the situation. For example, in some embodiments the disclosed antibodies or functional fragments may be administered once, twice, three, four, five, six, or seven times a week by subcutaneous or intravenous injection. In some embodiments, the disclosed antibodies or functional fragments may be administered once, twice, three, or four times a month by subcutaneous injection. In some embodiments, the disclosed antibodies or functional fragments may be administered once a day, once every other day, once every week, once every other week, once every three weeks, once every four weeks, once every other month, once every three months, once every four months, once every five months, or once every six months.
[0088] Exemplary doses can vary according to the size and health of the individual being treated, as well as the condition being treated. For example, in some embodiments, the disclosed antibodies or functional fragments may be administered in a dose of 0.1-150 mg/kg, or any range in between. For instance, an applicable dose range may be 1-100 mg/kg, 5-50 mg/kg, 10-25 mg/kg, or any ranges therein. In some embodiments, the disclosed antibodies and functional fragments may be administered in a dose of about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, or about 150 mg/kg.
[0089] Particular treatment regimens may be evaluated according to whether they will improve a given patient's outcome, meaning it will reduce the risk of recurrence of infection or increase the likelihood of survival of the patient with a SPN infection.
[0090] Thus, for the purposes of this disclosure, a subject is treated if one or more beneficial or desired results, including desirable clinical results, are obtained. For example, beneficial or desired clinical results include, but are not limited to, one or more of the following: decreasing one or more symptoms resulting from the infection, increasing the quality of life of those suffering from an infection, decreasing the dose of other medications required to treat the infection, delaying the progression of the infection, reducing the bacterial load (i.e., CFU count) of a patient with an infection and/or curing the patient of the SPN infection.
[0091] Furthermore, while the subject of the methods is generally a patient with a SPN infection, the age and health of the patient is not limited. The disclosed methods are useful for treating immunocompromised patients or patients with allergies to antibiotic. In some embodiments, the patient may be a pediatric patient, while in other embodiments, the subject may be an adult patient or an elderly patient (i.e. a patient that is 70 years of age or older).
VI. Methods and Devices for Diagnosing SPN
[0092] Disclosed herein are methods and devices for diagnosing SPN using the disclosed anti-SPN BsAbs or functional fragments thereof. Correctly determine the serotype of SPN that is infecting a given individual can play an integral role in how the individual should be treated and inform the caring physicians as to which of the disclosed anti-SPN BsAbs would be useful in treating the patient's infection.
[0093] Accordingly, the disclosed anti-SPN BsAbs can be incorporated into any antibody- based diagnostic known in the art in order to identify the specific serotype or serotypes present in a given patient's infection. Thus, in some embodiments, the disclosed anti-SPN BsAbs may be affixed to a bead, a dipstick, a chip, or a plate that can be contacted with a sample from an individual suspected of having a SPN infection. Further explanation of exemplary diagnostics can be found in U.S. Patent No. 9,279,815, which is hereby incorporated by reference.
[0094] In some embodiments, the diagnostic may comprise a lateral-flow test or kit. Lateral-flow tests are simple devices intended to detect the presence (or absence) of a specific serotype in sample (e.g., blood, plasma, saliva, urine, etc.) without the need for specialized and costly equipment. The test may comprise a at least one or a series of capillary beds, such as pieces of porous paper, microstructured polymer, or sintered polymer that are capable of transporting fluid. The disclosed anti-SPN BsAbs can be immobilized on these capillary beds such that they will bind the SPN serotype for which they possess specificity, thus indicating that a given serotype is present in the sample. In some embodiments, the lateral flow test may be a competitive test, while in some embodiments, the lateral flow test may be a sandwich assay test. [0095] In some embodiments, the diagnostic may be a point-of-care (POC) diagnostic, while in some embodiments, the diagnostic may require operation or interpretation by a specialty laboratory.
[0096] The disclosed devices and kits may be used in methods of diagnosing an individual as having SPN or, more specifically, a given serotype of SPN.
[0097] The following examples are given to illustrate the present invention. It should be understood, however, that the invention is not to be limited to the specific conditions or details described in these examples.
