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WO2025010424A1 - Anticorps dirigés contre des antigènes de staphylococcus et leurs méthodes d'utilisation - Google Patents

Anticorps dirigés contre des antigènes de staphylococcus et leurs méthodes d'utilisation Download PDF

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Publication number
WO2025010424A1
WO2025010424A1 PCT/US2024/036949 US2024036949W WO2025010424A1 WO 2025010424 A1 WO2025010424 A1 WO 2025010424A1 US 2024036949 W US2024036949 W US 2024036949W WO 2025010424 A1 WO2025010424 A1 WO 2025010424A1
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antigen
antibody
binding
binding fragment
fragment
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PCT/US2024/036949
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English (en)
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Martina BELTRAMELLO
Davide Corti
Francesco Muoio
Luca PICCOLI
Anna Brotcke ZUMSTEG
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Vir Biotechnology, Inc.
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Publication of WO2025010424A1 publication Critical patent/WO2025010424A1/fr

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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
    • C07K16/1271—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Micrococcaceae (F), e.g. Staphylococcus
    • 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
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/72—Increased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • Staphylococcus aureus causes diverse infections that often overlap with Enterococci and coagulase-negative staphylococci (CoNS), including skin/soft tissue infections, bacteremia/sepsis, hospital -associated pneumonia, endocarditis, neutropenia, hemodialysis associated/line associated bacteremia, osteomyelitis/joint infections, menstrual toxic shock syndrome, and peritonitis.
  • CoNS coagulase-negative staphylococci
  • antibiotics are the only option for treatment, which has been limited by antibiotic resistance. Attempted vaccines and monoclonal antibodies to-date have not been protective.
  • S. aureus Protein A can prevent serum antibodies and IgGl opsonizing monoclonal antibodies from harnessing Fc-mediated immunity. Protein A immune evasion may thwart development of a humoral-based vaccine for S. aureus.
  • the lack of an effective vaccine for S. aureus and related Gram-positive bacteria leads to significant morbidity and mortality. There is an unmet need for modalities to prevent and/or treat infection by Staphylococcus and related Gram-positive bacteria.
  • Figures 1A-1D show a workflow for discovery, recombinant expression, and characterization of antigen-specific monoclonal antibodies from human donors who recovered from S. aureus infection, and examples of results from the workflow.
  • Figures 2A-2P relate to antibody targeting of lipoteichoic acid (LTA).
  • Figure 2A shows (left) a table summarizing types of LTA and bacterial species expressing the indicated LTA is found, and (right) a schematic illustration of LTA types (I-V) at a cell membrane).
  • Gro glycerol
  • Glc glucose
  • Gal galactose
  • P-Glu P-glucosamine
  • AATGal 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose
  • GalNAc N-acetylgalactosamine
  • Rto ribitol
  • GlcNAc Nacetylglucosamine
  • P phosphate
  • Figure 2B provides a table showing binding (quantified as EC50 ng/ml) of certain anti-LTA antibodies of the present disclosure (SSI18-SSA8) to bacteria expressing Type I, Type II, or Type IV LTA (E coli was included as a reference), and variable domain gene usage of these antibodies.
  • the humanized anti-LTA antibody pagibaximab e.g., Patel and Kaufman, Expert Opin Biol Ther 75(4):595-600 (2015); doi: 10.1517/14712598.2015.1019857
  • pagibaximab did not reduce sepsis or death.
  • Figure 2C shows sensorgram curves showing that antibody SSA12 of the present disclosure does not compete with pagibaximab for LTA binding.
  • Figure 2D shows that SSA12 has more consistent binding across Type I LTA bacteria than pagibaximab.
  • Figure 2E shows binding (ELISA on LTA) of certain anti-LTA antibodies of the present disclosure, with pagibaximab as a comparator.
  • Figure 2F shows binding (EC50 ng/mL on LTA), gene usage, and IMGT CDRH3 amino acid sequence and length of the anti-LTA antibodies.
  • VH3-23 is used by 4 monoclonal antibodies produced by 3 donors.
  • VH3-7/VK1-6 are used by 10 clonally related monoclonal antibodies produced by 3 donors.
  • Figure 2G shows binding (ELISA on fixed bacteria) by certain anti-LTA antibodies of the present disclosure, with pagibaximab as a comparator.
  • Figure 2H shows quantified binding by the antibodies to bacteria expressing Type I, Type II, or Type IV LTA, or E. coli.
  • Figure 21 shows (left) FACS data showing binding by SSA12 to the indicated bacteria and (right) affinity for LTA, as compared to pagibaximab.
  • Figure 2J shows neutralization of infection on TLR2- expressing HEK293 cells. The foregoing data show that SSA12 is a broadly reactive anti-LTA antibody that binds with high affinity to a site on LTA that is not recognized by pagibaximab and can neutralize LTA activity.
  • Figure 2K shows a schematic of signaling initiated by binding of LTA to TLR2.
  • Figure 2L shows neutralization of LTA activity on TLR2-expressing HEK293 cells by certain anti-LTA antibodies of the present disclosure, and by pagibaximab.
  • HEK-Blue hTLR2 cells were co-transfected with hTLR2 and SEAP (secreted embryonic alkaline phosphatase) reporter genes under the control of the IFN-b minimal promoter fused to NF-kB and AP-l-binding sites. Stimulation of TLR2 activates NF-kB and AP-1 which induces the production of SEAP.
  • SEAP secreted embryonic alkaline phosphatase
  • Figure 2M shows (left) a schematic for a Cl -binding assay and (right) binding to Cl by certain opsonized Fc engineered anti-LTA antibodies of the present disclosure.
  • LS M428L/N434S mutations in Fc (increasing affinity for FcRn and in vivo half-life).
  • RF H435R/Y436F mutations in Fc (reducing binding to Protein A).
  • LS-RF M428L/N434S/H435R/Y436F, wherein Fc amino acid numbering is according to the EU numbering system, with reference to human IgGl . Pagibaximab does not avoid Protein A binding.
  • Figure 2N shows (left) a schematic for a Cl -binding assay and (right) binding to Cl by opsonized SSA12 bearing either LS or LS/RF mutations in the Fc.
  • FIG. 2P shows (left) Cl binding by SSA12 variant antibodies (pagibaximab and S2X303-LS-RF included as comparators) on S. epidermidis and (right) Cl binding AUC with S. epidermidis versus with S. aureus.
  • RPYL R292P/Y300L.
  • GARPYL G236A/R292P/Y300L.
  • GAYL G236A/Y300L.
  • Fc amino acid numbering is according to the EU numbering system, with reference to human IgGl.
  • KAEA KAEA
  • EFTAE EFTAE mutations
  • Figure 3 relates to antibody targeting of glucosaminidase.
  • glucosaminidase is a peptidoglycan-modifying enzyme that is highly conserved among Staphylococci and is necessary for cell division.
  • Glucosaminidase (Gmd or GlcA) is a domain of the autolysin (Atl) protein, the most predominant peptidoglycan hydrolase in S. aureus. Gmd is located on the cell surface and is highly conserved among S. aureus and S. epidermidis. Gmd catalyzes the hydrolysis of glucosidic linkages and plays a role in cell wall turnover, division, and separation.
  • FIG. 3 shows (right) a schematic illustration of a Gmd neutralization assay and (left, middle) results from ELISA binding studies using antibody SSG20 of the present disclosure (with 1C11 as comparator) against Gmd (left) and fixed bacteria (middle).
  • Figures 4A-4F relate to antibody targeting of alphatoxin or bicomponent toxins.
  • Staphylococcus secretes two types of P-barrel pore-forming toxins: 1. Alpha toxin; 2. Bicomponent (LukSF, LukED, LukAB, HlgAB and HlgCB). These toxins cause disruption of the plasma membrane, leading to osmotic imbalance and cell death, and are highly inflammatory.
  • the vast majority of S. aureus clinical isolates express Hla, HlgABC, and Luk.
  • the Luk S- and F-components are highly related structurally and share up to 80% amino acid identity.
  • Alpha toxin is a significant contributor to pathogenesis in some S.
  • Figures 4C-4F show: (Figure 4C) a schematic activity of bicomponent pore-forming cytotoxins (PFTs) at a cell membrane; (Figure 4D) binding (ELISA, SPR (Octet)) by certain antibodies of the present disclosure against Luk and HIg antigens; (Figure 4E) toxin neutralization on human monocytes; and (Figure 4F) binding competition assays.
  • PFTs bicomponent pore-forming cytotoxins
  • a variant of SpA Protein A contains substitution mutations in each of the five Ig-binding domains (Q9K, Q10K, D36A, D37A), abolishing the ability to bind Fey or Fab VH3 and promote B cell apoptosis (see Kim et al. J Exp Med, 207(9): 1863-1870 (2010); doi: 10.1084/jem.20092514).
  • Figure 5B shows (top) baiting of SPAKKAA memory B cells sorting and (bottom) binding (ELISA; SPR (Octet)) by certain antibodies of the present disclosure.
  • Figures 6A-6C relate to targeting of Clumping factor A (ClfA) by cross-reactive antibodies.
  • Clumping factor A Clumping factor A
  • S. aureus and S. epidermidis express sortase-attached SD repeat (SDR) proteins.
  • Clumping factor (Clf) promotes binding of fibrinogen to the bacterial cell and facilitates bacterial clumping.
  • Serine aspartate repeat proteins (Sdr) also promote bacterial adhesion.
  • Figure 6A shows a schematic of domains of Clf and Sdr proteins of S. aureus and S. epidermidis.
  • Figure 6B shows binding (ELISA) by certain antibodies of the present disclosure to ClfA.
  • Figures 7A-7C relate to antibody targeting of alpha-toxin.
  • Figure 7A shows (left graph) binding and (two graphs at right) neutralization by anti-alpha-toxin antibodies SSE1 and SSE158 of the present disclosure, with MEDI4893 as a comparator.
  • MEDI4893 also known as AR-320
  • Figure 7B shows results from a synergy/antagonism neutralization study combining anti-alpha-toxin antibodies SSE1 and SSE158.
  • Figure 7C shows survival of BALB/c mice prophylactically administered SSE1, SSE158, and/or comparator MEDI4893 at the indicated dose followed by infection (i.n.) with S. aureus. The combination study is repeated and in silica analysis of SSE1 and SSE158 is performed.
  • Figures 8A-8C relate to targeting of Gmd by certain antibodies.
  • Figures 8A-8B show results from ELISA binding studies using certain anti-Gmd antibodies of the present disclosure against Gmd from S. aureus and S. epidermidis.
  • Figure 8C provides a table showing quantified binding of certain anti-Gmd antibodies of the present disclosure to Gmd (S. aureus and S. epidermidis), quantified binding to bacterial strains, and variable domain gene usage of these antibodies. 1C11 was included as a comparator.
  • Figures 9A and 9B relate to antibody targeting of LTA.
  • Figure 9A LTA neutralization (reported as absolute EC50 ng/mL) for the antibodies shown in Figure 2F.
  • Figures 10A-10F relate to targeting of ClfA by certain antibodies.
  • Figure 10A shows binding by certain anti -ClfA antibodies of the present disclosure across strains of S. aureus with different ClfA genotypes, with E. coli included as a negative control.
  • the humanized anti-ClfA antibody tefibazumab e.g., Weems JJ Jr, Steinberg JP, Filler S, et al. Antimicrob Agents Chemother. 2006;50(8):2751-2755. doi: 10.1128/AAC.00096-06
  • Tefibazumab has been tested in a Phase 2 study for treatment of S. aureus in cystic fibrosis patients and treatment of S.
  • Figure 13 shows a graph comparing half-life of antibodies “FYl-rlgGl-LS” and “FY1- rlgGl-LS-RF” in Tg32 SCID mice expressing human FcRn. FYI recognizes influenza HA.
  • FIG. 14B shows (left) a schematic of FcyR binding measured by SPR and (right) a table showing FcyR binding by the indicated SSA12-LS-RF (or variant) antibodies, quantified by SPR. The values provided show a Log2 fold change compared to SSA12-LS-RF binding.
  • FIG 141 shows Staphylococcus survival in a whole blood assay after exposure to isotype control (S2X303-LS-RF) and SSA12-LS-RF (or variant) antibodies, tested by donor. In each graph, Staphylococcal survival for isotype control and the tested mAb is shown for each donor. Each line represents the change in survival with addition of the tested mAb.
  • Figure 14J shows (left) percent survival of S.
  • Figure 19 shows binding to Cl by anti-LTA antibodies SSA8 and SSA12 of the present disclosure.
  • Pagibaximab was included as a comparator.
  • Figure 20 shows neutralization, by anti-Gmd antibodies SSG20 and SSF11 of the present disclosure, in a cell wall digestion assay. 1C11 was included as a comparator.
  • Figure 21 shows quantified binding values (EC50 ng/mL to ClfA_001, ClfA_002, ClfA_004, and S. aureus strains). Tefibazumab was included as a comparator.
  • Figure 22 shows survival of BALB/c mice prophylactically administered SSE1, SSE158, MEDI4893, or an isotype control at the indicated dose followed by infection (i.n.) with S. aureus, as indicated.
  • Figure 23 relates to antibody targeting of Hla.
  • Figure 24 provides a table showing results from Hla-binding (quantified as EC50 ng/mL) and Hla neutralization (on THP-1 cells, quantified as IC50 ng/mL) assays by certain anti-Hla antibodies of the present disclosure.
  • Figure 25 relates to study design for testing efficacy of monoclonal antibodies against Staphylococcus targets in FPR3757 bacteremia model in BL6 mice.
  • Figure 26 provides a schematic (top) of the study design and a table showing the descriptions of the various study groups, antibody dose, antibody delivery route, antibody dosing time, group size, Staphylococcus aureus strain and dosage used to challenge, route of infection, take down day and read outs, and days for clinical observation.
  • Figures 27A-27B relate to promotion of clearance of Staphylococcus aureus infection by select n -Staphylococcus antibodies within a subset of treated mice in a disseminated infection model.
  • Figure 27A provides a graph showing effects of antibody treatment on bacterial kidney burden as measured by kidney CFU.
  • Figure 27B provides a graph showing effects of antibody treatment on weight loss.
  • Figure 28 provides graphs showing individual mouse weight loss within each antibody study group.
  • the antigen is an antigen that is also expressed by one or more (e.g., gram-positive) bacteria that is not of genus Staphylococcus, and the antibody or antigenbinding fragment is capable of binding thereto.
  • a "Staphylococcus antigen” is not limited to antigens expressed solely by bacteria of genus Staphylococcus and includes antigens that are expressed by Staphylococcus and by bacteria of one or more other genus.
  • the antigen is also expressed by a bacteria of genus Enterococcus (e.g., E. faecalis), a bacteria of genus Lactococcus (e.g., L.
  • the antibody or antigen-binding fragment is capable of binding to S. aureus e.g., MRS A), Coagulase-negative staphylococci (CoNS) such as S. epidermidis, S. pneumoniae, S. lugdunensis, S. hominis, or any combination thereof.
  • the antibody or antigen-binding fragment is capable of binding to: S. aureus FPR3757, S. aureus NE284 TE2 mutant, S. epidermidis RP62A, S. epidermidis 1200, S. epidermidis NIH04008, S. lugdunensis e.g., N860297), S. pyogenes, E. faecalis, S. agalactiae, S. hominis, L. garvieae, S. pneumoniae, E. coli, or any combination of thereof.
  • the antibody or antigen-binding fragment binds to LTA with higher affinity than does pagibaximab.
  • pagibaximab binds to an LTA with a Kd of 1.75E-1 IM and an antibody or antigen-binding fragment of the present disclosure binds to the LTA with a Kd of 3.48E-12M, as determined by surface plasmon resonance (SPR, e.g. using an Octet instrument).
  • the antibody or antigen-binding fragment binds to Hla with a Kd of 1.96E-10 M or of 6.14E-11 M, and MEDI4893 binds to the Hla with a Kd of 5.1E-10 by the same assay.
  • the antibody or antigen-binding fragment neutralizes Hla (e.g., in an assay using rabbit RBCs).
  • an antibody or antigen-binding fragment of the present disclosure is capable of binding to S. aureus HIgB.
  • an antibody or antigen-binding fragment of the present disclosure is capable of binding to a SpA.
  • the antibody or antigen-binding fragment is capable of binding to SpA with a Kd of about 2.46E-09 M, about 1.1 IE-08 M, about 7.633E- 10 M, 1.77E-09 M, or about 1.44E-09 M, as determined by surface plasmon resonance using an Octet instrument.
  • an antibody or antigen-binding fragment of the present disclosure is capable of binding to a ClfA.
  • the antibody or antigen-binding fragment is capable of binding to ClfA OOl, to ClfA_002 to ClfA_004, or to any combination thereof.
  • an antibody or antigen-binding fragment of the present disclosure is capable of binding to a Sbi.
  • an antibody or antigen-binding fragment is human, humanized, or chimeric.
  • the antibody or antigen-binding fragment comprises human amino acid sequences, e.g. : one, two, three, four, five, or six human CDR sequences; one, two, three, four, five, six, seven, or eight human variable domain framework region sequences.
  • the antibody or antigen-binding fragment comprises an Fc polypeptide, which can be, for example, a human Fc polypeptide or an engineered variant of a human Fc polypeptide.
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness are to be understood to include any integer within the recited range, unless otherwise indicated.
  • the term “about” means ⁇ 20% of the indicated range, value, or structure, unless otherwise indicated. It should be understood that the terms “a” and “an” as used herein refer to “one or more” of the enumerated components.
  • a “conservative substitution” refers to amino acid substitutions that do not significantly affect or alter binding characteristics of a particular protein. Generally, conservative substitutions are ones in which a substituted amino acid residue is replaced with an amino acid residue having a similar side chain. Conservative substitutions include a substitution found in one of the following groups: Group 1 : Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); Group 2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3: Asparagine (Asn or N), Glutamine (Gin or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (He or I), Leucine (Leu or L), Methionine (Met or M), Valine (Vai or V); and Group 6: Phenylalanine (Phe or F), Tyrosine (Tyr or
  • Modified nucleosides may include pseudouridine, such as N 1 - methylpseudouridine, 5-methylcytidine, 2-thiouridine, N6-methyladenonsine.
  • methylation of a naturally occurring (unmodified) nucleotide may be used alone or in combination with a modified nucleotide to achieve any of the above effects achievable using a modified nucleotide.
  • the proportion of nucleotides having a specific base or located in a specific sequence that are methylated may be used to achieve such effects.
  • methylation may be used to reduce recognition of the mRNA (or, in the case of DNA therapeutics, the DNA) as foreign in a mammalian host cell.
  • the mRNA, particularly saRNA or taRNA may include a cap or a cap analog, more specifically a 7-methylguanosine moiety linked via a phosphate, particularly a trisphosphate to an end nucleotide.
  • the including of a cap or cap analog may help prevent exonuclease cleavage of the mRNA, particularly saRNA or taRNA, and/or initiate translation of the mRNA, particularly saRNA or taRNA, in a mammalian host cell.
  • the cap or cap analog may initiate translation of the replication protein or peptide.
  • circRNA may lack a suitable location for a cap or cap analog because of the absence of an otherwise unbound 5’ end.
  • linear mRNA, prior to circularization to form circRNA may contain a cap or cap analog to, for example, increase stability and/or amplification of the linear mRNA prior to circularization.
  • UTRs may be 5’ and 3’ of the sequence encoding at least one, typically each, protein or peptide, or 5’ and 3’ of the sequence encoding at least two proteins or peptides, all proteins or peptides that function together once expressed (e.g. theVH and VL-containing sequences), or all proteins and peptides encoded by the mRNA.
  • the polyA tail prior to initial translation of mRNA, particularly saRNA or taRNA, particularly taRNA sequences encoding amplification proteins, containing the polyA tail, may have a length of at least or approximately 250, 200, 100, 50, 20, or 10 nucleotides, or a length in a range between 10 and 250, 10 and 200, 10 and 100, 10 and 50, 10 and 20, 20 and 250, 20 and 200, 20 and 100, 20 and 50, 50 and 250, 50 and 200, 50 and 100, 100 and 250, 100 and 200, or 200 and 250 nucleotides.
  • the mRNA, including circRNA, taRNA, or saRNA may be produced in a production host cell, which is a type of host cell.
  • the mRNA, including circRNA, taRNA, or saRNA may be produced in a cell-free system, such as a system using a DNA template and enzymes.
  • isolated can, in some embodiments, also describe an antibody, antigen-binding fragment, polynucleotide, vector, host cell, or composition that is outside of a human body. In certain embodiments, an isolated antibody, antigen-binding fragment, polynucleotide, vector, host cell, or composition is provided.
  • gene means the segment of DNA or RNA involved in producing a polypeptide chain; in certain contexts, it includes regions preceding and following the coding region (e.g., 5’ untranslated region (UTR) and 3’ UTR) as well as intervening sequences (introns) between individual coding segments (exons).
  • regions preceding and following the coding region e.g., 5’ untranslated region (UTR) and 3’ UTR
  • intervening sequences introns between individual coding segments (exons).
  • a “functional variant” refers to a polypeptide or polynucleotide that is structurally similar or substantially structurally similar to a parent or reference compound of this disclosure, but differs slightly in composition (e.g., one base, atom or functional group is different, added, or removed), such that the polypeptide or encoded polypeptide is capable of performing at least one function of the parent polypeptide with at least 50% efficiency, preferably at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or 100% level of activity of the parent polypeptide.
  • a functional variant of a polypeptide or encoded polypeptide of this disclosure has “similar binding,” “similar affinity” or “similar activity” when the functional variant displays no more than a 50% reduction in performance in a selected assay as compared to the parent or reference polypeptide, such as an assay for measuring binding affinity (e.g., Biacore® or tetramer staining measuring an association (Ka) or a dissociation (KD) constant).
  • binding affinity e.g., Biacore® or tetramer staining measuring an association (Ka) or a dissociation (KD) constant.
  • a “functional portion” or “functional fragment” refers to a polypeptide or polynucleotide that comprises only a domain, portion or fragment of a parent or reference compound, and the polypeptide or encoded polypeptide retains at least 50% activity associated with the domain, portion or fragment of the parent or reference compound, preferably at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least or 99.9%, or 100% level of activity of the parent polypeptide, or provides a biological benefit (e.g., effector function).
  • a biological benefit e.g., effector function
  • a “functional portion” or “functional fragment” of a polypeptide or encoded polypeptide of this disclosure has “similar binding” or “similar activity” when the functional portion or fragment displays no more than a 50% reduction in performance in a selected assay as compared to the parent or reference polypeptide (preferably no more than 20% or 10%, or no more than a log difference as compared to the parent or reference with regard to affinity).
  • homologous refers to a gene, protein, compound, nucleic acid molecule, or activity found in or derived from a host cell, species, or strain.
  • a heterologous or exogenous polynucleotide or gene encoding a polypeptide may be homologous to a native polynucleotide or gene and encode a homologous polypeptide or activity, but the polynucleotide or polypeptide may have an altered structure, sequence, expression level, or any combination thereof.
  • a non-endogenous polynucleotide or gene, as well as the encoded polypeptide or activity may be from the same species, a different species, or a combination thereof.
  • a nucleic acid molecule or portion thereof native to a host cell will be considered heterologous to the host cell if it has been altered or mutated, or a nucleic acid molecule native to a host cell may be considered heterologous if it has been altered with a heterologous expression control sequence or has been altered with an endogenous expression control sequence not normally associated with the nucleic acid molecule native to a host cell.
  • heterologous can refer to a biological activity that is different, altered, or not endogenous to a host cell.
  • endogenous or “native” refers to a polynucleotide, gene, protein, compound, molecule, or activity that is normally present in a host cell or a subject.
  • expression refers to the process by which a polypeptide is produced based on the encoding sequence of a nucleic acid molecule, such as a gene.
  • the process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof.
  • An expressed nucleic acid molecule is typically operably linked to an expression control sequence (e.g., a promoter).
  • operably linked refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other.
  • a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter).
  • Unlinked means that the associated genetic elements are not closely associated with one another and the function of one does not affect the other.
  • construct refers to any polynucleotide that contains a recombinant nucleic acid molecule (or, when the context clearly indicates, a fusion protein of the present disclosure).
  • a (polynucleotide) construct may be present in a vector (e.g., a bacterial vector, a viral vector) or may be integrated into a genome.
  • a “vector” is a nucleic acid molecule that is capable of transporting another nucleic acid molecule.
  • Vectors may be, for example, plasmids, cosmids, viruses, a RNA vector or a linear or circular DNA or RNA molecule that may include chromosomal, non-chromosomal, semi -synthetic or synthetic nucleic acid molecules.
  • Vectors of the present disclosure also include transposon systems (e.g., Sleeping Beauty, see, e.g., Geurts et al., Mol. Ther. 5: 108, 2003: Mates et al., Nat. Genet. 41'.753, 2009).
  • the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself or deliver the polynucleotide contained in the vector into the genome without the vector sequence.
  • plasmid “expression plasmid,” “virus,” and “vector” are often used interchangeably.
  • the “vector” may comprise or consist of mRNA, such as saRNA, taRNA, or circRNA.
  • the term “introduced” in the context of inserting a nucleic acid molecule into a cell means “transfection”, “transformation,” or “transduction” and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell wherein the nucleic acid molecule may be incorporated into the genome of a cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon (e.g. a replicon formed by saRNA), or transiently expressed e.g., transfected mRNA, such as circRNA, taRNA, or saRNA).
  • a cell e.g., chromosome, plasmid, plastid, or mitochondrial DNA
  • an autonomous replicon e.g. a replicon formed by saRNA
  • transiently expressed e.g., transfected mRNA, such as
  • “Retroviruses” are viruses having an RNA genome, which is reverse-transcribed into DNA using a reverse transcriptase enzyme, the reverse-transcribed DNA is then incorporated into the host cell genome.
  • “Gammaretrovirus” refers to a genus of the retroviridae family. Examples of gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses.
