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WO2024206126A1 - Cd16-binding antibodies and uses thereof - Google Patents

Cd16-binding antibodies and uses thereof Download PDF

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Publication number
WO2024206126A1
WO2024206126A1 PCT/US2024/021104 US2024021104W WO2024206126A1 WO 2024206126 A1 WO2024206126 A1 WO 2024206126A1 US 2024021104 W US2024021104 W US 2024021104W WO 2024206126 A1 WO2024206126 A1 WO 2024206126A1
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WIPO (PCT)
Prior art keywords
antibody
binding
human
antigen
amino acid
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PCT/US2024/021104
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French (fr)
Inventor
Graham FARRINGTON
Zhen Zhang
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ModernaTx Inc
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ModernaTx Inc
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Publication of WO2024206126A1 publication Critical patent/WO2024206126A1/en
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present disclosure relates generally to antibodies and binding polypeptides that specifically bind CD16 and nucleic acids encoding the same.
  • the present disclosure further relates to methods of producing the disclosed antibodies, binding domains, proteins (e.g., purified anti-CD16 binding proteins or chimeric molecules comprising such binding proteins), and nucleic acid molecules encoding such binding protein, as well as medical applications and treatments utilizing the disclosed antibodies, binding domains, proteins, and nucleic acid molecules.
  • CD16 also known as FcyRIII
  • CD16a exists as two isoforms, CD16a and CD16b, that share 96% sequence identity in their extracellular immunoglobulin-binding regions.
  • CD16a is expressed on macrophages, mast cells, and NK cells as a Attorney Docket No.: 45817-0156WO1 transmembrane receptor.
  • CD16b is present on neutrophils as a glycosyl- phosphatidylinositol (GPI)-anchored receptor, as well as a soluble receptor in serum.
  • GPI glycosyl- phosphatidylinositol
  • CD16 is a low affinity receptor for the Fc portion of some IgGs known to be involved in antibody-dependent cellular cytotoxicity (ADCC), and has been associated with triggering of target cell lysis by NK cells (Mandelboim et al, 1999, PNAS 96:5640-5644). CD16 is thus an important therapeutic target for activation of NK cells and targeting tumor cells. Accordingly, there remains a need for antibodies, binding domains, and related proteins that bind CD16 and nucleic acids encoding the same. SUMMARY OF THE DISCLOSURE The present disclosure provides, among other things, antibodies, binding domains, and related proteins that bind CD16 and nucleic acids encoding the same. In some instances, the CD16 is human CD16a.
  • the present disclosure provides a single-domain antibody the antibody specifically binds CD16 and comprises the following complementary determining regions (CDRs): CDR1 comprising the amino acid sequence GRTDSIYA (SEQ ID NO: 2), CDR2 comprising the amino acid sequence INSNTGRT (SEQ ID NO: 3), and CDR3 comprising the amino acid sequence AAGRGYGLLSISSNWYNY (SEQ ID NO: 4).
  • the antibody comprises a heavy chain comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 1 or 5-24.
  • the antibody comprises a heavy chain comprising an amino acid sequence that is at least 95% identical to the Attorney Docket No.: 45817-0156WO1 amino acid sequence of any one of SEQ ID NOs: 1 or 5-24. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence of any one of SEQ ID NOs: 1 or 5-24. In some embodiments, the antibody binds CD16 with a K D of 20 nM or less.
  • the disclosed antibodies, antigen-binding fragments, and binding proteins do not compete with human IgG for binding to CD16a.
  • the present disclosure provides a conjugate comprising a biologic and a single-domain antibody described herein.
  • the biologic and the single-domain antibody of the conjugate are covalently bound to one another as part of a single polypeptide chain.
  • the biologic and the single-domain antibody of the conjugate are connected via a chemical linker.
  • the biologic of the conjugate is selected from an antibody, a cytokine, a growth factor, an enzyme, a polypeptide, a protein, a carbohydrate, and a nucleic acid.
  • the conjugate when administered to a human subject, possesses a longer circulating half-life relative to the corresponding biologic that is not conjugated to the single-domain antibody.
  • the present disclosure provides an antibody or binding protein that specifically binds CD16 and comprises a heavy chain comprising a CDR1 comprising the amino acid sequence GRTDSIYA (SEQ ID NO: 2), a CDR2 comprising the amino acid sequence INSNTGRT (SEQ ID NO: 3), and a CDR3 comprising the amino acid sequence AAGRGYGLLSISSNWYNY (SEQ ID NO: 4).
  • the antibody or binding protein comprises heavy chain comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 1 or 5-24. In some embodiments, the antibody or binding protein comprises a heavy chain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 1 or 5- 24. In some embodiments, the antibody or binding protein comprises heavy chain comprising an amino acid sequence of any one of SEQ ID NOs: 1 or 5-24. In some embodiments, the antibody or binding protein binds CD16 with a K D of 20 nM or less.
  • the antibody or binding protein binds CD16 with a K D of 10 nM or less, optionally the antibody or binding protein binds CD16 with a KD of 5 nM or less, optionally the antibody or binding protein binds CD16 with a K D of 1 nM or less, optionally the antibody or binding protein binds CD16 with a KD of 0.5 nM or less, optionally the antibody or binding protein binds CD16 with a K D of 0.1 nM or less, optionally the antibody or binding protein binds CD16 with a KD of 0.05 nM or less, optionally the antibody or binding protein binds CD16 with a KD of 0.01 nM or less.
  • the antibody or binding protein is a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen- binding fragment thereof, a human antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a primatized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a multi-specific antibody or antigen-binding fragment thereof, a dual-variable immunoglobulin domain, a monovalent antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a single-chain Fv molecule (scFv), a diabody, a triabody, an antibody-like protein scaffold, a domain antibody, a Fv fragment, a Fab fragment, a F(ab’) 2 molecule, a tandem scFv (taFv), or a fusion protein.
  • scFv single-chain Fv molecule
  • the antibody or binding protein is a fusion protein comprising a biologic conjugated, either directly or indirectly, to the antibody or binding protein.
  • the antibody or binding protein is a multi-specific antibody comprising at least one binding domain that specifically binds to an antigen other than CD16.
  • the present disclosure provides a nucleic acid encoding a single-domain antibody, a conjugate, or an antibody or binding protein disclosed herein.
  • the nucleic acid is an mRNA.
  • the nucleic acid comprises an open reading frame of nucleosides selected from the group consisting of (i) a modified uridine, (ii) cytidine, (iii) adenosine, and (iv) guanosine.
  • the nucleic acid comprises one or more modified uridines selected from 1-methylpseudouridine, pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4- thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine, 5- aminoallyl-uridine, 5-halo-uridine, 3-methyl-uridine, 5-methoxy-uridine, uridine 5- oxyacetic acid, uridine 5-oxyacetic acid methyl ester, 5-carboxymethyl-uridine, 1- carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine, 5- carboxyhydroxymethyl-uridine methyl ester, 5-methoxycarbonylmethyl-uridine, 5- Attorney Docket No.: 45817-0156WO1 methoxycarbonylmethyl-2-thio-uridine
  • the nucleic acid comprises a modified uridine that is 1- methylpseudouridine.
  • the nucleic acid comprises one or more modified cytidines selected from 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl- cytidine, N4-acetyl-cytidine, 5-formyl-cytidine, N4-methyl-cytidine, 5-methyl- cytidine, 5-halo-cytidine, 5-hydroxymethyl-cytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1- deaza-
  • the nucleic acid comprises one or more modified adenosines selected from 2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine, 6-halo-purine, 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7- deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7- deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine, 2- methyl-adenine, N6-methyl-adenosine, 2-methylthio-N6-methyl-adenosine, N6- isopentenyl-adenosine, 2-methylthio-N6-isopentenyl-adenosine, N6-(cis-
  • the nucleic acid comprises one or more modified guanosines selected from inosine, 1-methyl-inosine, wyosine, methylwyosine, 4- demethyl-wyosine, isowyosine, wybutosine, peroxywybutosine, hydroxywybutosine, 7-deaza-guanosine, queuosine, epoxyqueuosine, galactosyl-queuosine, mannosyl- queuosine, 7-cyano-7-deaza-guanosine, 7-aminomethyl-7-deaza-guanosine, archaeosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6- thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7- Attorney Docket
  • the present disclosure provides a pharmaceutical composition comprising a single domain antibody, a conjugate, an antibody or binding protein, or a nucleic acid disclosed herein.
  • the pharmaceutical composition additionally comprises one or more pharmaceutically acceptable carriers, diluents, excipients, or any combination thereof.
  • the pharmaceutical composition comprises a plurality of lipid nanoparticles encapsulating the nucleic acid.
  • the pharmaceutical composition comprises a plurality of lipid nanoparticles having a mean particle size of from 80 nm to 160 nm.
  • the pharmaceutical composition comprises a plurality of lipid nanoparticles having a polydispersity index (PDI) of from 0.02 to 0.2 and/or a lipid:nucleic acid ratio of from 10 to 20.
  • the pharmaceutical composition comprises a plurality Attorney Docket No.: 45817-0156WO1 of lipid nanoparticles comprising a neutral lipid, a cationic lipid, a polyethyleneglycol (PEG) lipid, and/or a sterol.
  • the pharmaceutical composition comprises a plurality of lipid nanoparticles comprising 1,2-distearoyl-sn-glycero-3- phosphocholine.
  • the pharmaceutical composition comprises a plurality of lipid nanoparticles comprising a compound of Formula (I). In some embodiments, the pharmaceutical composition comprises a plurality of lipid nanoparticles comprising PEG 2000 dimyristoyl glycerol.
  • the pharmaceutical composition comprises a sterol selected from cholesterol, adosterol, agosterol A, atheronals, avenasterol, azacosterol, blazein, cerevisterol, colestolone, cycloartenol, daucosterol, 7-dehydrocholesterol, 5- dehydroepisterol, 7-dehydrositosterol, 20 ⁇ ,22R-dihydroxycholesterol, dinosterol, epibrassicasterol, episterol, ergosterol, ergosterol, fecosterol, fucosterol, fungisterol, ganoderenic acid, ganoderic acid, ganoderiol, ganodermadiol, 7 ⁇ -hydroxycholesterol, 22R-hydroxycholesterol, 27-hydroxycholesterol, inotodiol, lanosterol, lathosterol, lichesterol, lucidadiol, lumisterol, oxycholesterol,
  • the pharmaceutical composition comprises cholesterol.
  • the present disclosure provides a host cell comprising a single domain antibody, a conjugate, an antibody or binding protein, or a nucleic acid disclosed herein.
  • the host cell is a eukaryotic cell.
  • the host cell is a mammalian cell.
  • the host cell is a CHO cell or HEK cell.
  • the present disclosure provides a method of treating cancer comprising administering to a subject in need thereof a single domain antibody described herein or the antibody, a conjugate described herein, an antigen-binding fragment, or binding protein described herein, a nucleic acid described herein, a pharmaceutical composition described herein, or a host cell described herein.
  • the subject is human.
  • Attorney Docket No.: 45817-0156WO1 a VHH single domain antibody of the invention is incorporated into a binding molecule comprising at least one additional binding specificity (e.g., at least one binding moiety that binds to a molecule expressed on a cancer cell) and facilitates treatment of cancer.
  • the present disclosure provides a kit comprising (i) a single domain antibody, a conjugate, an antibody, antigen-binding fragment, binding protein, a nucleic acid, a pharmaceutical composition or host cell disclosed herein, and (ii) a package insert instructing a user of the kit to administer the single-domain antibody, conjugate, antibody, antigen-binding fragment, binding protein, nucleic acid, or pharmaceutical composition to a subject in need thereof.
  • the disclosure features a binding molecule comprising a polypeptide that specifically binds CD16, wherein the polypeptide comprises the amino acid sequences of a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:1 or SEQ ID NO:7.
  • the CD16 is human CD16a.
  • the human CD16a is CD16aF176.
  • the human CD16a is CD16aV176.
  • the CD16 is human CD16b NA2.
  • the CD16 is cynomolgus CD16.
  • the CD16 is rat CD16.
  • the polypeptide binds to human and cynomolgus CD16. In other cases, the polypeptide binds to human, cynomolgus, and rat CD16. In some cases, the polypeptide binds human, cynomolgus, and rat CD16, but not mouse CD16 or human CD16b NA1 variant.
  • the VHH-CDR1, VHH-CDR2, and VHH-CDR3 are based on Kabat, Chothia, enhanced Chothia, Contact, Aho, or IMGT CDR definitions. In certain instances, the binding molecule binds to human NK cells and/or human neutrophils.
  • the disclosure features a binding molecule comprising a polypeptide that specifically binds human CD16a, wherein the polypeptide comprises the amino acid sequences of a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of VHH1 based on any single CDR definition as set forth in Table 5.
  • Attorney Docket No.: 45817-0156WO1 the polypeptide is a VHH.
  • the VHH is humanized.
  • 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 8, 7, 6, 5, 43, 2, or 1 amino acids of the VHH are humanized.
  • the humanization technique is resurfacing/veneering. In some cases, the humanization technique is CDR grafting. In some instances, one or more (1, 2, 3, 4, 5, 6) of positions 37, 44, 45, 47, 95, and 117 (numbering based on Kabat numbering) are humanized. In other instances, one or more (1, 2, 3, 4, 5, 6) of positions 37, 44, 45, 47, 95, and 117 (numbering based on Kabat numbering) are not humanized. In some cases, positions 37 and 47 (numbering based on Kabat numbering) are not humanized. In some cases, positions 44 and 45 (numbering based on Kabat numbering) are not humanized.
  • the binding molecule further comprises an agent selected from the group consisting of a purification tag, a fluorophore, a drug, a photosensitizer, a nanoparticle, a toxic agent, a radionuclide, a VHH, an Fab, a scFv, a multimerization module, a moiety that facilitates the polypeptide crossing the blood brain barrier, and a half-life extension moiety.
  • the agent is a VHH, an Fab, or a scFv, and the agent is attached or linked to the N-terminus of the polypeptide.
  • the agent is a VHH, an Fab, or a scFv, and the agent is attached or linked to the C-terminus of the polypeptide.
  • the binding molecule further comprises a human Ig Fc domain.
  • the binding molecule further comprises a human Ig hinge domain.
  • the human Ig is a human IgG1, human IgG2, human IgG3, or human IgG4.
  • the binding molecule comprises an amino acid of a human IgG4 PAA hinge and IgG4 Fc domain sequence.