Examples
[0098] Example 1 - Testing an Anti-19A/3 BsAb in Mice Infected with Serotype 3
[0099] The efficacy of an exemplary anti-19A/3 BsAb (p4A05L/p6E03K-bi) was tested in a murine pulmonary infection model. Fully immunocompetent ICR mice were infected with 5.6 logio CFU of serotype 3 SPN (ATCC 10813) instilled intranasally. The mice then were administered with a single injection of either the BsAb 19A/3 at doses 10, 5.0 or 0.5 mg/kg, mAb 3 at 10 mg/kg, or Linezolid at 75 mg/kg and survival was tracked over the course of 10 days. Compared to mice receiving Linezolid, an antibiotic broadly effective against Gram-positive bacteria, mice receiving 10 mg/kg mAb 3 and BsAb 19 A/3 had reduced bacterial burden in their lungs 24 hr post-infection (Figure 3A) and improved survival rates (Figure 3B).
[0100] Example 2— Testing an Anti-19A/3 BsAb in Mice Infected with Serotype 19A
[0101] Success will be measured as BsAb-mediated 3-log kill, comparable to untreated SPN-infected mice, and that the CFU reduction is similar to or better than that of antibiotic- treated SPN-infected mice. Success will also be measured as prolonged survival of the BsAb treated mice compared to untreated mice. Survival rates of BsAb treated mice will be compared to that of mAb treated and antibiotic treated mice.
[0102] Although unlikely, the anti-19A/3 BsAb (p4A05L/p6E03K-bi), which has good OP A activity for 19A and 3 and in vivo activity against serotype 3 infection, may not prove efficacious in vivo against serotype 19A infection. In this case, the treatment plan can be modified to include repeated Ab dosing or another of the disclosed 19A-specific variable regions can be substituted in. The disclosed Ab library allows for the potential development of at least one additional 19A/3 BsAb and 3 22F/6A BsAbs.
[0103] The susceptibility of the SPN strains to antibiotics will be determined. The MIC for Levofloxacin and Linezolid against SPN 19A will be determined using the broth microdilution method described by the Clinical and Laboratory Standards Institute. Resistance will be defined as MIC values > than the breakpoints. If the 19A SPN strain is resistant to both antibiotics, a different strain of 19A serotype will be obtained (readily available from JMI) and tested for susceptibility.
[0104] The mice will be challenged with SPN 19A at the Appropriate Challenge Dose (ACD) intranasally or intravenously (determined previously). BsAb 19 A/3 and mAb 19A will be given intravenously at 10 mg/kg (dose selected based on previous mAb and BsAb studies). Controls will be SPN infection with no treatment, treatment with antibiotics, and treatment with parental mAb. Lung tissue will be harvested at 24 hr after SPN challenge and bacterial burden per gram of lung tissue will be determined. Success will be Ab-mediated reduction in bacterial load by 3 loglO CFU {see Table 5 for example of a BsAb in vivo study outline). The 3 logio CFU reduction metric is based on a similar reduction seen with antibiotics resulting in optimal bactericidal action.
Table 5 - Measuring CFU and 10-day mortality in a murine lung infection model using SPN serot es 3, 6 A, 19 A, or 22F and treatment with varying concentrations of BsAb s
Figure imgf000036_0001
[0105] Finally, the mice (CBA/J) will be challenged with 19A SPN at the Appropriate Challenge Dose intranasally. As described in Table 5, control will be treated with Levaquin or Linezolid (antibiotic dose will be selected based on literature and MIC results). Mice will be treated with lOmg/kg of the parental mAb 19A or with a dose range of BsAb 19 A/3. After 10 days, percent survival will be calculated. Success is BsAb-mediated survival comparable to antibiotics and mAb.
[0106] Example 3— Testing an Anti-6A/22F BsAb in Mice [0107] This study will be performed similar to those described in Examples 1 and 2, but utilizing SPN 6A and SPN 22F.
[0108] Success will be measured as BsAb-mediated 3-log kill, comparable to untreated SPN-infected mice, and that the CFU reduction is similar to that of antibiotic-treated SPN- infected mice. Success will also be measured as prolonged survival of the BsAb treated mice compared to untreated mice. Survival rates of BsAb treated mice will be compared to that of mAb treated and antibiotic treated mice.