  • Lentiviral vectors include HIV-based lentiviral vectors for gene delivery, which can be integrative or non-integrative, have relatively large packaging capacity, and can transduce a range of different cell types. Lentiviral vectors are usually generated following transient transfection of three (packaging, envelope, and transfer) or more plasmids into producer cells. Like HIV, lentiviral vectors enter the target cell through the interaction of viral surface glycoproteins with receptors on the cell surface. On entry, the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The product of reverse transcription is a double-stranded linear viral DNA, which is the substrate for viral integration into the DNA of infected cells.
  • a host cell may include any individual cell or cell culture which may receive a vector or the incorporation of nucleic acids or express proteins. The term also encompasses progeny of the host cell, whether genetically or phenotypically the same or different. Suitable host cells may depend on the vector and may include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells. These cells may be induced to incorporate the vector or other material by use of a viral vector, transformation via calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection, or other methods. See, for example, Sambrook el al., Molecular Cloning: A Laboratory Manual 2d ed. (Cold Spring Harbor Laboratory, 1989).
  • epitope includes any molecule, structure, amino acid sequence, or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin, or other binding molecule, domain, or protein.
  • Epitopic determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • a bacteria of genus Streptococcus e.g., S. pyogenes, S. agalacliae
  • a bacteria of genus Listeria e.g., L. monocytogenes
  • a bacteria of genus Clostridium e.g., C. inocuum, C. difficile
  • a Type I LTA can be found on Staphylococcus aureus and other Staphylococci, as well as on Enterococci (e.g., E. faecalis), L.
  • binding can be determined by recombinantly expressing an antigen in a host cell (e.g, by transfection) and immunostaining the (e.g, fixed, or fixed and permeabilized) host cell with antibody and analyzing binding by flow cytometry (e.g., using a ZE5 Cell Analyzer (BioRad®) and FlowJo software (TreeStar).
  • positive binding can be defined by differential staining by antibody of antigen-expressing cells versus control (e.g., mock) cells.
  • the antibody or antigen-binding fragment is capable of binding to the antigen with a KD in a range from about 1.0E-9 M to about 3.5E-12 M, such as, for example, about 1.0E-9 M, about 1.5E-9 M, about 2.0E-9 M, about 2.5E-9 M, about 3.0E-9 M, about 3.5E-9 M, about 4.0E-9 M, about 4.5E-9 M, about 5.0E-9 M, about 5.5E-9 M, about 6.0E-
  • the binding is as assessed by biolayer interferometry (BLI). In other embodiments, the binding is as assessed by surface plasmon resonance (SPR).
  • the antibody or antigen-binding fragment is capable of activating a human FcyRIIIa.
  • activation is as determined using a host cell (optionally, a Jurkat cell) comprising: (i) the human FcyRIIIa (optionally, a F158 allele); and (ii) a NF AT expression control sequence operably linked to a sequence encoding a reporter, such as a luciferase reporter, following incubation (e.g., of 23 hours) of the antibody or antigen-binding fragment with a target cell (e.g., a A549 cell) infected with an antigen-expressing bacteria.
  • the antibody or antigen-binding fragment is capable of treating and/or preventing an infection by an antigen-expressing bacteria in a subject.
  • antibody refers to an intact antibody comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as any antigen-binding portion or fragment of an intact antibody that has or retains the ability to bind to the antigen target molecule recognized by the intact antibody, such as an scFv, Fab, or Fab’2 fragment.
  • antibody herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen binding (Fab) fragments, F(ab’)2 fragments, Fab’ fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments.
  • Fab fragment antigen binding
  • rlgG recombinant IgG
  • scFv single chain variable fragments
  • single domain antibodies e.g., sdAb, sdFv, nanobody
  • the term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, and tandem tri-scFv.
  • antibody should be understood to encompass functional antibody fragments thereof.
  • the term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof (IgGl, IgG2, IgG3, IgG4), IgM, IgE, IgA, and IgD.
  • variable binding region also called variable region or variable binding domain or variable domain
  • a VL is a kappa (K) class (also “VK” herein).
  • a VL is a lambda ( ) class.
  • the variable binding regions comprise discrete, well-defined sub-regions known as “complementarity determining regions” (CDRs) and “framework regions” (FRs).
  • CDR complementarity determining region
  • HVR hypervariable region
  • an antibody VH comprises four FRs and three CDRs as follows: FR1- HCDR1-FR2-HCDR2-FR3-HCDR3-FR4; and an antibody VL comprises four FRs and three CDRs as follows: FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4.
  • the VH and the VL together form the antigen-binding site (or domain) through their respective CDRs.
  • one or more CDRs do not contact antigen and/or do not contribute energetically to antigen binding (but at least one CDR does contact antigen).
  • a “variant” of a CDR refers to a functional variant of a CDR sequence having up to 1-3 amino acid substitutions (e.g., conservative or non-conservative substitutions), deletions, or combinations thereof.
  • Numbering of CDR and framework regions may be according to any known method or scheme, such as the Kabat, Chothia, EU, IMGT, Contact, North, Martin, AbM, and Aho numbering schemes see, e.g., Kabat et al., “Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5 th ed.; Chothia and Lesk, J. Mol. Biol. 796:901-917 (1987)); Lefranc etal., Dev. Comp. Immunol. 27:55, 2003; Honegger and Pliickthun, J. Mol. Bio. 309:657-670 (2001); North et al. J Mol Biol.
  • an antibody or an antigen-binding fragment of the present disclosure comprises a CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2, and a CDRL3, wherein each CDR is independently selected from a corresponding CDR of an antigen-specific antibody as provided in Table 1, Table 2, Table 3, or Table 4. That is, all combinations of CDRs from antigen-specific antibodies provided in Table 1 and/or Table 2 and/or Table 3 and/or Table 4 are contemplated.
  • an antibody or an antigen-binding fragment of the present disclosure comprises a CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2, and a CDRL3 of any one of the antibodies shown in Table 1 or 2.
  • CDRs are in accordance with the IMGT numbering method.
  • Table 2 summarizes IMGT CDR amino acid sequences (SEQ ID NOs.) of certain antibodies of the present disclosure.
  • Table 3 summarizes variable domain amino acid sequences and examples of corresponding codon-optimized polynucleotide sequences (SEQ ID NOS.) of certain antibodies of the present disclosure.
  • Table 3 summarizes variable domain amino acid sequences and examples of corresponding codon-optimized polynucleotide sequences (SEQ ID NOS.) of certain antibodies of the present disclosure.
  • Table 3 Amino acid and certain polynucleotide sequences (SEQ ID NOs.) of variable domains of certain antibodies
  • an antibody or antigen-binding fragment comprises a CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2, and/or a CDRL3, or functional variants thereof (and in certain embodiments, comprises a CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2, and a CDRL3, or functional variants thereof) of the VH and VL amino acid sequences set forth in SEQ ID NOs.: (i) 41 and 45, respectively; (ii) 31 and 35, respectively; (iii) 121 and 125, respectively; (iv) 21 and 25, respectively; (v) 131 and 135, respectively; (vi) 141 and 145, respectively; (vii) 151 and 155, respectively; (viii) 161 and 165, respectively; (ix) 241 and 245, respectively; (x) 171 and 175, respectively; (xi) 1 and 5, respectively; (xii) 11 and 15, respectively; (xiii)
  • an antibody or antigen-binding fragment comprises a CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2, and a CDRL3, and optionally a VH and a VL, of one of the following antibodies as set forth in Table 2 and Table 3: SSG20; SSC35; SSE73; SSA12; SSA8; SSA9; SSA10; SSC1; SSC10; SSC15; SSE1; SSE158; SSF2; SSF11; SSF10; SSB18; SSB24; SSB54; SSB65; SSB67; SSH3; SSF33; SSL5; SSL17; SSL35; SSG50; SSG54; SSM47; SSG44; SSG48; SSM37; SSH4; SSH7; SSG27; SSG39; SSM45;
  • the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are according to the IMGT numbering system (optionally using the junction definitions for CDR3 sequences). In other embodiments, the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are according to the Kabat numbering system. In other embodiments, the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are according to the Chothia numbering system. In other embodiments, the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are according to the Enhanced Chothia (also referred to as “Martin”) numbering system.
  • Enhanced Chothia also referred to as “Martin”
  • the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are according to the AHo numbering system. In other embodiments, the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are according to the North numbering system. In other embodiments, the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are according to the Contact numbering system. In other embodiments, the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are according to the EU numbering system.
  • the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are according to the AbM numbering system (AbM antibody modelling software from Oxford Molecular). In some embodiments, the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are according to a combination of any two or more of IMGT, Kabat, Chothia, Enhanced Chothia, AHo, EU, North, AbM, and Contact. In some embodiments, the two or more numbering systems combined produce the CDR definition having the greatest length of the CDR according to any numbering system or combination of numbering systems.
  • an antibody or antigen-binding fragment comprises:
  • a CDRH1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs.: 42, 32, 122, 22, 132, 142, 152, 162, 242, 172, 2, 12, 182, 52, 192, 212, 102, 222, 112, 232, 82, 92, 62, 72, 202, 281, 291, 301, 311, 321, 331, 341, 351, 361, 371, 381, 391, 401, 411, 421, 433, 443, 453, 463, 473, 483, 493, 503, 513, 523, 533, 543, 553, 563, 573, 583, 593, 603, 613, 623, 633, 643, 653, 663, 673, 683, 693, 703, 713, 723, 733, 743, and 753, or a sequence variant thereof comprising one, two, or three acid substitutions, one or more of which substitutions is optionally a conservative substitution and/
  • a CDRH2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs. :43, 33, 123, 23, 133, 143, 153, 163, 243, 173, 3, 13, 183, 53, 193, 213, 103, 223, 113, 233, 83, 93, 63, 73, 203, 282, 292, 302, 312, 322, 332, 342,
  • a CDRH3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs.: 44, 34, 124, 24, 134, 144, 154, 164, 244, 174, 4,
  • a CDRL1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs.:46, 36, 126, 26, 136, 146, 156, 166, 246, 176, 6,
  • a CDRL2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs.:47, 37, 127, 27, 137, 147, 157, 167, 247, 177, 7,
  • a CDRL3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs. :48, 38, 128, 28, 138, 148, 158, 168, 248, 178, 8,
  • the antibody or antigen-binding fragment comprises a CDRH3 and a CDRL3, wherein the CDRH3 and the CDRL3 comprise, consist essentially of, or consist of the amino acid sequences set forth in SEQ ID NOS.: (i) 44 and 48, respectively; (ii) 34 and 38, respectively; (iii) 124 and 128, respectively; (iv) 24 and 28, respectively; (v) 134 and 138, respectively; (vi) 144 and 148, respectively; (vii) 154 and 158, respectively; (viii) 164 and 168, respectively; (ix) 244 and 248, respectively; (x) 174 and 178, respectively; (xi) 4 and 8, respectively; (xii) 14 and 18, respectively; (xiii) 184 and 188, respectively; (xiv) 54 and 58, respectively; (xv) 194 and 198, respectively; (xvi) 214 and 218, respectively; (xvii) 104 and 108, respectively; (xviii)
  • the antibody or antigen-binding fragment comprises a CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise, consist essentially of, or consist of the amino acid sequences set forth in SEQ ID NOS.: (i) 42-44 and 46-48, respectively; (ii) 32-34 and 36- 38, respectively; (iii) 122-124 and 126-128, respectively; (iv) 22-24 and 26-28, respectively; (v) 132-134 and 136-138, respectively; (vi) 142-144 and 146-148, respectively; (vii) 152-154 and 156-158, respectively; (viii) 162-164 and 166-168, respectively; (ix) 242-244 and 246-248, respectively; (x) 172-174 and 176-178, respectively; (xi) 2-4 and 6-8, respectively; (xii)
  • an antibody or antigen-binding fragment comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity or similarity to, or comprising, consisting essentially of, or consisting of, the amino acid sequence encoded by: IGHV3-23; IGHV3-7; IGHJ1; IGHJ3; IGLV3-21; IGKV1-5; IGKV1- 6; IGLJ2; IGKJ2; IGKJ1; IGHV1-8; IGHV3-30; IGHV3-49; IGHV4-39; IGHJ6; IGHJ3; IGHJ4; IGKV1-27; IGLV3-21; IGKV1-5; IGKV1D-12; IGKV1-9; IGLV1-40;
  • an antibody or antigen-binding fragment comprises amino acid sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity or similarity to, or comprising, consisting essentially of, or consisting of, the amino acid sequences encoded by: (i) IGHV3-23, IGHJ1, IGLV3-21, and IGLJ2; (ii) IGHV3-23, IGHJ3, IGKV1-5, and IGKJ1; (iii) IGHV3-7, IGHJ3, IGKV1-6, and IGKJ1; (iv) IGHV3-7, IGHJ3, IGKV1-6, and IGKJ2; (v) IGHV3-23, IGHJ1, IGLV3-21, and IGLJ2; (vi) IGHV3-23, IGHJ3, IGHJ3, I
  • an antibody or antigen-binding fragment comprises a framework amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity or similarity to, or comprising or consisting of, an amino acid sequence encoded by: IGHV3-23; IGHV3-7; IGHJ1; IGHJ3; IGLV3-21; IGKV1-5; IGKV1-6; IGLJ2; IGKJ2; IGKJ1; IGHV1-8; IGHV3-30; IGHV3-49; IGHV4-39; IGHJ6; IGHJ3; IGHJ4; IGKV1- 27; IGLV3-21; IGKV1-5; IGKV1D-12; IGKV1-9; IGLV1-40; IGVK1-6;
  • an antibody or antigen-binding fragment comprises framework amino acid sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity or similarity to, or comprising or consisting essentially of or consisting of, the framework amino acid sequences encoded by: (i) IGHV3-23, IGHJ1, IGLV3-21, and IGLJ2; (ii) IGHV3-23, IGHJ3, IGKV1-5, and IGKJ1; (iii) IGHV3-7, IGHJ3, IGKV1-6, and IGKJ1; (iv) IGHV3-7, IGHJ3, IGKV1-6, and IGKJ2; (v) IGHV3-23, IGHJ1, IGLV3-21, and IGLJ2; (vi) IGHV3-23, IGHJ3, IGHJ3,
  • Framework amino acid sequences can be identified by a same numbering scheme that is used to identify CDRs; e.g., IMGT, Kabat, Chothia, Enhanced Chothia, AbM, AHo, North, Martin, Contact, or any combination thereof.
  • an antibody or antigen-binding fragment comprises a VH framework region (VHFR)1, a VHFR2, a VHFR3, a VL framework region (VLFR)1, a VLFR2, a VLFR3, and/or a VLFR4 (or a variant of the VHFR1, VHFR2, VHFR3, VHFR4, VLFR1, VLFR2, VLFR3, or VLFR4 comprising one, two, three, four, or five substitutions, insertions, and/or deletions, or a variant having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity or similarity to the VHFR1, VHFR2, VHFR3, VHFR4, VLFR1, VLFR2, VLFR3, or VLFR4, respectively) of the VH and VL amino
  • the framework region or regions are according to the IMGT numbering scheme. In some embodiments, the framework region or regions are according to the Kabat numbering scheme. In some embodiments, the framework region or regions are according to the Chothia numbering scheme. In some embodiments, the framework region or regions are according to the Enhanced Chothia numbering scheme. In some embodiments, the framework region or regions are according to the AbM numbering scheme. In some embodiments, the framework region or regions are according to the EU numbering scheme. In some embodiments, the framework region or regions are according to the North numbering scheme. In some embodiments, the framework region or regions are according to the Contact numbering scheme. In some embodiments, the framework region or regions are according to the AHo numbering scheme. In some embodiments, the framework region or regions are according to a combination of any two or more of the following numbering schemes: IMGT, Kabat Chothia, Enhanced Chothia, AbM, AHo, EU, North, Contact.
  • the antibody or antigen-binding fragment comprises a VH and a VL, wherein the VH and the VL comprise, consist essentially of, or consist of, amino acid sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity or similarity to, or comprising or consisting essentially of or consisting of, the VH and VL amino acid sequences set forth in SEQ ID NOs.: (i) 41 and 45, respectively; (ii) 31 and 35, respectively; (iii) 121 and 125, respectively; (iv) 21 and 25, respectively; (v) 131 and 135, respectively; (vi) 141 and 145, respectively; (vii) 151 and 155, respectively; (viii) 161 and 165, respectively; (ix) 241 and 245, respectively; (x) 171 and
  • DNA in the germline variable (V), joining (J), and diversity (D) gene loci may be rearranged and insertions and/or deletions of nucleotides in the coding sequence may occur. Somatic mutations may be encoded by the resultant sequence, and can be identified by reference to a corresponding known germline sequence.
  • somatic mutations that are not critical to a desired property of the antibody e.g., binding to a herein-disclosed antigen
  • that confer an undesirable property upon the antibody e.g., an increased risk of immunogenicity in a subject administered the antibody
  • the antibody or antigenbinding fragment of the present disclosure comprises at least one more germline-encoded amino acid in a variable region as compared to a parent antibody or antigen-binding fragment, provided that the parent antibody or antigen binding fragment comprises one or more somatic mutations.
  • Variable region and CDR amino acid sequences (SEQ ID NOs.) of certain antibodies of the present disclosure are provided in Tables 2-4 herein.
  • CL refers to an “immunoglobulin light chain constant region” or a “light chain constant region,” z.e., a constant region from an antibody light chain.
  • CH refers to an “immunoglobulin heavy chain constant region” or a “heavy chain constant region,” which is further divisible, depending on the antibody isotype, into CHI, CH2, and CH3 (IgA, IgD, IgG), or CHI, CH2, CH3, and CH4 domains (IgE, IgM).
  • CHI immunoglobulin heavy chain constant region
  • an antibody or antigen-binding fragment of the present disclosure comprises any one or more of CL, a CHI, a CH2, and a CH3. In any of the presently disclosed embodiments, an antibody or antigen-binding fragment of the present disclosure may comprise any one or more of CL, a CHI, a CH2, and a CH3.
  • a CL comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity (or similarity) to the amino acid sequence of a human IgG kappa constant domain (e.g., to SEQ ID NO.:278) or to a human IgG lambda constant domain (e.g., to SEQ ID NO.:279).
  • a CH1-CH2-CH3 comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity (or similarity) to the amino acid sequence of a human IgGl isotype, an engineered human IgGl isotype, a human IgG3 isotype, or an engineered human IgG3 isotype.
  • a CH1-CH2-CH3 comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity (or similarity) to the amino acid sequence of any one of SEQ ID NOs.:251-277 and 765. It will be understood that, for example, production in a mammalian cell line can remove one or more C-terminal lysine of an antibody heavy chain (see, e.g., Liu et al. mAbs 6(5): 1145-1154 (2014)).
  • an antibody or antigen-binding fragment of the present disclosure can comprise a heavy chain, a CH1-CH3, a CH3, or an Fc polypeptide wherein a C-terminal lysine residue is present or is absent; in other words, encompassed are embodiments where the C-terminal residue of a heavy chain, a CH1-CH3, or an Fc polypeptide is not a lysine, and embodiments where a lysine is the C-terminal residue.
  • a composition comprises a plurality of an antibody and/or an antigenbinding fragment of the present disclosure, wherein one or more antibody or antigen-binding fragment does not comprise a lysine residue at the C-terminal end of the heavy chain, CH1-CH3, or Fc polypeptide, and wherein one or more antibody or antigen-binding fragment comprises a lysine residue at the C-terminal end of the heavy chain, CH1-CH3, or Fc polypeptide.
  • an antibody or antigen-binding fragment of the present disclosure can comprise a heavy chain, a CH1-CH3, a CH3, or an Fc polypeptide wherein a C-terminal glycine-lysine sequence e.g., the last two amino acids of SEQ ID NO.:251) is present or is absent.
  • a “Fab” fragment antigen binding is the part of an antibody that binds to antigens and includes the variable region and CHI of the heavy chain linked to the light chain via an interchain disulfide bond. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site (or domain). Pepsin treatment of an antibody yields a single large F(ab’)2 fragment that roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen.
  • Fab fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region.
  • Fab’-SH is the designation herein for Fab’ in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab’)2 antibody fragments originally were produced as pairs of Fab’ fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • Fv is a small antibody fragment that contains a complete antigen-recognition and antigen-binding site (or domain). This fragment generally consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although typically at a lower affinity than the entire binding site (or domain).
  • Single-chain Fv also abbreviated as “sFv” or “scFv”, are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain.
  • the scFv polypeptide comprises a polypeptide linker disposed between and linking the VH and VL domains that enables the scFv to retain or form the desired structure for antigen binding.
  • a peptide linker can be incorporated into a fusion polypeptide using standard techniques well known in the art.
  • the antibody or antigenbinding fragment comprises a scFv comprising a VH domain, a VL domain, and a peptide linker linking the VH domain to the VL domain.
  • a scFv comprises a VH domain linked to a VL domain by a peptide linker, which can be in a VH-linker-VL orientation or in a VL-linker-VH orientation.
  • Peptide linker sequences may be chosen, for example, based on: (1) their ability to adopt a flexible extended conformation; (2) their inability or lack of ability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides and/or on a target molecule; and/or (3) the lack or relative lack of hydrophobic or charged residues that might react with the polypeptides and/or target molecule.
  • linker design e.g., length
  • linker design can include the conformation or range of conformations in which the VH and VL can form a functional antigen-binding site (or domain).
  • peptide linker sequences contain, for example, Gly, Asn and Ser residues.
  • linker sequence may also be included in a linker sequence.
  • Other amino acid sequences which may be usefully employed as linker include those disclosed in Maratea et al., Gene 40:39 46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258 8262 (1986); U.S. Pat. No. 4,935,233, and U.S. Pat. No. 4,751,180.
  • linkers may include, for example, Glu-Gly-Lys-Ser-Ser-Gly-Ser-Gly-Ser-Glu-Ser-Lys-Val-Asp (Chaudhary et al., Proc. Natl. Acad. Sci.
  • Any suitable linker may be used, and in general can be about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 15 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100 amino acids in length, or less than about 200 amino acids in length, and will preferably comprise a flexible structure (can provide flexibility and room for conformational movement between two regions, domains, motifs, fragments, or modules connected by the linker), and will preferably be biologically inert and/or have a low risk of immunogenicity in a human.
  • ScFvs can be constructed using any combination of the VH and VL sequences or any combination of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences disclosed herein.
  • linker sequences are not required; for example, when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
  • an antibody or antigen-binding fragment of the present disclosure is monospecific (e.g., binds to a single epitope) or is multispecific (e.g., binds to multiple epitopes and/or target molecules).
  • Antibodies and antigen binding fragments may be constructed in various formats. Exemplary antibody formats disclosed in Spiess et al., Mol. Immunol.
  • FIT-Ig e.g., PCT Publication No.
  • the antibody or antigen-binding fragment comprises two or more of VH domains, two or more VL domains, or both (i.e., two or more VH domains and two or more VL domains).
  • an antigen-binding fragment comprises the format (N-terminal to C-terminal direction) VH-linker-VL-linker-VH-linker-VL, wherein the two VH sequences can be the same or different and the two VL sequences can be the same or different.
  • Such linked scFvs can include any combination of VH and VL domains arranged to bind to a given target, and in formats comprising two or more VH and/or two or more VL, one, two, or more different epitopes or antigens may be bound. It will be appreciated that formats incorporating multiple antigen-binding domains may include VH and/or VL sequences in any combination or orientation.
  • the antigen-binding fragment can comprise the format VL-linker-VH-linker-VL-linker-VH, VH-linker-VL-linker-VL-linker-VH, or VL-linker-VH- linker-VH-linker-VL.
  • Monospecific or multispecific antibodies or antigen-binding fragments of the present disclosure can comprise any combination of the VH and VL sequences and/or any combination of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences disclosed herein.
  • a bispecific or multispecific antibody or antigen-binding fragment may, in some embodiments, comprise one, two, or more antigen-binding domains (e.g., a VH and a VL) of the instant disclosure.
  • Two or more binding domains may be present that bind to the same or a different antigen epitope, and a bispecific or multispecific antibody or antigen-binding fragment as provided herein can, in some embodiments, comprise a further antigen-specific binding domain, and/or can comprise a binding domain that binds to a different antigen or pathogen altogether.
  • the antibody or antigen-binding fragment can be multispecific; e.g., bispecific, trispecific, or the like.
  • an antibody, or an antigen-binding fragment thereof which is a multi-specific antibody or antigen-binding fragment thereof, which is capable of binding to: an LTA and a Gmd; an LTA and an alpha toxin; an LTA and a gamma toxin; an LTA and a leucocidin; an LTA and a SpA; an LTA and a ClfA; an alpha toxin and a Gmd; an alpha toxin and a gamma toxin; an alpha toxin and a leucocidin; an alpha toxin and a SpA; an alpha toxin and a ClfA; a gamma toxin and a Gmd; a gamma toxin and a leucocidin; a gamma toxin and a SpA; a gamma toxin and a ClfA; a leucocidin and a Sp
  • each antigen-binding domain of a multispecific antibody or antigen-binding fragment is capable of targeting a different binding site of the same target selected from: an LTA, an alpha toxin, a gamma toxin, a SpA, a ClfA, a Gmd, a Sbi, and a leucocidin.
  • a multispecific antibody or antigen-binding fragment comprises at least two antigen-binding sites, wherein at least one antigen-binding site is capable of targeting a first binding site of a target selected from: an LTA, an alpha toxin, a gamma toxin, a SpA, a ClfA, a Gmd, a Sbi, and a leucocidin; and wherein at least one antigen-binding site is capable of targeting a second binding site of the target.
  • a first antigen-binding domain of a multispecific antibody or antigen-binding fragment binds to an opsonizing target and a second antigen-binding domain of the multispecific antibody or antigen-binding fragment binds to a neutralizing target.
  • a first antigen-binding domain of a multispecific antibody or antigen-binding fragment binds to a target selected from LTA, ClfA, Protein A, and Gmd, and a second antigenbinding domain of the multispecific antibody or antigen-binding fragment binds to an alpha toxin.