  • the human IgG4PAA hinge + Fc sequence is at the N-terminus of the polypeptide.
  • the Fc domain includes mutations that promote heterodimerization (e.g., knob- into-hole mutations, electrosteering mutations).
  • the polypeptide is a VHH and comprises the VHH-CDR1, VHH-CDR2, and VHH-CDR3 of any one of the CDR definitions set forth in Table 5.
  • the VHH comprises a sequence that is at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any amino acid sequence set forth in Table 8.
  • the VHH comprises a sequence that is identical to any amino acid sequence set forth in Table 8 except for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions.
  • the substitutions are conservative substitutions.
  • one or more (1, 2, 3, 4, 5, 6) of positions 37, 44, 45, 47, 95, and 117 are not substituted.
  • positions 37 and 47 are not substituted.
  • position 37 and/or 47 is/are substituted.
  • positions 44 and 45 are not substituted.
  • the disclosure features a bispecific antibody comprising (i) a binding molecule described above; and (ii) a second binding molecule that binds to a different epitope of human CD16 than the binding molecule, or that binds to a different antigen.
  • the different antigen is an antigen on a T cell, a neutrophil, an NK cell, or a tumor cell.
  • the second binding molecule is a VHH. In other cases, the second binding molecule is a scFv. In certain cases, the second binding molecule is a Fab. In other cases, the second binding molecule is a F(ab)2. In certain instances, the binding molecule and the second binding molecule are connected via a linker. In some cases, the linker is a polypeptide linker.
  • the polypeptide linker comprises an IgG Fc domain and optionally a human Attorney Docket No.: 45817-0156WO1 IgG hinge region (which may be mutated – e.g., IgG4PAA hinge).
  • the IgG is a human IgG1, human IgG2, human IgG3, or human IgG4.
  • the polypeptide linker comprises an IgG4PAA hinge+ Fc region.
  • the linker is a glycine linker, a serine linker, or a glycine-serine linker.
  • the disclosure provides a bispecific antibody comprising a means for binding CD16 and a second binding molecule that binds to a different antigen.
  • the CD16 is human CD16a.
  • the human CD16a is CD16aF176.
  • the human CD16a is CD16aV176.
  • the CD16 is human CD16b NA2.
  • the CD16 is cynomolgus CD16.
  • the CD16 is rat CD16.
  • the polypeptide binds to human and cynomolgus CD16. In other cases, the polypeptide binds to human, cynomolgus, and rat CD16. In some cases, the polypeptide binds human, cynomolgus, and rat CD16, but not mouse CD16 or human CD16b NA1 variant. In some cases, the different antigen is an antigen on a T cell or NK cell.
  • the means for binding CD16 is a VHH comprising the three VHH CDRs (according to any CDR definition such as Kabat, Chothia, enhanced Chothia, Aho, contact, IMGT) of any one clone listed in Table 8, or a VHH comprising the amino acid sequence of any clone of Table 8.
  • the disclosure provides a human CD16a binding chimeric antigen receptor (CAR) comprising a binding molecule described above.
  • the human CD16a binding CAR further comprises a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.
  • the transmembrane domain is derived from an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9.
  • TLR1 Toll-like receptor 1
  • TLR2 TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9.
  • the costimulatory domain comprises a costimulatory region of CD3, CD4, CD8, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-l, ICOS, Attorney Docket No.: 45817-0156WO1 lymphocyte function-associated antigen-l (LFA-l), CD2, CD7, LIGHT, NKG2C, B7- H3, or a fragment thereof.
  • the intracellular signaling domain comprises a fragment or domain from one or more molecules selected from the group consisting of a T cell receptor (TCR), CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma Rlla, DAP 10, DAP 12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, 0X40, CD30, CD40, PD-l, ICOS, a KIR family protein, lymphocyte function-associated antigen-l (LFA-l), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-l, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127,
  • the human CD16a binding CAR further comprises CD28 and CD137 signaling domains and CD3 ⁇ (CD16-28-137-3 ⁇ ).
  • the disclosure features a T cell or a NK cell expressing a human CD16a binding CAR described above.
  • the disclosure provides a human CD16a binding CAR comprising a means for binding human CD16a as well as a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.
  • the means for binding human CD16a is a VHH comprising the three VHH CDRs (according to any CDR definition such as Kabat, Chothia, enhanced Chothia, Aho, contact, IMGT) of any one clone listed in Table 8, or a VHH comprising the amino acid sequence of any clone of Table 8.
  • Attorney Docket No.: 45817-0156WO1 the disclosure relates to a nucleic acid or nucleic acids encoding a binding molecule, a bispecific antibody, or the human CD16a binding CAR described above.
  • a vector or vectors comprising the nucleic acid or nucleic acids.
  • a host cell comprising the nucleic acid or nucleic acids, or vector or vectors described herein.
  • the host cell is a COS cell, a CHO cell, a 293 cell, or a NIH3T3 cell.
  • the host cell is a yeast cell.
  • the disclosure also provides a method of making a binding molecule or a bispecific antibody. The method comprises culturing the host cell described herein under conditions that facilitate expression of the binding molecule or the bispecific antibody, and isolating the binding molecule or the bispecific antibody. In some cases, the method further comprises formulating the binding molecule or the bispecific antibody as a sterile pharmaceutical composition.
  • the disclosure provides a pharmaceutical composition comprising a binding molecule, a bispecific antibody, or a T cell or NK cell expressing the human CD16a binding CAR described herein, and a pharmaceutically acceptable carrier.
  • the disclosure features a pharmaceutical composition comprising a means for binding human CD16a and a pharmaceutically acceptable carrier.
  • the means for binding human CD16a is a VHH comprising the three VHH CDRs (according to any CDR definition such as Kabat, Chothia, enhanced Chothia, Aho, contact, IMGT) of any one clone listed in Table 8, or a VHH comprising the amino acid sequence of any clone of Table 8.
  • the disclosure relates to a method of treating a cancer in a human subject in need thereof, killing a tumor cell in a human subject in need thereof, or decreasing the rate of tumor growth in a human subject in need thereof.
  • the method comprises administering to the human subject a therapeutically effective amount of a binding molecule, a bispecific antibody, a T cell or NK cell expressing the human CD16a binding CAR, or a pharmaceutical composition described herein.
  • Attorney Docket No.: 45817-0156WO1 the cancer is one which benefits from NK cell targeting and killing of a tumor.
  • the disclosure features polynucleotide comprising an mRNA comprising: (i) a 5' UTR; (ii) an open reading frame (ORF) encoding a binding molecule, a bispecific antibody, or a human CD16a binding CAR described herein; (iii) a stop codon; and (iv) a 3' UTR.
  • the mRNA comprises a microRNA (miR) binding site.
  • the microRNA is expressed in an immune cell of hematopoietic lineage or a cell that expresses TLR7 and/or TLR8 and secretes pro-inflammatory cytokines and/or chemokines.
  • the microRNA binding site is for a microRNA selected from miR-126, miR-142, miR-144, miR-146, miR-150, miR-155, miR-16, miR-21, miR-223, miR-24, miR-27, miR-26a, or any combination thereof.
  • the microRNA binding site is for a microRNA selected from miR126-3p, miR- 142-3p, miR-142-5p, miR-155, or any combination thereof.
  • the microRNA binding site is located in the 3' UTR of the mRNA.
  • the mRNA comprises a 5' terminal cap.
  • the 5' terminal cap comprises a Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA- guanosine, 2-azidoguanosine, Cap2, Cap4, 5' methylG cap, or an analog thereof.
  • the mRNA comprises a poly-A region.
  • the poly-A region is at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90 nucleotides in length, or at least about 100 nucleotides in length. In certain cases, the poly-A region is about 10 to about 200, about 20 to about 180, about 50 to about 160, about 70 to about 140, or about 80 to about 120 nucleotides in length. In some instances, the mRNA comprises at least one chemically modified nucleobase, sugar, backbone, or any combination thereof.
  • the at least one chemically modified nucleobase is selected from the group consisting of Attorney Docket No.: 45817-0156WO1 pseudouracil ( ⁇ ), N1-methylpseudouracil (m1 ⁇ ), 1-ethylpseudouracil, 2-thiouracil (s2U), 4’-thiouracil, 5-methylcytosine, 5-methyluracil, 5-methoxyuracil, and any combination thereof.
  • the open reading frame consists of nucleosides selected from the group consisting of (i) uridine or a modified uridine, (ii) cytidine or a modified cytidine, (iii) adenosine or a modified adenosine, and (iv) guanosine or a modified guanosine.
  • the modified uridine is 1-methylpseudouridine.
  • the mRNA comprises a 5’terminal cap comprising Cap1 and a poly-A region 100 nucleotides in length (SEQ ID NO: 37).
  • all uracils of the polynucleotide are N1- methylpseudouracils.
  • the disclosure provides a pharmaceutical composition comprising a polynucleotide described above, and a delivery agent.
  • the delivery agent comprises a lipid nanoparticle.
  • the lipid nanoparticle has a mean particle size of from 80 nm to 160 nm.
  • the lipid nanoparticle has a polydispersity index (PDI) of from 0.02 to 0.2 and/or a lipid:nucleic acid ratio of from 10 to 20.
  • the lipid nanoparticle comprises a neutral lipid, an ionizable amino lipid, a polyethyleneglycol (PEG) lipid, and/or a sterol.
  • the neutral lipid is 1,2-distearoyl-sn-glycero-3- phosphocholine.
  • the ionizable amino lipid is a compound of Formula (I).
  • the PEG lipid is PEG 2000 dimyristoyl glycerol or 134-hydroxy- 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87 ,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132- tetratetracontaoxatetratriacontahectyl stearate or a salt thereof.
  • the sterol is cholesterol, adosterol, agosterol A, atheronals, avenasterol, azacosterol, blazein, cerevisterol, colestolone, cycloartenol, daucosterol, 7-dehydrocholesterol, 5- Attorney Docket No.: 45817-0156WO1 dehydroepisterol, 7-dehydrositosterol, 20 ⁇ ,22R-dihydroxycholesterol, dinosterol, epibrassicasterol, episterol, ergosterol, ergosterol, fecosterol, fucosterol, fungisterol, ganoderenic acid, ganoderic acid, ganoderiol, ganodermadiol, 7 ⁇ -hydroxycholesterol, 22R-hydroxycholesterol, 27-hydroxycholesterol, inotodiol, lanosterol, lathosterol, lichesterol, lucidadiol, lumisterol
  • the disclosure features a method of treating a cancer in a human subject in need thereof, killing a tumor cell in a human subject in need thereof, or decreasing the rate of tumor growth in a human subject in need thereof.
  • the method comprises administering to the human subject a therapeutically effective amount of a polynucleotide, or a pharmaceutical composition described above.
  • the cancer is one that benefits from NK cell targeting to kill a tumor.
  • the disclosure relates to a kit comprising a binding molecule, bispecific antibody, human CD16a binding CAR, polynucleotide, or a pharmaceutical composition described above and a package insert instructing a user of the kit to administer the binding molecule, bispecific antibody, human CD16a binding CAR, polynucleotide, or a pharmaceutical composition to a human subject in need thereof.
  • the disclosure features a lipid nanoparticle comprising an mRNA that encodes a binding molecule comprising a polypeptide that specifically binds CD16, wherein the polypeptide is a VHH and comprises the amino acid sequences of a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO: 1 or 7, optionally wherein the polypeptide binds human, cynomolgus and rat CD16, but not mouse CD16, or human CD16b NA1 variant, and further optionally wherein the binding molecule binds to human NK cells and/or human neutrophils.
  • the polypeptide is a VHH and comprises the amino acid sequences of a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO: 1 or 7, optionally wherein the polypeptide binds human, cynomolgus and rat CD16, but not
  • VHH-CDR1, VHH-CDR2, and VHH-CDR3 comprise the sequences set forth in SEQ ID NOs: (i) 2, 3, 4, respectively; (ii) 25, 26, 27, respectively; (iii) 28, 29, 27, respectively; (iv) 28, 26, 27, respectively; (v) 30, 31, Attorney Docket No.: 45817-0156WO1 32, respectively; (vi) 30, 36, 32, respectively; or (vii) 33, 34, 27, respectively.
  • the VHH comprises a sequence that is 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%, at least 99%, or 100% identical to any amino acid sequence set forth in Table 8; or wherein the VHH comprises a sequence with 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 8, 7, 6, 5, 4, 3, 2, or 1 substitution relative to any amino acid sequence set forth in Table 8.
  • the polypeptide further comprises an agent selected from the group consisting of a purification tag, a fluorophore, a drug, a photosensitizer, a nanoparticle, a toxic agent, a radionuclide, a VHH, an Fab, a scFv, a multimerization module, a moiety that facilitates the polypeptide crossing the blood brain barrier, and a half-life extension moiety, optionally wherein when the agent is a VHH, an Fab, or a scFv, it is attached or linked to the N-terminus of the polypeptide.
  • an agent selected from the group consisting of a purification tag, a fluorophore, a drug, a photosensitizer, a nanoparticle, a toxic agent, a radionuclide, a VHH, an Fab, a scFv, a multimerization module, a moiety that facilitates the polypeptide crossing the blood brain barrier, and a
  • the polypeptide further comprises a human Ig Fc domain, optionally further comprising a human Ig hinge domain, and further optionally wherein the human Ig is a human IgG1, human IgG2, human IgG3, or human IgG4.
  • the polypeptide comprises an amino acid of an human IgG4 PAA hinge and Fc domain sequence, optionally wherein the human IgG4PAA sequence is at the N-terminus of the polypeptide, optionally wherein the Fc domain includes mutations that promote heterodimerization.
  • the lipid nanoparticle comprises an ionizable amino lipid, a PEG-lipid, a structural lipid, and a phospholipid.
  • the lipid nanoparticle comprises: about 47.5 mol % of ionizable amino lipid; about 39 mol % of cholesterol; about 10.5 mol % of DSPC; and about 3 mol % of PEG-lipid. In certain cases, the lipid nanoparticle comprises: 47.5 mol % of an ionizable amino lipid; 39 mol % of cholesterol; 10.5 mol % of DSPC; and 3 mol % of a PEG-lipid.
  • the ionizable amino acid lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(8- (nonyloxy)-8-oxooctyl)amino)octanoate which has the formula Attorney Docket No.: 45817-0156WO1 (Compound I-1), or a salt thereof. is heptadecan-9-yl 8-((2- oxo- amino)octanoate or a salt thereof.