[0109] SPN serotypes 6A and 3 can cause pulmonary infection in ICR mice and SPN serotype 19A in CBA/J mice; SPN serotype 22F is not able to cause a pulmonary infection in ICR mice (data not shown). However, since SPN serotype 19A can cause a pulmonary infection in CBA/J mice, it is likely that SPN serotype 22F will also able to cause a pulmonary infection in CBA/J mice. Yet, it may be determined that the SPN serotype 22F is not able to cause a pulmonary infection, in which case the study will be performed in an intravenous bacteremia murine model in a 10-day mortality study.
[0110] Mice will be challenged with SPN 22F at approximately 3, 4, 5, or 6 loglO CFU via intranasal administration. Success Metric: Finding a challenge dose resulting in 5-7 loglO CFUs in the lungs after 24 h post-infection will represent the ACD. The effectiveness of Levofloxacin and Linezolid against SPN serotype 6A and 22F will also be determined. This will be done as described in Example 2 above, but for SPN serotype 6A and SPN serotype 22F.
[0111] The ability of the anti-22F/6A BsAb to reduce bacterial load in the lung of an infected mouse will be tested. This will be performed as described in Task 4 as separate experiments with SPN serotype 6A and SPN serotype 22F (see Table 5). Finally, the ability of the anti-22F/6A BsAb to improve survival of an infected mouse comparable to standard of care (treatment with antibiotic), will be tested utilizing a standard pulmonary infection murine model. This will be performed as described in Example 2 as separate experiments with SPN serotype 6 A and SPN serotype 22F.
[0112] As in Example 2, if the anti-6A/22F BsAb is ineffective, the treatment plan can be modified to include additional or alternative doses, or the BsAb can be redesigned to include alternative 6A- or 22F-specific variable regions. [0113] These exemplary experimental methods could be used to test any other bispecific antibody disclosed herein with any combination of serotype specificities. The use of further antibodies alone and in combination with standard-of-care antibiotics is planned for subsequent studies.
[0114] All patents and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the disclosure pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
[0115] Further, one skilled in the art readily appreciates that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the disclosure and are defined by the scope of the claims, which set forth non-limiting embodiments of the disclosure.

Claims

What is claimed:
1. A bispecific anti -streptococcus pneumoniae antibody or a functional fragment thereof that specifically binds to at least two serotypes selected from a group consisting of serotypes 19A, 3, 22F, 6A, 6B, 6C, 19F, 15A, 23 A, 11 A, 35B, 12F, 22A and 7F.
2. The bispecific antibody or functional fragment thereof of claim 1, wherein the antibody or functional fragment thereof specifically binds to serotypes 19A and 3.
3. The bispecific antibody or functional fragment thereof of claim 1, wherein the antibody or functional fragment thereof specifically binds to serotypes 6 A and 22F.
4. The bispecific antibody or functional fragment thereof of claim 3, wherein the antibody is cross-reactive with serotypes 6B or 6C.
5. The bispecific antibody or functional fragment thereof of claim 3, wherein the antibody is cross-reactive with serotypes 6B and 6C
6. The bispecific antibody or functional fragment thereof of claim 1, wherein the antibody or functional fragment thereof specifically binds to at least two of serotypes 19F, 15A, 23 A, 11 A, 35B, 12F, 22A and 7F.
7. The bispecific antibody or functional fragment thereof of claim 1, wherein the antibody or functional fragment thereof has a KD for the serotypes that it binds that is at least at least 1.0 x 10"9 M.
8. The bispecific antibody or functional fragment thereof of claim 1, wherein the antibody or functional fragment thereof has an OP A titer of greater than 8.