  • a first antigen-binding domain of a multispecific antibody or antigen-binding fragment binds to an opsonizing target and a second antigen-binding domain of the multispecific antibody or antigen-binding fragment binds to an alpha toxin.
  • a first antigen-binding domain of a multispecific antibody or antigen-binding fragment binds to a target selected from LTA, ClfA, Protein A, and Gmd and a second antigen-binding domain of the multispecific antibody or antigen-binding fragment binds to a neutralizing target.
  • fusion proteins that comprise an antibody or antigen-binding fragment of the present disclosure.
  • a fusion protein comprises (i) an extracellular component comprising the antibody or antigen-binding fragment, (ii) a transmembrane component, and (iii) an intracellular component comprising one or more signaling domains (e.g., from CD3( ⁇ , CD28, 4-1BB, and/or TLR8).
  • a fusion protein comprises a chimeric antigen receptor.
  • a fusion protein comprises a chimeric engulfment receptor (see, e.g., Corey el al. , Molecular Therapy Methods & Clinical Development 28: 1-10 (2023); doi . org/ 10.1016/j . omtim .2022.11.004).
  • the antibody or antigen-binding fragment comprises a Fc polypeptide, or a fragment thereof.
  • the “Fc” fragment or Fc polypeptide comprises the carboxyterminal portions i.e., the CH2 and CH3 domains of IgG) of both antibody H chains held together by disulfides.
  • An Fc may comprise a dimer comprised of two Fc polypeptides (i.e., two CH2-CH3 polypeptides).
  • Antibody “effector functions” refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype.
  • antibody effector functions include: Clq binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.
  • modifications e.g., amino acid substitutions
  • Fc domain in order to modify (e.g., improve, reduce, or ablate) one or more functionality of an Fc-containing polypeptide (e.g., an antibody of the present disclosure).
  • Such functions include, for example, Fc receptor (FcR) binding, antibody half-life modulation (e.g., by binding to FcRn), ADCC function, protein A binding, protein G binding, and complement binding.
  • Amino acid modifications that modify (e.g., improve, reduce, or ablate) Fc functionalities include, for example, the T250Q/M428L, M252Y/S254T/T256E, H433K/N434F, M428L/N434S, M428L/434A, E233P/L234V/L235A/G236 + A327G/A330S/P331S, E333A, S239D/A330L/I332E, P257VQ311, K326W/E333S, S239D/I332E/G236A, N297Q, K322A, S228P, L235E + E318A/K320A/K322A, L234A/L235A (also referred to herein as “LALA”), and L234A/L235A/P329G mutations, and other mutations described herein, certain of which mutations are summarized and annotated in
  • the Clq protein complex can bind to at least two molecules of IgGl or one molecule of IgM when the immunoglobulin molecule(s) is attached to the antigenic target (Ward, E. S., and Ghetie, V., Ther. Immunol. 2 (1995) 77-94).
  • Burton, D. R. described (Mol. Immunol. 22 (1985) 161-206) that the heavy chain region comprising amino acid residues 318 to 337 is involved in complement fixation.
  • FcR binding can be mediated by the interaction of the Fc moiety (of an antibody) with Fc receptors (FcRs), which are specialized cell surface receptors on cells including hematopoietic cells.
  • Fc receptors belong to the immunoglobulin superfamily, and shown to mediate both the removal of antibody-coated pathogens by phagocytosis of immune complexes, and the lysis of erythrocytes and various other cellular targets (e.g. tumor cells) coated with the corresponding antibody, via antibody dependent cell mediated cytotoxicity (ADCC; Van de Winkel, J. G., and Anderson, C. L., J. Leukoc. Biol. 49 (1991) 511-524).
  • ADCC antibody dependent cell mediated cytotoxicity
  • FcRs are defined by their specificity for immunoglobulin classes; Fc receptors for IgG antibodies are referred to as FcyR, for IgE as FcsR, for IgA as FcaR and so on and neonatal Fc receptors are referred to as FcRn.
  • Fc receptor binding is described for example in Ravetch, J. V., and Kinet, J. P., Annu. Rev. Immunol. 9 (1991) 457-492; Capel, P. J., et al., Immunomethods 4 (1994) 25-34; de Haas, M., et al., J Lab. Clin. Med. 126 (1995) 330-341; and Gessner, J. E., et al., Ann. Hematol. 76 (1998) 231-248.
  • FcyR Fc domain of native IgG antibodies
  • FcyR In humans, three classes of FcyR have been characterized to-date, which are: (i) FcyRI (CD64), which binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils and eosinophils; (ii) FcyRII (CD32), which binds complexed IgG with medium to low affinity, is widely expressed, in particular on leukocytes, is believed to be a central player in antibody-mediated immunity, and which can be divided into FcyRIIA, FcyRIIB and FcyRIIC, which perform different functions in the immune system, but bind with similar low affinity to the IgG-Fc, and the ectodomains of these receptors are highly homologuous; and (iii) FcyRIII (CD 16), which binds IgG with medium to low affinity and has been found in two forms: FcyRIIIA, which has been found on NK cells, macrophages,
  • FcyRIIA is found on many cells involved in killing (e.g. macrophages, monocytes, neutrophils) and seems able to activate the killing process.
  • FcyRIIB seems to play a role in inhibitory processes and is found on B-cells, macrophages and on mast cells and eosinophils. Importantly, it has been shown that 75% of all FcyRIIB is found in the liver (Ganesan, L. P. et al., 2012: “FcyRIIb on liver sinusoidal endothelium clears small immune complexes,” loumal of Immunology 189: 4981-4988).
  • FcyRIIB is abundantly expressed on Liver Sinusoidal Endothelium, called LSEC, and in Kupffer cells in the liver and LSEC are the major site of small immune complexes clearance (Ganesan, L. P. et al., 2012: FcyRIIb on liver sinusoidal endothelium clears small immune complexes. lournal of Immunology 189: 4981-4988).
  • the antibodies disclosed herein and the antigen-binding fragments thereof comprise an Fc polypeptide or fragment thereof for binding to FcyRIIb, in particular an Fc region, such as, for example IgG-type antibodies.
  • FcyRIIb an Fc region
  • it is possible to engineer the Fc moiety to enhance FcyRIIB binding by introducing the mutations S267E and L328F as described by Chu, S. Y. et al., 2008: Inhibition of B cell receptor-mediated activation of primary human B cells by co-engagement of CD19 and FcgammaRIIb with Fc-engineered antibodies.
  • Molecular Immunology 45, 3926-3933 are examples of the FcyRIIb.
  • the antibodies of the present disclosure comprise an engineered Fc moiety with the mutations S267E and L328F, in particular as described by Chu, S. Y. et al., 2008: Inhibition of b cell receptor-mediated activation of primary human B cells by co-engagement of CD19 and FcgammaRIIb with Fc-engineered antibodies.
  • FcyRIIB may function to suppress further immunoglobulin production and isotype switching to, for example, the IgE class.
  • FcyRIIB On macrophages, FcyRIIB is thought to inhibit phagocytosis as mediated through FcyRIIA.
  • the B form On eosinophils and mast cells, the B form may help to suppress activation of these cells through IgE binding to its separate receptor.
  • modification in native IgG of at least one of E233-G236, P238, D265, N297, A327 and P329 reduces binding to FcyRI.
  • IgG2 residues at positions 233-236, substituted into corresponding positions IgGl and IgG4, reduces binding of IgGl and IgG4 to FcyRI by 10 3 -fold and eliminated the human monocyte response to antibody-sensitized red blood cells (Armour, K. L., et al. Eur. J. Immunol. 29 (1999) 2613-2624).
  • FcyRIIA reduced binding for FcyRIIA is found, e.g., for IgG mutation of at least one of E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292 and K414.
  • FcyRIII binding reduced binding to FcyRIIIA is found, e.g., for mutation of at least one of E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338 and D376. Mapping of the binding sites on human IgGl for Fc receptors, the above-mentioned mutation sites, and methods for measuring binding to FcyRI and FcyRIIA, are described in Shields, R. L., et al., J. Biol. Chem. 276 (2001) 6591-6604.
  • FcyRIIIA Two allelic forms of human FcyRIIIA are the “Fl 58” variant, which binds to IgGl Fc with lower affinity, and the “V158” variant, which binds to IgGl Fc with higher affinity. See, e.g., Bruhns et al., Blood 11331X6-3125 (2009).
  • two regions of native IgG Fc appear to be involved in interactions between FcyRIIs and IgGs, namely (i) the lower hinge site of IgG Fc, in particular amino acid residues L, L, G, G (234 - 237, EU numbering), and (ii) the adjacent region of the CH2 domain of IgG Fc, in particular a loop and strands in the upper CH2 domain adjacent to the lower hinge region, e.g. in a region of P331 (Wines, B.D., et al., I. Immunol. 2000; 164: 5313 - 5318).
  • FcyRI appears to bind to the same site on IgG Fc
  • FcRn and Protein A bind to a different site on IgG Fc, which appears to be at the CH2-CH3 interface
  • mutations that increase binding affinity of an Fc polypeptide or fragment thereof of the present disclosure to a (i.e., one or more) Fey receptor (e.g., as compared to a reference Fc polypeptide or fragment thereof or containing the same that does not comprise the mutation(s)). See, e.g., Delillo and Ravetch, Cell 161(5): 1035-1045 (2015) and Ahmed et al., J. Struc. Biol. 194(1):78 (2016), the Fc mutations and techniques of which are incorporated herein by reference.
  • an antibody or antigen-binding fragment can comprise a Fc polypeptide or fragment thereof comprising a mutation selected from G236A; S239D; A330L; and I332E; or a combination comprising any two or more of the same; e.g., S239D/I332E; S239D/A330L/I332E; G236A/S239D/I332E; G236A/A330L/I332E (also referred to herein as “GAALIE”); or G236A/S239D/A330L/I332E.
  • the Fc polypeptide or fragment thereof does not comprise S239D.
  • the Fc polypeptide or fragment thereof comprises S at position 239 (EU numbering).
  • the Fc polypeptide or fragment thereof may comprise or consist of at least a portion of an Fc polypeptide or fragment thereof that is involved in FcRn binding.
  • the Fc polypeptide or fragment thereof comprises one or more amino acid modifications that improve binding affinity for (e.g., enhance binding to) FcRn (e.g., at a pH of about 6.0) and, in some embodiments, thereby extend in vivo half-life of a molecule comprising the Fc polypeptide or fragment thereof (e.g., as compared to a reference Fc polypeptide or fragment thereof or antibody that is otherwise the same but does not comprise the modification(s)).
  • the Fc polypeptide or fragment thereof comprises or is derived from a IgG Fc and a half-life-extending mutation comprises any one or more of: M428L; N434S; N434H; N434A; N434S; M252Y; S254T; T256E; T250Q; P257I; Q311I; D376V; T307A; E380A (EU numbering).
  • a half-life-extending mutation comprises M428L/N434S (also referred to herein as “MLNS”, “LS”, “_LS”, and “-LS”).
  • a half-life-extending mutation comprises M252Y/S254T/T256E.
  • a half-life-extending mutation comprises T250Q/M428L. In certain embodiments, a half-life-extending mutation comprises P257EQ311I. In certain embodiments, a half-life-extending mutation comprises P257I/N434H. In certain embodiments, a half-life- extending mutation comprises D376V/N434H. In certain embodiments, a half-life-extending mutation comprises T307A/E380A/N434A. In certain embodiments, a half-life-extending mutation comprises M428L/N434A.
  • an antibody or antigen-binding fragment includes a Fc moiety that comprises the substitution mutations M428L/N434S. In some embodiments, an antibody or antigen-binding fragment includes a Fc moiety that comprises the substitution mutations M428L/N434A. In some embodiments, an antibody or antigen-binding fragment includes a Fc polypeptide or fragment thereof that comprises the substitution mutations G236A/A330L/I332E.
  • an antibody or antigen-binding fragment includes a (e.g., IgG) Fc moiety that comprises a G236A mutation, an A330L mutation, and a I332E mutation (GAALIE), and does not comprise a S239D mutation (e.g., comprises a native S at position 239).
  • an antibody or antigen-binding fragment includes an Fc polypeptide or fragment thereof that comprises the substitution mutations: M428L/N434S and G236A/A330L/I332E, and optionally does not comprise S239D (e.g., comprises S at 239).
  • an antibody or antigen-binding fragment includes a Fc polypeptide or fragment thereof that comprises the substitution mutations: M428L/N434S and G236 A/S239D/A330L/I332E.
  • an antibody or antigen-binding fragment comprises an IgGl Fc polypeptide (or an engineered variant thereof) comprising an H435R mutation, a Y436F mutation, or an H435R mutation and a Y436F mutation. See, e.g., Jendeberg et al. J Immunol Methods 207(l):25-34 (1997); doi: 10.1016/s0022-1759(96)00215-3.
  • an antibody or antigen-binding fragment includes an (e.g., IgGl) Fc polypeptide that comprises the following mutations: (i) M428L, N434S, H435R; (ii) M428L, N434S, Y436F; or (iii) (ii) M428L, N434S, H435R, and Y436F.
  • an antibody or antigen-binding fragment may be expressed as (e.g., recombinant) human IgG3 (IgG3 contains an arginine “R” residue at EU position 435).
  • an antibody has a native IgGl isotype and is expressed recombinantly as an IgG3 isotype.
  • the antibody or antigen-binding fragment comprises a mutation that alters glycosylation, wherein the mutation that alters glycosylation comprises N297A, N297Q, or N297G, and/or the antibody or antigen-binding fragment is partially or fully aglycosylated and/or is partially or fully afucosylated.
  • Host cell lines and methods of making partially or fully aglycosylated or partially or fully afucosylated antibodies and antigen-binding fragments are known (see, e.g., PCT Publication No. WO 2016/181357; Suzuki et al. Clin. Cancer Res. 73(6): 1875-82 (2007); Huang et al.
  • an antibody or antigen-binding fragment comprises a heavy chain that comprises one or more mutations in the hinge, CH2, and/or CH3 (or in the Fc), wherein the antibody or antigen-binding fragment has one or more improved characteristics over, for example, the antibody or antigenbinding fragment comprising reference wild-type Fc polypeptide and/or comprising a known variant Fc polypeptide).
  • Fc variants possess, for example: increased binding to one or more human FcyRA (e.g., a FcyRIIA and/or a FcyRIIIA; decreased/reduced binding to a human FcyRIIB; increased binding to one or more human FcyRA as compared to binding to a human FcyRIIB; increased thermostability as compared to known Fc polypeptides; increased binding to human Clq; increased human FcyRIIIA signaling in a host cell expressing the FcyRIIIA, increased human FcyRIIIA signaling in a host cell expressing the FcyRIIA, decreased human FcyRIIB signaling in a host cell expressing the FcyRIIB, a relative increase in binding to FcyRA as compared to FcyRIIB, improved qualities for production as compared to known Fc polypeptides; and combinations of such features.
  • human FcyRA e.g., a FcyRIIA
  • antibodies comprising a variant Fc polypeptide of the present disclosure provide surprising advantages, such as any one or more of the following: increased binding affinity (e.g. as determined by surface plasmon resonance, e.g. using a Biacore instrument and/or as determined by a electrochemiluminescence assay, such as a meso scale discovery (MSD) assay) for and/or inducing increased signaling (e.g.
  • increased binding affinity e.g. as determined by surface plasmon resonance, e.g. using a Biacore instrument and/or as determined by a electrochemiluminescence assay, such as a meso scale discovery (MSD) assay
  • MSD meso scale discovery
  • an Fc variant antibody (2) antigen-expressing target cells and (3) reporter cells expressing one or more human FcyRA, optionally driving expression of a reporter gene such as, for example, GFP or luciferase) by one or more human FcyRA, as compared to the antibody comprising a reference Fc polypeptide not comprising the mutation(s) and/or fucosylation state; decreased binding affinity for and/or inducing decreased signaling of human FcyRIIB, as compared to the antibody comprising a reference Fc polypeptide not comprising the mutation(s) and/or fucosylation state; a unique and optionally improved binding profile across human FcyRIIA-H, human FcyRIIA-R, human FcyRIIB, human FcyRIIIA-F, and human FcyRIIIA-V, wherein improved binding comprises an overall increase in binding to and/or activation of FcyRA signaling relative to binding to and/or activation of inhibitory FcyR signaling, as
  • moDCs and/or increasing expression of CD83 by moDCs in a sample when provided in combination with the antigen, as compared to the antibody comprising a reference Fc polypeptide not comprising the mutation(s) and/or fucosylation state, when provided in combination with the antigen; increasing production of one or more cytokine (optionally selected from the group consisting of IL-ip, IFN-y, IL-6, and TNF-a) by moDCs in a sample when provided in combination with the antigen, as compared to the antibody comprising a reference Fc polypeptide not comprising the mutation(s) and/or fucosylation state, when provided in combination with the antigen; and/or increasing the ability of moDCs to stimulate antigenspecific CD4+ T cells when provided to the moDCs in combination with the antigen, as compared to the antibody comprising a reference Fc polypeptide not comprising the mutation(s) and/or fucosylation state, when provided to the moDCs in combination with the antigen, wherein, optionally,
  • an engineered Fc or Fc fragment of the present disclosure (or a polypeptide comprising the same) comprises two or more substitution mutations as compared to a reference wild-type Fc or Fc fragment, and the combined effect of the two or more substitutions is different than, and is optionally greater than, would be expected based on the effects of the individual component substitution mutations and/or based on the effects of a subset of the two or more substitution mutations.
  • combination mutations comprise a non-additive or synergistic effect with reference to the individual component mutations and/or to a subset thereof.
  • antibodies or antigen-binding fragments comprising an Fc variant possess characteristics such as effector functions, ability to bind human Clq, ability to induce FcyRA-mediated cell signaling, ability to bind to human FcRn, ability to promote ADCP, ability to promote ADCC, ability to promote activation of CD4+ T cells, and the like.
  • polypeptides include those that comprise a variant of: an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises one or more modifications as compared to the IgG Fc polypeptide or fragment thereof.
  • a “reference” polypeptide or antibody e.g., reference IgG Fc polypeptide or fragment thereof, reference antibody, reference CH2 polypeptide, reference IgG hinge-CH2, reference IgG hinge-Fc polypeptide, reference CH3 polypeptide
  • is preferably identical to the recited molecule e.g., variant of an Fc polypeptide or fragment thereof; polypeptide comprising such a variant; antibody comprising a variant of an Fc polypeptide except for the recited difference or differences.
  • a reference Fc polypeptide includes an IgGl Fc polypeptide that is otherwise identical to the variant except that a native glycine (G) amino acid is found at EU position 236.
  • a reference Fc polypeptide fragment is preferably of an identical length to the variant and preferably differs from the variant only by the recited features (e.g., amino acid mutation or mutations present in the variant).
  • a reference Fc polypeptide, Fc polypeptide fragment, or antibody comprises a wild-type amino acid sequence (e.g., wild-type human IgGl). Excepting the recited differences present in the variant, a reference Fc polypeptide, Fc polypeptide fragment, or antibody will be of the same isotype, and, preferably, of the same allotype, as the variant. In the case of a reference antibody, the Fabs or other antigen-binding domains will preferably be identical to those present in the specified antibody comprising a variant Fc polypeptide or fragment thereof.
  • variants of IgG Fc polypeptides or fragments thereof include one or more amino acid substitution as compared to a reference (e.g.
  • the position of an amino acid in a variant IgG Fc polypeptide or fragment may be described by referencing the “EU position”; it will be understood that “the EU position” follows the EU numbering system as set forth in Kabat.
  • EU position follows the EU numbering system as set forth in Kabat.
  • two or more amino acid substitutions present in a variant can be expressed in a variety of ways, for example, as G236A_Y300L, or as G236A/Y300L.
  • a mutation or combination mutation may be referenced using a short form including the original amino acid(s) and the amino acid(s) resulting from the substitution(s).
  • G236A may be described as “GA” or “236A”; G236A_Y300L may be described as “GAYL”; G236A_L328V_Q295E may be described as “GALVQE”; G236A_R292P_Y300L may be described as “GARPYL”, G236A_R292P_I377N may be described as “GARPIN”, or the like.
  • a variant of an Fc polypeptide or fragment thereof can be derived from or comprise a human Fc polypeptide or fragment thereof, and/or can be derived from or comprise a human IgGl, a human IgG2, a human IgG3, or a human IgG4 isotype.
  • the expression “derived from” means that the variant is the same as the referenced polypeptide or isotype, with the exception of the specified modification(s) (e.g., amino acid substitution(s)).
  • a variant Fc polypeptide which comprises a wild-type human IgGl Fc amino acid sequence with the exception of the amino acid substitution mutations G236A L328V Q295E (and, optionally, other amino acid substitutions) can be said to be “derived from” wild-type human IgGl Fc.
  • a polypeptide, CH2, Fc, Fc fragment, or antibody may comprise human Ig sequence, such as human IgGl sequence.
  • the polypeptide, CH2, Fc, Fc fragment, or antibody can comprise a native or wild-type human Ig sequence with the exception of the described mutation(s), or can comprise a human Ig (e.g. IgG) sequence that contains one or more additional mutations.
  • An antibody or antigen-binding fragment may be of any allotype or combination of allotypes.
  • Allotype refers to the allelic variation found among the IgG subclasses.
  • an allotype may comprise Glml (or Glm(a)), Glm2 (or Glm(x)), Glm3 (or Glm(f)), Glml7 (or Gm(z))m), Glm27, and/or Glm28 (Glm27 and Glm28 have been described as “alloallotypes”).
  • the Glm3 and Glml7 allotypes are located at the same position in the CHI domain (position 214 according to EU numbering).
  • Glm3 comprises R214 (EU)
  • Glml7 comprises K214 (EU).
  • the Glml allotype is located in the CH3 domain (at positions 356 and 358 (EU)) and refers to the replacements E356D and M358L.
  • the Glm2 allotype refers to a replacement of the alanine in position 431 (EU) by a glycine.
  • Glm allotypes, alloallotypes, and features thereof are known in the art and described at, for example, www.imgt.org/IMGTrepertoire/Proteins/allotypes/human/IGH/IGHC/Glm_allotypes.html and Lefranc, M.-P. and Lefranc, G. Human Gm, Km and Am allotypes and their molecular characterization: a remarkable demonstration of polymorphism In: B. Tait, F. Christiansen (Eds.), Immunogenetics, chap. 34, Humana Press, Springer, New York, USA. Methods Mol. Biol. 2012; 882, 635-680. PMID: 22665258, LIGM: 406, the contents and allotypes and allotype information of which are incorporated herein by reference.
  • the Glml allotype may be combined, for example, with the Glm3, Glm 17, Glm27, Glm2, and/or Glm28 allotype.
  • an allotype is Glm3 with no Glml (Glm3,-1).
  • an allotype is Glml7,l allotype.
  • an allotype is Glm3,l.
  • an allotype is Glml7 with no Glml (Glml7,-1).
  • these allotypes may be combined (or not combined) with the Glm2, Glm27 or Glm28 allotype.
  • an allotype may be Glml7,l,2.
  • an antibody or antigen-binding fragment of the present disclosure comprises a Glm3 allotype or a Glm3,l allotype. In some embodiments, an antibody or antigen-binding fragment of the present disclosure comprises a Glm3 allotype and comprises M428L and N434S or M428L and N434A mutations or any other mutation(s) that enhance binding to a human FcRn, such as those described herein.
  • an antibody or antigen-binding fragment of the present disclosure comprises a Glm3,l allotype and comprises M428L and N434S or M428L and N434A mutations or any other mutation(s) that enhance binding to a human FcRn, such as those described herein.
  • an antibody or antigen-binding fragment of the present disclosure comprises a Glml7, 1 allotype.
  • an antibody or antigen-binding fragment of the present disclosure comprises a Glml7, 1 allotype and comprises M428L and N434S or M428L and N434A mutations or any other mutation(s) that enhance binding to a human FcRn, as described further herein.
  • the antibody or antigen-binding fragment comprises an IgG3 isotype. In some embodiments, the antibody or antigen-binding fragment comprises an IgG3m(b*) allotype, an IgG3m(b**) allotype, an IgG3m(c3*) allotype, an IgG3m(c3c5*) allotype, an IgG3m(g*) allotype, an IgG3m(s*) allotype, an IgG3m(st*) allotype, an IgG3 GM5 allotype, an IgG3 G4m5 allotype, or any combination thereof.
  • an antibody or antigen-binding fragment comprises a CH3 sequence engineered to reduce potential immunogenicity of the antibody or antigen-binding fragment by replacing specific amino acids of one allotype with those of another allotype (these may be referred to as isoallotype mutations), as described in more detail in Stickler et al. (Genes Immun. 2011 Apr; 12(3): 213-221), which is herein incorporated by reference in its entirety, including, for example, the isoallotype mutations disclosed therein.
  • specific amino acids of the Glml allotype are replaced.
  • isoallotype mutations D356E and L358M are made in the CH3 sequence.
  • a variant of an Fc polypeptide comprises only the specified or recited amino acid mutations (e.g. substitutions), and does not comprise any further amino acid substitutions or mutations; e.g., relative to the reference polypeptide (e.g., a wild-type Fc polypeptide or fragment thereof).
  • a variant Fc polypeptide comprising the amino acid substitutions G236A Y300L does not comprise any other amino acid substitutions; i.e., comprises an amino acid sequence that is wild-type except for G236A and Y300L.
  • a variant of an Fc polypeptide may comprise one or more additional amino acid mutations (e.g. substitutions), which can be specified (e.g., M428L_N434S; M428L_N434A).
  • additional amino acid mutations e.g. substitutions
  • a further amino acid mutation or mutations is physically remote to the recited amino acid positions in tertiary structure, and/or is of such nature (e.g.
  • variant of an Fc polypeptide comprises the mutations M428L and N434S or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, including those described herein.
  • an antibody or antigen-binding fragment (described further herein) is provided that comprises, in a(n e.g.