  • the PEG lipid is 134-hydroxy- 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,8 4,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132- tetratetracontaoxatetratriacontahectyl stearate which has the formula cases, all uridines of the mRNA are 1-methylpseudouridine.
  • the mRNA comprises a 5’ terminal cap, a 5’UTR, a 3’UTR, and a poly A tail.
  • the disclosure features a method of treating a cancer in a human subject in need thereof; killing a tumor cell in a human subject in need thereof; or decreasing the rate of tumor growth in a human subject in need thereof.
  • the method comprises administering to the human subject a therapeutically effective amount of a LNP described above.
  • the cancer is one that benefits from NK cell targeting to kill a tumor.
  • the LNP is administered subcutaneously.
  • the LNP is administered intravenously.
  • the LNP is administered intramuscularly.
  • the disclosure relates to a kit comprising LNP described above and a package insert instructing a user of the kit to administer the LNP to a human subject in need thereof.
  • FIG.1 shows results of FACS binding affinity assay of His labelled anti- CD16 sdAb in CHO cell lines stably expressing CD16a and CD16b compared to control antibody, in the presence and absence of HSA.
  • FIG.2 shows results of FACS binding affinity assay of His labelled anti- CD16 sdAb on neutrophils and NK cells. Anti-His or anti-VHH antibodies were used to detect CD16 binding on each cell type.
  • compositions and methods of the disclosure feature CD16 heavy chain variable domains (anti-CD16 VHH domains) and complementarity determining regions (CDRs) thereof, as well as antibodies, antigen-binding fragments, and other related binding proteins that comprise the disclosed VHH domains or CDRs.
  • the disclosure also provides nucleic acids encoding the disclosed proteins, and methods of using such antibodies, antigen-binding fragments, binding proteins, and nucleic acids.
  • the antibodies are single domain antibodies (e.g., a VHH).
  • the antibodies are full-length antibodies that include a pair of heavy chains and a pair of light chains, each containing a variable domain and a constant region.
  • the antibodies are single-domain antibodies (sdAbs) or single chain Fv (scFv) molecules, among other antigen-binding fragments described herein.
  • the antibodies are bispecific antibodies (i.e., engagers) that bind to CD16 and another antigenic target.
  • these binding molecules bind human CD16a.
  • these binding molecules bind human and cynomolgus CD16.
  • these binding molecules bind human, cynomolgus, and rat CD16.
  • these binding molecules bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1.
  • compositions and methods of the disclosure exhibit a series of beneficial biochemical properties.
  • VHH domains described herein and antibodies and binding polypeptides are capable of binding CD16 with high affinity. More Attorney Docket No.: 45817-0156WO1 specifically, the anti-CD16 VHH domains described herein is not affected by the presence of human serum. Because the binding of the instant VHH domains does not compete with human IgG for binding to CD16a, these VHH domains are particularly suited for therapies where binding to CD16a in vivo is desired. Definitions As used herein, the term “about” refers to a stated numerical term and a value that is no more than 10% above or below the value being described.
  • CD16 antibody or “CD16-antibody” refers to an antibody or fragment thereof that specifically binds to, or is immunologically reactive with, CD16.
  • CD16 binding protein or “anti-CD16 binding protein” refers to any protein comprising at least one domain (such as a VHH domain disclosed herein) that specifically binds to or is immunologically reactive with CD16.
  • a “CD16 binding protein” or “anti-CD16 binding protein” includes, for example, anti-CD16 antibodies (both monospecific and bispecific), and other constructs that bind to CD16.
  • antibody refers to an immunoglobulin molecule, or a molecule having an immunoglobulin-like scaffold, that specifically binds to, or is immunologically reactive with, a particular antigen.
  • antibody includes polyclonal, monoclonal, genetically engineered, and otherwise modified forms of antibodies, including, but not limited to, chimeric antibodies, humanized antibodies, and heteroconjugate antibodies (e.g., bi- tri-, quad-, and multispecific antibodies, diabodies, triabodies, and tetrabodies).
  • heteroconjugate antibodies e.g., bi- tri-, quad-, and multispecific antibodies, diabodies, triabodies, and tetrabodies.
  • antigen-binding fragment refers to one or more fragments of an antibody that retain the ability to specifically bind to a target antigen.
  • the antigen-binding function of an antibody can be performed by fragments of a full- length antibody.
  • the antibody fragments can be, e.g., a single-domain antibody Attorney Docket No.: 45817-0156WO1 (sdAb), Fab, F(ab’)2, Fab Fv, VHH, scFv, SMIP, diabody, a triabody, an affibody, an aptamer, or recombinant fragments thereof.
  • binding fragments encompassed by the term “antigen-binding fragment” of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V H and C H 1 domains; (iv) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (v) a dAb including VH and VL domains; (vi) a dAb fragment (Ward et al., Nature 341:544-546, 1989), which consists of a V H domain; (vii) a dAb which consists of a VH or a VL domain; (viii) an isolated complementarity determining region (CDR); and (ix) a
  • the two domains of the Fv fragment, V L and V H are coded for by separate genes, they can be joined, using recombinant methods, by a linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426 (1988), and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)).
  • scFv single chain Fv
  • These antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments can be screened for utility in the same manner as intact antibodies.
  • Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in some embodiments, by chemical peptide synthesis procedures known in the art.
  • biological refers to a medicinal preparation that is created by biological processes rather than chemical synthesis.
  • Exemplary biologics include certain vaccines, antibodies, cell preparations, tissue preparations, recombinant proteins, nucleic acids, cytokines, growth factors, enzymes, peptides, proteins, carbohydrates, or combinations thereof.
  • Biologics also include biosimilar molecules (or biosimilars), which are molecular entities that are structurally similar to and have no clinically meaningful differences in terms of safety, purity, and potency from known biologics.
  • bispecific antibodies refers to monoclonal, often human or humanized antibodies that have binding specificities for at least two different antigens.
  • Bispecific CD16 antibodies of the disclosure may have binding specificities that are directed towards CD16 and any other antigen, e.g., for a cell- surface protein, receptor, receptor subunit, or tissue-specific antigen.
  • a bispecific antibody may also be an antibody or antigen-binding fragment thereof that includes two separate antigen-binding domains (e.g., two scFvs joined by a linker). The scFvs may bind the same antigen or different antigens.
  • multispecific antibodies refers to monoclonal, often human or humanized antibodies that have binding specificities for at least two different antigens, e.g., bispecific antibodies.
  • Multispecific CD16 antibodies of the disclosure may have binding specificities that are directed towards CD16 and any other antigen(s), e.g., for a cell-surface protein, receptor, receptor subunit, or tissue- specific antigen.
  • a multispecific antibody may also be an antibody or antigen-binding fragment thereof that includes multiple separate antigen-binding domains (e.g., two scFvs joined by a linker).
  • the scFvs may bind the same antigen or different antigens.
  • Such structures can include, but not limited to, IgG-Fv, IgG-(scFv) 2 , DVD-Ig, (scFv)2-(scFv)2-Fc and (scFv)2-Fc-(scFv)2.
  • the scFv can be attached to either the N-terminal or the C- terminal end of either the heavy chain or the light chain.
  • antibody fragments can be components of multi-specific molecules without Fc regions, based on fragments of IgG or DVD or scFv.
  • chimeric antibody refers to an antibody having portions of its sequence derived from at least two different sources, such as variable domain sequences (e.g., CDR sequences) derived from an immunoglobulin of one source organism, such as rat or mouse, and constant regions derived from an immunoglobulin of a different organism (e.g., a human, another primate, pig, goat, rabbit, hamster, cat, dog, guinea pig, member of the bovidae family (such as cattle, bison, buffalo, elk, and yaks, among others), cow, sheep, horse, bison, llama, camel, or shark among others).
  • variable domain sequences e.g., CDR sequences
  • constant regions derived from an immunoglobulin of a different organism e.g., a human, another primate, pig, goat, rabbit, hamster, cat, dog, guinea pig, member of the bovidae family (such as cattle, bison, buffalo, e
  • CDR complementarity determining region
  • variable domains can be viewed as hybrid hypervariable positions in that these positions can be deemed to be within a hypervariable region under one set of criteria while being deemed to be outside a hypervariable region under a different set of criteria. One or more of these positions can also be found in extended hypervariable regions.
  • the disclosure includes antibodies comprising modifications in these hybrid hypervariable positions.
  • the variable domains of native heavy and light chains each comprise four framework regions that primarily adopt a ⁇ -sheet configuration, connected by three CDRs, which form loops that connect, and in some cases form part of, the ⁇ -sheet structure.
  • the CDRs in each chain are held together in Attorney Docket No.: 45817-0156WO1 close proximity by the FR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3- FR4 and, with the CDRs from the other antibody chains, contribute to the formation of the target binding site of antibodies (see, Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. (1987); incorporated herein by reference). As used herein, numbering of immunoglobulin amino acid residues is performed according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated.
  • the terms “conservative mutation,” “conservative substitution,” “conservative amino acid substitution,” and the like refer to a substitution of one or more amino acids for one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and/or steric volume. These properties are summarized for each of the twenty naturally-occurring amino acids in Table 1 below.
  • conjugate refers to a compound formed by the chemical bonding of a reactive functional group of one molecule with an appropriately reactive functional group of another molecule. Conjugates may additionally be produced, e.g., as two polypeptide domains covalently bound to one another as part of a single polypeptide chain that is synthesized by the translation of a single RNA transcript encoding both polypeptides in frame with one another.
  • Attorney Docket No.: 45817-0156WO1 As used herein in the context of a CD16-binding protein, the term “construct” refers to a fusion protein containing a first polypeptide domain bound to a second polypeptide domain.
  • the polypeptide domains may each independently be anti-CD16 single chain polypeptides, for instance, as described herein.
  • the first polypeptide domain may be covalently bound to the second polypeptide domain, for instance, by way of a linker, such as a peptide linker or a disulfide bridge, among others.
  • a linker such as a peptide linker or a disulfide bridge, among others.
  • Exemplary linkers that may be used to join the polypeptide domains of a CD16 construct include, without limitation, those that are described in Leriche et al., Bioorg. Med. Chem., 20:571-582 (2012), the disclosure of which is incorporated herein by reference in its entirety.
  • the term “derivatized antibodies” refers to antibodies that are modified by a chemical reaction so as to cleave residues or add chemical moieties not native to an isolated antibody. Derivatized antibodies can be obtained by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by addition of known chemical protecting/blocking groups, proteolytic cleavage, and/or linkage to a cellular ligand or other protein. Any of a variety of chemical modifications can be carried out by known techniques, including, without limitation, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. using established procedures.
  • the derivative can contain one or more non-natural amino acids, e.g., using amber suppression technology (see, e.g., US Patent No.6,964,859; incorporated herein by reference).
  • the term “diabodies” refers to bivalent antibodies comprising two polypeptide chains, in which each polypeptide chain includes VH and VL domains joined by a linker that is too short (e.g., a linker composed of five amino acids) to allow for intramolecular association of VH and VL domains on the same peptide chain. This configuration forces each domain to pair with a complementary domain on another polypeptide chain so as to form a homodimeric structure.
  • triabodies refers to trivalent antibodies comprising three peptide chains, each of which contains one V H domain and one V L domain joined by a linker that is exceedingly short (e.g., a linker composed of 1-2 amino acids) to permit Attorney Docket No.: 45817-0156WO1 intramolecular association of VH and VL domains within the same peptide chain.
  • linker that is exceedingly short (e.g., a linker composed of 1-2 amino acids) to permit Attorney Docket No.: 45817-0156WO1 intramolecular association of VH and VL domains within the same peptide chain.
  • peptides configured in this way typically trimerize so as to position the VH and VL domains of neighboring peptide chains spatially proximal to one another to permit proper folding (see, Holliger et al., Proc. Natl. Acad. Sci.
  • a “tetravalent bispecific antibody” or “tetravalent bsAB” refers to an antibody comprising two peptide chains.
  • Each of the peptide chains contains two sdAB sequences.
  • each peptide chain may include an anti- CD16 sdAB and a second sdAB that binds to a different target than the first sdAB.
  • the peptide chains can dimerize at the hinge region of their respective Fc domains, thus resulting in a tetravalent construct.
  • the term “endogenous” describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell).
  • a molecule e.g., a polypeptide, nucleic acid, or cofactor
  • exogenous describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is not found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell).
  • Exogenous materials include those that are provided from an external source to an organism or to cultured matter extracted there from.
  • the term “framework region” or “FW region” includes amino acid residues that are adjacent to the CDRs. FW region residues may be present in, for example, human antibodies, rodent-derived antibodies (e.g., murine antibodies), humanized antibodies, primatized antibodies, chimeric antibodies, antibody fragments (e.g., Fab fragments), single-chain antibody fragments (e.g., scFv fragments), antibody domains, and bispecific antibodies, among others.
  • the term “fusion protein” refers to a protein that is joined via a covalent bond to another molecule.
  • a fusion protein can be chemically synthesized by, e.g., an amide-bond forming reaction between the N-terminus of one protein to the C-terminus of another protein.
  • a fusion protein containing one protein covalently bound to another protein can be expressed recombinantly in a cell (e.g., a eukaryotic cell or prokaryotic cell) by expression of a polynucleotide encoding the fusion protein, for example, from a vector or the genome of the cell.
  • a fusion protein may contain one protein that is covalently bound to a linker, which in turn is covalently bound to another molecule.
  • linker it may be desirable to include D-amino acids in the linker, as these residues are not present in naturally-occurring proteins and are thus more resistant to degradation by endogenous proteases.
  • Linkers can be prepared using a variety of strategies that are well known in the art, and depending on the reactive components of the linker, can be cleaved by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (Leriche et al., Bioorg. Med. Chem., 20:571-582 (2012)).
  • heterospecific antibodies refers to monoclonal (e.g., human or humanized) antibodies that have binding specificities for at least two different antigens.
  • heterospecific antibodies are based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Milstein et al., Nature 305:537 (1983)). Similar procedures are disclosed, e.g., in WO 93/08829, U.S. Pat.
  • Heterospecific antibodies can include Fc mutations that enforce correct chain Attorney Docket No.: 45817-0156WO1 association in multi-specific antibodies, as described by Klein et al., mAbs 4(6):653- 663 (2012); incorporated herein by reference.
  • the term “human antibody” refers to an antibody in which substantially every part of the protein (e.g., CDR, framework, C L , C H domains (e.g., CH1, CH2, CH3), hinge, (VL, VH)) is substantially non-immunogenic in humans, with only minor sequence changes or variations.