9. A bispecific antibody or functional fragment thereof that specifically binds to at least two serotypes of streptococcus pneumoniae, wherein the bispecific antibody or functional fragment thereof comprises: a. a first heavy chain variable region comprising: a complementarity determining region (CDR) HI selected from the group consisting of SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, and 43; a CDRH2 selected from the group consisting of SEQ ID NOs: 2, 8, 14, 20, 26, 32, 38, and 44; and a CDRH3 selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, and 45; b. a second heavy chain variable region comprising: a CDRH1 selected from the group consisting of SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, and 43; a CDRH2 selected from the group consisting of SEQ ID NOs: 2, 8, 14, 20, 26, 32, 38, and 44; and a CDRH3 selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, and 45; c. a first light chain variable region comprising: a CDRL1 selected from the group consisting of SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, and 46; a CDRL2 selected from the group consisting of SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, and 47; and a CDRL3 selected from the group consisting of SEQ ID NOs: 6, 12, 18, 24, 30, 36, 42, and 48; and d. a second light chain variable region comprising: a CDRL1 selected from the group consisting of SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, and 46; a CDRL2 selected from the group consisting of SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, and 47; and a CDRL3 selected from the group consisting of SEQ ID NOs: 6, 12, 18, 24, 30, 36, 42, and 48; wherein the first and second heavy chain do not comprise the same CDR sequences, and wherein the first and second light chain do not comprise the same CDR sequences.
10. The bispecific antibody or functional fragment thereof of claim 9, wherein the antibody or functional fragment thereof specifically binds to at least two of serotypes 19 A, 3, 22F, 6A, 6B, or 6C.
11. The bispecific antibody or functional fragment thereof of claim 10, wherein the antibody or functional fragment thereof specifically binds to serotypes 19A and 3.
12. The bispecific antibody or functional fragment thereof of claim 10, wherein the antibody or functional fragment thereof specifically binds to serotype 22F and at least one of serotypes 6A, 6B, or 6C.
13. The bispecific antibody or functional fragment thereof of claim 9, wherein the first and second heavy chain variable regions comprise an amino acid sequence comprising SEQ ID NOs: 49, 51, 53, 55, 57, 59, 61, or 63, and the first and second light chain variable regions comprise an amino acid sequence comprising SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, or 64, wherein the first and second heavy chains do not comprise the same sequences and the first and second light chains do not comprise the same sequences.
14. The bispecific antibody or functional fragment thereof of claim 9, wherein a. the first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 3; b. the second heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 25, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 26, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 27; c. the first light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 4, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 6; and d. the second light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 28, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 29, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 30.
15. The bispecific antibody or functional fragment thereof of claim 14, wherein the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 49, the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 57, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 50, and the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 58.
16. The bispecific antibody or functional fragment thereof of claim 9, wherein a. the first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 7, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 9; b. the second heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 31, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 33; c. the first light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 10, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 12; and d. the second light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 34, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 36.
17. The bispecific antibody or functional fragment thereof of claim 16, wherein the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 51, the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 59, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 52, and the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 60.
18. The bispecific antibody or functional fragment thereof of claim 9, wherein a. the first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 7, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 9; b. the second heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 37, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 39; c. the first light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 10, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 12; and d. the second light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 40, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 42.
19. The bispecific antibody or functional fragment thereof of claim 18, wherein the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 51, the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 61, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 52, and the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 62.
20. The bispecific antibody or functional fragment thereof of claim 9, wherein a. the first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 13, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 14, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 15; b. the second heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 43, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 45; c. the first light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 16, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 17, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 18; and d. the second light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 46, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 48.
21. The bispecific antibody or functional fragment thereof of claim 20, wherein the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 53, the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 63, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 54, and the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 64.
22. The bispecific antibody or functional fragment thereof of claim 9, wherein a. the first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 19, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 21; b. the second heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 43, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 45; c. the first light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 22, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 23, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 24; and d. the second light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 46, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 48.
23. The bispecific antibody or functional fragment thereof of claim 22, wherein the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 55, the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 63, the first heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 56, and the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 64.
24. The bispecific antibody or functional fragment thereof of claim 9, wherein the antibody or functional fragment thereof has a KD for the serotypes that it binds that is at least at least 1.0 x 10"9 M.
25. The bispecific antibody of claim 9, wherein the antibody or functional fragment thereof has an OP A titer of greater than 8.
26. A bispecific antibody or functional fragment thereof that specifically binds to at least two serotypes of streptococcus pneumoniae, wherein the bispecific antibody or functional fragment thereof comprises two different heavy chain variable regions and two different light chain variable regions, wherein the two different heavy chain variable regions and the two different light chain variable regions are encoded by four different nucleic acid sequences chosen from Table 4.