  • the antibody or antigen-binding fragment is afucosylated.
  • the antibody or antigen-binding fragment further comprises one or more mutation that enhances binding to a human FcRn, such as M428L and N434S mutations or M428L and N434A mutations (EU numbering) or any other mutation(s) that enhance binding to a human FcRn, such as those described herein.
  • the antibody or antigen-binding fragment is afucosylated.
  • the IgGl heavy chain comprises a CH1-CH3 or a CH2-CH3 or a hinge-CH2-CH3, wherein the CH1-CH3 or CH2-CH3 or hinge-CH2-CH3 has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity (or similarity) to a wild-type human IgGl CH1-CH3 or CH2-CH3 or hinge-CH2-CH3, respectively.
  • an antibody or antigen-binding fragment of the present disclosure comprises an Fc variant comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity (or similarity) to the amino acid sequence set forth in any one of SEQ ID NOs.:251-277 and 765.
  • the antibody or antigen-binding fragment comprises the amino acid sequence set forth in any one of SEQ ID NOs.:251-277 and 765, or a variant thereof, e.g. that further comprises one or more mutation that enhances binding to a human FcRn, such as M428L and N434S mutations or M428L and N434A mutations (EU numbering) or any other mutation(s) that enhance binding to a human FcRn, including those described herein.
  • the antibody or antigen-binding fragment comprises an amino acid sequence that differs from the amino acid sequence set forth in any one of SEQ ID NOs.:251-277 and 765 only by one or more IgGl allotype-specific mutations and/or by the presence of M428L and N434S mutations or M428L and N434A mutations or other mutation(s) that enhance binding to a human FcRn.
  • an antibody or antigen-binding fragment comprises an Fc polypeptide comprising one or more of the following mutations to, e.g., in a human IgGl background, can promote binding to Cl : K236WZE333S; K326A/E333A; S267E/H268F/S324T; S267E/H268F/S324T/G236A/I332E; Y300L.
  • an Fc polypeptide can comprise any mutation or mutations described in Table A to increase binding to Cl (or Clq).
  • an antibody or antigen-binding fragment comprises an Fc polypeptide comprising E345R/E430G/S440Y mutations to, e.g., in a human IgGl background, can promote oligomerization.
  • An antibody or antigen-binding fragment of the present disclosure can be fucosylated (e.g., comprising one or more fucosyl moiety, and typically comprising a native (wild-type) fucosylation pattern or a fucosylation pattern that includes one or more additional, or fewer, fucosyl moieties as compared to native), or can be afucosylated.
  • native IgGl antibodies carry a glycan site at N297, and this is typically the only site where a core fucose moiety may be found in the antibody, though some glycan sites may arise through mutation (e.g. in the variable domains) during antibody development.
  • Fucosylation of an antibody or antigenbinding fragment can be affected by introducing amino acid mutations to introduce or disrupt a fucosylation site (e.g. a mutation at N297, such as N297Q or N297A, to disrupt formation of a glycan that can include a core fucose moiety), though typically it is preferred to maintain N297 and the glycan thereof, such as by expressing the antibody or antigen-binding fragment in a host cell which has been genetically engineered to lack the ability (or have an inhibited or compromised ability) to fucosylate the antibody or antigen-binding fragment; by expressing the antibody or antigen-binding fragment under conditions in which a host cell is impaired in its ability to fucosylate the polypeptide (e.g, in the presence of 2-fluoro-L-fucose (2FF)), or the like.
  • a fucosylation site e.g. a mutation at N297, such as N297Q or N297A, to disrupt formation of a
  • An afucosylated antibody or antigen-binding fragment can comprise no fucose moieties, or substantially no fucose moieties, and/or can be expressed by a host cell that is genetically engineered to lack the ability (or have an inhibited or compromised ability) to fucosylate the antibody or antigen-binding fragment and/or can be expressed under conditions in which a host cell is impaired in its ability to fucosylate the antibody or antigen-binding fragment (e.g, in the presence of 2-fluoro-L-fucose (2FF)).
  • an antibody or antigen-binding fragment does not comprise a core fucose moiety at Asn297.
  • afucosylated antibodies or antigen-binding fragments have increased binding to FcyRIIIA.
  • addition of 2FF to a culture media comprising host cells expressing an antibody results in about 85% or more of the antibodies or antigen-binding fragments not carrying a fucose moiety. Accordingly, a plurality of antibodies or antigen-binding fragments may be described as “afucosylated” when the plurality was produced in the presence of 2FF or like reagent.
  • a plurality of antibodies or antigen-binding fragments may be described as, for example, afucosylated, meaning that about 85% or more of the single antibody or antigen-binding fragment molecules of the plurality do not comprise a fucose moiety.
  • an afucosylated antibody or antigen-binding fragment or a population or a plurality thereof comprises an asparagine (N) at EU position 297. Fucosylation or lack thereof can be assessed using, for example, mass spectrometry (e.g. Electrospray mass spectrometry (ESI-MS)).
  • compositions are provided that comprise a plurality of any one or more of the presently disclosed antibodies or antigen-binding fragments, wherein the composition comprises afucosylated antibodies or antigen-binding fragments.
  • IgG Fc polypeptides or fragments thereof possess one or more function that is distinct from (e.g. improved as compared to) the corresponding function of a reference Fc polypeptide that comprises the following mutation or mutations: G236A; G236S; G236A_A330L_I332E; G236A_A330L_I332E_M428L_N434S; A330L I332E; or G236A_S239D_A330L_I332E.
  • a presently disclosed variant of an IgG Fc polypeptide or fragment thereof possesses one or more of the following properties, as compared to a reference Fc polypeptide that comprises the following mutation or mutations: G236A; G236S; G236A_A330L_I332E; G236A_A330L_I332E_M428L_N434S; A330LJ332E; or G236A_S239D_A330L_I332E: increased binding (e.g. affinity) to and/or signaling via a human FcyRIIa H131; increased binding (e.g.
  • affinity to and/or signaling via a human FcyRIIa R131
  • decreased binding to (e.g. affinity) and/or signaling via human FcyRIIb an increased ratio of binding to (e.g. affinity) and/or signaling via a human FcyRIIa (H131, R131, or both) versus the ratio of binding to or signaling via (respectively) a human FcyRIIb
  • increased binding e.g.
  • binding of a variant Fc polypeptide or fragment may be described as increased (or “greater than”, or the like) or decreased (or “reduced” or “less than”, or the like) as compared to the binding of a comparator (e.g., to a reference wild-type IgGl Fc, or to a reference IgGl Fc that is wild-type except for M428L and N434S mutations or except for M428L and N434A mutations or to a variant IgGl Fc comprising G236A_A330L_I332E mutations) to a same binding partner.
  • a comparator e.g., to a reference wild-type IgGl Fc, or to a reference IgGl Fc that is wild-type except for M428L and N434S mutations or except for M428L and N434A mutations or to a variant IgGl Fc comprising G236A_A330L_I332E mutations
  • Binding interactions between a variant Fc polypeptide or fragment (or an antibody or polypeptide comprising the same) and a binding partner can preferably be determined using an electrochemiluminescence assay, more preferably using the Meso Scale Discovery (“MSD”; mesoscale.com) platform.
  • MSD binding assay is similar to ELISA though MSD uses electrochemiluminescence, as opposed to colorimetry, as a detection technique.
  • Other techniques for measuring binding interactions are known and include, for example, ELISA, surface plasmon resonance (SPR), biolayer interferometry (BLI), and the like.
  • binding includes affinity, avidity, or both.
  • Affinity refers to the strength of a bond between a binding molecule and its binding partner.
  • binding can include affinity and/or avidity.
  • avidity refers to the total binding strength of a molecule to a binding partner, and reflects binding affinity, valency of binding sites (or domains) (e.g., whether an Fc polypeptide comprises one, two, or more binding sites (or domains)), and, for example, whether another agent is present that can affect the binding (e.g., a non-competitive inhibitor of the Fc polypeptide).
  • a binding interaction between a variant molecule of the present disclosure and a binding partner can be expressed in terms of fold-change relative to the binding interaction between a reference molecule and the binding partner.
  • binding of a presently disclosed antibody comprising a variant Fc to a human FcyRIIa may be stronger than the binding of the antibody comprising a wild-type Fc to the human FcyRIIa, and the relative increased strength of the variant can be expressed in terms of fold-change e.g., linear scale of area-under-the-curve) relative to the reference molecule binding using the same assay.
  • a variant Fc polypeptide or fragment may bind to a FcyRIIa with a 2-fold, 3-fold, 4-fold, or 5-fold greater binding strength than a reference Fc polypeptide or fragment binds to the FcyRIIa.
  • a variant Fc polypeptide or fragment thereof may bind less strongly to a FcyRIIb as compared to a reference Fc or fragment thereof; e.g., may have a 0.9-fold binding, 0.8-fold binding, 0.7-fold binding, 0.6-fold binding, or the like, as compared to the reference Fc polypeptide or fragment thereof.
  • the expression “2-fold greater binding as compared to the binding of a reference” means a 2-fold increase in binding as compared to the reference.
  • binding of a variant Fc molecule of the present disclosure to two different partner molecules can be described in terms of a ratio, and this ratio can be compared to a like ratio obtained using a reference molecule with the same assay.
  • a variant Fc polypeptide may bind to a human FcyRIIa H131 five times more strongly than it binds to a human FcyRIIb, while a reference wild-type Fc polypeptide binds to FcyRIIa H131 as strongly as it binds to a human FcyRIIb.
  • the variant Fc polypeptide can be said to have a 5: 1 (binding FcyRIIIa H131 :binding FcyRIIb) binding ratio, which can be compared to the 1 : 1 (binding FcyRIIIa H131 :binding FcyRIIb) binding ratio of the reference wild-type Fc polypeptide.
  • Variant Fc molecules of the present disclosure may also be described in terms of ability to induce signaling in a host cell, wherein the host cell expresses or over-expresses one or more FcyR (e.g., FcyRIIa H131, FcyRIIa R131, FcyRIIb, FcyRIIIa F158, or FcyRIIIa V158) and the signaling is induced by binding of the variant molecule to the FcyR.
  • Reporter cells useful for determining signaling include, for example, cells in which NF AT drives expression of a luciferase reporter (e.g., available from Promega®).
  • FcyRs, FcRn, and Cl e.g., Clq
  • FcyRs, FcRn, and Cl e.g., Clq
  • an antibody or antigen-binding fragment comprising a variant Fc polypeptide or fragment is preferably capable of inducing one or more of: antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP); and complementdependent cytotoxicity. Assays for measuring these functions are known.
  • a variant Fc polypeptide or fragment preferably has comparable binding to a human FcRn (e.g., at pH 6.0) and/or a comparable in vivo half-life in a mammal as compared to a reference Fc polypeptide, fragment, antibody, or antigen-binding fragment, respectively.
  • a variant Fc polypeptide or fragment preferably has increased binding to a human FcRn (e.g., at pH 6.0) and/or increased in vivo half-life in a mammal as compared to a reference Fc polypeptide, fragment, antibody, or antigen-binding fragment, respectively.
  • a variant Fc polypeptide or fragment preferably has a melting temperature (Tm) that is less than 12°C, less than 11°C, less than 10°C, less than 9°C, less than 8°C, less than 7°C, less than 6°C, less than 5°C, less than 4°C, less than 3°C, less than 2°C, or less than 1°C below the Tm of a reference Fc polypeptide (or antibody or antigen-binding fragment comprising the same), or has a Tm that is higher than the Tm of the reference Fc polypeptide or fragment (or polypeptide or antibody comprising the same).
  • the reference polypeptide or fragment is or comprises a wild-type human Fc polypeptide (or antibody comprising the same).
  • a variant Fc polypeptide or fragment (or antibody or antigenbinding fragment comprising the same) has a melting temperature that is higher than the melting temperature of a reference Fc polypeptide or fragment (or antibody or antigen-binding fragment comprising the same) that comprises the mutations G236A, A330L, I332E, and, optionally, M428L and N434S,
  • a variant Fc polypeptide or fragment is preferably capable of being produced in a host cell line (e.g., a CHO cell line) at least about as efficiently (e.g., produces at least about the same titer and/or within less than O. l-fold, less than 0.09-fold, less than 0.08-fold, less than 0.07-fold, less than 0.06-fold, less than 0.05-fold, less than 0.04-fold, less than 0.03-fold, less than 0.02-fold, or less than 0.02 -fold less) as compared to a reference Fc polypeptide or fragment (or antibody or antigen-binding fragment comprising the same).
  • a host cell line e.g., a CHO cell line
  • an antibody or antigen-binding fragment of the present disclosure comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, a valine (V) at EU position 328, and a glutamic acid (E) at EU position 295.
  • the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GALVQE”).
  • the antibody or antigen-binding fragment further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein.
  • the antibody or antigen-binding fragment is afucosylated.
  • an antibody or antigen-binding fragment of the present disclosure comprises a variant of: (i) an IgG hinge-CH2 polypeptide; or (ii) an IgG hinge-Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, an alanine (A) at EU position 230, and a glutamic acid (E) at EU position 295.
  • the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wildtype) IgGl Fc polypeptide or fragment thereof (“GAPAQE”).
  • the antibody or antigen-binding fragment further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein.
  • the antibody or antigen-binding fragment is afucosylated.
  • an antibody or antigen-binding fragment of the present disclosure comprises a variant of: an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, a proline (P) at EU position 292, and an asparagine (N) at EU position 377.
  • the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GARPIN”).
  • the antibody or antigen-binding fragment further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein.
  • the antibody or antigen-binding fragment is afucosylated.
  • an antibody or antigen-binding fragment comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, an alanine (A) at EU position 334, and a glutamic acid (E) at EU position 295.
  • the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GAKAQE”).
  • the antibody or antigen-binding fragment further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein.
  • the antibody or antigen-binding fragment is afucosylated.
  • an antibody or antigen-binding fragment of the present disclosure comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises a serine (S) at EU position 236, a proline (P) at EU position 292, and a leucine (L) at EU position 300.
  • the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GSRPYL”).
  • the antibody or antigen-binding fragment further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein.
  • the antibody or antigen-binding fragment is afucosylated.
  • an antibody or antigen-binding fragment of the present disclosure comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, a proline (P) at EU position 292, and a leucine (L) at EU position 300.
  • the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GARPYL”).
  • the antibody or antigen-binding fragment further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein.
  • the antibody or antigen-binding fragment is afucosylated.
  • an antibody or antigen-binding fragment of the present disclosure comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236 and a leucine (L) at EU position 300.
  • the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GAYL”).
  • the antibody or antigen-binding fragment further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein.
  • the antibody or antigen-binding fragment is afucosylated.
  • an antibody or antigen-binding fragment of the present disclosure comprises a variant of: (i) an IgG CH2 polypeptide; or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, an aspartic acid (D) at EU position 239, and a glutamic acid (E) at EU position 268.
  • the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GASDHE”).
  • the antibody or antigenbinding fragment further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein.
  • the antibody or antigen-binding fragment is afucosylated.
  • the antibody or antigen-binding fragment has increased binding to a human FcyRIIa and/or has decreased binding to a human FcyRIIb, as compared to the binding of a reference antibody or antigen-binding fragment to the human FcyRIIa or the human FcyRIIb, respectively, wherein, optionally, binding is as determined using an electrochemiluminescence assay, further optionally Meso Scale Discovery.
  • the increased binding to a human FcyRIIa comprises more than 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater binding to the human FcyRIIa as compared to the binding of a reference antibody or antigen-binding fragment (optionally comprising a wild-type human IgG e.g. IgGl) Fc polypeptide or a fragment thereof) to the human FcyRIIa.
  • a reference antibody or antigen-binding fragment optionally comprising a wild-type human IgG e.g. IgGl
  • the human FcyRIIa comprises H131 and, optionally, the increased binding to the human FcyRIIa H131 comprises at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater binding to the human FcyRIIa H131 as compared to the binding of a reference antibody or antigen-binding fragment (optionally comprising a wild-type human IgG (e.g. IgGl) Fc polypeptide or a fragment thereof) to the human FcyRIIa H131.
  • a reference antibody or antigen-binding fragment optionally comprising a wild-type human IgG (e.g. IgGl) Fc polypeptide or a fragment thereof
  • the human FcyRIIa comprises R131 and, optionally, the increased binding to the human FcyRIIa R131 comprises more than 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater binding to the human FcyRIIa R131 as compared to the binding of a reference antibody or antigen-binding fragment (optionally comprising a wild-type human IgG (e.g. IgGl) Fc polypeptide or a fragment thereof) to the human FcyRIIa R131.
  • a reference antibody or antigen-binding fragment optionally comprising a wild-type human IgG (e.g. IgGl) Fc polypeptide or a fragment thereof
  • the decreased binding to a human FcyRIIb comprises less than 0.9-fold, less than 0.8-fold, less than 0.7-fold, less than 0.6-fold, or between 0.5-fold and 0.9- fold, of the binding of a reference antibody or antigen-binding fragment (optionally comprising a wild-type human IgG (e.g. IgGl) Fc polypeptide or a fragment thereof) to the human FcyRIIb.
  • a reference antibody or antigen-binding fragment optionally comprising a wild-type human IgG (e.g. IgGl) Fc polypeptide or a fragment thereof
  • a ratio of (i) the binding of the antibody or antigen-binding fragment to a human FcyRIIa to (ii) the binding of the antibody or antigen-binding fragment, respectively, to a human FcyRIIb is greater than (2) a ratio of (iii) the binding of a reference polypeptide to the human FcyRIIa to (iv) the binding of the reference antibody or antigen-binding fragment to the human FcyRIIb, wherein the reference antibody or antigen-binding fragment optionally comprises a wild-type human IgG (e.g.
  • the human FcyRIIa comprises H131, R131, or both.
  • the ratio in (1) is more than 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11 -fold, at least 12-fold, at least 13-fold, or at least 14-fold greater than the ratio in (2).
  • an antibody or antigen-binding fragment of the present disclosure comprising a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236 and a leucine (L) at EU position 300.
  • the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GAYL”).
  • the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present.
  • the antibody or antigen-binding fragment is afucoyslated.
  • the antibody or antigen-binding fragment has increased binding to a human FcyRIIa as compared to the binding of a reference antibody or antigen-binding fragment to the human FcyRIIa, wherein, optionally, binding is as determined using an electrochemiluminescence assay, further optionally Meso Scale Discovery.
  • the increased binding to a human FcyRIIa comprises at least 4- fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, or at least 18-fold greater binding to the human FcyRIIa as compared to the binding of a reference antibody or antigen-binding fragment (optionally comprising a wild-type human IgG (e.g. IgGl) Fc polypeptide or a fragment thereof) to the human FcyRIIa.
  • a reference antibody or antigen-binding fragment optionally comprising a wild-type human IgG (e.g. IgGl) Fc polypeptide or a fragment thereof
  • the human FcyRIIa comprises H131 and, optionally, the increased binding to the human FcyRIIa H131 comprises at least 4-fold, at least 5-fold, at least 6- fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold, at least 11-fold, at least 12- fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, or at least 18-fold greater binding to the human FcyRIIa H131 as compared to the binding of a reference antibody or antigen-binding fragment (optionally comprising a wild-type human IgG (e.g. IgGl) Fc polypeptide or a fragment thereof) to the human FcyRIIa H131.
  • a reference antibody or antigen-binding fragment optionally comprising a wild-type human IgG (e.g. IgGl) Fc polypeptide or a fragment thereof
  • the human FcyRIIa comprises R131 and, optionally, the increased binding to the human FcyRIIa R131 comprises at least 4-fold greater binding to the human FcyRIIa R131 as compared to the binding of a reference antibody or antigen-binding fragment (optionally comprising a wild-type human IgG (e.g IgGl) Fc polypeptide or a fragment thereof) to the human FcyRIIa R131.
  • a reference antibody or antigen-binding fragment optionally comprising a wild-type human IgG (e.g IgGl) Fc polypeptide or a fragment thereof
  • (1) a ratio of (i) the binding of the antibody or antigen-binding fragment to a human FcyRIIa to (ii) the binding of the antibody or antigen-binding fragment, respectively, to a human FcyRIIb is greater than (2) a ratio of (iii) the binding of a reference antibody or antigen-binding fragment to the human FcyRIIa to (iv) the binding of the reference polypeptide to the human FcyRIIb, wherein the reference antibody or antigen-binding fragment comprises a wild-type human IgG Fc polypeptide or a fragment thereof.
  • the human FcyRIIa comprises H131, R131, or both.
  • the ratio in (1) is at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, or at least 17-fold greater than the ratio in (2).
  • an antibody or antigen-binding fragment of the present disclosure that comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, a proline (P) at EU position 292, and a leucine (L) at EU position 300.
  • the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GARPYL”).
  • the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present.
  • the antibody or antigen-binding fragment is afucoyslated.
  • the antibody or antigen-binding fragment has increased binding to a human FcyRIIIa, as compared to the binding of a reference antibody or antigen-binding fragment to the human FcyRIIIa, wherein, optionally, binding is as determined using an electrochemiluminescence assay, further optionally Meso Scale Discovery.
  • the increased binding to a human FcyRIIa comprises at least 2- fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11 -fold, at least 12-fold, at least 13 -fold, or at least 14-fold greater binding to the human FcyRIIa as compared to the binding of a reference antibody or antigen-binding fragment optionally comprising a wild-type human IgG Fc polypeptide or a fragment thereof to the human FcyRIIa.
  • the human FcyRIIa comprises H131 and, optionally, the increased binding to the human FcyRIIa H131 comprises at least 2-fold, at least 3-fold, at least 4- fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, or at least 14-fold greater binding to the human FcyRIIa H131 as compared to the binding of a reference antibody or antigen-binding fragment optionally comprising a wild-type human IgG Fc polypeptide or a fragment thereof to the human FcyRIIa H131.
  • the human FcyRIIa comprises R131 and, optionally, the increased binding to the human FcyRIIa H131 comprises at least 2-fold greater binding to the human FcyRIIa R131 as compared to the binding of a reference antibody or antigen-binding fragment optionally comprising a wild-type human IgG Fc polypeptide or a fragment thereof to the human FcyRIIa R131.
  • (1) a ratio of (i) the binding of the antibody or antigen-binding fragment to a human FcyRIIa to (ii) the binding of the antibody or antigen-binding fragment, respectively, to a human FcyRIIb is greater than (2) a ratio of (iii) the binding of a reference antibody or antigen-binding fragment to the human FcyRIIa to (iv) the binding of the reference antibody or antigen-binding fragment to the human FcyRIIb, wherein the reference antibody or antigen-binding fragment optionally comprises a wild-type human IgG Fc polypeptide or a fragment thereof, wherein, optionally, binding is as determined using an electrochemiluminescence assay, further optionally Meso Scale Discovery.
  • the human FcyRIIa comprises H131, R131, or both.
  • the ratio in (1) is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13- fold, at least 14-fold, or at least 15-fold greater than the ratio in (2).
  • the antibody or antigen-binding fragment has increased binding to a human FcyRIIIa, as compared to the binding of a reference antibody or antigen-binding fragment to the human FcyRIIIa, wherein, optionally, binding is as determined using an electrochemiluminescence assay, further optionally Meso Scale Discovery.
  • the human FcyRIII comprises V158, F158, or both.
  • the increased binding to a human FcyRIIIa comprises greater than 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3.0 fold, at least 3.1-fold, at least 3.2- fold, at least 3.3-fold, at least 3.4-fold, at least 3.5-fold, at least 3.6-fold, or at least 3.7-fold greater binding to the human FcyRIIIa as compared to the binding of a reference antibody or antigen-binding fragment optionally comprising a wild-type human IgG Fc polypeptide or a fragment thereof to the human FcyRIIIa.
  • the antibody or antigen-binding fragment is capable of binding to a human complement component Iq (Clq), wherein, optionally, binding is as determined using an electrochemiluminescence assay, further optionally Meso Scale Discovery.
  • an antibody or antigen-binding fragment of the present disclosure that comprises a variant of an IgG Fc polypeptide, wherein the variant comprises a serine (S) at EU position 236, a valine (V) at EU position 420, a glutamic acid (E) at EU position 446, and a threonine (T) at EU position 309.
  • the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GSGVGELT”).
  • the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present.
  • the antibody or antigenbinding fragment is afucoyslated.
  • an antibody or antigen-binding fragment of the present disclosure that comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide, wherein the variant comprises an alanine (A) at EU position 236 and a proline (P) at EU position 292.
  • the antibody or antigen-binding fragment comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GARP”).
  • the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present.
  • the antibody or antigen-binding fragment is afucoyslated.
  • the antibody or antigen-binding fragment has decreased binding to a human FcyRIIb as compared to the binding of a reference antibody or antigen-binding fragment to the human FcyRIIb, wherein, optionally, binding is as determined using an electrochemiluminescence assay, further optionally Meso Scale Discovery.
  • the decreased binding to a human FcyRIIb comprises less than 0.9-fold, less than 0.8-fold, less than 0.7-fold, less than 0.6-fold, less than 0.5-fold, or less than 0.4-fold as compared to the binding of a reference antibody or antigen-binding fragment optionally comprising a wild-type human IgG Fc polypeptide or a fragment thereof to the human FcyRIIb.
  • the antibody or antigen-binding fragment has increased binding to a human FcyRIIa as compared to the binding of a reference antibody or antigen-binding fragment to the human FcyRIIa, wherein, optionally, binding is as determined using an electrochemiluminescence assay, further optionally Meso Scale Discovery.
  • the increased binding to the human FcyRIIa comprises greater than 1-fold, at least 2-fold, at least 3 -fold, at least 4-fold, or at least 5 -fold greater binding to the human FcyRIIa as compared to the binding of a reference antibody or antigen-binding fragment comprising a wild-type human IgG Fc polypeptide or a fragment thereof to the human FcyRIIa.
  • the human FcyRIIa comprises H131, R131, or both.