  • a human antibody can be produced in a human cell (e.g., by recombinant expression), or by a non-human animal or a prokaryotic or eukaryotic cell that is capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and/or light chain) genes.
  • a human antibody when a human antibody is a single-chain antibody, it can include a linker peptide that is not found in native human antibodies.
  • an Fv can comprise a linker peptide, such as two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods using antibody libraries derived from human immunoglobulin sequences. See, U.S. Patent Nos.4,444,887 and 4,716,111; and PCT publications WO 1998/46645; WO 1998/50433; WO 1998/24893; WO 1998/16654; WO 1996/34096; WO 1996/33735; and WO 1991/10741; incorporated herein by reference. Human antibodies can also be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.
  • humanized antibodies refers to forms of non-human (e.g., murine) antibodies that are chimeric immunoglobulins, or immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other target-binding subdomains of antibodies), which contain minimal sequences derived from non-human immunoglobulin.
  • a humanized antibody will contain substantially all of at least one, and typically two, variable domains, in which all or Attorney Docket No.: 45817-0156WO1 substantially all of the CDRs correspond to those of a non-human immunoglobulin. All or substantially all of the FRs may also be those of a human immunoglobulin sequence.
  • the humanized antibody may also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence.
  • Fc immunoglobulin constant region
  • Methods of antibody humanization are known in the art. See, e.g., Riechmann et al., Nature 332:323-7 (1988); U.S. Patent Nos: 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370 to Queen et al; EP239400; PCT publication WO 91/09967; U.S. Patent No.5,225,539; EP592106; and EP519596; the disclosure of each of which is incorporated herein by reference.
  • lipid nanoparticle refers to a transfer vehicle including one or more lipids (e.g., cationic lipids, non-cationic lipids, and PEG- modified lipids).
  • lipids e.g., cationic lipids, non-cationic lipids, and PEG- modified lipids.
  • Exemplary lipid nanoparticles are formulated to deliver one or more mRNA to one or more target cells.
  • suitable lipids include, for example, the phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides).
  • Lipid nanoparticles may contain a cationic lipid, or a lipid species with a net positive charge at a selected pH (e.g., physiological pH), to encapsulate and/or enhance the delivery of mRNA into the target cells.
  • a selected pH e.g., physiological pH
  • the terms “messenger RNA” or “mRNA” refer to any polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ, or ex vivo.
  • the basic components of an mRNA molecule include a coding region, a 5’UTR, a 3’UTR, a 5’ cap, and a poly-A tail.
  • modified messenger RNA or “modified mRNA” refer to mRNA polynucleotides that include naturally occurring and/or non-naturally occurring modifications, for example, of a sugar, a nucleobase, or an internucleoside linkage (e.g., to a linking phosphate, to a phosphodiester linkage, or to the phosphodiester backbone).
  • Non-natural modified nucleotides may be introduced during synthesis of post-synthesis of the polynucleotides to achieve desired functions Attorney Docket No.: 45817-0156WO1 or properties.
  • the modifications may be present on an internucleoside linkage, purine or pyrimidine base, or sugar.
  • the modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a polynucleotide may be chemically modified.
  • the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • nucleic acid includes any compound containing a continuous segment of nucleosides joined by way of one or more internucleoside linkages (e.g., polymers of nucleosides linked by way of phosphodiester bonds).
  • nucleic acids include ribonucleic acids (RNA, in particular mRNA), deoxyribonucleic acids (DNA), threose nucleic acids (TNA), glycol nucleic acids (GNA), peptide nucleic acids (PNA), locked nucleic acids (LNA), or hybrids thereof.
  • Nucleic acids also include RNAi inducers, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNAs, tRNAs, RNAs that induce triple spiral formation, aptamers, vectors, and the like.
  • the nucleic acid is one or more modified messenger RNAs (modified mRNAs).
  • percent (%) sequence identity As used herein, the terms “percent (%) sequence identity,” “percent (%) identity,” and the like, with respect to a reference polynucleotide or polypeptide sequence, is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software.
  • percent sequence Attorney Docket No.: 45817-0156WO1 identity values may be generated using the sequence comparison computer program BLAST.
  • the percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, is calculated as: 100 multiplied by (the fraction X/Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program’s alignment of A and B, and where Y is the total number of nucleic acids in B.
  • nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B
  • percent sequence identity of A to B will not equal the percent sequence identity of B to A.
  • primary antibody refers to an antibody comprising framework regions from primate-derived antibodies and other regions, such as CDRs and/or constant regions, from antibodies of a non-primate source. Methods for producing primatized antibodies are known in the art. See, e.g., U.S. Patent Nos.5,658,570; 5,681,722; and 5,693,780; incorporated herein by reference.
  • a primatized antibody or antigen-binding fragment thereof described herein can be produced by inserting the CDRs of a non-primate antibody or antigen- binding fragment thereof into an antibody or antigen-binding fragment thereof that contains one or more framework regions of a primate.
  • the term “operatively linked” in the context of a polynucleotide fragment is intended to mean that the two polynucleotide fragments are joined such that the amino acid sequences encoded by the two polynucleotide fragments remain in-frame.
  • the term “pharmacokinetic profile” refers to the absorption, distribution, metabolism, and clearance of a therapeutic agent (e.g., a polypeptide, such as an anti-CD16 antibody, antigen-binding fragment thereof, single-chain Attorney Docket No.: 45817-0156WO1 polypeptide, or construct of the disclosure) over time following administration of the drug to a patient.
  • a therapeutic agent e.g., a polypeptide, such as an anti-CD16 antibody, antigen-binding fragment thereof, single-chain Attorney Docket No.: 45817-0156WO1 polypeptide, or construct of the disclosure
  • regulatory sequence includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation, e.g., of antibody chain genes.
  • scFv refers to a single-chain Fv antibody in which the variable domains of the heavy chain and the light chain from an antibody have been joined to form one chain.
  • ScFv fragments contain a single polypeptide chain that includes the variable region of an antibody light chain (V L ) (e.g., CDR-L1, CDR-L2, and/or CDR-L3) and the variable region of an antibody heavy chain (VH) (e.g., CDR- H1, CDR-H2, and/or CDR-H3) separated by a linker.
  • V L an antibody light chain
  • VH an antibody heavy chain
  • the linker that joins the V L and VH regions of a scFv fragment can be a peptide linker composed of proteinogenic amino acids.
  • linkers can be used to so as to increase the resistance of the scFv fragment to proteolytic degradation (e.g., linkers containing D-amino acids), in order to enhance the solubility of the scFv fragment (e.g., hydrophilic linkers such as polyethylene glycol-containing linkers or polypeptides containing repeating glycine and serine residues), to improve the biophysical stability of the molecule (e.g., a linker containing cysteine residues that form intramolecular or intermolecular disulfide bonds), or to attenuate the immunogenicity of the scFv fragment (e.g., linkers containing glycosylation sites).
  • linkers containing D-amino acids e.g., hydrophilic linkers such as polyethylene glycol-containing linkers or polypeptides containing repeating glycine and serine residues
  • hydrophilic linkers such as polyethylene glycol-containing linkers or polypeptides containing repeating
  • VL and VH domains of a scFv molecule can be derived from one or more antibody molecules.
  • variable regions of the scFv molecules described herein can be modified such that they vary in amino acid sequence from the antibody molecule from which they were Attorney Docket No.: 45817-0156WO1 derived.
  • nucleotide or amino acid substitutions leading to conservative substitutions or changes at amino acid residues can be made (e.g., in CDR and/or framework residues).
  • mutations are made to CDR amino acid residues to optimize antigen binding using art recognized techniques. ScFv fragments are described, for example, in WO 2011/084714; incorporated herein by reference.
  • single-domain antibody As used herein, the terms “single-domain antibody,” “sdAb,” “nanobody,” and “VHH antibody” are used interchangeably to refer to a single-chain antibody fragment that contains only a single heavy-chain variable domain. Unlike a traditional, full-length antibody, which includes heavy chains and light chains, each containing a corresponding variable domain (i.e., a heavy chain variable domain, VH, and a light chain variable domain, V L ) having three CDRs, a single-domain antibody only includes one heavy-chain variable domain having a total of three CDRs (referred to herein as CDR-H1, CDR-H2, and CDR-H3).
  • the phrase “specifically binds” refers to a binding reaction which is determinative of the presence of an antigen in a heterogeneous population of proteins and other biological molecules that is recognized, e.g., by an antibody or antigen-binding fragment thereof, with particularity.
  • An antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a K D of less than 100 nM.
  • an antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen via the antigen binding domain with a K D of up to 100 nM (e.g., between 1 pM and 100 nM).
  • An antibody or antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or epitope thereof will exhibit a K D of greater than 100 nM (e.g., greater than 500 nm, 1 ⁇ M, 100 ⁇ M, 500 ⁇ M, or 1 mM) for that particular antigen or epitope thereof.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein or carbohydrate.
  • solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein or carbohydrate.
  • the terms “subject” and “patient” refer to an organism that receives treatment (e.g., by administration of a CD16 polypeptide, such as an antibody, antigen-binding fragment thereof, single-chain polypeptide, or construct described herein) for a particular disease or condition, such as a cancer or an immunological disorder (e.g., an autoimmune disease).
  • a CD16 polypeptide such as an antibody, antigen-binding fragment thereof, single-chain polypeptide, or construct described herein
  • a particular disease or condition such as a cancer or an immunological disorder (e.g., an autoimmune disease).
  • subjects and patients include mammals, such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the bovidae family (such as cattle, bison, buffalo, and yaks, among others), sheep, and horses, among others, receiving treatment for a cancer, immunological diseases or conditions, such as autoimmune disorders (e.g., allograft rejection) and graft-versus-host disease, among others.
  • a patient that may be treated using the compositions and methods described herein may have an established disease, in which case the patient has been diagnosed as having the disease and has shown symptoms of the disease for a prolonged period of time (e.g., over the course of days, weeks, months, or years).
  • a patient may be symptomatic for a particular disease, but has yet to be diagnosed with the disease by a physician.
  • Other patients that may be treated using the compositions and methods described herein include those that have been diagnosed as having a disease or disorder, and may or may not be showing symptoms of the disease as of yet.
  • the term “transfection” refers to any of a wide variety of techniques commonly used for the introduction of a nucleic acid molecule, e.g., exogenous DNA or RNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, lipofection, calcium- phosphate precipitation, DEAE- dextran transfection and the like.
  • the terms “treat” or “treatment” refer to therapeutic treatment, in which the object is to inhibit or slow down (lessen) an undesired physiological change or disorder, such as a cancer or an immunological disorder (e.g., autoimmune Attorney Docket No.: 45817-0156WO1 disorders (e.g., allograft rejection) and graft-versus-host disease, among others).
  • an immunological disorder e.g., autoimmune Attorney Docket No.: 45817-0156WO1 disorders (e.g., allograft rejection) and graft-versus-host disease, among others.
  • Beneficial or desired clinical results of treatment include, without limitation, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • variable region CDR includes amino acids in a CDR or complementarity determining region as identified using sequence or structure-based methods.
  • CDR complementarity determining region refers to the noncontiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol.
  • CDR is a CDR as defined by Kabat based on sequence comparisons.
  • vector includes a nucleic acid vector, e.g., a DNA vector, such as a plasmid, an RNA vector, virus or other suitable replicon (e.g., viral vector).
  • a DNA vector such as a plasmid, an RNA vector, virus or other suitable replicon (e.g., viral vector).
  • Expression vectors described herein contain a polynucleotide sequence as well as, e.g., additional sequence elements used for the expression of proteins and/or the integration of these polynucleotide sequences into the genome of a mammalian cell.
  • Certain vectors that can be used for the expression of antibodies, antibody fragments, and/or binding proteins described herein include plasmids that contain Attorney Docket No.: 45817-0156WO1 regulatory sequences, such as promoter and enhancer regions, which direct gene transcription.
  • Other useful vectors for expression of antibodies, antibody fragments, and/or binding proteins contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription.
  • sequence elements include, e.g., 5’ and 3’ untranslated regions, an internal ribosomal entry site (IRES), and polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector.
  • the expression vectors described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker include genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.
  • V H refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, or Fab.
  • V L refers to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv or Fab.
  • Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules which lack target specificity.
  • Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain of a native antibody has at the amino terminus a variable domain (VH) followed by a number of constant domains.
  • Each light chain of a native antibody has a variable domain at the amino terminus (VL) and a constant domain at the carboxy terminus.
  • VL amino terminus
  • CDRs are those regions that predominantly dictate the CD16-binding properties of the molecule. This selection provides amino acid Attorney Docket No.: 45817-0156WO1 sequence information for the CDRs of anti-CD16 VHH domains and antibodies comprising such anti-CD16 VHH domains of the disclosure.
  • the disclosure provides an anti-CD16 antibody or binding protein having one, two, or three of the CDRs described in Table 4, below.
  • the disclosure features an anti-CD16 binding molecule comprising a VHH that binds to human CD16a, the VHH comprising a VHH-CDR1, a VHH- CDR2, and a VHH-CDR3 of any one of SEQ ID NO: 1 or 5-24.
  • the VHH-CDRs can be based on any known definition in the art.
  • the anti-CD16 binding molecule comprises the three VHH-CDRs of SEQ ID NO:1 based on any single definition as shown in Table 5.
  • Exemplary CDRs of SEQ ID NO:7 are shown in Table A below.
  • Table A Exemplary CDR Definitions of anti-CD16 VHH, VHH1-H3 CDR VHH-CDR1 VHH-CDR2 VHH-CDR3 Definition
  • the binding molecule comprises the three VHH-CDRs of SEQ ID NO:7 based on any single CDR definition as shown in Table A.
  • an anti-CD16 antibody or binding protein of the disclosure is an antibody (e.g., a VHH or bispecific antibody) or antigen- binding fragment thereof (e.g., a scFv) having one or more of the following CDRs: Attorney Docket No.: 45817-0156WO1 (a) a CDR1 having the amino acid sequence GRTDSIYA (SEQ ID NO: 2) or an amino acid sequence having up to two amino acid substitutions (e.g., conservative amino acid substitutions) relative to SEQ ID NO: 2; (b) a CDR2 having the amino acid sequence INSNTGRT (SEQ ID NO: 3), or an amino acid sequence having up to two amino acid substitutions (e.g., conservative amino acid substitutions) relative to SEQ ID NO: 3; and (c) a CDR3 having the amino acid sequence AAGRGYGLLSISSNWYNY (SEQ ID NO: 4), or an amino acid sequence having up to two amino acid substitutions (e.g.,
  • the anti-CD16 antibody or binding protein comprises the three CDRs based on any single definition as shown in Table A or Table 5.