27. A method of treating a Streptococcus pneumoniae infection in an individual comprising, administering to an individual with a S. pneumoniae infection (i) a therapeutically effective amount of a bispecific antibody or a functional fragment thereof that specifically binds to at least two serotypes selected from a group consisting of serotypes 19A, 3, 22F, 6A, 6B, 6C, 19F, 15A, 23 A, 11 A, 35B, 12F, 22A and 7F; and (ii) an antibiotic.
28. The method of claim 27, wherein the antibody or functional fragment thereof specifically binds to at least two of serotypes 19 A, 3, 22F, 6 A, 6B, or 6C.
29. The method of claim 27, wherein the bispecific antibody or functional fragment thereof comprises: a. a first heavy chain variable region comprising: a complementarity determining region (CDR) HI selected from the group consisting of SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, and 43; a CDRH2 selected from the group consisting of SEQ ID NOs: 2, 8, 14, 20, 26, 32, 38, and 44; and a CDRH3 selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, and 45; b. a second heavy chain variable region comprising: a complementarity determining region (CDR) HI selected from the group consisting of SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, and 43; a CDRH2 selected from the group consisting of SEQ ID NOs: 2, 8, 14, 20, 26, 32, 38, and 44; and a CDRH3 selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, and 45; c. a first light chain variable region comprising: a CDRLl selected from the group consisting of SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, and 46; a CDRL2 selected from the group consisting of SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, and 47; and a CDRL3 selected from the group consisting of SEQ ID NOs: 6, 12, 18, 24, 30, 36, 42, and 48; and d. a second light chain variable region comprising: a CDRLl selected from the group consisting of SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, and 46; a CDRL2 selected from the group consisting of SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, and 47; and a CDRL3 selected from the group consisting of SEQ ID NOs: 6, 12, 18, 24, 30, 36, 42, and 48; wherein the first and second heavy chain do not comprise the same sequence, and wherein the first and second light chain do not comprise the same sequence.
30. The method of claim 29, wherein a. the first heavy chain variable region comprises a CDRHl comprising the amino acid sequence of SEQ ID NO: 1, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 3; b. the second heavy chain variable region comprises a CDRHl comprising the amino acid sequence of SEQ ID NO: 25, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 26, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 27; c. the first light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 4, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 6; and d. the second light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 28, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 29, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 30.
31. The method of claim 29, wherein a. the first heavy chain variable region comprises a CDRHl comprising the amino acid sequence of SEQ ID NO: 7, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 9; b. the second heavy chain variable region comprises a CDRHl comprising the amino acid sequence of SEQ ID NO: 31, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 33; c. the first light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 10, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 12; and d. the second light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 34, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 36.
32. The method of claim 29, wherein a. the first heavy chain variable region comprises a CDRHl comprising the amino acid sequence of SEQ ID NO: 7, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 9; b. the second heavy chain variable region comprises a CDRHl comprising the amino acid sequence of SEQ ID NO: 37, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 39; c. the first light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 10, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 12; and d. the second light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 40, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 42.
33. The method of claim 29, wherein a. the first heavy chain variable region comprises a CDRHl comprising the amino acid sequence of SEQ ID NO: 13, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 14, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 15; b. the second heavy chain variable region comprises a CDRHl comprising the amino acid sequence of SEQ ID NO: 43, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 45; c. the first light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 16, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 17, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 18; and d. the second light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 46, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 48
34. The method of claim 29, wherein a. the first heavy chain variable region comprises a CDRHl comprising the amino acid sequence of SEQ ID NO: 19, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 21; b. the second heavy chain variable region comprises a CDRHl comprising the amino acid sequence of SEQ ID NO: 43, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 45; c. the first light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 22, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 23, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 24; and d. the second light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 46, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 48.
35. The method of claim 27, wherein the antibiotic is a β-lactam antibiotic, a tetracycline, a macrolide, a quinolone, or a fluoroquinolone.
36. The method of claim 27, wherein the antibiotic is Levofloxacin or Linezolid.
37. The method of claim 27, wherein it was determined prior to administration of the bispecific antibody or functional fragment thereof that the individual's S. pneumoniae infection comprises at least one of the serotypes specifically bound by the bispecific antibody or functional fragment thereof.