  • (1) a ratio of (i) the binding of the antibody or antigen-binding fragment to a human FcyRIIa to (ii) the binding of the antibody or antigen-binding fragment, respectively, to a human FcyRIIb is greater than (2) a ratio of (iii) the binding of a antibody or antigen-binding fragment to the human FcyRIIa to (iv) the binding of the reference antibody or antigen-binding fragment to the human FcyRIIb, wherein the reference antibody or antigenbinding fragment optionally comprises a wild-type human IgG Fc polypeptide or a fragment thereof, wherein, optionally, binding is as determined using an electrochemiluminescence assay, further optionally Meso Scale Discovery.
  • the human FcyRIIa comprises H131, R131, or both.
  • the ratio in (1) is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 10-fold, at least 11-fold, or at least 12-fold greater than the ratio in (2).
  • an antibody or antigen-binding fragment of the present disclosure that comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide, wherein the variant comprises a proline (P) at EU position 292 and a leucine (L) at EU position 300, and wherein, optionally, variant and, further optionally, the antibody or antigen-binding fragment has increased binding to a human FcyRIIIa with as compared to the binding of a reference antibody or antigen-binding fragment to the human FcyRIIIa, wherein, optionally, the binding is as determined using an electrochemiluminescence assay, further optionally Meso Scale Discovery.
  • the antibody or antigen-binding fragment comprises an (e.g., otherwise wild-type) IgGl CH2 polypeptide or IgG Fc polypeptide (“RPYL”).
  • RYL IgG Fc polypeptide
  • the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present.
  • the antibody or antigen-binding fragment is afucoyslated.
  • the human FcyRIIIa comprises V158, F158, or both, and wherein the increased binding to the human FcyRIIIa comprises at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.1-fold, or at least 5.2-fold greater binding as compared to the binding of a reference antibody or antigen-binding fragment optionally comprising a wild-type human IgG Fc polypeptide or a fragment thereof to the human FcyRIIa.
  • an antibody or antigen-binding fragment of the present disclosure that comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises a leucine (L) at EU position 300.
  • the IgG CH2 polypeptide or IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“YL”).
  • the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present.
  • the antibody or antigen-binding fragment is afucoyslated.
  • an antibody or antigen-binding fragment of the present disclosure that comprises a variant of: an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises a lysine (K) at EU position 345, a serine (S) at EU position 236, tyrosine (Y) at EU position 235, and a glutamic acid (E) at EU position 267.
  • the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GSEKLYSE”).
  • the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present.
  • the antibody or antigen-binding fragment is afucoyslated.
  • antibody or antigen-binding fragment of the present disclosure that comprises a variant of: (i) an IgG hinge-CH2 polypeptide or (ii) an IgG hinge-Fc polypeptide or a fragment thereof, wherein the variant comprises an arginine (R) at EU position 272, a threonine (T) at EU position 309, a tyrosine (Y) at EU position 219, and a glutamic acid (E) at EU position 267.
  • the IgG hinge-CH2 polypeptide or an IgG hinge-Fc polypeptide or a fragment thereof comprises an (e.g.
  • IgGl hinge-CH2 polypeptide or IgG hinge-Fc polypeptide or a fragment thereof (“SYSEERLT”).
  • the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present.
  • the antibody or antigen-binding fragment is afucoyslated.
  • an antibody or antigen-binding fragment of the present disclosure that comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises a tyrosine (Y) at EU position 236.
  • the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GY”).
  • GY tyrosine
  • the antibody or antigen-binding fragment is afucoyslated.
  • an antibody or antigen-binding fragment of the present disclosure that comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises a tryptophan (W) at EU position 236.
  • the IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof comprises an (e.g., otherwise wild-type) IgGl CH2 polypeptide or Fc polypeptide or fragment thereof (“GW”).
  • the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present.
  • the antibody or antigenbinding fragment is afucoyslated.
  • an antibody or antigen-binding fragment of the present disclosure that comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, wherein the IgG Fc polypeptide or fragment thereof, and optionally the polypeptide, is afucosylated, and wherein, further optionally, the variant comprises a leucine (L) at EU position 330 and a glutamic acid (E) at EU postion 332, wherein, still further optionally, the variant does not comprise an aspartic acid (D) at EU position 239, and, even further optionally, comprises a serine (S) at EU position 239.
  • the IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof comprises an (e.g., otherwise wild-type) IgGl CH2 polypeptide or Fc polypeptide or fragment thereof (“GA-afuc” or “GAALIE-afuc”, respectively).
  • the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present.
  • an antibody or antigen-binding fragment of the present disclosure that comprises a variant of: an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises a leucine (L) at EU position 243, a glutamic acid (E) at EU position 446, a leucine (L) at EU position 396, and a glutamic acid (E) at EU position 267.
  • the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“FLSEPLGE”).
  • the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present.
  • the antibody or antigen-binding fragment is afucoyslated.
  • an antibody or antigen-binding fragment that comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, an aspartic acid (D) at EU position 239, a glutamic acid (E) and EU position 332, a leucine (L) at EU position 428, and a serine (S) or an alanine (A) at EU position 434.
  • the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GASDIEMLNS” or “GASDIEMLNA”).
  • an antibody or antigen-binding fragment of the present disclosure comprises a variant of an (e.g. IgGl) IgG Fc polypeptide, wherein the variant comprises the following mutations, according to EU numbering: (i) M428L, N434S, G236A, L328V, and Q295E; (ii) M428L, N434S, G236A, R292P, and I377N; (iii) M428L, N434S, G236A, and Y300L; (iv) M428L, N434S, G236A, R292P, and Y300L; (v) M428L, N434S, G236A, L328V, and Q295E, wherein the antibody or antigen-binding fragment is afucosylated; (vi) M428L, N434S, G236A, R292P, and I377N, wherein the antibody or
  • the variant of an (e.g. IgGl) IgG Fc polypeptide comprises amino acid substitutions that consist essentially of the substitution mutations in (i), (ii), (iii), (iv), (v), (vi), (vii), or (viii) above.
  • the antibody or antigen-binding fragment comprises a kappa light chain.
  • an antibody or antigen-binding fragment of the present disclosure comprises a variant of an (e.g. IgGl) IgG Fc polypeptide, wherein the variant comprises the following mutations, according to EU numbering: (i) M428L, N434A, G236A, L328V, and Q295E; (ii) M428L, N434A, G236A, R292P, and I377N; (iii) M428L, N434A, G236A, and Y300L; (iv) M428L, N434A, G236A, R292P, and Y300L; (v) M428L, N434A, G236A, L328V, and Q295E, wherein the antibody or antigen-binding fragment is afucosylated; (vi) M428L, N434A, G236A, R292P, and I377N, wherein the antibody or
  • the variant of an IgG Fc polypeptide comprises amino acid substitutions that consist essentially of the substitution mutations in (i), (ii), (iii), (iv), (v), (vi), (vii), or (viii) above.
  • the antibody comprises a kappa light chain.
  • the antibody or antigenbinding fragment is capable of eliciting continued protection in vivo in a subject even once no detectable levels of the antibody or antigen-binding fragment can be found in the subject (z.e., when the antibody or antigen-binding fragment has been cleared from the subject following administration). Such protection is referred to herein as a vaccinal effect.
  • an antibody or antigen-binding fragment comprises one or more modifications, such as, for example, mutations in the Fc comprising G236A, A330L, and I332E, that are capable of activating dendritic cells that may induce, e.g., T cell immunity to the antigen.
  • the antibody or antigen-binding fragment comprises a Fc polypeptide or a fragment thereof, including a CH2 (or a fragment thereof, a CH3 (or a fragment thereof), or a CH2 and a CH3, wherein the CH2, the CH3, or both can be of any isotype and may contain amino acid substitutions or other modifications as compared to a corresponding wild-type CH2 or CH3, respectively.
  • a Fc of the present disclosure comprises two CH2-CH3 polypeptides that associate to form a dimer.
  • an antibody or antigen-binding fragment of the present disclosure comprises a heavy chain comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity (or similarity) to, or comprising or consisting of, the amino acid sequence set forth in any one of SEQ ID NOs.:251-277 and 765.
  • the antibody or antigen-binding fragment can be monoclonal.
  • the term “monoclonal antibody” (mAb) as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, z.e., individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present, in some cases in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different epitopes, each monoclonal antibody is directed against a single epitope of the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies.
  • monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al., Nature 256 :495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal, or plant cells (see, e.g., U.S. Pat. No. 4,816,567).
  • Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.
  • Monoclonal antibodies may also be obtained using methods disclosed in PCT Publication No. WO 2004/076677A2.
  • Antibodies and antigen-binding fragments of the present disclosure include “chimeric antibodies” in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, U.S. Pat. Nos. 4,816,567; 5,530,101 and 7,498,415; and Morrison et al., Proc. Natl. Acad. Sci. USA, 57:6851-6855 (1984)).
  • chimeric antibodies may comprise human and non-human residues.
  • chimeric antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. For further details, see Jones etal., Nature 321 :522-525 (1986); Riechmann etal., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). Chimeric antibodies also include primatized and humanized antibodies.
  • a “humanized antibody” is generally considered to be a human antibody that has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are typically taken from a variable domain. Humanization may be performed following the method of Winter and co-workers (Jones et al., Nature, 321 : 522-525 (1986); Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534- 1536 (1988)), by substituting non-human variable sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. Nos.
  • a “humanized” antibody is one which is produced by a non-human cell or animal and comprises human sequences, e.g., He domains.
  • human antibody is an antibody containing only sequences that are present in an antibody that is produced by a human (i.e., sequences that are encoded by human antibodyencoding genes).
  • human antibodies may comprise residues or modifications not found in a naturally occurring human antibody (e.g., an antibody that is isolated from a human), including those modifications and variant sequences described herein. These are typically made to further refine or enhance antibody performance.
  • human antibodies are produced by transgenic animals. For example, see U.S. Pat. Nos. 5,770,429; 6,596,541 and 7,049,426.
  • an antibody or antigen-binding fragment of the present disclosure is chimeric, humanized, or human.
  • PK pharmacokinetic
  • ti/2 or “half-life” refers to the elimination half-life of the antibody or antigen-binding fragment included in the pharmaceutical composition administered to a subject.
  • Clast generally refers to the last measurable plasma concentration (z.e., subsequent thereto, the substance is not present at a measurable concentration in plasma).
  • the antibody, or the antigen-binding fragment comprises a human antibody, a monoclonal antibody, a purified antibody, a single chain antibody, a Fab, a Fab’, a F(ab’)2, or Fv.
  • the antibody or antigen-binding fragment is a multi-specific antibody or antigen-binding fragment, wherein, optionally, the antibody or antigen-binding fragment is a bi-specific antibody or antigen-binding fragment.
  • the antibody or antigen-binding fragment comprises an (e.g., IgGl) Fc polypeptide or a fragment thereof.
  • the antibody or antigenbinding fragment comprises an IgG, IgA, IgM, IgE, or IgD isotype.
  • the antibody or antigen-binding fragment comprises an IgG isotype selected from IgGl, IgG2, IgG3, and IgG4. In certain embodiments, the antibody or antigen-binding fragment comprises an IgGl isotype.
  • the antibody or antigen-binding fragment comprises an IgGlm3 allotype, an IgGlml7 allotype, an IgGl ml allotype, or any combination thereof. In some embodiments, the antibody or antigen-binding fragment comprises an IgG3 isotype.
  • the antibody or antigen-binding fragment comprises an IgG3m(b*) allotype, an IgG3m(b**) allotype, an IgG3m(c3*) allotype, an IgG3m(c3c5*) allotype, an IgG3m(g*) allotype, an IgG3m(s*) allotype, an IgG3m(st*) allotype, an IgG3 GM5 allotype, an IgG3 G4m5 allotype, or any combination thereof.
  • the Fc polypeptide or fragment thereof comprises: (i) a mutation that increases binding affinity to a human FcRn (e.g., as measured using surface plasmon resonance (SPR) (e.g., Biacore, e.g., T200 instrument, using manufacturer’s protocols)), as compared to a reference Fc polypeptide that does not comprise the mutation; and/or (ii) a mutation that increases binding affinity to a human FcyR (e.g., as measured using surface plasmon resonance (SPR) (e.g., Biacore, e.g., T200 instrument, using manufacturer’s protocols, and/or as measured using mesoscale discovery (MSD))) as compared to a reference Fc polypeptide that does not comprise the mutation.
  • SPR surface plasmon resonance
  • MSD mesoscale discovery
  • the mutation that increases binding affinity to a human FcRn comprises: M428L; N434S; N434H; N434A; N434S; M252Y; S254T; T256E; T250Q; P257I; Q31 II; D376V; T307A; E380A; or any combination thereof.
  • the mutation that enhances binding to a FcyR comprises S239D; I332E; A330L; G236A; or any combination thereof.
  • the mutation that enhances binding to a FcyR comprises: (i) S239D/I332E; (ii) S239D/A330L/I332E; (iii) G236A/S239D/I332E; or (iv) G236A/A330L/I332E, wherein the Fc polypeptide or fragment thereof optionally comprises Ser at position 239.
  • the antibody or antigen-binding fragment comprises a mutation that alters glycosylation, wherein the mutation that alters glycosylation comprises N297A, N297Q, or N297G, and/or which is aglycosylated, and/or which is afucosylated.
  • antibody conjugates that comprise an antibody or antigen-binding fragment of the present disclosure conjugated to a payload molecule or moiety, such as, for example, a drug compound.
  • Antibody or antigen-binding fragment structures amenable to conjugation include, for example, lysine amino acid residues, cysteine amino acid residues, nonnatural amino acids (e.g., amino acid analogs), carbohydrate moi eties, and N-termini of heavy and light chains (or of VH and VL).
  • Such structures may be native to the antibody or antigenbinding fragment, or may be introduced (e.g., a native non-lysine or non-cysteine amino acid of the antibody may be replaced by a lysine or cysteine, and/or a lysine or cysteine may be inserted into the amino acid sequence of the antibody or antigen-binding fragment, as an additional amino acid).
  • conjugation techniques in which a protein with a strong interaction with an antibody or antigen-binding fragment (e.g., FcRn, Protein A, Protein G, or fragments of these) is conjugated to a compound and non-covalent interaction between the protein and the antibody or antigen-binding fragment effectively couples the compound to the antibody or antigen-binding fragment, as well as modification in nucleotide-binding sites (e.g., for modification by photoaffinity labeling), or in catalytic sites (if present).
  • an antibody or antigen-binding fragment e.g., FcRn, Protein A, Protein G, or fragments of these
  • modification in nucleotide-binding sites e.g., for modification by photoaffinity labeling
  • catalytic sites if present.
  • WO 2006/034488 WO 2011/005481, WO 2011/156328, WO 2016/040856, WO 2015/157595, WO 2013/093809, WO 2005/018572, WO 1996/014339, WO 2008/070593, WO 2009/092011, WO 2020/191306, WO 2006/074397, WO 2008/038024, WO 2010/141902, and WO 2004/042017, as well as in Sang et al., Methods Mol. Biol. 2078:235-250 (2020), Haque et al. Chem. Commun. 57:10689-10702 (2021), Brun and Gauzy -Lazo, Methods Mol. Biol.
  • Non-limiting examples of mutations include the following (all residue positions according to EU numbering of human IgGl): Al 18C, S239C, T289C, A330C, and S442C in the heavy chain, and K149C in the light chain.
  • An antibody or antigen-binding fragment of a conjugate can comprise one or more native lysine, one or more native cysteine, one or more introduced lysine, one or more introduced cysteine, or any combination thereof.
  • an antibody or antigen-binding fragment comprises its native lysines, its native cysteines, or both.
  • an antibody or antigen-binding fragment does not comprise modifications in its amino acid sequence for conjugation.
  • Conjugation may comprise forming conjugates at all, or at fewer than all, of the available conjugation sites.
  • a payload molecule is covalently linked by a linker to the antibody or antigen-binding fragment thereof.
  • the linker is selected from a cleavable linker and a non-cleavable linker.
  • the cleavable linker is a protease-sensitive linker, a pH-sensitive linker, or a glutathione-sensitive linker.
  • the cleavable linker is a protease-sensitive linker comprising a valine-citrulline dipeptide.
  • the linker comprises a maleimide group.
  • the herein disclosed antibody or antigen-binding fragment thereof comprises a reduced disulfide bridge in a hinge region and the reduced disulfide bridge is coupled to the maleimide group.
  • the linker further comprises a self-demolishing group, such as, for example, para-amino benzyl alcohol (PABC).
  • PABC para-amino benzyl alcohol
  • an antibody antigen-binding fragment conjugate comprises payload molecule that is covalently linked by a linker to the antibody or antigenbinding fragment thereof.
  • Linkers used in antibody antigen-binding fragment conjugates may be organic compounds that fall into one of two groups, organized according to the mechanism by which the payload molecule is released from the carrier molecule.
  • Cleavable linkers are designed to be selectively degraded or cleaved according to an inherent property of the target cell: three types of cleavable linkers are protease-sensitive linkers (whereby cleavage of the linker, e.g., a linker comprising a valine-citrulline or phenylalanine-lysine dipeptide or a tetrapeptide (e.g., GFLG or ALAL), by proteases present in a tumor cell lysosome releases the payload molecule); pH-sensitive linkers, containing an acid labile group that is selectively hydrolyzed by the lower pH of endosomal and lysosomal compartments, relative to cytosolic pH; and glutathione-sensitive linkers, which comprise a disulfide bridge that is reduced by intracellular glutathione.
  • Non-cleavable linkers rely on non-specific degradation of the antibody conjugate to release the payload molecule.
  • an antibody or antigen-binding fragment conjugate comprises a linker is selected from a cleavable linker and a non-cleavable linker.
  • the linker is a cleavable linker selected from a protease-sensitive linker, a pH-sensitive linker, or a glutathionesensitive linker.
  • a cleavable linker is a protease-sensitive linker comprising a valine-citrulline dipeptide.
  • a linker may be connected or coupled to the antibody or antigen-binding fragment thereof using any appropriate technique or mechanism.
  • a linker comprises a maleimide group (optionally PEGylated) capable of reacting with a reduced disulfide bridge in a hinge region of the antibody or antigen-binding fragment thereof.
  • Other sites on the carrier molecule (i.e., the antibody or antigen-binding fragment thereof) suitable for conjugation to a linker may be introduced or engineered using recombinant techniques, such as introducing cysteine residues or non-natural amino acids for site-specific conjugation. Methods for introducing such modifications include, for example, the method described in Examples 6.3-7 of PCT Publication No. WO 2012/032181.
  • a linker further comprises a self-demoli shing group, also referred to as a self-immolative group or a self-immolative spacer, to assist in a selective cleavage reaction.
  • the self-demoli shing group is para-amino benzyl alcohol (PABC).
  • Click chemistries useful for generating antibody conjugates include those described in Meyer et al.. Bioconjug. Chem. 27(12):2791-2807 (2016), and are herein incorporated by reference in their entirety.
  • the payload molecule may be selected from a therapeutic agent and a detectable indicator.
  • the present disclosure provides isolated polynucleotides that encode any of the presently disclosed antibodies or an antigen-binding fragment thereof, or a portion thereof (e.g., a CDR, a VH, a VL, a Fab, a Fd, a heavy chain, or a light chain, or a heavy chain and a light chain), or that encode a presently disclosed polypeptide.
  • the polynucleotide comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), wherein the RNA optionally comprises messenger RNA (mRNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • the polynucleotide comprises a modified nucleoside, a cap-1 structure, a cap-2 structure, or any combination thereof.
  • the polynucleotide comprises a pseudouridine, a N6-methyladenonsine, a 5-methylcytidine, a 2- thiouridine, or any combination thereof.
  • the pseudouridine comprises N1 -methylpseudouridine.
  • the polynucleotide is codon-optimized for expression in a host cell (e.g., a human cell, or a CHO cell).
  • a host cell e.g., a human cell, or a CHO cell.
  • codon optimization can be performed using known techniques and tools, e.g., using the GenScript® OptimumGeneTM tool, or the like).
  • Codon-optimized sequences include sequences that are partially codon-optimized (z.e., one or more codon is optimized for expression in the host cell) and those that are fully codon-optimized.
  • polynucleotides encoding antibodies and antigen-binding fragments of the present disclosure may possess different nucleotide sequences while still encoding a same antibody or antigen-binding fragment due to, for example, the degeneracy of the genetic code, splicing, and the like.
  • the polynucleotide can comprise deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • the RNA comprises messenger RNA (mRNA), more specifically circRNA, taRNA, or saRNA.
  • mRNA messenger RNA
  • the polynucleotide may be optimized for expression in a host cell.
  • the polynucleotide comprises taRNA or saRNA, one or more sequences encoding a replication protein or peptide, one or more sequences encoding an antibody or antigen-binding fragment, or combinations thereof may be optimized for expression in human cells.
  • a polynucleotide comprises (i) a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99% identity (or similarity) to the VH polynucleotide sequence of an antibody as set forth in Table 3, and (ii) a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99% identity (or similarity) to the corresponding VL polynucleotide sequence of the antibody as set
  • a polynucleotide comprises (i) a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99% identity (or similarity) to SEQ ID NO.:29, and (ii) a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99% identity (or similarity) to SEQ ID NO.:30.
  • the polynucleotide can comprise deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • the RNA comprises messenger RNA (mRNA).
  • the first polynucleotide may further comprise a nucleic acid sequence encoding a replication protein or peptide.
  • a second polynucleotide is provided that comprises a nucleic acid sequence encoding a replication protein or peptide suitable to cause replication of the first polynucleotide.
  • the first polynucleotide comprises or consists of a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99% identity (or similarity) to the VH polynucleotide sequence of an antibody as set forth in Table 3 and the second polynucleotide comprises or consists of a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 98%, or at
  • the polynucleotides are mRNAs at least one and optionally both of the first and second polynucleotide is a saRNA, and further comprise a nucleic acid sequence encoding a replication protein or peptide.
  • a third polynucleotide is provided that comprises a nucleic acid sequence encoding a replication protein or peptide suitable to cause replication of the first polynucleotide and second polynucleotide.
  • Vectors are also provided, wherein the vectors comprise or contain a polynucleotide as disclosed herein (e.g., a polynucleotide that encodes an antibody or antigen-binding fragment).
  • a vector can comprise any one or more of the vectors disclosed herein.
  • a vector is provided that comprises a DNA plasmid construct encoding the antibody or antigen-binding fragment, or a portion thereof (e.g., so-called “DMAb”; see, e.g., Muthumani et al., J Infect Dis. 2/-/(3):369-378 (2016); Muthumani etal., Hum Vaccin Immunother 9:2253-2262 (2013)); Flingai et al., Sci Rep.
  • a DNA plasmid construct comprises a single open reading frame encoding a heavy chain and a light chain (or a VH and a VL) of the antibody or antigen-binding fragment, wherein the sequence encoding the heavy chain and the sequence encoding the light chain are optionally separated by polynucleotide encoding a protease cleavage site and/or by a polynucleotide encoding a self-cleaving peptide.
  • the substituent components of the antibody or antigen-binding fragment are encoded by a polynucleotide comprised in a single plasmid. In other embodiments, the substituent components of the antibody or antigen-binding fragment are encoded by a polynucleotide comprised in two or more plasmids (e.g, a first plasmid comprises a polynucleotide encoding a heavy chain, VH, or VH+CH1, and a second plasmid comprises a polynucleotide encoding the cognate light chain, or VL).
  • a single plasmid comprises a polynucleotide encoding a heavy chain and/or a light chain from two or more antibodies or antigen-binding fragments of the present disclosure.
  • An exemplary expression vector is pVaxl, available from Invitrogen®.
  • a DNA plasmid of the present disclosure can be delivered to a subject by, for example, electroporation (e.g., intramuscular electroporation), or with an appropriate formulation (e.g., hyaluronidase).
  • the vector may comprise or consist of circRNA, taRNA, or saRNA.
  • the RNA therapeutic consists of a naked circRNA, taRNA, or saRNA vector
  • the cirRNA, taRNA, or saRNA may, in particular, contain modified nucleosides or other modifications disclosed herein to facilitate delivery to and expression in a human host cell in vivo.
  • naked DNA vectors or other naked DNA therapeutics may contain modifications to facilitate delivery to and expression in a human host cell in vivo.
  • DNA therapeutics and RNA therapeutics including circRNA therapeutics, taRNA therapeutics, or saRNA therapeutics, are also provided, wherein the DNA therapeutics or RNA therapeutics comprise or contain a polynucleotide as disclosed herein (e.g., a polynucleotide that encodes an antibody or antigen-binding fragment that binds to two or more bacterial antigens as described herein).
  • a polynucleotide as disclosed herein e.g., a polynucleotide that encodes an antibody or antigen-binding fragment that binds to two or more bacterial antigens as described herein.
  • the present disclosure also provides a host cell expressing an antibody or antigen-binding fragment according to the present disclosure; or comprising or containing a DNA therapeutic, RNA therapeutic, vector, or polynucleotide according the present disclosure.
  • a method comprises administering to a subject a first polynucleotide (e.g., mRNA) encoding an antibody heavy chain, a VH, or a Fd (VH + CHI), and administering to the subject a second polynucleotide (e.g., mRNA) encoding the cognate antibody light chain, or VL.
  • a first polynucleotide e.g., mRNA
  • VH + CHI Fd
  • a method comprises administering to a subject a first plasmid or vector that comprises a polynucleotide (e.g. mRNA) encoding a VH, a VH + CHI, or a heavy chain, and a second plasmid or vector is provided that comprises a polynucleotide (e.g. mRNA) encoding the cognate VL or light chain.
  • a first plasmid or vector and/or the second plasmid or vector can be comprised in a composition that further comprises a pharmaceutically acceptable carrier, excipient, or diluent.
  • a polynucleotide e.g., mRNA
  • a polynucleotide that encodes a heavy chain and a light chain of an antibody or antigen-binding fragment thereof.