  • the anti-CD16 antibody or binding protein binds human CD16a.
  • the anti- CD16 antibody or binding protein binds cynomolgus CD16.
  • the anti- CD16 antibody or binding protein binds human CD16a and cynomolgus CD16.
  • the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1.
  • the anti-CD16 antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH1, shown below (CDR sequences shown in bold): QVQLVESGGGLVQAGGSLRLSCAASGRTDSIYAMGWFRQAPGKERDFVAAI NSNTGRTYHADSVKGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAAGRG YGLLSISSNWYNYWGQGTQVTVSS (VHH1, SEQ ID NO: 1).
  • the anti-CD16 antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 1.
  • the anti-CD16 Attorney Docket No.: 45817-0156WO1 antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 1.
  • the anti-CD16 antibody or binding protein contains a VHH domain having the amino acid sequence of SEQ ID NO: 1.
  • the anti-CD16 antibody or binding protein binds human CD16a.
  • the anti-CD16 antibody or binding protein binds cynomolgus CD16.
  • the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16.
  • the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H1, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKELDFVSAIN SNTGRTYHADSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCAAGRGY GLLSISSNWYNYWGQGTLVTVSS (VHH1-H1, SEQ ID NO: 5).
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 5.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 5.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 5.
  • the anti- CD16 antibody or binding protein binds human CD16a.
  • the anti-CD16 antibody or binding protein binds cynomolgus CD16.
  • the anti-CD16 Attorney Docket No.: 45817-0156WO1 antibody or binding protein binds human CD16a and cynomolgus CD16.
  • the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H2, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKELEFVAAI NSNTGRTYHADSVKGRFTISRDNAKNTLYLQMNSLKAEDTAVYYCAAGRG YGLLSISSNWYNYWGQGTLVTVSS (VHH1-H2, SEQ ID NO: 6).
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 6.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 6.
  • the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 6.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H3, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKEREFVSAIN SNTGRTYHADSVKGRFTISRDNAKNMVYLQMNSLRAEDTAVYYCAAGRGY GLLSISSNWYNYWGQGTLVTVSS (VH1-H3, SEQ ID NO: 7).
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 7.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 7.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 7.
  • the anti- CD16 antibody or binding protein binds human CD16a.
  • the anti-CD16 antibody or binding protein binds cynomolgus CD16.
  • the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16.
  • the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H4, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKELEFVSAIN SNTGRTYHADSVKGRFTISRDNAKNMVYLQMNSLKAEDTAVYYCAAGRGY GLLSISSNWYNYWGQGTLVTVSS (VHH1-H4, SEQ ID NO: 8).
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 8.
  • the aanti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 8.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 8.
  • the anti- CD16 antibody or binding protein binds human CD16a.
  • the anti-CD16 antibody or binding protein binds cynomolgus CD16.
  • the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16.
  • the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H5, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKELEFVSAIN SNTGRTYHADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAAGRGY GLLSISSNWYNYWGQGTLVTVSS (VHH1-H5, SEQ ID NO: 9).
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 9.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 9.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 9.
  • the anti- CD16 antibody or binding protein binds human CD16a.
  • the anti-CD16 antibody or binding protein binds cynomolgus CD16.
  • the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16.
  • the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H6, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKEREFVSAIN SNTGRTYHADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAGRGY GLLSISSNWYNYWGQGTLVTVSS (VHH1-H6, SEQ ID NO: 10).
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 10.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 10.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 10.
  • the anti- CD16 antibody or binding protein binds human CD16a.
  • the anti-CD16 antibody or binding protein binds cynomolgus CD16.
  • the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16.
  • the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H7, shown below (CDR sequences shown in bold): Attorney Docket No.: 45817-0156WO1 EVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKEREFVSAIN SNTGRTYHADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAAGRGY GLLSISSNWYNYWGQGTLVTVSS (VHH1-H7, SEQ ID NO: 11).
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 11.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 11.
  • the anti-CD1 ⁇ 6 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 11.
  • the anti- CD16 antibody or binding protein binds human CD16a.
  • the anti-CD16 antibody or binding protein binds cynomolgus CD16.
  • the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16.
  • the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H8, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKELEFVSAIN SNTGRTYHADSVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCAAGRGY GLLSISSNWYNYWGQGTLVTVSS (VHH1-H8, SEQ ID NO: 12).
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 12.
  • the Attorney Docket No.: 45817-0156WO1 anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 12.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 12.
  • the anti- CD16 antibody or binding protein binds human CD16a.
  • the anti-CD16 antibody or binding protein binds cynomolgus CD16.
  • the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16.
  • the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H9, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKERDFVSAI NSNTGRTYHADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAGRG YGLLSISSNWYNYWGQGTLVTVSS (VHH1-H9, SEQ ID NO: 13).
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 13.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 13.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 13.
  • the anti- CD16 antibody or binding protein binds human CD16a.
  • the anti-CD16 antibody or binding protein binds cynomolgus CD16.
  • the anti-CD16 Attorney Docket No.: 45817-0156WO1 antibody or binding protein binds human CD16a and cynomolgus CD16.
  • the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H10, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKERDFVSAIN SNTGRTYHADSVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCAAGRGY GLLSISSNWYNYWGQGTLVTVSS (VHH1-H10, SEQ ID NO: 14).
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 14.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 14.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 14. In some cases, the anti- CD16 antibody or binding protein binds human CD16a. In other cases, the anti-CD16 antibody or binding protein binds cynomolgus CD16. In certain cases, the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16. In some cases, the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Attorney Docket No.: 45817-0156WO1 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H11, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKELDFVSAIN SNTGRTYHADSVKGRFTISRDNAKNMVYLQMNSLRPEDTAVYYCAAGRGY GLLSISSNWYNYWGQGTLVTVSS (VHH1-H11, SEQ ID NO: 15).
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 15.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 15.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 15.
  • the anti- CD16 antibody or binding protein binds human CD16a.
  • the anti-CD16 antibody or binding protein binds cynomolgus CD16.
  • the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16.
  • the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H12, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKERDFVSAI NSNTGRTYHADSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCAAGRG YGLLSISSNWYNYWGQGTLVTVSS (VHH1-H12, SEQ ID NO: 16).
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 16.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 16.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 16.
  • the anti- CD16 antibody or binding protein binds human CD16a.
  • the anti-CD16 antibody or binding protein binds cynomolgus CD16.
  • the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16.
  • the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H13, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKERDFVAAI NSNTGRTYHADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAAGRG YGLLSISSNWYNYWGQGTLVTVSS (VHH1-H13, SEQ ID NO: 17).
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 17.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 17.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 17.
  • the anti- CD16 antibody or binding protein binds human CD16a.
  • the anti-CD16 antibody or binding protein binds cynomolgus CD16.
  • the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16.
  • the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H14, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKEREFVSAIN SNTGRTYHADSVKGRFTISRDNAKNMLYLQMNSLKAEDTAVYYCAAGRGY GLLSISSNWYNYWGQGTLVTVSS (VHH1-H14, SEQ ID NO: 18).
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 18.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 18.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 18.
  • the anti- CD16 antibody or binding protein binds human CD16a.
  • the anti-CD16 antibody or binding protein binds cynomolgus CD16.
  • the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16.
  • the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H15, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKEREFVAAI NSNTGRTYHADSVKGRFTISRDNAKNTLYLQMNSLKAEDTAVYYCAAGRG YGLLSISSNWYNYWGQGTLVTVSS (VHH1-H15, SEQ ID NO: 19).
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 19.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 19.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 19. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H16, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKERDFVSAI NSNTGRTYHADSVKGRFTISRDNAKNTLYLQMNSLKAEDTAVYYCAAGRG YGLLSISSNWYNYWGQGTLVTVSS (VHH1-H16, SEQ ID NO: 20).
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% Attorney Docket No.: 45817-0156WO1 identical) to the amino acid sequence of SEQ ID NO: 20.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 20.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 20.
  • the anti- CD16 antibody or binding protein binds human CD16a.
  • the anti-CD16 antibody or binding protein binds cynomolgus CD16.
  • the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16.
  • the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H17, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKELDFVAAI NSNTGRTYHADSVKGRFTISRDNAKNMLYLQMNSLRAEDTAVYYCAAGRG YGLLSISSNWYNYWGQGTLVTVSS (VHH1-H17, SEQ ID NO: 21).
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 21.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 21.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 21.
  • the anti- CD16 antibody or binding protein binds human CD16a.
  • the anti-CD16 Attorney Docket No.: 45817-0156WO1 antibody or binding protein binds cynomolgus CD16.
  • the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16.
  • the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H18, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKERDFVSAI NSNTGRTYHADSVKGRFTISRDNAKNMLYLQMNSLKAEDTAVYYCAAGRG YGLLSISSNWYNYWGQGTLVTVSS (VHH1-H18, SEQ ID NO: 22).
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 22.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 22.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 22.
  • the anti- CD16 antibody or binding protein binds human CD16a.
  • the anti-CD16 antibody or binding protein binds cynomolgus CD16.
  • the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16.
  • the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Attorney Docket No.: 45817-0156WO1 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH1-H19, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKERDFVAAI NSNTGRTYHADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAGRG YGLLSISSNWYNYWGQGTLVTVSS (VHH1-H19, SEQ ID NO: 23).
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 23.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 23.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 23.
  • the anti- CD16 antibody or binding protein binds human CD16a.
  • the anti-CD16 antibody or binding protein binds cynomolgus CD16.
  • the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16.
  • the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H20, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKERDFVAAI NSNTGRTYHADSVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCAAGRG YGLLSISSNWYNYWGQGTLVTVSS (VHH1-H20, SEQ ID NO: 24).
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 24.
  • the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 24.
  • the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 24.
  • the anti-CD16 antibody or binding protein binds human CD16a.
  • the anti-CD16 antibody or binding protein binds cynomolgus CD16.
  • the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16.
  • the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1.
  • an antibody or antigen-binding fragment is a murine- specific antibody or antigen-binding fragment, e.g., the antibody or binding protein specifically binds the murine antigen.
  • an antibody or antigen- binding fragment is a rat-specific antibody or antigen-binding fragment, e.g., the antibody or binding protein specifically binds the rat antigen.
  • an antibody or antigen-binding fragment is a llama-specific antibody or antigen- binding fragment, e.g., the antibody or binding protein specifically binds the llama antigen.
  • an antibody or antigen-binding fragment is a human- specific antibody or antigen-binding fragment, e.g., the antibody or binding protein specifically binds the human antigen.
  • an antibody or antigen- binding fragment is human-specific even if the antibody or binding protein is not human or humanized.
  • Multispecific antibodies for example, bispecific antibodies (BsAbs), that may have binding specificities that are Attorney Docket No.: 45817-0156WO1 directed towards CD16 and any other antigen, e.g., for a cell-surface protein, receptor, receptor subunit, or tissue-specific antigen, or other non-CD16 antigen.
  • Multispecific antibodies typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen (i.e., CD16 and any other antigen).
  • Each antigen-binding domain of a bispecific antibody can comprise a heavy chain variable domain (VH), a light chain variable domain (VL), or a VHH and a VL.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • VHH VHH and a VL.
  • each antigen binding domain comprises at least one CDR that alone, or in combination with one or more additional CDRs and/or framework regions, specifically binds to a particular antigen (i.e., CD16, any other antigen).
  • the first antigen-binding domain and the second antigen-binding domain may be directly or indirectly connected to one another to form a bispecific antigen-binding fragment (i.e., bispecific scFv) further bound to an Fc domain.
  • the first antigen-binding domain and the second antigen-binding domain may each be connected to a separate Fc domain.
  • Bispecific antigen-binding fragments of the present disclosure may comprise two Fc domains that are each individually part of a separate antibody heavy chain.
  • the first and second Fc domains may be of the same sequence, or the Fc domains may have a mutation in the CH3 domain intended for the facilitation or ease of purification of heterodimeric (i.e., bispecific) molecules.
  • multispecific antibodies of the present disclosure are secreted (e.g., released from a cell, for example, into the extracellular milieu).
  • Multispecific antibodies of the present disclosure can include any anti-CD16 CDRs, or VHH domains described herein.
  • Multispecific antibodies of the present disclosure can comprise binding specificities that are directed towards CD16 and any other antigen. Any other antigen may be or comprise, for example, a cancer cell antigen/marker, an immune cell antigen (e.g., a T cell activation marker), a pathogenic antigen, or any other non- CD16 antigen.
  • an antibody or fragment thereof having a first antigen binding specificity can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity to produce Attorney Docket No.: 45817-0156WO1 a bispecific antigen-binding molecule.
  • bispecific formats that can be used in the context of the present disclosure include, without limitation, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into- holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgGl/IgG2, dual acting Fab (DAF)-IgG, and Mab2 bispecific formats (see, e.g., Klein et al.2012, mAbs 4:6, 1-11 , and references cited therein, for a review of the foregoing formats).
  • the disclosed multispecific antibodies can be made from or incorporate the CDRs or variable regions from polyclonal, monoclonal, chimeric, human, partially or fully humanized, and/or recombinant antibodies.
  • the “parent” antibodies for the disclosed multispecific antibodies are not particularly limited; however, they are preferably fully human or humanized.
  • the parent antibody can be a polyclonal antibody.
  • the parent antibody can be a monoclonal.
  • the parent antibody can be a human antibody.
  • Antibodies, antigen-binding fragments, or binding proteins of the disclosure may have an affinity for CD16 of, for example, from 1 nM to 100 nM (e.g., from 10 nM to 90 nM, from 20 nM to 80 nM, from 30 nM to 70 nM, from 40 nM to 60 nM, or about 50 nM). In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of from about 1 nM to about 100 nM.
  • antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of from about 1 nM to about 90 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity CD16 of from about 1 nM to about 80 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of from about 1 nM to 60 nM. In Attorney Docket No.: 45817-0156WO1 some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of from about 1 nM to 40 nM.
  • antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of from about 1 nM to 20 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 100 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 95 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 90 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 85 nM.
  • antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 80 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 75 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 70 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 65 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 60 nM.
  • antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 55 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 50 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 45 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 40 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 35 nM.
  • antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 30 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 25 Attorney Docket No.: 45817-0156WO1 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 20 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 15 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 10 nM.
  • antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 5 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 1 nM.
  • the specific binding of an antibody, antigen-binding fragments, or binding proteins described herein to CD16 can be determined by any of a variety of established methods. The affinity can be represented quantitatively by various measurements, including the concentration of antibody or binding protein needed to achieve the equilibrium constant (K D ) of the antibody- , antigen-binding fragment-, or binding proteins - antigen complex dissociation.
  • the equilibrium constant, KD which describes the interaction of CD16 with an antibody or binding proteins described herein is the chemical equilibrium constant for the dissociation reaction of an antigen- antibody, –antigen-binding fragment, or – binding protein complex into solvent- separated antigen and antibody or binding proteins that do not interact with one another.
  • Antibodies, antigen-binding fragments, or binding proteins described herein include those that specifically bind to CD16 with a K D value of less than 100 nM (e.g., less than 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM).
  • K D value of less than 100 nM (e.g., less than 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM,
  • Antibodies, antigen-binding fragments, or binding proteins described herein can also be characterized by a variety of in vitro binding assays. Examples of experiments that can be used to determine the K D or EC 50 of an anti-CD16 antibody or binding protein include, e.g., surface plasmon resonance, isothermal titration calorimetry, fluorescence anisotropy, ELISA-based assays, gene expression assays, and protein expression assays, among others.
  • Antibodies, antigen-binding fragments, or binding proteins described herein may additionally bind isolated peptides derived from CD16 that structurally pre-organize various residues in a manner that simulates the conformation of the above fragments in the native protein.
  • this binding can be quantified, e.g., by analyzing the luminescence that occurs upon incubation of an HRP substrate (e.g., 2,2’-azino-di-3- ethylbenzthiazoline sulfonate) with an antigen-antibody, antigen-antigen-binding fragment, or antigen-binding protein complex bound to a HRP-conjugated secondary antibody.
  • an HRP substrate e.g., 2,2’-azino-di-3- ethylbenzthiazoline sulfonate
  • SPR surface plasmon resonance
  • the antigen is typically immobilized on a solid metallic surface which is treated in pulses with solutions of increasing concentrations of antibody or binding proteins.
  • Antibody-, antigen-binding fragment-, or binding protein-antigen binding induces distortion in the angle of reflection of incident light at the metallic surface, and this change in refractive index over time as antibody or binding protein is introduced to the system can be fit to established regression models in order to calculate the association and dissociation rate constants of an antibody- or antigen- binding-fragment- or binding protein- antigen interaction.
  • Antibodies, antigen-binding fragments, or binding proteins described herein may exhibit high kon and low koff values upon interaction with CD16.
  • antibodies, antigen-binding fragments, or binding proteins described herein may exhibit k on values in the presence of CD16 of greater than 10 4 M -1 s -1 (e.g., 1.0 x 10 4 M -1 s -1 , 1.5 x 10 4 M -1 s -1 , 2.0 x 10 4 M -1 s -1 , 2.5 x 10 4 M -1 s -1 , 3.0 x 10 4 M -1 s -1 , 3.5 x 10 4 Attorney Docket No.: 45817-0156WO1 M -1 s -1 , 4.0 x 10 4 M -1 s -1 , 4.5 x 10 4 M -1 s , 5.0 x 10 4 M -1 s , 5.5 x 10 4 M -1 s -1 , 6.0 x 10 4 M- 1 s -1 , 6.5 x 10 4 M -1 s -1 , 7.0 x 10 4 M -1 s -1 ,
  • Antibodies, antigen-binding fragments, or binding proteins may exhibit low koff values when bound to CD16.
  • antibodies, antigen-binding fragments, or binding proteins described herein may exhibit koff values of less than 10 -3 s -1 when complexed to CD16 (e.g., 1.0 x 10 -3 s -1 , 9.5 x 10 -4 s -1 , 9.0 x 10 -4 s -1 , 8.5 x 10 -4 s -1 , 8.0 x 10 -4 s -1 , 7.5 x 10 -4 s -1 , 7.0 x 10 -4 s -1 , 6.5 x 10 -4 s -1 , 6.0 x 10 -4 s -1 , 5.5 x 10 -4 s -1 , 5.0 x 10 -4 s -1 , 4.5 x 10 -4 s -1 , 4.0 x 10 -4 s -1 , 3.5 x 10 -4 s
  • Antibodies, antigen-binding fragments, or binding proteins described herein can include fully human, humanized, primatized, and chimeric antibodies that contain one or more of the CDR sequences shown in Table 4, below.
  • one strategy that can be used to design humanized antibodies, antigen-binding fragments, or binding proteins described herein is to align the sequences of the V H and/or V L of an antibody or binding protein (e.g., of the present disclosure) with the VH and/or VL of a consensus human antibody.
  • Consensus human antibody heavy chain and light chain sequences are known in the art (see, e.g., the “VBASE” human germline sequence database; see also Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.91 -3242 (1991); Tomlinson et al., J. Mol. Biol.227:776-98 (1992); and Cox et al., Eur. J. Immunol.24:827-836 (1994); the disclosure of which is incorporated herein by reference).
  • variable domain framework Attorney Docket No.: 45817-0156WO1 residues and CDRs can be identified by sequence alignment (see, Kabat, supra).
  • this strategy can also be used to produce primatized anti-CD16 antibodies, antigen-binding fragments, or binding proteins, as one can substitute, for example, one or more, or all, of the CDRs of a primate antibody consensus sequence with, for example, one or more, or all, of the CDRs of an antibody or binding protein of the disclosure.
  • Consensus primate antibody sequences known in the art (see, e.g., U.S. Patent Nos.5,658,570; 5,681,722; and 5,693,780; the disclosures of each of which are incorporated herein by reference).
  • US Patent No.6,054,297 identifies several instances when it may be advantageous to retain certain framework residues from a particular antibody heavy chain or light chain variable region in the resulting humanized antibody or binding proteins.
  • framework residues may engage in non-covalent interactions with the antigen and thus contribute to the affinity of the antibody or binding proteins for the target antigen.
  • individual framework residues may modulate the conformation of a CDR, and thus indirectly influence the interaction of the antibody, antigen-binding domain, or binding proteins with the antigen.
  • Certain framework residues may form the interface between V H and V L domains, and may therefore contribute to the global antibody, antigen-binding domain, or binding protein structure.
  • framework residues may constitute functional glycosylation sites (e.g., Asn-X- Ser/Thr) which may dictate antibody, antigen-binding domain, or binding protein structure and antigen affinity upon attachment to carbohydrate moieties.
  • an anti-CD16 antibody or binding protein in, e.g., a humanized or primatized antibody Attorney Docket No.: 45817-0156WO1 or antigen-binding fragment or binding protein thereof, as various framework residues may promote high epitope affinity and improved biochemical activity of the antibody or antigen-binding fragment or binding protein thereof.
  • humanized variant sequences of the antibodies, antigen- binding fragments, or binding proteins described herein can be found in Table 7 below.
  • Antibodies described herein also include antibody fragments, Fab domains, F(ab’) molecules, F(ab’)2 molecules, single-chain variable fragments (scFvs), tandem scFv fragments, diabodies, triabodies, dual variable domain immunoglobulins, multi- specific antibodies, bispecific antibodies, VHH, and heterospecific antibodies that contain one or more of the CDRs in Table 4, below, or a CDR having at least 85% sequence identity thereto (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto).
  • Anti-CD16 antibodies, antigen-binding fragments, or binding proteins described herein can be prepared by any of a variety of established techniques. For instance, an anti- CD16 antibody or antigen-binding fragment described herein can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell.
  • a host cell can be transfected with one or more recombinant expression vectors carrying DNA fragments encoding the desired antibody chain(s), antigen-binding fragments, and/or additional binding protein domains (e.g., transmembrane domains, hinge domains).
  • additional binding protein domains e.g., transmembrane domains, hinge domains.
  • the light and/or heavy chains of an antibody or an antigen-binding fragment can be expressed in the host cell and, Attorney Docket No.: 45817-0156WO1 optionally, secreted into the medium in which the host cells are cultured, from which medium the antibodies can be recovered.
  • Standard recombinant DNA methodologies are used to obtain antibody heavy chain genes, light chain genes, and binding protein domains and to incorporate these genes into recombinant expression vectors and introduce the vectors into host cells, such as those described in Molecular Cloning; A Laboratory Manual, Second Edition (Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, N. Y., 1989), Current Protocols in Molecular Biology (Ausubel et al., eds., Greene Publishing Associates (1989), and in U.S. Patent No.4,816,397; the disclosures of each of which are incorporated herein by reference.
  • Suitable vectors include, but are not limited to, viral vectors and non-viral vectors.
  • Viral vectors can include retrovirus, adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., Measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox).
  • adenovirus e.g., Ad5, Ad26, Ad
  • viruses useful for delivering polynucleotides encoding antibody light and heavy chains or antibody fragments or binding proteins described herein include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.
  • Non-viral vectors can include plasmids.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • TALEN transcription activator-like effector nucleases
  • Additional genome editing techniques that can be used to incorporate polynucleotides encoding antibodies, antigen-binding fragments, or binding proteins described herein into the genome of a prokaryotic or eukaryotic cell include the use of ARCUS TM meganucleases that can be rationally designed so as to site-specifically cleave genomic DNA.
  • polynucleotides encoding partial or full-length light and heavy chains e.g., polynucleotides that encode a one or more of the CDR sequences of an antibody or binding protein described herein, can be inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences.
  • the expression vector and expression control sequences are chosen to be compatible with the expression host cell used.
  • Polynucleotides encoding the light chain gene and the heavy chain of an anti-CD16 antibody or binding protein can be inserted into separate vectors, or, optionally, both polynucleotides can be incorporated into the same expression vector using established techniques described herein or known in the art.
  • the recombinant expression vectors described herein may carry regulatory sequences that Attorney Docket No.: 45817-0156WO1 control the expression of the antibody chain genes or binding protein domains in a host cell.
  • the design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed or the level of expression of protein desired.
  • suitable regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma.
  • CMV cytomegalovirus
  • SV40 Simian Virus 40
  • AdMLP adenovirus major late promoter
  • Viral regulatory elements, and sequences thereof are described in detail, for instance, in U.S. Patent No.5, 168,062, U.S. Patent No.4,510,245, and U.S.
  • Patent No.4,968,615 the disclosures of each of which are incorporated herein by reference.
  • the recombinant expression vectors described herein can carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes.
  • a selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Patents Nos.4,399,216, 4,634,665 and 5,179,017).
  • the selectable marker gene confers resistance to cytotoxic drugs, such as G418, puromycin, blasticidin, hygromycin or methotrexate, to a host cell into which the vector has been introduced.
  • cytotoxic drugs such as G418, puromycin, blasticidin, hygromycin or methotrexate
  • Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in DHFR ” host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
  • DHFR dihydrofolate reductase
  • the expression vector(s) containing polynucleotides encoding the heavy and light chains can be transfected into a host cell by standard techniques.
  • Host cells for expression of anti-CD16 antibodies, antigen-binding fragments, or binding proteins It is possible to express the antibodies, antigen-binding fragments, or binding proteins described herein in either prokaryotic or eukaryotic host cells.
  • expression of antibodies, antigen-binding fragments, or binding proteins is performed in eukaryotic cells, e.g., mammalian host cells, for high secretion of a properly folded and immunologically active antibody or antigen- binding fragments.
  • Exemplary mammalian host cells for expressing the recombinant antibodies, antigen-binding fragments, or binding proteins described herein include Chinese Hamster Ovary (CHO cells) (including DHFR CHO cells, described in Urlaub and Chasin (1980, Proc. Natl. Acad. Sci. USA 77:4216-4220), used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982, Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, 293 cells (e.g., expi293), and SP2/0 cells.
  • Chinese Hamster Ovary CHO cells
  • DHFR CHO cells described in Urlaub and Chasin (1980, Proc. Natl. Acad. Sci. USA 77:4216-4220
  • a DHFR selectable marker e.g., as described in Kaufman and Sharp (1982, Mol. Biol. 159:601-621
  • Additional cell types that may be useful for the expression of antibodies, antigen-binding fragments, or binding proteins include bacterial cells, such as BL- 21(DE3) E. coli cells, which can be transformed with vectors containing foreign DNA according to established protocols.
  • Additional eukaryotic cells that may be useful for expression of antibodies, antigen-binding fragments, or binding proteins include yeast cells, such as auxotrophic strains of S. cerevisiae, which can be transformed and selectively grown in incomplete media according to established procedures known in the art.
  • an anti-CD16 antibody or antigen-binding fragment of the disclosure is conjugated to a second molecule, e g., to extend the half-life of the anti-CD16 antibody or antigen-binding fragment in vivo.
  • a second molecule e g., to extend the half-life of the anti-CD16 antibody or antigen-binding fragment in vivo.
  • PEG polyethylene glycol
  • Anti-CD16 antibodies can be conjugated to various molecules for the purpose of improving the half-life, solubility, and stability of the protein in aqueous solution.
  • examples of such molecules include polyethylene glycol (PEG), murine serum albumin (MSA), bovine serum albumin (BSA), and human serum albumin (HSA), among others.
  • PEG polyethylene glycol
  • MSA murine serum albumin
  • BSA bovine serum albumin
  • HSA human serum albumin
  • one can conjugate an anti-CD16 antibody or antigen- binding fragment to carbohydrate moieties in order to evade detection of the antibody antigen-binding fragment by the immune system of the patient receiving treatment. This process of hyperglycosylation reduces the immunogenicity of therapeutic proteins by sterically inhibiting the interaction of the protein with B cell receptors in circulation.
  • anti-CD16 antibodies, antigen-binding fragments, or binding proteins can be conjugated to molecules that prevent clearance from human serum and improve the pharmacokinetic profile of the antibodies, antigen-binding fragments, or binding proteins.
  • Anti-CD16 antibodies, antigen-binding fragments, or binding proteins can be covalently appended directly to a half-life extending or other molecule by chemical conjugation as described.
  • fusion proteins containing anti-CD16 antibodies, antigen-binding fragments, or binding proteins can be expressed recombinantly from a cell (e.g., a eukaryotic cell or prokaryotic cell).
  • linkers that can be used for the Attorney Docket No.: 45817-0156WO1 formation of a conjugate include polypeptide linkers, such as those that contain naturally occurring or non-naturally occurring amino acids.