38. The method of claim 27, wherein the bispecific antibody or functional fragment thereof is administered before the antibiotic.
39. The method of claim 27, wherein the bispecific antibody or functional fragment thereof is administered after the antibiotic.
40. The method of claim 27, wherein the bispecific antibody or functional fragment thereof is administered concurrently with the antibiotic.
41. A method of treating an antibiotic-resistant Streptococcus pneumoniae infection in an individual comprising, administering to an individual with an antibiotic-resistant S. pneumoniae infection a therapeutically effective amount of a bispecific antibody or a functional fragment thereof that specifically binds to at least two serotypes selected from a group consisting of serotypes 19A, 3, 22F, 6A, 6B, 6C, 19F, 15A, 23 A, 11 A, 35B, 12F, 22A and 7F.
42. The method of claim 41, wherein the individual has previously received an antibiotic to treat the S. pneumoniae infection.
43. The method of claim 41, wherein the antibody or functional fragment thereof specifically binds to at least two of serotypes 19 A, 3, 22F, 6 A, 6B, or 6C.
44. The method of claim 41, wherein the bispecific antibody or functional fragment thereof comprises: a. a first heavy chain variable region comprising: a complementarity determining region (CDR) HI selected from the group consisting of SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, and 43; a CDRH2 selected from the group consisting of SEQ ID NOs: 2, 8, 14, 20, 26, 32, 38, and 44; and a CDRH3 selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, and 45; b. a second heavy chain variable region comprising: a complementarity determining region (CDR) HI selected from the group consisting of SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, and 43; a CDRH2 selected from the group consisting of SEQ ID NOs: 2, 8, 14, 20, 26, 32, 38, and 44; and a CDRH3 selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, and 45; c. a first light chain variable region comprising: a CDRL1 selected from the group consisting of SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, and 46; a CDRL2 selected from the group consisting of SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, and 47; and a CDRL3 selected from the group consisting of SEQ ID NOs: 6, 12, 18, 24, 30, 36, 42, and 48; and d. a second light chain variable region comprising: a CDRL1 selected from the group consisting of SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, and 46; a CDRL2 selected from the group consisting of SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, and 47; and a CDRL3 selected from the group consisting of SEQ ID NOs: 6, 12, 18, 24, 30, 36, 42, and 48; wherein the first and second heavy chain do not comprise the same sequence, and wherein the first and second light chain do not comprise the same sequence.
45. The method of claim 44, wherein a. the first heavy chain variable region comprises a CDRHl comprising the amino acid sequence of SEQ ID NO: 1, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 3; b. the second heavy chain variable region comprises a CDRHl comprising the amino acid sequence of SEQ ID NO: 25, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 26, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 27; c. the first light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 4, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 6; and d. the second light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 28, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 29, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 30.
46. The method of claim 44, wherein a. the first heavy chain variable region comprises a CDRHl comprising the amino acid sequence of SEQ ID NO: 7, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 9; b. the second heavy chain variable region comprises a CDRHl comprising the amino acid sequence of SEQ ID NO: 31, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 33; c. the first light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 10, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 12; and d. the second light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 34, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 36.
47. The method of claim 44, wherein a. the first heavy chain variable region comprises a CDRHl comprising the amino acid sequence of SEQ ID NO: 7, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 9; b. the second heavy chain variable region comprises a CDRHl comprising the amino acid sequence of SEQ ID NO: 37, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 39; c. the first light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 10, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 12; and d. the second light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 40, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 42.
48. The method of claim 44, wherein a. the first heavy chain variable region comprises a CDRHl comprising the amino acid sequence of SEQ ID NO: 13, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 14, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 15; b. the second heavy chain variable region comprises a CDRHl comprising the amino acid sequence of SEQ ID NO: 43, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 45; c. the first light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 16, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 17, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 18; and d. the second light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 46, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 48
49. The method of claim 44, wherein a. the first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 19, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 21; b. the second heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 43, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 45; c. the first light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 22, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 23, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 24; and d. the second light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 46, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 48.