  • a polynucleotide e.g., mRNA
  • a polynucleotide is provided that encodes two heavy chains and two light chains of an antibody or antigen-binding fragment thereof. See, e.g. Li, JQ., Zhang, ZR., Zhang, HQ. et al. Intranasal delivery of replicating mRNA encoding neutralizing antibody against SARS-CoV-2 infection in mice. Sig Transduct Target Ther 6, 369 (2021).
  • a polynucleotide is delivered to a subject via an alphavirus replicon particle (VRP) delivery system.
  • VRP alphavirus replicon particle
  • a replicon comprises a modified VEEV replicon comprising two subgenomic promoters.
  • a polynucleotide or replicon can translate simultaneously the heavy chain (or VH, or VH+1) and the light chain (or VL) of an antibody or antigen-binding fragment thereof.
  • a method is provided that comprises delivering to a subject such a polynucleotide or replicon.
  • the present disclosure also provides a host cell expressing an antibody or antigen-binding fragment according to the present disclosure; or comprising or containing a vector or polynucleotide according the present disclosure.
  • the cells include but are not limited to, eukaryotic cells, e.g., yeast cells, animal cells, insect cells, plant cells; and prokaryotic cells, including A. coli.
  • the cells are mammalian cells, such as human B cells.
  • the cells are a mammalian cell line such as CHO cells (e.g., DHFR- CHO cells (Urlaub et al., PNAS 77:4216 (1980)), human embryonic kidney cells (e.g., HEK293T cells), PER.C6 cells, Y0 cells, Sp2/0 cells.
  • NS0 cells human liver cells, e.g.
  • Hepa RG cells myeloma cells or hybridoma cells.
  • mammalian host cell lines include mouse sertoli cells (e.g., TM4 cells); monkey kidney CV1 line transformed by SV40 (COS-7); baby hamster kidney cells (BHK); African green monkey kidney cells (VERO-76); monkey kidney cells (CV1); human cervical carcinoma cells (HELA); human lung cells (W138); human liver cells (Hep G2); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3 A); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells.
  • mouse sertoli cells e.g., TM4 cells
  • COS-7 monkey kidney CV1 line transformed by SV40
  • BHK baby hamster kidney cells
  • VERO-76 African green monkey kidney cells
  • CV1 monkey kidney cells
  • HELA human cervical carcinoma cells
  • W138 human lung cells
  • Hep G2 human liver cells
  • canine kidney cells MDCK; buffalo
  • Mammalian host cell lines suitable for antibody production also include those described in, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003).
  • a host cell is a prokaryotic cell, such as an E. coli.
  • a prokaryotic cell such as an E. coli.
  • the expression of peptides in prokaryotic cells such as A. coli is well established (see, e.g., Pluckthun, A. Bio/Technology 9:545-551 (1991).
  • antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed.
  • For expression of antibody fragments and polypeptides in bacteria see, e.g., U.S. Pat. Nos. 5,648,237; 5,789,199; and 5,840,523.
  • the cell may be transfected with a vector, DNA therapeutic, or RNA therapeutic according to the present description with an expression vector.
  • transfection refers to the introduction of nucleic acid molecules, such as DNA or RNA (e.g. mRNA) molecules, into cells, such as into eukaryotic cells.
  • RNA e.g. mRNA
  • transfection encompasses any method known to the skilled person for introducing nucleic acid molecules into cells, such as into eukaryotic cells, including into mammalian cells.
  • the present disclosure also provides recombinant host cells that heterologously express an antibody or antigen-binding fragment of the present disclosure.
  • the cell may be of a species that is different to the species from which the antibody was fully or partially obtained (e.g., CHO cells expressing a human antibody or an engineered human antibody).
  • the cell type of the host cell does not express the antibody or antigen-binding fragment in nature.
  • the present disclosure provides methods for producing an antibody, or antigen-binding fragment, wherein the methods comprise culturing a host cell of the present disclosure under conditions and for a time sufficient to produce the antibody, or the antigenbinding fragment.
  • Methods useful for isolating and purifying recombinantly produced antibodies may include obtaining supernatants from suitable host cell/vector systems that secrete the recombinant antibody into culture media and then concentrating the media using a commercially available filter. Following concentration, the concentrate may be applied to a single suitable purification matrix or to a series of suitable matrices, such as an affinity matrix or an ion exchange resin.
  • One or more reverse phase HPLC steps may be employed to further purify a recombinant polypeptide. These purification methods may also be employed when isolating an immunogen from its natural environment. Methods for large scale production of one or more of the isolated/recombinant antibody described herein include batch cell culture, which is monitored and controlled to maintain appropriate culture conditions. Purification of soluble antibodies may be performed according to methods described herein and known in the art and that comport with laws and guidelines of domestic and foreign regulatory agencies.
  • compositions that comprise any one or more of the presently disclosed antibodies, antigen-binding fragments, polypeptides, polynucleotides, vectors, DNA therapeutics, RNA therapeutics) (including mRNA therapeutics, circ RNA therapeutics, taRNA therapeutics, and saRNA therapeutics), or host cells, singly or in any combination, and can further comprise a pharmaceutically acceptable carrier, excipient, or diluent.
  • RNA therapeutics including mRNA therapeutics, circ RNA therapeutics, taRNA therapeutics, and saRNA therapeutics
  • a composition comprises a first vector comprising a first plasmid, and a second vector comprising a second plasmid, wherein the first plasmid comprises a polynucleotide encoding a heavy chain, VH, or VH+CH1, and a second plasmid comprises a polynucleotide encoding the cognate light chain or VL of the antibody or antigen-binding fragment thereof.
  • a composition comprises a polynucleotide (e.g., mRNA) coupled to a suitable delivery vehicle or carrier.
  • a composition comprises a first polynucleotide (e.g., mRNA) encoding an antibody heavy chain, a VH, or a Fd (VH + CHI), and a second polynucleotide (e.g., mRNA) encoding the cognate antibody light chain or VL.
  • a first polynucleotide e.g., mRNA
  • VH + CHI a Fd
  • VH + CHI Fd
  • Exemplary vehicles or carriers for administration to a human subject include a lipid or lipid-derived delivery vehicle, such as a liposome, solid lipid nanoparticle, oily suspension, submicron lipid emulsion, lipid microbubble, inverse lipid micelle, cochlear liposome, lipid microtubule, lipid microcylinder, or lipid nanoparticle (LNP) or a nanoscale platform see, e.g., Li et al. Wilery Interdiscip Rev. Nanomed Nanobiotechnol. 77(2):el530 (2019)). Principles, reagents, and techniques for designing appropriate mRNA and formulating mRNA-LNP and delivering the same are described in, for example, Pardi et al.
  • lipid nanoparticles e.g., ionizable cationic lipid/phosphatidylcholine/cholesterol/PEG-lipid; ionizable lipid:distearoyl PC:cholesterol:polyethylene glycol lipid
  • subcutaneous, intramuscular, intradermal, intravenous, intraperitoneal, and intratracheal administration of the same, are incorporated herein by reference.
  • a composition comprises a first antibody or antigen-binding fragment of the present disclosure and a second antibody or antigen-binding fragment of the present disclosure, wherein of the first antibody or antigen-binding fragment and the second antibody or antigen-binding fragment are different.
  • the first antibody or antigen-binding fragment and the second antibody or antigen-binding fragment bind to the following, respectively: an LTA and a Gmd; an LTA and a PFT (e.g., an alpha toxin, a gamma toxin, or a leukotoxin); an LTA and a SpA; an LTA and a ClfA; a Gmd and a PFT; a Gmd and a SpA; a Gmd and a ClfA; a PFT and a SpA; a PFT and a ClfA; a SpA and a ClfA; a Sbi and an LTA; a Sbi and a Gmd; a Sbi and a PFT; a Sbi and a SpA; or a Sbi and a ClfA.
  • an LTA and a Gmd an LTA and a PFT (e.g., an alpha toxin,
  • a first antibody or antigen-binding fragment thereof comprises a first antibody which binds to an opsonizing target and a second antibody or antigen-binding fragment thereof binds to a neutralizing target.
  • a first antibody or antigen-binding fragment thereof comprises a first antibody which binds to a target selected from LTA, ClfA, Protein A, and Gmd and a second antibody or antigen-binding fragment thereof binds to an alpha toxin.
  • a first antibody or antigen-binding fragment thereof comprises a first antibody which binds to a target selected from LTA, ClfA, Protein A, and Gmd and a second antibody or antigen-binding fragment thereof binds to a neutralizing target.
  • a first antibody or antigen-binding fragment thereof comprises a first antibody which binds to an opsonizing target and a second antibody or antigen-binding fragment thereof binds to an alpha toxin.
  • a combination or composition comprises a first antibody or antigen-binding fragment thereof and a second antigen-binding fragment thereof, wherein the first antibody or antigen-binding fragment thereof or the second antibody or antigen-binding fragment thereof comprises the six CDR sequences (e.g., according to IMGT), and optionally the VH and VL, of (i) SSA12; (ii) SSG20; (iii) SSH3; (iv) SSL17; (v) SSE1; (vi) SSE158; (vii) SSB24; (viii) SSB65; (ix) SSG50; (x) SSG54; (xi) SSM47; (xii) SSG44; (xiii) SSG48; (xiv) SSM37; (xv) SSH4; (xvi) SSH7; (xvii) SSG27; (xviii) SSG39; (xix) SSM45; (xx) SSF52; (xxi) S
  • a combination or composition comprises a first antibody or antigen-binding fragment thereof and a second antigen-binding fragment thereof, wherein the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof comprises the six CDR sequences (e.g., according to IMGT), and optionally the VH and VL, of (i) SSA12 and SSG20, respectively; (ii) SSA12 and SSL17, respectively; (iii) SSA12 and SSH3, respectively; (iv) SSG20 and SSL17, respectively; (v) SSG20 and SSH3, respectively; (vi) SSL17 and SSH3, respectively; (vii) SSE1 and SSE158, respectively; or (viii) SSB24 and SSB65, respectively.
  • the six CDR sequences e.g., according to IMGT
  • VH and VL of (i) SSA12 and SSG20, respectively; (ii) SSA12 and SSL17, respectively; (iii) SSA12 and S
  • the first antibody or antigen-binding fragment and/or the second antibody or antigen-binding fragment comprises a human IgGl Fc polypeptide comprising M428L/N434S/H435R/Y436F mutations.
  • a composition comprises polynucleotide, a vector, a RNA therapeutic, or an DNA therapeutic.
  • the composition comprises a single polynucleotide encoding i) a heavy chain, VH, or VH+CH; and ii) a cognate light chain, VL, or VL+CL, of the antibody or antigen-binding fragment.
  • the polynucleotide is an saRNA and further encodes a replication protein or peptide.
  • the polynucleotide is a circRNA.
  • the composition comprises a first taRNA encoding i) a heavy chain, VH, or VH+CH; and ii) a cognate light chain, VL, or VL+CL, of the antibody or antigenbinding fragment, and a second taRNA that encodes a replication protein or peptide.
  • at least one, or both the first and second taRNA is a circRNA.
  • the composition comprises a first polynucleotide encoding a heavy chain, VH, or VH+CH, and a second polynucleotide encoding a cognate light chain, VL, or VL+CL, of the antibody or antigen-binding fragment.
  • the polynucleotides are mRNAs
  • the first polynucleotide, the second polynucleotide, or both are saRNAs or taRNAs an further encode a replication protein or peptide.
  • at least one, at least two, or all of the polynucleotides is a circRNA.
  • the composition comprises a first mRNA encoding a heavy chain, VH, or VH+CH, and a second mRNA encoding a cognate light chain, VL, or VL+CL, of the antibody or antigen-binding fragment.
  • the composition in which the polynucleotides are mRNAs, is a taRNA composition that comprises a third taRNA that encodes a replication protein or peptide.
  • at least one, at least two, or all of the polynucleotides is a circRNA.
  • LTA laminol
  • Gmd glucosaminidase
  • PFT Pore Forming Toxin
  • a leukocidin e.g., LukED, a LukSF, or both
  • an alpha toxin e.g., Hla
  • a gamma toxin or gamma hemolysin e.g., HIgAB
  • SpA Staphylococcal Protein A
  • ClfA clumping factor A
  • an infection comprises infection by bacteria of any or more of: genus Staphylococcus,' genus Enterococcus,' genus Streptococcus,' and genus Lactococcus.
  • an infection comprises infection by: S. aureus (e.g., MRS A); Coagulase-negative staphylococci (CoNS); S. epidermidis,' S. pneumoniae,' S. lugdunensis,' or S. hominis,' S. aureus FPR3757; S. az//vz/.s NE284 TE2 mutant; S. epidermidis RP62A; S. epidermidis 1200; S.
  • S. aureus e.g., MRS A
  • CoNS Coagulase-negative staphylococci
  • S. epidermidis S. pneumoniae,' S. lugdunensis,' or S. hominis,' S. aureus FPR3757
  • an infection comprises infection by a gram-positive bacteria.
  • Methods of diagnosis may include contacting an antibody, antibody fragment (e.g., antigen binding fragment) with a sample.
  • samples may be isolated from a subject, for example an isolated tissue sample taken from, for example, nasal passages, sinus cavities, salivary glands, lung, liver, pancreas, kidney, ear, eye, placenta, alimentary tract, heart, ovaries, pituitary, adrenals, thyroid, brain, skin, or blood.
  • the methods of diagnosis may also include the detection of an antigen/antibody complex, in particular following the contacting of an antibody or antibody fragment with a sample.
  • a detection step can be performed at the bench, i.e. without any contact to the human or animal body. Examples of detection methods are well-known to the person skilled in the art and include, e.g., ELISA (enzyme-linked immunosorbent assay), including direct, indirect, and sandwich ELISA.
  • “Treat,” “treatment,” or “ameliorate” refers to medical management of a disease, disorder, or condition of a subject e.g., a human or non-human mammal, such as a primate, horse, cat, dog, goat, mouse, or rat).
  • an appropriate dose or treatment regimen comprising an antibody or composition of the present disclosure is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit.
  • Therapeutic or prophylactic/preventive benefit includes improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease, stabilization of disease state; delay or prevention of disease progression; remission; survival; prolonged survival; or any combination thereof.
  • therapeutic or prophylactic/preventive benefit includes reduction or prevention of hospitalization for treatment of a bacterial infection i.e., in a statistically significant manner).
  • therapeutic or prophylactic/preventive benefit includes a reduced duration of hospitalization for treatment of a bacterial infection (z.e., in a statistically significant manner).
  • a “therapeutically effective amount” or “effective amount” of an antibody, antigenbinding fragment, polynucleotide, vector, DNA therapeutic, RNA therapeutic, host cell, or composition of this disclosure refers to an amount of the composition or molecule sufficient to result in a therapeutic effect, including improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease, stabilization of disease state; delay of disease progression; remission; survival; or prolonged survival in a statistically significant manner.
  • a therapeutically effective amount refers to the effects of that ingredient or cell expressing that ingredient alone.
  • a therapeutically effective amount refers to the combined amounts of active ingredients or combined adjunctive active ingredient with a cell expressing an active ingredient that results in a therapeutic effect, whether administered serially, sequentially, or simultaneously.
  • a human subject treated according to the present disclosure is an infant, a child, a young adult, an adult of middle age, or an elderly person. In certain embodiments, a human subject treated according to the present disclosure is less than 1 year old, or is 1 to 5 years old, or is between 5 and 125 years old (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 125 years old, including any and all ages therein or therebetween).
  • a human subject treated according to the present disclosure is 0-19 years old, 20-44 years old, 45-54 years old, 55-64 years old, 65-74 years old, 75-84 years old, or 85 years old, or older. Persons of middle, and especially of elderly age are can be at particular risk.
  • the human subject is 45-54 years old, 55-64 years old, 65-74 years old, 75-84 years old, or 85 years old, or older.
  • the human subject is male. In some embodiments, the human subject is female.
  • a subject treated according to the present disclosure has received a vaccine for a bacterial infection and the vaccine is determined to be ineffective, e.g., by postvaccine infection or symptoms in the subject, by clinical diagnosis or scientific or regulatory consensus.
  • Prophylaxis of a bacterial infection refers in particular to prophylactic settings, wherein the subject was not diagnosed with infection by the bacteria (either no diagnosis was performed or diagnosis results were negative) and/or the subject does not show or experience symptoms of bacterial infection.
  • Prophylaxis of bacterial infection is particularly useful in subjects at greater risk of severe disease or complications when infected, such as pregnant women, children (such as children under 59 months), the elderly, subjects with chronic medical conditions (such as chronic cardiac, pulmonary, renal, metabolic, neurodevelopmental, liver or hematologic diseases) and individuals with immunosuppressive conditions (such as HIV/AIDS, receiving chemotherapy or steroids, or malignancy), subjects using a ventilator, subjects undergoing or having recently undergone surgery, subjects with a blood stream infection or history of blood stream infections, subjects receiving or having received a prosthetic joint or other implant, subjects undergoing or having undergone dialysis, subjects, subjects with bacteremia or a history of bacteremia, subjects having wound, or the like.
  • prophylaxis of bacterial infection is also particularly useful in subjects at greater risk acquiring bacterial infection, e.g., due to increased exposure, for example subjects working or staying in unsanitary areas.
  • treatment is administered as peri-exposure or pre-exposure prophylaxis.
  • treatment is administered as post-exposure prophylaxis.
  • the subject has a bacterial infection, is diagnosed with a bacterial infection, and/or shows symptoms of a bacterial infection.
  • treatment and “therapy”/” therapeutic” of bacterial infection can refer to (complete) cure as well as attenuation/reduction of bacterial infection and/or related symptoms (e.g., attenuation/reduction of severity of infection and/or symptoms, number of symptoms, duration of infection and/or symptoms, or any combination thereof).
  • a reference subject can be, for example, (i) the same subject during an earlier period of time, (ii) a subject of a same or a similar: age or age group; gender; pregnancy status; chronic medical condition (such as chronic cardiac, pulmonary, renal, metabolic, neurodevelopmental, liver or hematologic diseases) or lack thereof; and/or immunosuppressive condition or lack thereof; or (iii) a typical subject within a population (e.g., local, regional, or national, including of a same or similar age or age range and/or general state of health) during a period of time.
  • Prophylaxis can be determined by, for example, the failure to develop a diagnosed bacterial infection and/or the lack of symptoms associated with a bacterial infection over a period of time.
  • treatment and/or prevention comprises post-exposure prophylaxis.
  • the subject has received, is receiving, or will receive an antibiotic agent.
  • the antibiotic agent comprises a penicillin, a fluoroquinolone, a cephalosporin, a macrolide, a beta-lactam with increased activity (e.g. amoxicillin-clavulanate), a tetracycline, a trimethoprim-sulfamethoxazole, a lincosamide (e.g. clindamycin), a urinary anti- infectives, or any combination thereof.
  • the antibiotic agent comprises vancomycin, cefazolin, nafcillin, oxacillin, daptomycin, linezolid, penicillin, amoxicillin, azithromycin, erythromycin, cephalexin, cefdinir, ciproflaxin, levofloxacin, amoxicillin- clavulanate, ceftazidime/avibactam, tetracycline, doxycycline, trimethoprim-sulfamethoxazole, nitrofurantoin, clindamycin, or any combination thereof.
  • a subject has received is receiving, or will receive, wound drainage, removal of a medical device, or both.
  • Typical routes of administering the presently disclosed compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal.
  • parenteral includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.
  • administering comprises administering by a route that is selected from oral, intravenous, parenteral, intragastric, intrapleural, intrapulmonary, intrarectal, intradermal, intraperitoneal, intratumoral, subcutaneous, topical, transdermal, intracisternal, intrathecal, intranasal, and intramuscular.
  • a method comprises orally administering the antibody, antigen-binding fragment, polynucleotide, vector, DNA therapeutic, RNA therapeutic, host cell, or composition to the subject.
  • composition to be administered will, in any event, contain an effective amount of an antibody or antigen-binding fragment, polynucleotide, vector, DNA therapeutic, RNA therapeutic, host cell, or composition of the present disclosure, for treatment of a disease or condition of interest in accordance with teachings herein.
  • a composition may be in the form of a solid or liquid.
  • the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form.
  • the carrier(s) may be liquid, with the compositions being, for example, an oral oil, injectable liquid or an aerosol, which is useful in, for example, inhalatory administration.
  • the pharmaceutical composition is preferably in either solid or liquid form, where semi solid, semi liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
  • the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like.
  • a solid composition will typically contain one or more inert diluents or edible carriers.
  • binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, com starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent.
  • a liquid carrier such as polyethylene glycol or oil.
  • the composition may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension.
  • the liquid may be for oral administration or for delivery by injection, as two examples.
  • preferred compositions contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer.
  • a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.
  • Liquid pharmaceutical compositions may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • Physiological saline is a preferred adjuvant.
  • a liquid composition intended for either parenteral or oral administration should contain an amount of an antibody or antigen-binding fragment as herein disclosed such that a suitable dosage will be obtained. Typically, this amount is at least 0.01% of the antibody or antigenbinding fragment in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition. Certain oral pharmaceutical compositions contain between about 4% and about 75% of the antibody or antigen-binding fragment. In certain embodiments, pharmaceutical compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains between 0.01 to 10% by weight of antibody or antigen-binding fragment prior to dilution.
  • the composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base.
  • the base may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device.
  • the pharmaceutical composition may be intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the drug.
  • the composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient.
  • bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.
  • a composition may include various materials which modify the physical form of a solid or liquid dosage unit.
  • the composition may include materials that form a coating shell around the active ingredients.
  • the materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents.
  • the active ingredients may be encased in a gelatin capsule.
  • the composition in solid or liquid form may include an agent that binds to the antibody or antigen-binding fragment of the disclosure and thereby assists in the delivery of the compound. Suitable agents that may act in this capacity include monoclonal or polyclonal antibodies, one or more proteins or a liposome.
  • the composition may consist essentially of dosage units that can be administered as an aerosol.
  • aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols may be delivered in single phase, bi phasic, or tri phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit. One of ordinary skill in the art, without undue experimentation, may determine preferred aerosols.
  • compositions of the present disclosure also encompass carrier molecules for polynucleotides, as described herein (e.g., lipid nanoparticles, nanoscale delivery platforms, and the like).
  • compositions may be prepared by methodology well known in the pharmaceutical art.
  • a composition intended to be administered by injection can be prepared by combining a composition that comprises an antibody, antigen-binding fragment thereof, or antibody conjugate as described herein and optionally, one or more of salts, buffers and/or stabilizers, with sterile, distilled water so as to form a solution.
  • a surfactant may be added to facilitate the formation of a homogeneous solution or suspension.
  • Surfactants are compounds that non-covalently interact with the peptide composition so as to facilitate dissolution or homogeneous suspension of the antibody or antigen-binding fragment thereof in the aqueous delivery system.
  • an appropriate dose and treatment regimen provide the composition(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (such as described herein, including an improved clinical outcome (e.g., a decrease in frequency, duration, or severity of diarrhea or associated dehydration, or inflammation, or longer disease-free and/or overall survival, or a lessening of symptom severity).
  • a dose should be sufficient to prevent, delay the onset of, or diminish the severity of a disease associated with disease or disorder.
  • Prophylactic benefit of the compositions administered according to the methods described herein can be determined by performing pre-clinical (including in vitro and in vivo animal studies) and clinical studies and analyzing data obtained therefrom by appropriate statistical, biological, and clinical methods and techniques, all of which can readily be practiced by a person skilled in the art.
  • compositions are administered in an effective amount (e.g., to treat a bacterial (e.g., Staphylococcus infection), which will vary depending upon a variety of factors including the activity of the specific compound employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the subject; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy.
  • a bacterial e.g., Staphylococcus infection
  • test subjects will exhibit about a 10% up to about a 99% reduction in one or more symptoms associated with the disease or disorder being treated as compared to placebo-treated or other suitable control subjects.
  • a therapeutically effective dose of an antibody or antigen binding fragment is (for a 70 kg mammal) from about 0.001 mg/kg (z.e., 0.07 mg) to about 100 mg/kg (i.e., 7.0 g); preferably a therapeutically effective dose is (for a 70 kg mammal) from about 0.01 mg/kg (i.e., 0.7 mg) to about 50 mg/kg (i.e., 3.5 g); more preferably a therapeutically effective dose is (for a 70 kg mammal) from about 1 mg/kg (i.e., 70 mg) to about 25 mg/kg (i.e., 1.75 g).
  • a therapeutically effective dose may be different than for an antibody or antigen-binding fragment.
  • the dose may be sufficient to cause endogenous production of the antibody or antigen-binding fragment in a human patient that results, within a specified period of time, in serum concentrations equivalent to that of a therapeutically effective daily dose of the antibody or antigen-binding fragment.
  • the specified period of time may be 1, 2, 5, 7, 14, or 30 days from the time of administration.
  • the serum concentration equivalent to that of a therapeutically effective daily dose of the antibody or antigen-binding fragment may be maintained for at least 2 weeks, 1 month, 2 months, 3 months, or 6 months, or for at least a time between 2 weeks and 6 months, 2 weeks and 3 months, 2 weeks and 2 months, 2 weeks and 1 month, 1 and 6 months, 1 and 3 months, 1 and 2 months, 2 and 6 months, 2 and 3 months, or 3 and 6 months from the time of administration.
  • the polynucleotide may not integrate into the human host cell genome or persist in the human host, such that the antibody or antigen-binding fragment may be undetectable in serum by at least 1, 2, 3, 6, or 12 months from the time of administration.
  • Embodiment 70 The polynucleotide of any one of Embodiments 67-69, comprising a modified nucleoside, a cap-1 structure, a cap-2 structure, or any combination thereof, wherein, optionally, the polynucleotide comprises a pseudouridine, aN6-methyladenonsine, a 5 -methylcytidine, a 2-thiouridine, or any combination thereof, wherein, further optionally, the pseudouridine comprises Nl- methylpseudouridine .