  • D-amino acids in the linker, as these residues are not present in naturally-occurring proteins and are thus more resistant to degradation by endogenous proteases.
  • Fusion proteins containing polypeptide linkers can be made using chemical synthesis techniques, such as those described herein, or through recombinant expression of a polynucleotide encoding the fusion protein in a cell (e.g., a prokaryotic or eukaryotic cell).
  • Linkers can be prepared using a variety of strategies that are well known in the art, and depending on the reactive components of the linker, can be cleaved by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (Leriche et al., Bioorg. Med. Chem., 20:571-582 (2012)). Nucleic Acids Encoding Anti-CD16 Antibodies or Binding Proteins This section provides exemplary nucleic acids that may be used to encode antibodies or antigen-binding fragments of the disclosure.
  • the nucleic acid molecules of the disclosure may include one or more alterations.
  • nucleotide such as the nucleic acids of the disclosure (e.g., an mRNA or an oligonucleotide)
  • alteration or, as appropriate, “alternative” refer to alteration with respect to A, G, U or C ribonucleotides.
  • the alterations may be various distinct alterations.
  • the nucleic acid is an mRNA
  • the coding region, the flanking regions, and/or the terminal regions may contain one, two, or more (optionally different) nucleoside or nucleotide alterations.
  • an alternative polynucleotide introduced to a cell may exhibit reduced degradation in the cell, as compared to an unaltered polynucleotide.
  • the polynucleotides can include any useful alteration, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g., to a linking phosphate, to a phosphodiester linkage, or to the phosphodiester backbone).
  • Attorney Docket No.: 45817-0156WO1 alterations are present in each of the sugar and the internucleoside linkage.
  • RNAs ribonucleic acids
  • DNAs deoxyribonucleic acids
  • TAAs threose nucleic acids
  • GAAs glycol nucleic acids
  • PNAs peptide nucleic acids
  • LNAs locked nucleic acids
  • the disclosure provides an alternative nucleic acid molecule containing a degradation domain, which is capable of being acted on in a directed manner within a cell.
  • the polynucleotides can optionally include other agents (e.g., RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamers, vectors, etc.).
  • the polynucleotides may include one or more messenger RNAs (mRNAs) having one or more alternative nucleoside or nucleotides (i.e., mRNA molecules).
  • the polynucleotides may include one or more oligonucleotides having one or more alternative nucleoside or nucleotides.
  • a composition of the disclosure includes an mRNA and/or one or more oligonucleotides having one or more alternative nucleoside or nucleotides. Modified nucleic acids According to Aduri et al., (Aduri, R.
  • nucleosides including 1-methyladenosine, 2-methylthio-N6-hydroxynorvalyl carbamoyladenosine, 2-methyladenosine, 2-O-ribosylphosphate adenosine, N6-methyl-N6- threonylcarbamoyladenosine, N6-acetyladenosine, N6-glycinylcarbamoyladenosine, N6-isopentenyladenosine, N6-methyladenosine, N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, N6- Attorney Docket No.: 45817-0156WO1 hydroxynorvalylcarbamoyladenosine
  • Nucleosides containing modified sugars The alternative nucleosides and nucleotides (e.g., building block molecules), which may be incorporated into a polynucleotide (e.g., RNA or mRNA, as described herein), can be altered on the sugar of the ribonucleic acid.
  • a polynucleotide e.g., RNA or mRNA, as described herein
  • the 2′ hydroxyl group (OH) can be modified or replaced with a number of different substituents.
  • substitutions at the 2′-position include, but are not limited to, H, halo, optionally substituted C 1-6 alkyl; optionally substituted C 1-6 alkoxy; optionally substituted C6-10 aryloxy; optionally substituted C3-8 cycloalkyl; optionally substituted C 3-8 cycloalkoxy; optionally substituted C 6-10 aryloxy; optionally substituted C 6-10 aryl- C1-6 alkoxy, optionally substituted C1-12 (heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any described herein); a polyethyleneglycol (PEG), - O(CH2CH2O)nCH2CH2OR, where R is H or optionally substituted alkyl, and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 16,
  • Exemplary, non-limiting alternative nucleotides include replacement of the oxygen in ribose (e.g., with S, Se, or alkylene, such as methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or Attorney Docket No.: 45817-0156WO1 cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone); multicyclic forms (e.g., tricyclo; and “unlocked” forms,
  • nucleoside is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”).
  • nucleotide is defined as a nucleoside including a phosphate group.
  • exemplary non-limiting alterations include an amino group, a thiol group, an alkyl group, a halo group, or any described herein.
  • the alternative nucleotides may by synthesized by any useful method, as described herein (e.g., chemically, enzymatically, or recombinantly to include one or more alternative or alternative nucleosides).
  • a nucleic acid of the disclosure includes one or more 2’-OMe nucleotides, 2’-methoxyethyl nucleotides (2’-MOE nucleotides), 2’-F nucleotide, 2’-NH2 nucleotide, Attorney Docket No.: 45817-0156WO1 2’fluoroarabino nucleotides (FANA nucleotides), locked nucleic acid nucleotides (LNA nucleotides), or 4’-S nucleotides.
  • 2’-OMe nucleotides 2’-methoxyethyl nucleotides
  • 2’-F nucleotide 2’-NH2 nucleotide
  • Attorney Docket No.: 45817-0156WO1 2’fluoroarabino nucleotides FANA nucleotides
  • LNA nucleotides locked nucleic acid nucleotides
  • 4’-S nucleotides 4’-S nu
  • the alternative nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, and guanosine-cytosine base pairs, but also base pairs formed between nucleotides and/or alternative nucleotides including non- standard or alternative bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures.
  • non-standard base pairing is the base pairing between the alternative nucleotide inosine and adenine, cytosine, or uracil.
  • the alternative nucleosides and nucleotides can include an alternative nucleobase.
  • nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil.
  • nucleobase found in DNA include, but are not limited to, adenine, guanine, cytosine, and thymine. These nucleobases can be altered or wholly replaced to provide polynucleotide molecules having enhanced properties (e.g., resistance to nucleases and stability), and these properties may manifest through disruption of the binding of a major groove binding partner.
  • the alternative nucleobase is an alternative uracil.
  • nucleobases and nucleosides having an alternative uracil include pseudouridine ( ⁇ ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio- 5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio- pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridineor 5-bromo-uridine), 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-oxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5- carboxyhydroxymethyl-uridine (chm 5 U
  • the nucleic acid is modified to contain 1- methylpseudouridine (m 1 ⁇ ) in lieu of uridine at each instance.
  • the alternative nucleobase is an alternative cytosine.
  • Exemplary nucleobases and nucleosides having an alternative cytosine include 5-aza- cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m 3 C), N4-acetyl- cytidine (ac 4 C), 5-formyl-cytidine (f 5 C), N4-methyl-cytidine (m 4 C), 5-methyl- Attorney Docket No.: 45817-0156WO1 cytidine (m 5 C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm 5 C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine,
  • the alternative nucleobase is an alternative adenine.
  • Exemplary nucleobases and nucleosides having an alternative adenine include 2- amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro- purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido- adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7- deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6- diaminopurine, 1-methyl-adenosine (m 1 A), 2-methyl-adenine (m 2 A), N6-methyl- adenosine (m 6 A),
  • the alternative nucleobase is an alternative guanine.
  • Exemplary nucleobases and nucleosides having an alternative guanine include inosine (I), 1-methyl-inosine (m 1 I), wyosine (imG), methylwyosine (mimG), 4-demethyl- wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o 2 yW), hydroxywybutosine (OhyW), undermodified hydroxywybutosine (OhyW*), 7-deaza- guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7- deaza-guanosine (
  • the nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine, or pyrimidine analog.
  • the nucleobase can each be independently selected from adenine, cytosine, guanine, uracil, or hypoxanthine.
  • the nucleobase can also include, for example, naturally-occurring and synthetic derivatives of a base, including pyrazolo[3,4-d]pyrimidines, 5- methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl, and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5- Attorney Docket No.: 45817-0156WO1 uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8- thioalkyl,
  • each letter refers to the representative base and/or derivatives thereof (e.g., A includes adenine or adenine analogs (e.g., 7-deaza adenine)).
  • the polynucleotides of the disclosure contain 5- methoxy-uracil, uracil, 5-methyl-cytosine, and cytosine as the only uracils and cytosines.
  • the polynucleotides of the disclosure contain 5- methoxy-uracil, uracil, 5-trifluoromethyl-cytosine, and cytosine as the only uracils and cytosines.
  • the polynucleotides of the disclosure contain 5- methoxy-uracil, uracil, 5-hydroxymethyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uracil, uracil, 5-bromo-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uracil, uracil, 5-iodo-cytosine, and cytosine as the only uracils and cytosines.
  • the polynucleotides of the disclosure contain 5-methoxy- uracil, uracil, 5-methoxy-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-ethyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-phenyl-cytosine, and cytosine as the only uracils and cytosines.
  • the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-ethnyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, N4-methyl-cytosine, and cytosine as the only uracils and cytosines. In some Attorney Docket No.: 45817-0156WO1 embodiments, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-fluoro-cytosine, and cytosine as the only uracils and cytosines.
  • the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, N4-acetyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, pseudoisocytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-formyl-cytosine, and cytosine as the only uracils and cytosines.
  • the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-aminoallyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-carboxy-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl- pseudouracil, uracil, 5-methyl-cytosine, and cytosine as the only uracils and cytosines.
  • the polynucleotides of the disclosure contain 1-methyl- pseudouracil, uracil, 5-trifluoromethyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 1- methyl-pseudouracil, uracil, 5-hydroxymethyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-bromo-cytosine, and cytosine as the only uracils and cytosines.
  • the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-iodo-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-methoxy-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-ethyl-cytosine, and cytosine as the only uracils and cytosines.
  • the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-phenyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-ethnyl-cytosine, and cytosine as the only uracils and cytosines.
  • the polynucleotides of the disclosure Attorney Docket No.: 45817-0156WO1 contain 1-methyl-pseudouracil, uracil, N4-methyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-fluoro-cytosine, and cytosine as the only uracils and cytosines.
  • the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, N4-acetyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, pseudoisocytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-formyl-cytosine, and cytosine as the only uracils and cytosines.
  • the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-aminoallyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-carboxy-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, 5-methyl-cytidine, and cytidine as the only uridines and cytidines.
  • the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, 5-trifluoromethyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, 5-hydroxymethyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, 5-bromo-cytidine, and cytidine as the only uridines and cytidines.
  • the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, 5-iodo-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, 5-methoxy-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, 5-ethyl-cytidine, and cytidine as the only uridines and cytidines.
  • the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, 5-phenyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 5- Attorney Docket No.: 45817-0156WO1 methoxy-uridine, uridine, 5-ethnyl-cytidine, and cytidine as the only uridines and cytidines.
  • the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, N4-methyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, 5-fluoro-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, N4-acetyl-cytidine, and cytidine as the only uridines and cytidines.
  • the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, pseudoisocytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, 5-formyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, 5-aminoallyl-cytidine, and cytidine as the only uridines and cytidines.
  • the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, 5-carboxy-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl- pseudouridine, uridine, 5-methyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 1- methyl-pseudouridine, uridine, 5-trifluoromethyl-cytidine, and cytidine as the only uridines and cytidines.
  • the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-hydroxymethyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-bromo-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-iodo-cytidine, and cytidine as the only uridines and cytidines.
  • the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-methoxy-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-ethyl-cytidine, and cytidine as the only uridines and cytidines.
  • the polynucleotides of the Attorney Docket No.: 45817-0156WO1 disclosure contain 1-methyl-pseudouridine, uridine, 5-phenyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-ethnyl-cytidine, and cytidine as the only uridines and cytidines.
  • the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, N4-methyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-fluoro-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, N4-acetyl-cytidine, and cytidine as the only uridines and cytidines.
  • the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, pseudoisocytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-formyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-aminoallyl-cytidine, and cytidine as the only uridines and cytidines.
  • the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-carboxy-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain the uracil of one of the nucleosides of Table 2 and uracil as the only uracils. In other embodiments, the polynucleotides of the disclosure contain a uridine of Table 2 and uridine as the only uridines.
  • the polynucleotides of the disclosure contain the cytosine of one of the
  • the polynucleotides of the disclosure contain a cytidine of Table 3 and cytidine as the only cytidines.
  • Attorney Docket No.: 45817-0156WO1 Nucleoside Name 2'-Deoxy-2'-b-mercaptocytidine ' ' Alterations on the internucleoside linkage
  • the alternative nucleotides, which may be incorporated into a polynucleotide molecule can be altered on the internucleoside linkage (e.g., phosphate backbone).
  • Backbone phosphate groups can be altered by replacing one or more of the oxygen atoms with a different substituent.
  • the alternative nucleosides and nucleotides can include the wholesale replacement of an unaltered phosphate moiety with another internucleoside linkage as described herein.
  • Examples of alternative phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters.
  • Phosphorodithioates have both non-linking oxygens replaced by sulfur.
  • the phosphate linker can also be altered by the replacement of a linking oxygen with Attorney Docket No.: 45817-0156WO1 nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).
  • the alternative nucleosides and nucleotides can include the replacement of one or more of the non-bridging oxygens with a borane moiety (BH 3 ), sulfur (thio), methyl, ethyl and/or methoxy.
  • two non-bridging oxygens at the same position can be replaced with a sulfur (thio) and a methoxy.
  • the replacement of one or more of the oxygen atoms at the ⁇ position of the phosphate moiety is provided to confer stability (such as against exonucleases and endonucleases) to RNA and DNA through the unnatural phosphorothioate backbone linkages.
  • Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment.
  • an alternative nucleoside includes an alpha-thio- nucleoside (e.g., 5′-O-(1-thiophosphate)-adenosine, 5′-O-(1-thiophosphate)-cytidine ( ⁇ -thio-cytidine), 5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine, or 5′-O-(1-thiophosphate)-pseudouridine).
  • alpha-thio- nucleoside e.g., 5′-O-(1-thiophosphate)-adenosine, 5′-O-(1-thiophosphate)-cytidine ( ⁇ -thio-cytidine), 5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine, or 5′-O-(1-thiophosphate)-pseu
  • internucleoside linkages that may be employed according to the present disclosure, including internucleoside linkages which do not contain a phosphorous atom, are described herein below.