50. A method of treating a Streptococcus pneumoniae infection in an individual comprising, administering to an individual with a S. pneumoniae infection a therapeutically effective amount of a bispecific antibody or a functional fragment thereof that specifically binds to at least two serotypes selected from a group consisting of serotypes 19 A, 3, 22F, 6A, 6B, and 6C, wherein the bispecific antibody or functional fragment thereof has a KD for the serotypes that it binds that is at least at least 1.0 x 10"9 M.
51. The method of claim 50, wherein the bispecific antibody or functional fragment thereof comprises: a. a first heavy chain variable region comprising: a complementarity determining region (CDR) HI selected from the group consisting of SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, and 43; a CDRH2 selected from the group consisting of SEQ ID NOs: 2, 8, 14, 20, 26, 32, 38, and 44; and a CDRH3 selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, and 45; b. a second heavy chain variable region comprising: a complementarity determining region (CDR) HI selected from the group consisting of SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, and 43; a CDRH2 selected from the group consisting of SEQ ID NOs: 2, 8, 14, 20, 26, 32, 38, and 44; and a CDRH3 selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, and 45; c. a first light chain variable region comprising: a CDRL1 selected from the group consisting of SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, and 46; a CDRL2 selected from the group consisting of SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, and 47; and a CDRL3 selected from the group consisting of SEQ ID NOs: 6, 12, 18, 24, 30, 36, 42, and 48; and d. a second light chain variable region comprising: a CDRL1 selected from the group consisting of SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, and 46; a CDRL2 selected from the group consisting of SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, and 47; and a CDRL3 selected from the group consisting of SEQ ID NOs: 6, 12, 18, 24, 30, 36, 42, and 48; wherein the first and second heavy chain do not comprise the same sequence, and wherein the first and second light chain do not comprise the same sequence.
52. The method of claim 51, wherein a. the first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 3; b. the second heavy chain variable region comprises a CDRHl comprising the amino acid sequence of SEQ ID NO: 25, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 26, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 27; c. the first light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 4, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 6; and d. the second light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 28, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 29, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 30.
53. The method of claim 51, wherein a. the first heavy chain variable region comprises a CDRHl comprising the amino acid sequence of SEQ ID NO: 7, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 9; b. the second heavy chain variable region comprises a CDRHl comprising the amino acid sequence of SEQ ID NO: 31, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 33; c. the first light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 10, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 12; and d. the second light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 34, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 36.
The method of claim 51, wherein a. the first heavy chain variable region comprises a CDRHl comprising the amino acid sequence of SEQ ID NO: 7, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 9; b. the second heavy chain variable region comprises a CDRHl comprising the amino acid sequence of SEQ ID NO: 37, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 39; c. the first light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 10, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 12; and d. the second light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 40, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 42.
The method of claim 51, wherein a. the first heavy chain variable region comprises a CDRHl comprising the amino acid sequence of SEQ ID NO: 13, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 14, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 15; b. the second heavy chain variable region comprises a CDRHl comprising the amino acid sequence of SEQ ID NO: 43, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 45; c. the first light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 16, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 17, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 18; and d. the second light chain variable region comprises a CDRLl comprising the amino acid sequence of SEQ ID NO: 46, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 48
56. The method of claim 51, wherein a. the first heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 19, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 21; b. the second heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 43, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 45; c. the first light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 22, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 23, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 24; and d. the second light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 46, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 48.
57. The method of claim 50, further comprising administering an antibiotic to the individual.
58. The method of claim 57, wherein the antibiotic is a β-lactam antibiotic, a tetracycline, a macrolide, a quinolone, or a fluoroquinolone.
59. The method of claim 57, wherein the antibiotic is Levofloxacin or Linezolid.
60. The method of claim 50, wherein the S. pneumoniae infection is antibiotic- resistant.
61. The method of claim 50, wherein it was determined prior to administration of the bispecific antibody or functional fragment thereof that the individual's S. pneumoniae infection comprises at least one of the serotypes specifically bound by the bispecific antibody or functional fragment thereof.
62. The method of claim 50, wherein the antibody or functional fragment thereof has an OP A titer of greater than 8.
PCT/US2018/035170 2017-05-31 2018-05-30 Bispecific antibodies for the treatment of streptococcus pneumonia Ceased WO2018222741A1 (en)

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