  • Embodiment 82 The composition or combination of Embodiment 81, wherein the first antibody or antigen-binding fragment and the second antibody or antigen-binding fragment are capable of binding to: an LTA and a Gmd, respectively; an LTA and an alpha toxin, respectively; an LTA and a gamma toxin, respectively; an LTA and a leucocidin, respectively; an LTA and a SpA, respectively; an LTA and a ClfA, respectively; an alpha toxin and a Gmd, respectively; an alpha toxin and a gamma toxin, respectively; an alpha toxin and a leucocidin, respectively; an alpha toxin and a SpA, respectively; an alpha toxin and a ClfA, respectively; a gamma toxin and a Gmd, respectively; a gamma toxin and a leucocidin, respectively; a gamma toxin and
  • Embodiment 83 The composition or combination of any one of Embodiments 80-82, comprising a first antibody or antigen-binding fragment and a second antibody or antigen-binding fragment, wherein the first antibody or antigen-binding fragment or the second antibody or antigen-binding fragment comprises the six CDR sequences (e.g., according to IMGT), and optionally the VH and VL, of: (i) SSA12; (ii) SSG20; (iii) SSH3; (iv) SSL17; (v) SSE1; (vi) SSE158; (vii) SSB24; (viii) SSB65; (ix) SSG50; (x) SSG54; (xi) SSM47; (xii) SSG44; (xiii) SSG48; (xiv) SSM37; (xv) SSH4; (xvi) SSH7; (xvii) SSG27; (xviii) SSG39; (xix) SSM45
  • Embodiment 88 The method of making an antibody or antigen-binding fragment of Embodiment 87, wherein the host cell of Embodiment 78 or the human B cell of Embodiment 79 comprises the polynucleotide of any one of Embodiments 67-74 and/or recombinant vector, DNA therapeutic, or RNA therapeutic of any one of Embodiments 75-77.
  • Embodiment 89 The method of Embodiment 87 or 88, further comprising isolating the antibody or antigen-binding fragment.
  • Embodiment 90 A method of treating or preventing a bacterial infection in a(n, e.g., human) subject, wherein the bacterial infection is optionally bacteremia, wherein further optionally the bacteremia is hemodialysis associated/line associated bacteremia, the method comprising administering to the subject an effective amount of: (i) the antibody or antigen -binding fragment of any one of Embodiments 1-66; (ii) the polynucleotide of any one of Embodiments 67-74; (iii) the recombinant vector, DNA therapeutic, or RNA therapeutic of any one of Embodiments 75-77; (iv) the host cell of Embodiment 78; (v) the human B cell of Embodiment 79; and/or (vi) the composition or combination of any one of Embodiments 80-86, wherein the bacteria expresses the LTA, Gmd, ClfA, SpA, alpha toxin, leucocidin, gamma to
  • Embodiment 91 The method of Embodiment 90, wherein the bacteria comprises a bacteria of genus Staphylococcus and/or a bacteria of genus Enterococcus and/or a bacteria of genus Streptococcus and/or a bacteria of genus Lactococcus, wherein, optionally, the bacteria comprises: (1) .S', aureus, wherein, further optionally, the .S', aureus comprises a methicillin-resistant .S'. Aureus (MRSA); (2) a Coagulase-negative Staphylococcus (CoNS); (3) .S', epidermidis,' (4) E.
  • MRSA methicillin-resistant .S'. Aureus
  • CoNS Coagulase-negative Staphylococcus
  • Embodiment 92 The method of Embodiment 90 or 91, comprising administering a single dose of the antibody or antigen-binding fragment, polypeptide, polynucleotide, recombinant vector, DNA therapeutic, RNA therapeutic, host cell, or composition to the subject.
  • Embodiment 93 The method of any one of Embodiments 90-92, comprising administering two or more doses of the antibody or antigen-binding fragment, polypeptide, polynucleotide, recombinant vector, DNA therapeutic, RNA therapeutic, host cell, or composition to the subject.
  • Embodiment 94 The method of any one of Embodiments 90-93, comprising administering the antibody or antigen-binding fragment, polypeptide, polynucleotide, recombinant vector, DNA therapeutic, RNA therapeutic, host cell, or composition intramuscularly, subcutaneously, or intravenously.
  • Embodiment 95 The method of any one of Embodiments 90-94, wherein the treatment and/or prevention comprises pre-exposure or post-exposure prophylaxis.
  • Embodiment 96 The method of any one of Embodiments 90-95, wherein the subject: is using a ventilator; is undergoing, will undergo, or has recently undergone surgery; has a blood stream infection or a history of blood stream infections; is receiving, will receive, or has received a prosthetic joint or other implant; is undergoing, will undergo, or has undergone dialysis; has bacteremia or a history of bacteremia; has neutropenia or a history of neutropenia; has hemodialysis associated/line associated bacteremia or a history of hemodialysis associated/line associated bacteremia; has a wound; or any combination of the foregoing.
  • Embodiment 97 The method of any one of Embodiments 90-96, further comprising administering to the subject or more antibiotic agent, or wherein the subject has received one or more antibiotic agent.
  • Embodiment 98 The method of Embodiment 97, wherein the antibiotic agent comprises a penicillin, a fluoroquinolone, a cephalosporin, a macrolide, a beta-lactam with increased activity (e.g. amoxicillin-clavulanate), a tetracycline, a trimethoprim-sulfamethoxazole, a lincosamide (e.g. clindamycin), a urinary anti-infectives, or any combination thereof.
  • the antibiotic agent comprises a penicillin, a fluoroquinolone, a cephalosporin, a macrolide, a beta-lactam with increased activity (e.g. amoxicillin-clavulanate), a tetracycline, a trimethoprim-sulfamethoxazole, a lincosamide (e.g. clindamycin), a urinary anti-infectives, or
  • Embodiment 99 The method of Embodiment 97 or 98, wherein the antibiotic agent comprises vancomycin, cefazolin, nafcillin, oxacillin, daptomycin, linezolid, penicillin, amoxicillin, azithromycin, erythromycin, cephalexin, cefdinir, ciproflaxin, levofloxacin, amoxicillin-clavulanate, ceftazidime/avibactam, tetracycline, doxycycline, trimethoprim-sulfamethoxazole, nitrofurantoin, clindamycin, or any combination thereof.
  • the antibiotic agent comprises vancomycin, cefazolin, nafcillin, oxacillin, daptomycin, linezolid, penicillin, amoxicillin, azithromycin, erythromycin, cephalexin, cefdinir, ciproflaxin, levoflox
  • Embodiment 100 The antibody or antigen-binding fragment of any one of Embodiments 1- 61a, the polynucleotide of any one of Embodiments 62-66b, the recombinant vector, DNA therapeutic, or RNA therapeutic of any one of Embodiments 67-67b, the host cell of Embodiment 68, the human B cell of Embodiment 69, and/or the composition or combination of any one of Embodiments 70-71, for use in a method of treating or preventing a bacterial infection in a subject, wherein the bacteria expresses the LTA, Gmd, ClfA, SpA, alpha toxin, leucocidin, gamma toxin, or Sbi, wherein, optionally, the bacteria comprises .S'. aureus.
  • Embodiment 101 The antibody or antigen-binding fragment of any one of Embodiments 1- 66, the polynucleotide of any one of Embodiments 67-74, the recombinant vector, DNA therapeutic, or RNA therapeutic of any one of Embodiments 75-77, the host cell of Embodiment 78, the human B cell of Embodiment 79, and/or the composition or combination of any one of Embodiments 80-86, for use in the preparation of a medicament for the treatment or prevention of a bacterial infection in a subject, wherein the bacteria expresses the LTA, Gmd, ClfA, SpA, alpha toxin, leucocidin, gamma toxin, or Sbi, wherein, optionally, the bacteria comprises .S'. aureus.
  • Embodiment 102 A method for in vitro diagnosis of a bacterial infection, the method comprising:
  • Aureus MRSA
  • CoNS Coagulase -negative Staphylococcus
  • CoNS Coagulase -negative Staphylococcus
  • .S' epidermidis
  • E.faecalis E.faecalis
  • (5) .S', pyogenes (6) .S', agalactiae,' (7) L. garviae
  • Embodiment 104 A library comprising a plurality of antibodies or antigen-binding fragments comprising amino acid sequences from any one or more of, and/or comprising a plurality of polynucleotides encoding amino acid sequences encoded by any one or more of the following human immunoglobulin genes: IGHV3-23; IGHV3-7; IGHJ1; IGHJ3; IGLV3-21; IGKV1-5; IGKV1-6; IGLJ2; IGKJ2; IGKJ1; IGHV1-8; IGHV3-30; IGHV3-49; IGHV4-39; IGHJ6; IGHJ3; IGHJ4; IGKV1-27; IGLV3-21; IGKV1-5; IGKV1D-12; IGKV1-9; IGLV1-40; IGVK1-6; IGLV1-47; IGKJ1; IGLJ2; IGKJ2; IGKJ1; IGL
  • Embodiment 106 The library of Embodiment 104 or 105, wherein the library comprises a plurality of antibodies or antigen-binding fragments comprising amino acid sequences from any one or more of, and/or comprises a plurality of polynucleotides encoding amino acid sequences from any one or more of (i)-(xxiii): (i) IGHV3-23, IGHJ1, IGLV3-21, and IGLJ2; (ii) IGHV3-23, IGHJ3, IGKV1-5, and IGKJ1; (iii) IGHV3-7, IGHJ3, IGKV1-6, and IGKJ1; (iv) IGHV3-7, IGHJ3, IGKV1-6, and IGKJ2; (v) IGHV3-23, IGHJ1, IGLV3-21, and IGLJ2; (vi) IGHV3-23, IGHJ3, IGKV1-5, and IGKJ2; (vii) IGHV3- 23,
  • Embodiment 107 The library of Embodiment 106, wherein the plurality of antibodies or antigenbinding fragments, or the plurality of polynucleotides encoding the antibodies or antigen-binding fragments, further comprises or encodes additional amino acid sequences, wherein the additional amino acid sequences are optionally randomized and/or are limited to one or more CDR regions of an antibody or antigen-binding fragment.
  • Embodiment 108 A method comprising screening one or more antibodies of the library of any one of Embodiments 104-107 for binding to a bacterial (e.g., .S', aureus) antigen selected from: a lipoteichoic acid (LTA) (optionally a Type I, a Type II, and/or a Type IV LTA); a glucosaminidase (Gmd); a clumping factor A (ClfA); a Staphylococcus Protein A (SpA); an alpha toxin; a leukocidin; a gamma toxin; and a Sbi.
  • LTA lipoteichoic acid
  • Gmd glucosaminidase
  • ClfA clumping factor A
  • SpA Staphylococcus Protein A
  • Embodiment 109 A kit comprising a liquid composition comprising and antibody or antigenbinding fragment of any one of Embodiments 1-66, 100, 101, or 103, the polynucleotide of any one of Embodiments 67-74, the recombinant vector, DNA therapeutic, or RNA therapeutic of any one of Embodiments 75-77, the host cell of Embodiment 78, the human B cell of Embodiment 79, and/or the composition of any one of Embodiments 80-86, and instructions for use thereof in treating a bacterial (e.g., Staphylococcus) infection in a subject.
  • a bacterial e.g., Staphylococcus
  • Embodiment 201 An antibody, or an antigen-binding fragment thereof, comprising the complementarity determining region (CDR)H1, CDRH2, CDRH3, CDRL1, CDRL2, and/or CDRL3 amino acid sequence(s), or functional variants thereof (and optionally comprising the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences, or functional variants thereof), of the heavy chain variable domain (VH) and light chain variable domain (VL) amino acid sequences set forth in SEQ ID NOs.: (a) (i) 31 and 35, respectively; (ii) 121 and 125, respectively; (iii) 21 and 25, respectively; (iv) 131 and 135, respectively; (v) 141 and 145, respectively; (vi) 151 and 155, respectively; (vii) 161 and 165, respectively; (viii) 241 and 245, respectively; (ix) 171 and 175, respectively; (x) 522 and 526, respectively; (xi) 532
  • Embodiment 202 An antibody, or an antigen-binding fragment thereof, comprising the complementarity determining region (CDR)H1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences, and optionally the heavy chain variable domain (VH) and light chain variable domain (VL) amino acid sequences, of one of the following antibodies: (a) SSC35; SSE73; SSA12; SSA8; SSA9; SSA10; SSC1; SSC10; SSC15; SSAA5; SSAA7; SSAA15; SSAA21; SSAA60; SSAA65; SSAA68; SSAA69; SSAA71; SSAA72; SSAB24; SSAB30; SSAC1; SSE137; SSE150; SSE151; SSE153; or SSF69, wherein the antibody or antigen-binding fragment is capable of binding to a lipoteichoic acid (LTA); (b) SSG20; SSF
  • Embodiment 203 The antibody or antigen-binding fragment of embodiment 201 or 202, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences are according to: (i) the IMGT numbering system (optionally using the junction definitions for the CDRH3 and CDRL3 sequences); (ii) the Kabat numbering system; (iii) the Chothia numbering system; (iv) the Enhanced Chothia (also referred to as “Martin”) numbering system; (v) the AHo numbering system; (vi) the North numbering system; (vii) the Contact numbering system; (viii) the EU numbering system; (ix) the AbM numbering system; or (x) a combination of any two or more of: IMGT, Kabat, Chothia, Enhanced Chothia, AHo, EU, North, AbM, and Contact, wherein, optionally, the two or more numbering systems combined produce CDR definitions having the greatest length of the CDRs
  • Embodiment 204 An antibody or antigen-binding fragment thereof comprising a CDRH3 and a CDRL3, wherein the CDRH3 and the CDRL3 comprise, consist essentially of, or consist of the amino acid sequences set forth in SEQ ID NOS.: (a) (i) 34 and 38, respectively; (ii) 124 and 128, respectively; (iii) 24 and 28, respectively; (iv) 134 and 138, respectively; (v) 144 and 148, respectively; (vi) 154 and 158, respectively; (vii) 164 and 168, respectively; (viii) 244 and 248, respectively; (ix) 174 and 178, respectively; (x) 525 and 529, respectively; (xi) 535 and 539, respectively; (xii) 545 and 549, respectively; (xiii) 555 and 559, respectively; (xiv) 565 and 569, respectively; (xv) 575 and 579, respectively; (xvi) 585 and 589, respectively;
  • Staphylococcus Protein A (SpA); (e) (i) 4 and 8, respectively; (ii) 14 and 18, respectively; (iii) 435 and 439, respectively; (iv) 445 and 449, respectively; (v) 455 and 459, respectively; (vi) 465 and 469, respectively; (vii) 475 and 479, respectively; (viii) 485 and 489, respectively; (ix) 495 and 499, respectively; (x) 505 and 509, respectively; or (xi) 515 and 519, respectively; wherein the antibody or antigen-binding fragment is capable of binding to an alpha toxin; (f) (i) 214 and 218, respectively; (ii) 104 and 108, respectively; (iii) 224 and 228, respectively; (iv) 114 and 118, respectively; or (v) 234 and 238, respectively; wherein the antibody or antigen-binding fragment is capable of binding to a leucocidin and/or gamma toxin
  • Embodiment 205 An antibody, or an antigen-binding fragment thereof, comprising a complementarity determining region (CDR)H1, a CDRH2, a CDRH3, a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise, consist essentially of, or consist of the amino acid sequences set forth in SEQ ID NOS.: (a) (i) 32-34 and 36-38, respectively; (ii) 122-124 and 126-128, respectively; (iii) 22-24 and 26-28, respectively; (iv) 132-134 and 136-138, respectively; (v) 142-144 and 146-148, respectively; (vi) 152-154 and 156-158, respectively; (vii) 162-164 and 166-168, respectively; (viii) 242-244 and 246-248, respectively; (ix) 172-174 and 176- 178, respectively; (x) 523-525 and 527-529
  • Embodiment 206 The antibody or antigen-binding fragment of any one of embodiments 201- 205, comprising a heavy chain variable domain framework region (VHFR)1, a VHFR2, a VHFR3, a light chain variable domain framework region (VLFR)1, a VLFR2, a VLFR3, and/or a VLFR4 (or a variant of the VHFR1, VHFR2, VHFR3, VHFR4, VLFR1, VLFR2, VLFR3, or VLFR4 comprising one, two, three, four, or five substitutions, insertions, and/or deletions, or a variant having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity or similarity to the VHFR1, VHFR2, VHFR3, VHFR4, VLFR1, VLFR2,
  • Embodiment 207 The antibody or antigen-binding fragment of embodiment 206, wherein the framework region or regions are according to: (i)the IMGT numbering system (optionally using the junction definitions for the CDRH3 and CDRL3 sequences); (ii) the Kabat numbering system; (iii) the Chothia numbering system; (iv) the Enhanced Chothia (also referred to as “Martin”) numbering system; (v) the AHo numbering system; (vi) the North numbering system; (vii) the Contact numbering system; (viii) the EU numbering system; (ix) the AbM numbering system; or (x) a combination of any two or more of: IMGT, Kabat, Chothia, Enhanced Chothia, AHo, EU, North, AbM, and Contact, wherein, optionally, the two or more numbering systems combined produce CDR definitions having the greatest length of the CDRs according to any numbering system or combination of numbering systems.
  • the two or more numbering systems combined produce C
  • Embodiment 208 The antibody or antigen-binding fragment of any one of embodiments 201- 207, comprising a VH and a VL, wherein the VH and the VL comprise, consist essentially of, or consist of, amino acid sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity or similarity to, or comprising, consisting essentially of, or consisting of, the VH and VL amino acid sequences set forth in SEQ ID NOs.: (a) (i) 31 and 35, respectively; (ii) 121 and 125, respectively; (iii) 21 and 25, respectively; (iv) 131 and 135, respectively; (v) 141 and 145, respectively; (vi) 151 and 155, respectively; (vii) 161 and 165, respectively; (viii) 241 and
  • Staphylococcus Protein A (SpA); (e) (i) 1 and 5, respectively; (ii) 11 and 15, respectively; (iii) 432 and 436, respectively; (iv) 442 and 446, respectively; (v) 452 and 456, respectively; (vi) 462 and 466, respectively; (vii) 472 and 476, respectively; (viii) 482 and 486, respectively; (ix) 492 and 496, respectively; (x) 502 and 506, respectively; or (xi) 512 and 516, respectively, wherein the antibody or antigen-binding fragment is capable of binding to an alpha toxin; (f) (i) 211 and 215, respectively; (ii) 101 and 105, respectively; (iii) 221 and 225, respectively; (iv) 111 and 115, respectively; or (v) 231 and 235, respectively; wherein the antibody or antigen-binding fragment is capable of binding to a leucocidin and/or gamma toxin; or
  • Embodiment 209 The antibody or antigen-binding fragment of any one of embodiments 201- 208, wherein the antibody or antigen-binding fragment is capable of binding to a LTA, wherein, optionally, the antibody or antigen-binding binding fragment is capable of binding to: (i) a Type I LTA; (ii) a Type II LTA; (iii) a Type IV LTA; or (iv) any combination of (i)-(iii).
  • Embodiment 210 The antibody or antigen-binding fragment of any one of embodiments 201- 208, wherein the antibody or antigen-binding binding fragment is capable of binding to a Gmd, wherein, optionally, the antibody or antigen-binding fragment is capable of binding to a .S'. aureus, to .S'. epide rmidis. or to .S' aureus and to .S' epidermidis .
  • Embodiment 211 The antibody or antigen-binding fragment of any one of embodiments 201- 208, wherein the antibody or antigen-binding fragment is capable of binding to a ClfA.
  • Embodiment 212 The antibody or antigen-binding fragment of embodiment 211, wherein the antibody or antigen-binding binding is capable of binding to a ClfA OOl, a ClfA_002, and/or a ClfA_004.
  • Embodiment 213 The antibody or antigen-binding fragment of any one of embodiments 201- 208, wherein the antibody or antigen-binding fragment is capable of binding to a Staphylococcus Protein A (SpA), wherein, optionally, the antibody or antigen-binding fragment is capable of binding to a SPAKKAA-
  • SpA Staphylococcus Protein A
  • Embodiment 214 The antibody or antigen-binding fragment of any one of embodiments 201- 208, wherein the antibody or antigen-binding fragment is capable of binding to an alpha toxin, wherein, optionally, the alpha toxin comprises a-hemolysin (also called Hla).
  • a-hemolysin also called Hla
  • Embodiment 215. The antibody or antigen-binding fragment of any one of embodiments 201- 208, wherein the antibody or antigen-binding fragment is capable of binding to a gamma toxin.
  • Embodiment 216 The antibody or antigen-binding fragment of any one of embodiments 201-208 and 215, wherein the antibody or antigen-binding fragment is capable of binding to a leukocidin, wherein, optionally, the leukocidin comprises a LukD, a LukE, a LukF, a LukS, or any combination thereof.
  • Embodiment 217 The antibody or antigen-binding fragment of embodiment 216, wherein the antibody or antigen-binding fragment is capable of binding to a LukED, a LukSF, or to a LukED and a LukSF.
  • Embodiment 218 The antibody or antigen-binding fragment of any one of embodiments 201-208 and 215-217, wherein the antibody or antigen-binding fragment is capable of binding to a HIgA, a HIgB, or both.
  • Embodiment 219. The antibody or antigen-binding fragment of any one of embodiments 201- 208, wherein the antibody is capable of binding to a second immunoglobulin-binding protein (Sbi, also called a Staphylococcal binder of immunoglobulin.).
  • a second immunoglobulin-binding protein also called a Staphylococcal binder of immunoglobulin.
  • Embodiment 22 An anti-lipoteichoic acid (LTA) antibody, or an antigen-binding fragment thereof, comprising the complementarity determining region (CDR)H1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of the heavy chain variable domain (VH) and light chain variable domain (VL) amino acid sequences set forth in SEQ ID NOs.: 21 and 25, respectively.
  • LTA complementarity determining region
  • VH heavy chain variable domain
  • VL light chain variable domain
  • first antigen-binding domain comprising a first antigen-binding domain and second antigen-binding domain, wherein the first antigen-binding domain or the second antigen-binding domain comprises the six CDR sequences (e.g., according to IMGT), and optionally the VH and VL, of: (i) SSA12; (ii) SSG20; (iii) SSH3; (iv) SSL17; (v) SSE1; (vi) SSE158; (vii) SSB24; or (viii) SSB65; (ix) SSG50; (x) SSG54; (xi) SSM47; (xii) SSG44; (xiii) SSG48; (xiv) SSM37; (xv) SSH4; (xvi) SSH7; (xvii) SSG27; (xviii) SSG39; (xix) SSM45; (xx) SSE52; (xxi) SSG51; (xxii) SSE34
  • Embodiment 232 An antibody, or an antigen-binding fragment thereof, which is a multi-specific antibody or antigen-binding fragment thereof, which is capable of binding to: an LTA and a Gmd; an LTA and an alpha toxin; an LTA and a gamma toxin; an LTA and a leucocidin; an LTA and a SpA; an LTA and a ClfA; an alpha toxin and a Gmd; an alpha toxin and a gamma toxin; an alpha toxin and a leucocidin; an alpha toxin and a SpA; an alpha toxin and a ClfA; a gamma toxin and a Gmd; a gamma toxin and a leucocidin; a gamma toxin and a SpA; a gamma toxin and a ClfA; a leucocidin and a
  • 232 which comprises or is of a format selected from: a Bispecific T cell Engager (BiTE); a DART; a Knobs-Into-Holes (KIH) assembly; a scFv-CH3-KIH assembly; a KIH Common Light-Chain antibody; a TandAb; a Triple Body; a TriBi Minibody; a Fab-scFv; a scFv-CH-CL-scFv; a F(ab’)2-scFv2; a tetravalent Hcab; an intrabody; a CrossMab; a Dual Action Fab (DAF) (two-in-one or four-in-one); a DutaMab; a DT-IgG, a charge paired antibody; a Fab-arm Exchange antibody, a SEEDbody; a Triomab; a LUZ-Y assembly, an Fcab; a K/.-body: an orthogonal
  • the mutation that increases binding affinity to a human FcRn comprises M428L/N434S or M428L/N434A.
  • Embodiment 246 The antibody or antigen-binding fragment of any one of embodiments 201-
  • Embodiment 247 The antibody or antigen-binding fragment of any one of embodiments 201-
  • Embodiment 248 The antibody or antigen-binding fragment of any one of embodiments 201-
  • Embodiment 249. The antibody or antigen-binding fragment of any one of embodiments 201-
  • amino acid mutation that promotes complement activation comprises or consists of: K236W/E333S; K326A/E333A; S267E/H268F/S324T; S267E/H268F/S324T/G236A/I332E; Y300L; G236A/Y300L; or G236A/R292P/Y300L.
  • Embodiment 250 The antibody or antigen-binding fragment of any one of embodiments 201-
  • amino acid mutation that reduces binding by Staphylococcus Protein A comprises or consists of: H435R; Y436F; or H435R/Y436F.
  • Embodiment 251 The antibody or antigen-binding fragment of any one of embodiments 201-
  • Embodiment 252 The antibody or antigen-binding fragment of any one of embodiments 201-
  • Embodiment 251 which is capable of neutralizing an infection (in vitro, ex vivo, and/or in vivo) by a bacteria expressing the LTA, Gmd, ClfA, SpA, alpha toxin, leucocidin, gamma toxin, or Sbi.
  • Embodiment 253. The antibody or antigen-binding fragment of any one of embodiments 201-
  • Embodiment 254 The antibody or antigen-binding fragment of any one of embodiments 201-
  • the bacteria comprises .S', aureus.
  • Embodiment 255 The antibody or antigen-binding fragment of any one of embodiments 201-
  • Embodiment 256 The antibody or antigen-binding fragment of any one of embodiments 201-
  • Embodiment 257 An antibody, or an antigen-binding fragment, that is capable of binding to an .S', aureus antigen selected from: a lipoteichoic acid (LTA) (optionally a Type I, a Type II, and/or a Type IV LTA); a glucosaminidase (Gmd); a clumping factor A (ClfA); a Staphylococcus Protein A (SpA); an alpha toxin; a leukocidin; a gamma toxin; and a Sbi, wherein, optionally:
  • LTA lipoteichoic acid
  • Gmd glucosaminidase
  • ClfA clumping factor A
  • SpA Staphylococcus Protein A
  • the antibody or antigen-binding fragment binds to a LTA and does not complete with pagibaximab for binding to the LTA;
  • the antibody or antigen-binding fragment comprises a VH and a VL that comprise, consist essentially of, or consist of, amino acid sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity or similarity to, or comprising, consisting essentially of, or consisting of, the VH and VL amino acid sequences set forth in SEQ ID NOs.:
  • Embodiment 259 The polynucleotide of embodiment 258, wherein the polynucleotide comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), wherein the RNA optionally comprises messenger RNA (mRNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • Embodiment 260 The polynucleotide of embodiment 259, wherein the polynucleotide comprises self-amplifying RNA (saRNA), trans-amplifying RNA (taRNA), or circular RNA (circRNA).
  • saRNA self-amplifying RNA
  • taRNA trans-amplifying RNA
  • circRNA circular RNA
  • Embodiment 261 The polynucleotide of any one of embodiments 258-260, comprising a modified nucleoside, a cap-1 structure, a cap-2 structure, or any combination thereof, wherein, optionally, the polynucleotide comprises a pseudouridine, a N6-methyladenonsine, a 5 -methylcytidine, a 2- thiouridine, or any combination thereof, wherein, further optionally, the pseudouridine comprises Nl- methylpseudouridine .
  • Embodiment 264 The polynucleotide of embodiment 262, wherein the host cell is a production host cell.
  • Embodiment 265. The polynucleotide of embodiment 262, wherein the host cell is an in vivo human host cell.
  • Embodiment 266 A recombinant vector, DNA therapeutic, or RNA therapeutic comprising the polynucleotide of any one of embodiments 258-265.
  • Embodiment 267 A DNA therapeutic construct or RNA therapeutic construct comprising the polynucleotide of any one of embodiments 258-265 encapsulated in a carrier, wherein the carrier optionally comprises a lipid, a lipid-derived delivery vehicle, such as a liposome, a solid lipid nanoparticle, an oily suspension, a submicron lipid emulsion, a lipid microbubble, an inverse lipid micelle, a cochlear liposome, a lipid microtubule, a lipid microcylinder, lipid nanoparticle (LNP), or a nanoscale platform.
  • a lipid-derived delivery vehicle such as a liposome, a solid lipid nanoparticle, an oily suspension, a submicron lipid emulsion, a lipid microbubble, an inverse lipid micelle, a cochlear liposome, a lipid microtubule, a lipid microcylinder, lipid nanoparticle (LNP), or
  • Embodiment 268 A recombinant vector comprising the polynucleotide of any one of embodiments 258-265.
  • Embodiment 269. A host cell comprising the polynucleotide of any one of embodiments 258-265 and/or the vector, DNA therapeutic, or RNA therapeutic of any one of embodiments 266-268, wherein the polynucleotide is optionally heterologous to the host cell and/or wherein the host cell is capable of expressing the encoded antibody or antigen-binding fragment or polypeptide.
  • Embodiment 270 An isolated human B cell comprising the polynucleotide of any one of embodiments 258-265 and/or the vector, DNA therapeutic, or RNA therapeutic of any one of embodiments 266-268, wherein polynucleotide is optionally heterologous to the human B cell and/or wherein the human B cell is immortalized.
  • Embodiment 271 A composition or combination comprising: (i) the antibody or antigen-binding fragment of any one of embodiments 201-257; (ii) the polynucleotide of any one of embodiments 258- 265; (iii) the recombinant vector, DNA therapeutic, or RNA therapeutic of any one of embodiments 266- 268; (iv) the host cell of embodiment 269; and/or (v) the human B cell of embodiment 270, and, optionally, a pharmaceutically acceptable excipient, carrier, or diluent.
  • Embodiment 272 The composition or combination of embodiment 271, which comprises (1) a first antibody or antigen-binding fragment of any one of embodiments 201-257 and (2) a second, different antibody or antigen-binding fragment of any one of embodiments 201-257.
  • Embodiment 273 The composition or combination of embodiment 272, wherein the first antibody or antigen-binding fragment and the second antibody or antigen-binding fragment are capable of binding to: an LTA and a Gmd, respectively; an LTA and an alpha toxin, respectively; an LTA and a gamma toxin, respectively; an LTA and a leucocidin, respectively; an LTA and a SpA, respectively; an LTA and a ClfA, respectively; an alpha toxin and a Gmd, respectively; an alpha toxin and a gamma toxin, respectively; an alpha toxin and a leucocidin, respectively; an alpha toxin and a SpA, respectively; an alpha toxin and a ClfA, respectively; a gamma toxin and a Gmd, respectively; a gamma toxin and a leucocidin, respectively; a gamma toxin and a SpA,
  • Embodiment 274 The composition or combination of any one of embodiments 271-273, comprising a first antibody or antigen-binding fragment and a second antibody or antigen-binding fragment, wherein the first antibody or antigen-binding fragment or the second antibody or antigenbinding fragment comprises the six CDR sequences (e.g., according to IMGT), and optionally the VH and VL, of: (i) SSA12; (ii) SSG20; (iii) SSH3; (iv) SSL17; (v) SSE1; (vi) SSE158; (vii) SSB24; (viii) SSB65; (ix) SSG50; (x) SSG54; (xi) SSM47; (xii) SSG44; (xiii) SSG48; (xiv) SSM37; (xv) SSH4; (xvi) SSH7; (xvii) SSG27; (xviii) SSG39; (xix) SSM45; (xx
  • Embodiment 275 The composition or combination of any one of embodiments 271-274, comprising a first antibody or antigen-binding fragment and a second antibody or antigen-binding fragment, wherein the first antibody or antigen-binding fragment and the second antibody or antigenbinding fragment comprise the six CDR sequences (e.g., according to IMGT), and optionally the VH and VL, of: (i) SSA12 and SSG20, respectively; (ii) SSA12 and SSL17, respectively; (iii) SSA12 and SSH3, respectively; (iv) SSG20 and SSL17, respectively; (v) SSG20 and SSH3, respectively; (vi) SSL17 and SSH3, respectively; (vii) SSE1 and SSE158, respectively; or (viii) SSB24 and SSB65, respectively, wherein, optionally, the antibody or antigen-binding fragment comprises a human IgGl Fc polypeptide comprising M428L/N434S/H435R/Y436F
  • Embodiment 276 The composition or combination of embodiment 271, comprising a first polynucleotide and a second polynucleotide, each according to any one of embodiments 258-265, or a recombinant vector, DNA therapeutic, or RNA therapeutic of any one of embodiments 266-268, encoding two or more antibodies or antigen-binding fragments, wherein the first antibody or antigen-binding fragment encoded by the first polypeptide and second antibody or antigen-binding fragment encoded by the second polypeptide, respectively, or the multispecific antibody or antigen-binding fragment, comprise(s) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL, according to any two antibodies of the present disclosure, or any antigen-binding fragments thereof.
  • Embodiment 277 A composition comprising the polynucleotide of any one of embodiments 258- 265 or the vector, DNA therapeutic, or RNA therapeutic of any one of embodiments 266-268 encapsulated in a carrier molecule, wherein the carrier molecule optionally comprises a lipid, a lipidderived delivery vehicle, such as a liposome, a solid lipid nanoparticle, an oily suspension, a submicron lipid emulsion, a lipid microbubble, an inverse lipid micelle, a cochlear liposome, a lipid microtubule, a lipid microcylinder, lipid nanoparticle (LNP), or a nanoscale platform.
  • Embodiment 278 A(n, e.g.
  • in vitro or ex vivo method of making an antibody or antigen-binding fragment of any one of embodiments 201-257, comprising culturing the host cell of embodiment 269 or the human B cell of embodiment 270 for a time and under conditions sufficient for the host cell or human B cell, respectively, to express the antibody or antigen-binding fragment.
  • Embodiment 279. The method of making an antibody or antigen-binding fragment of embodiment 278, wherein the host cell of embodiment 269 or the human B cell of embodiment 270 comprises the polynucleotide of any one of embodiment 258-265 and/or recombinant vector, DNA therapeutic, or RNA therapeutic of any one of embodiments 266-268.
  • Embodiment 280 The method of embodiment 278 or 279, further comprising isolating the antibody or antigen-binding fragment.
  • Embodiment 28 A method of treating or preventing a bacterial infection in a(n, e.g. , human) subject, wherein the bacterial infection is optionally bacteremia, wherein further optionally the bacteremia is hemodialysis associated/line associated bacteremia, the method comprising administering to the subject an effective amount of: (i) the antibody or antigen-binding fragment of any one of embodiments 201-257;
  • Embodiment 282 The method of embodiment 281, wherein the bacteria comprises a bacteria of genus Staphylococcus and/or a bacteria of genus Enterococcus and/or a bacteria of genus Streptococcus and/or a bacteria of genus Lactococcus, wherein, optionally, the bacteria comprises: (1) .S', aureus, wherein, further optionally, the .S', aureus comprises a methicillin-resistant .S'. Aureus (MRSA); (2) a Coagulase-negative Staphylococcus (CoNS); (3) .S', epidermidis,' (4) E.
  • MRSA methicillin-resistant .S'. Aureus
  • CoNS Coagulase-negative Staphylococcus
  • Embodiment 283 The method of embodiment 281 or 282, comprising administering a single dose of the antibody or antigen-binding fragment, polypeptide, polynucleotide, recombinant vector, DNA therapeutic, RNA therapeutic, host cell, or composition to the subject.
  • Embodiment 284 The method of any one of embodiments 281-283, comprising administering two or more doses of the antibody or antigen-binding fragment, polypeptide, polynucleotide, recombinant vector, DNA therapeutic, RNA therapeutic, host cell, or composition to the subject.
  • Embodiment 285. The method of any one of embodiments 281-284, comprising administering the antibody or antigen-binding fragment, polypeptide, polynucleotide, recombinant vector, DNA therapeutic, RNA therapeutic, host cell, or composition intramuscularly, subcutaneously, or intravenously.
  • Embodiment 286 The method of any one of embodiments 281-285, wherein the treatment and/or prevention comprises pre-exposure or post-exposure prophylaxis.
  • Embodiment 287 The method of any one of embodiments 281-286, wherein the subject: is using a ventilator; is undergoing, will undergo, or has recently undergone surgery; has a blood stream infection or a history of blood stream infections; is receiving, will receive, or has received a prosthetic joint or other implant; is undergoing, will undergo, or has undergone dialysis; has bacteremia or a history of bacteremia; has neutropenia or a history of neutropenia; has hemodialysis associated/line associated bacteremia or a history of hemodialysis associated/line associated bacteremia; has a wound; or any combination of the foregoing.
  • Embodiment 288 The method of any one of embodiments 281-287, further comprising administering to the subject or more antibiotic agent, or wherein the subject has received one or more antibiotic agent.
  • Embodiment 289. The method of embodiment 288, wherein the antibiotic agent comprises a penicillin, a fluoroquinolone, a cephalosporin, a macrolide, a beta-lactam with increased activity (e.g. amoxicillin-clavulanate), a tetracycline, a trimethoprim-sulfamethoxazole, a lincosamide (e.g. clindamycin), a urinary anti-infectives, or any combination thereof.
  • the antibiotic agent comprises a penicillin, a fluoroquinolone, a cephalosporin, a macrolide, a beta-lactam with increased activity (e.g. amoxicillin-clavulanate), a tetracycline, a trimethoprim-sulfamethoxazole, a lincosamide (e.g. clindamycin), a urinary anti-infectives, or any combination thereof.
  • Embodiment 290 The method of embodiment 288 or 289, wherein the antibiotic agent comprises vancomycin, cefazolin, nafcillin, oxacillin, daptomycin, linezolid, penicillin, amoxicillin, azithromycin, erythromycin, cephalexin, cefdinir, ciproflaxin, levofloxacin, amoxicillin-clavulanate, ceftazidime/avibactam, tetracycline, doxycycline, trimethoprim-sulfamethoxazole, nitrofurantoin, clindamycin, or any combination thereof.
  • the antibiotic agent comprises vancomycin, cefazolin, nafcillin, oxacillin, daptomycin, linezolid, penicillin, amoxicillin, azithromycin, erythromycin, cephalexin, cefdinir, ciproflaxin, levofloxacin
  • Embodiment 291 The antibody or antigen-binding fragment of any one of embodiments 201- 257, the polynucleotide of any one of embodiments 258-265, the recombinant vector, DNA therapeutic, or RNA therapeutic of any one of embodiments 266-268, the host cell of embodiment 269, the human B cell of embodiment 270, and/or the composition or combination of any one of embodiments 271-277, for use in a method of treating or preventing a bacterial infection in a subject, wherein the bacteria expresses the LTA, Gmd, ClfA, SpA, alpha toxin, leucocidin, gamma toxin, or Sbi, wherein, optionally, the bacteria comprises .S', aureus.
  • Embodiment 292 The antibody or antigen-binding fragment of any one of embodiments 201- 257, the polynucleotide of any one of embodiments 258-265, the recombinant vector, DNA therapeutic, RNA or therapeutic of any one of embodiments 266-268, the host cell of embodiment 269, the human B cell of embodiment 270, and/or the composition or combination of any one of embodiments 271-277, for use in the preparation of a medicament for the treatment or prevention of a bacterial infection in a subject, wherein the bacteria expresses the LTA, Gmd, ClfA, SpA, alpha toxin, leucocidin, gamma toxin, or Sbi, wherein, optionally, the bacteria comprises .S'. aureus.
  • Embodiment 293. A method for in vitro diagnosis of a bacterial infection, the method comprising: (i) contacting a sample from a subject with an antibody or antigen-binding fragment of any one of embodiments 201-257; and (ii) detecting a complex comprising an antigen and the antibody, or comprising an antigen and the antigen-binding fragment, wherein the bacteria expresses the LTA, Gmd, ClfA, SpA, alpha toxin, leucocidin, gamma toxin, or Sbi, wherein, optionally, the bacteria comprises .S'. aureus.
  • Embodiment 294 The antibody, antigen-binding fragment, polynucleotide, recombinant vector, DNA therapeutic, RNA therapeutic, host cell, human B cell, or composition for use of embodiment 291 or 292, or the method of embodiment 293, wherein wherein the bacteria comprises a bacteria of genus Staphylococcus and/or a bacteria of genus Enterococcus and/or a bacteria of genus Streptococcus and/or a bacteria of genus Lactococcus, wherein, optionally, the bacteria comprises: (1) .S', aureus, wherein, further optionally, the .S', aureus comprises a methicillin-resistant .S'.
  • Aureus MRSA
  • CoNS Coagulase -negative Staphylococcus
  • CoNS Coagulase -negative Staphylococcus
  • .S' epidermidis
  • E. faecalis E. faecalis
  • (5) .S', pyogenes E. faecalis
  • (6) .S' pyogenes
  • (6) .S' agalactiae
  • L. garviae E.
  • Embodiment 295. A library comprising a plurality of antibodies or antigen-binding fragments comprising amino acid sequences from any one or more of, and/or comprising a plurality of polynucleotides encoding amino acid sequences encoded by any one or more of the following human immunoglobulin genes: IGHV3-23; IGHV3-7; IGHJ1; IGHJ3; IGLV3-21; IGKV1-5; IGKV1-6; IGLJ2; IGKJ2; IGKJ1; IGHV1-8; IGHV3-30; IGHV3-49; IGHV4-39; IGHJ6; IGHJ3; IGHJ4; IGKV1-27; IGLV3-21; IGKV1-5; IGKV1D-12; IGKV1-9; IGLV1-40; IGVK1-6; IGLV1-47; IGKJ1; IGLJ2; IGKJ2; IGKJ1; IGL
  • Embodiment 296 The library of embodiment 295, wherein the human immunoglobulin genes in the library consist essentially of, or consist of, any one or more of the human immunoglobulin genes of embodiment 295.
  • Embodiment 297 The library of embodiment 295 or 296, wherein the library comprises a plurality of antibodies or antigen-binding fragments comprising amino acid sequences from any one or more of, and/or comprises a plurality of polynucleotides encoding amino acid sequences from any one or more of (i)-(xxiii): (i) IGHV3-23, IGHJ1, IGLV3-21, and IGLJ2; (ii) IGHV3-23, IGHJ3, IGKV1-5, and IGKJ1; (iii) IGHV3-7, IGHJ3, IGKV1-6, and IGKJ1; (iv) IGHV3-7, IGHJ3, IGKV1-6, and IGKJ2; (v) IGHV3-23, IGHJ1, IGLV3-21, and IGLJ2; (vi) IGHV3-23, IGHJ3, IGKV1-5, and IGKJ2; (vii) IGHV3- 23, IGHJ4,
  • Embodiment 298 The library of embodiment 297, wherein the plurality of antibodies or antigenbinding fragments, or the plurality of polynucleotides encoding the antibodies or antigen-binding fragments, further comprises or encodes additional amino acid sequences, wherein the additional amino acid sequences are optionally randomized and/or are limited to one or more CDR regions of an antibody or antigen-binding fragment.
  • Embodiment 299. A method comprising screening one or more antibodies of the library of any one of embodiments 104-107 for binding to a bacterial (e.g. , .S', aureus) antigen selected from: a lipoteichoic acid (LTA) (optionally a Type I, a Type II, and/or a Type IV LTA); a glucosaminidase (Gmd); a clumping factor A (ClfA); a Staphylococcus Protein A (SpA); an alpha toxin; a leukocidin; a gamma toxin; and a Sbi.
  • LTA lipoteichoic acid
  • Gmd glucosaminidase
  • ClfA clumping factor A
  • SpA Staphylococcus Protein A
  • Embodiment 300 A kit comprising a liquid composition comprising and antibody or antigenbinding fragment of any one of embodiments 201-257, 291, 292, or 294, the polynucleotide of any one of embodiments 258-265, the recombinant vector, DNA therapeutic, or RNA therapeutic of any one of embodiments 266-268, the host cell of embodiment 269, the human B cell of embodiment 270, and/or the composition of any one of embodiment 271-277, and instructions for use thereof in treating a bacterial (e.g., Staphylococcus) infection in a subject.
  • a bacterial e.g., Staphylococcus
  • Monoclonal antibodies (mAbs) reactive against one or more Staphylococcus antigen were identified from a human tonsil bank and from PBMCs from human donors recovered from S. aureus, as shown and described for Figures 1A-1D.
  • MAbs were cloned, recombinantly expressed (in some contexts, carrying one or more Fc mutation), and characterized for binding specificity, neutralization, and other functions, as shown and described for Figures 2A-6C.
  • opsonizing antibodies include anti-LTA antibodies, anti-Gmd antibodies, anti-ClfA antibodies, and anti-SpA antibodies.
  • Neutralizing antibodies include anti- Hla antibodies and anti-bicomponent (Luk, HIgAB) antibodies.
  • anti-LTA antibodies further experiments assess breadth of binding to live bacteria (FACS) and epitope-binning.
  • anti-Gmd antibodies further experiments assess breadth of binding to different strains of Staphylococcus, epitope-binning, and effect on Gmd enzymatic activity.
  • anti-ClfA antibodies opsonophagocytic killing and fibrinogen inhibition of binding are assessed.
  • anti-Hla antibodies synergism of Hla neutralization and neutralization on THP1 cells are assessed.
  • anti-bicomponent toxins neutralization studies are performed.
  • mice are used to study bacteremia, pneumonia, dermonecrosis, a prosthetic joint infection model, a foreign body model, and a surgical site infection model.
  • a foreign body model beads coated with 500 CFU of S. aureus are injected into the skin, representative of a real-life infection dose.
  • S. aureus introduced on a foreign object is not cleared as easily as suspension cells and presents as biofilm-like.
  • a surgically placed stainless steel K-wire implant can be inserted into the knee joints of mice and inoculated with S. aureus to model a prosthetic joint infection.
  • Figure 7A shows results from SSE1 and SSE158 binding and neutralization studies, with MEDI4893 included as a comparator.
  • SSE1 and SSE158 bind to H1A and neutralize H1A activity on rabbit red blood cells (“RBCs”) and THP-1 cells.
  • Figure 7B shows results (neutralization of infection) from a synergy/antagonism study with SSE1 and SSE158. SSE1 and SSE158 did not show synergistic neutralization in this assay.
  • Figure 7C shows that SSE1 and SSE158 protect BALB/c mice in an in vivo model of S. aureus pneumonia.
  • the experimental set-up was as shown in the schematic in the bottom of Figure 7C. Percent survival was measured post-infection.
  • Figures 8A-8B show binding (ELISA) to Gmd S. aureus and Gmd S. epidermis, with 1C11 included as a comparator.
  • Figure 8C shows quantified binding EC50 values to Gmd S. aureus and Gmd S. epidermis,' to strains of S. aureus, S. epidermidis, and S. lugdunensis,' and V- domain gene usage of the indicated antibodies.
  • Figures 9A-9B relate to neutralization of LTA activity on TLR-2-expressing cells by certain antibodies of the present disclosure.
  • HEK-Blue hTLR2 cells were co-transfected with hTLR2 and SEAP (secreted embryonic alkaline phosphatase) reporter genes under the control of the IFN-b minimal promoter fused to NF-kB and AP-l-binding sites. Stimulation of TLR2 activates NF-kB and AP-1 which induces the production of SEAP. The hydrolysis of the substrate in the medium by SEAP produces an easily detected purple/blue color.
  • Figure 10A shows binding (ELISA) by certain antibodies of the present disclosure to strains of S. aureus with different ClfA genotypes (ClfA_001, ClfA_002, and ClfA_004), with E. coli included as a negative control.
  • Figure 10B shows quantified binding EC50 values to ClfA_001, ClfA_002, ClfA_004, and strains of S. aureus,' binding affinity values (Kato ClfA OOl); inhibition of ClfA binding to fibrinogen (IC50); and V-domain gene usage of certain antibodies of the present disclosure. Tefibazumab was included as a comparator.
  • Figure 10C shows inhibition of binding between ClfA and fibrinogen (Fg) by certain antibodies of the present disclosure, with tefibazumab included as a comparator.
  • Figure 10D shows sequence alignments between ClfA_001, ClfA_002, and ClfA_004 genotypes and a schematic of a representative recombinant ClfA expression construct.
  • Figure 10E shows inhibition of binding between ClfA and Fg by certain antibodies of the present disclosure.
  • Figure 11A shows binding (EC50 as determined by ELISA) to SpA-KKAA, percent inhibition of SpA binding to IgG, binding affinity values of antibodies of the present disclosure to Spa-KKAA (Ka), V-domain gene usage, and epitope binning of certain antibodies of the present disclosure.
  • Figure 11B shows neutralization of binding between SpA and IgGl by the indicated antibodies of the present disclosure (Octet).
  • Figure 11B shows antibody neutralization of Protein A binding of IgGs assessed using BLI (Octet).
  • FIG. 18A shows a schematic of Sbi structure.
  • Figure 18B shows (top) binding quantified by ELISA and (bottom) quantified binding values (EC50), percent IgG-binding inhibition, binding affinity (KD), and V-domain gene usage of SSH4 and SSH7.
  • Figure 18C shows sensorgram curves showing that antibodies SSH4 and SSH7 of the present disclosure do not compete with one another for Sbi binding.
  • anti-LTA, anti-ClfA, anti-SpA, and anti-Gmd mAbs were administered to 8-week-old female C57BL/6 mice at 10 mg/kg intravenously 2 days prior to challenge with 2xlO A 7 cfu of S. aureus strain FPR3757 via retroorbital injection (Figure 25). Mice were then monitored for weight loss and kidney burden was determined 4 days post infection by organ homogenization and serial dilution.
  • mice As a control, one group was treated with daptomycin (50 mg/kg subcutaneously) at days 2 and 3 post infection to compare the relative weight reduction and kidney burden of antibiotics in a treatment model. Additional control groups included mice dosed with non-binding isotype control mAbs and a histidine buffer group. Mouse serum Ig level was measured at day 1 post-dosing using meso scale discovery (goat antihuman IgG as capture antibody, sulfo tag anti-human CH2 as detection antibody; Figure 26). 514G3 RF antibody had 2.3-fold lower serum Ig level compared to isotype control, similar to results for 514G3 IgG3 antibody format. SSF11 antibody had 3-folder lower serum Ig level compared to isotype control.
  • FIGS 27A-27B show kidney burden at 4 days post infection for individual mice (Figure 27 A), weight loss over time and the weight loss calculated from the area under the curve (Figure 27B). Mice treated with isotype control antibodies had uniformly high kidney burden with pronounced weight loss, while daptomycin treated mice showed a 1-3 log reduction in S. aureus kidney burden associated with a minor reduction in weight loss. Individual mice treated with anti-Staphylococcal antibodies varied from no reduction to 6 log reduction in kidney burden ( Figure 28).

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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente divulgation concerne des anticorps et des fragments de liaison à l'antigène de ceux-ci qui peuvent se lier à un antigène bactérien choisi parmi un acide lipotéichoïque (LTA) ; une glucosaminidase (Gmd) ; un facteur d'agglutination A (ClfA) ; une protéine A Staphylococcus (SpA) ; une alpha-toxine ; une leucocidine ; une gamma-toxine ; ou un Sbi. L'antigène peut être un antigène qui est exprimé par Staphylococci (par exemple, S. aureus ) et, dans certains modes de réalisation, par une ou plusieurs autres bactéries.<i /> L'invention concerne également des polynucléotides qui codent un anticorps ou un fragment de liaison à l'antigène, des vecteurs qui comprennent de tels polynucléotides, des cellules hôtes qui peuvent exprimer les anticorps, des compositions associées, ainsi que des méthodes d'utilisation des compositions divulguées pour, par exemple, traiter ou prévenir une infection bactérienne.
PCT/US2024/036949 2023-07-06 2024-07-05 Anticorps dirigés contre des antigènes de staphylococcus et leurs méthodes d'utilisation WO2025010424A1 (fr)

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