  • Combinations of alternative sugars, nucleobases, and internucleoside linkages The polynucleotides of the disclosure can include a combination of alterations to the sugar, the nucleobase, and/or the internucleoside linkage. These combinations can include any one or more alterations described herein.
  • Attorney Docket No.: 45817-0156WO1 Synthesis of polynucleotides
  • the polynucleotide molecules for use in accordance with the disclosure may be prepared according to any useful technique, as described herein.
  • nucleosides and nucleotides used in the synthesis of polynucleotide molecules disclosed herein can be prepared from readily available starting materials using the following general methods and procedures. Where typical or preferred process conditions (e.g., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are provided, a skilled artisan would be able to optimize and develop additional process conditions. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures. The processes described herein can be monitored according to any suitable method known in the art.
  • product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography (e.g., high performance liquid chromatography (HPLC) or thin layer chromatography).
  • spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography (e.g., high performance liquid chromatography (HPLC) or thin layer chromatography).
  • spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography (e.g., high performance
  • Suitable solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out (i.e., temperatures which can range from the solvent’s freezing temperature to the solvent’s boiling temperature).
  • a given reaction can be carried out Attorney Docket No.: 45817-0156WO1 in one solvent or a mixture of more than one solvent.
  • suitable solvents for a particular reaction step can be selected.
  • Resolution of racemic mixtures of alternative polynucleotides or nucleic acids can be carried out by any of numerous methods known in the art.
  • An example method includes fractional recrystallization using a “chiral resolving acid” which is an optically active, salt-forming organic acid.
  • Suitable resolving agents for fractional recrystallization methods are, for example, optically active acids, such as the D and L forms of tartaric acid, acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or the various optically active camphorsulfonic acids.
  • Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine).
  • an optically active resolving agent e.g., dinitrobenzoylphenylglycine
  • Suitable elution solvent composition can be determined by one skilled in the art.
  • Alternative nucleosides and nucleotides e.g., building block molecules
  • nucleosides and nucleotides can be prepared according to the synthetic methods described in Ogata et al., J. Org. Chem.74:2585-2588 (2009); Purmal et al., Nucl.
  • polynucleotide includes one or more alternative nucleosides or nucleotides, the polynucleotides of the disclosure may or may not be uniformly altered along the entire length of the molecule.
  • nucleotide e.g., purine or pyrimidine, or any one or more or all of A, G, U, C
  • nucleotide may or may not be uniformly altered in a polynucleotide of the disclosure, or in a given predetermined sequence region thereof.
  • all nucleotides X in a polynucleotide of the disclosure (or in a given sequence region thereof) are altered, wherein X may any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.
  • nucleotide analogs or other alteration(s) may be located at any position(s) of a polynucleotide such that the function of the polynucleotide is not substantially decreased.
  • An alteration may also be a 5′ or 3′ terminal alteration.
  • the polynucleotide may contain from 1% to 100% alternative nucleosides, nucleotides, or internucleoside linkages (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e.
  • any one or more of A, G, U or C) or any intervening percentage e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 90% to 100%, and from 95% to 100.
  • any intervening percentage e.g.,
  • the remaining percentage is accounted for by the presence of A, G, U, or C.
  • the remaining percentage necessary to total 100% is accounted for by the corresponding natural nucleoside, nucleotide, or internucleoside linkage.
  • the remaining percentage necessary to total 100% is accounted for by a second alternative nucleoside, nucleotide, or internucleoside linkage.
  • compositions including one or more mRNAs, where each mRNA encodes a polypeptide (e.g., an anti-CD16 antibody or antigen- Attorney Docket No.: 45817-0156WO1 binding fragment described herein).
  • exemplary mRNAs of the disclosure each include (i) a 5’-cap structure; (ii) a 5’-UTR; (iii) an open reading frame encoding the polypeptide; (iv) a 3’-untranslated region (3’-UTR); and (v) a poly-A region.
  • the mRNA includes from about 30 to about 3,000 (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500
  • mRNA: 5’-cap The 5′-cap structure of an mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species.
  • CBP mRNA Cap Binding Protein
  • the cap further assists the removal of 5′ proximal introns removal during mRNA splicing.
  • Endogenous mRNA molecules may be 5′-end capped generating a 5′-ppp-5′- triphosphate linkage between a terminal guanosine cap residue and the 5′-terminal transcribed sense nucleotide of the mRNA.
  • This 5′-guanylate cap may then be methylated to generate an N7-methyl-guanylate residue.
  • Attorney Docket No.: 45817-0156WO1 Alterations to the nucleic acids of the present disclosure may generate a non- hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life.
  • nucleotides may be used during the capping reaction.
  • a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) may be used with ⁇ -thio-guanosine nucleotides according to the manufacturer’s instructions to create a phosphorothioate linkage in the 5′-ppp-5′ cap.
  • Additional alternative guanosine nucleotides may be used such as ⁇ -methyl-phosphonate and seleno-phosphate nucleotides.
  • Additional alterations include, but are not limited to, 2′-O-methylation of the ribose sugars of 5′-terminal and/or 5′-anteterminal nucleotides of the mRNA (as mentioned above) on the 2′-hydroxyl group of the sugar ring.
  • Multiple distinct 5′-cap structures can be used to generate the 5′-cap of a nucleic acid molecule, such as an mRNA molecule.
  • 5’-cap structures include those described in International Patent Publication Nos. WO2008/127688, WO2008/016473, and WO2011/015347, each of which is incorporated herein by reference in its entirety.
  • Cap analogs which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e., endogenous, wild-type or physiological) 5′-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e., non- enzymatically) or enzymatically synthesized and/linked to a nucleic acid molecule.
  • the Anti-Reverse Cap Analog (ARCA) cap contains two guanosines linked by a 5′-5′-triphosphate group, wherein one guanosine contains an N7 methyl group as well as a 3′-O-methyl group (i.e., N7,3′-O-dimethyl-guanosine-5′- triphosphate-5′-guanosine (m 7 G-3′mppp-G; which may equivalently be designated 3′ O-Me-m7G(5')ppp(5')G)).
  • N7,3′-O-dimethyl-guanosine-5′- triphosphate-5′-guanosine m 7 G-3′mppp-G; which may equivalently be designated 3′ O-Me-m7G(5')ppp(5')G
  • the 3′-O atom of the other, unaltered, guanosine becomes linked to the 5′-terminal nucleotide of the capped nucleic acid molecule (e.g., an Attorney Docket No.: 45817-0156WO1 mRNA or mmRNA).
  • the N7- and 3′-O-methlyated guanosine provides the terminal moiety of the capped nucleic acid molecule (e.g., mRNA or mmRNA).
  • mCAP is similar to ARCA but has a 2′-O- methyl group on guanosine (i.e., N7,2′-O-dimethyl-guanosine-5′-triphosphate-5′- guanosine, m 7 Gm-ppp-G).
  • the cap is a dinucleotide cap analog.
  • the dinucleotide cap analog may be modified at different phosphate positions with a boranophosphate group or a phophoroselenoate group such as the dinucleotide cap analogs described in US Patent No. US 8,519,110, the contents of which are herein incorporated by reference in its entirety.
  • the cap analog is a N7-(4-chlorophenoxyethyl) substituted dicnucleotide form of a cap analog known in the art and/or described herein.
  • Non-limiting examples of a N7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analog include a N7-(4-chlorophenoxyethyl)- G(5’)ppp(5’)G and a N7-(4-chlorophenoxyethyl)-m 3’-O G(5’)ppp(5’)G cap analog (see, e.g., the various cap analogs and the methods of synthesizing cap analogs described in Kore et al.
  • a cap analog of the present disclosure is a 4-chloro/bromophenoxyethyl analog. While cap analogs allow for the concomitant capping of a nucleic acid molecule in an in vitro transcription reaction, up to 20% of transcripts remain uncapped. This, as well as the structural differences of a cap analog from endogenous 5′-cap structures of nucleic acids produced by the endogenous, cellular transcription machinery, may lead to reduced translational competency and reduced cellular stability.
  • Nucleic acids of the disclosure may also be capped post-transcriptionally, using enzymes.5’ cap structures produced by enzymatic capping may enhance binding of cap binding proteins, increase half-life, Attorney Docket No.: 45817-0156WO1 reduce susceptibility to 5′ endonucleases and/or reduce 5′ decapping, as compared to synthetic 5′-cap structures known in the art (or to a wild-type, natural or physiological 5′-cap structure).
  • recombinant Vaccinia Virus Capping Enzyme and recombinant 2′-O-methyltransferase enzyme can create a canonical 5′-5′-triphosphate linkage between the 5′-terminal nucleotide of an mRNA and a guanosine cap nucleotide wherein the cap guanosine contains an N7 methylation and the 5′-terminal nucleotide of the mRNA contains a 2′-O-methyl.
  • Cap1 structure is termed the Cap1 structure.
  • Cap structures include 7mG(5')ppp(5')N,pN2p (cap 0), 7mG(5')ppp(5')NlmpNp (cap 1), 7mG(5')- ppp(5')NlmpN2mp (cap 2), and m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up (cap 4).
  • 5′ terminal caps may include endogenous caps or cap analogs.
  • a 5′ terminal cap may include a guanosine analog.
  • Useful guanosine analogs include inosine, N1-methyl- guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino- guanosine, LNA-guanosine, and 2-azido-guanosine.
  • the nucleic acids described herein may contain a modified 5’-cap. A modification on the 5’-cap may increase the stability of mRNA, increase the half-life of the mRNA, and could increase the mRNA translational efficiency.
  • the modified 5’-cap may include, but is not limited to, one or more of the following modifications: modification at the 2’ and/or 3’ position of a capped guanosine triphosphate (GTP), a replacement of the sugar ring oxygen (that produced the carbocyclic ring) with a methylene moiety (CH2), a modification at the triphosphate bridge moiety of the cap structure, or a modification at the nucleobase (G) moiety.
  • GTP capped guanosine triphosphate
  • CH2 methylene moiety
  • G nucleobase
  • protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of this present disclosure.
  • any protein fragment of a reference protein meaning a polypeptide sequence at least one amino acid residue shorter than a reference polypeptide sequence but otherwise identical
  • 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or greater than 100 amino acids in length 10.
  • any protein that includes a stretch of about 20, about 30, about 40, about 50, or about 100 amino acids which are about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% identical to any of the sequences described herein can be utilized in accordance with the present disclosure.
  • a protein sequence to be utilized in accordance with the present disclosure includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided or referenced herein.
  • mRNA Poly-A tail
  • poly(A) tail a long chain of adenosine nucleotides
  • poly(A) polymerase adds a chain of adenosine nucleotides to the RNA.
  • polyadenylation adds a poly-A tail that is between 100 and 250 residues long (SEQ ID NO: 38).
  • Methods for the stabilization of RNA by incorporation of chain-terminating nucleosides at the 3’-terminus include those described in International Patent Publication No. WO2013/103659, incorporated herein in its entirety.
  • Poly(A) tail deadenylation by 3′ exonucleases is a key step in cellular mRNA degradation in eukaryotes. By blocking 3' exonucleases, the functional half-life of mRNA can be increased, resulting in increased protein expression.
  • the poly(A)tail of the mRNA includes a 3’ LA10 or IdT modification.
  • LA10 or IdT modification for example, as described in International Patent Publication No. WO2017/049275, the tail modifications of which are incorporated by reference in their entirety.
  • Additional strategies have been explored to further stabilize mRNA, including: chemical modification of the 3’ nucleotide (e.g., conjugation of a morpholino to the 3’ end of the poly(A)tail); incorporation of stabilizing sequences after the poly(A) tail (e.g., a co-polymer, a stem-loop, or a triple helix); and/or annealing of structured oligos to the 3' end of an mRNA, as described, for example, in International Patent Publication No. WO2017/049286, the stabilized linkages of which are incorporated by reference in their entirety.
  • chemical modification of the 3’ nucleotide e.g., conjugation of a morpholino to the 3’ end of the poly(A)tail
  • stabilizing sequences after the poly(A) tail e.g., a co-polymer, a stem-loop, or a triple helix
  • annealing of structured oligos to the 3' end
  • Annealing an oligonucleotide (e.g., an oligonucleotide conjugate) with a complex secondary structure (e.g., a triple-helix structure or a stem-loop structure) at the 3’end may provide nuclease resistance and increase half-life of mRNA.
  • Unique poly(A) tail lengths may provide certain advantages to the RNAs of the present disclosure. Generally, the length of a poly(A) tail of the present disclosure is greater than 30 nucleotides in length (SEQ ID NO: 39). In some embodiments, the poly(A) tail is greater than 35 nucleotides in length. In some embodiments, the length is at least 40 nucleotides.
  • the length is at least 45 nucleotides. In some embodiments, the length is at least 50 nucleotides. In some embodiments, the length is at least 55 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 65 nucleotides. In another embodiment, the length is at least 70 nucleotides. In some embodiments, the length is at least 80 nucleotides. In some embodiments, the length is at least 90 nucleotides. In some embodiments, the length is at least 100 nucleotides. In some embodiments, the length is at least 120 nucleotides. In some embodiments, the length is at least 140 nucleotides.
  • the length is at least 160 nucleotides. In some embodiments, the length is at least 180 nucleotides. In some embodiments, the length Attorney Docket No.: 45817-0156WO1 is at least 200 nucleotides. In some embodiments, the length is at least 250 nucleotides. In some embodiments, the length is at least 300 nucleotides. In some embodiments, the length is at least 350 nucleotides. In some embodiments, the length is at least 400 nucleotides. In some embodiments, the length is at least 450 nucleotides. In some embodiments, the length is at least 500 nucleotides. In some embodiments, the length is at least 600 nucleotides.
  • the length is at least 700 nucleotides. In some embodiments, the length is at least 800 nucleotides. In some embodiments, the length is at least 900 nucleotides. In some embodiments, the length is at least 1000 nucleotides. In some embodiments, the length is at least 1100 nucleotides. In some embodiments, the length is at least 1200 nucleotides. In some embodiments, the length is at least 1300 nucleotides. In some embodiments, the length is at least 1400 nucleotides. In some embodiments, the length is at least 1500 nucleotides. In some embodiments, the length is at least 1600 nucleotides.
  • the length is at least 1700 nucleotides. In some embodiments, the length is at least 1800 nucleotides. In some embodiments, the length is at least 1900 nucleotides. In some embodiments, the length is at least 2000 nucleotides. In some embodiments, the length is at least 2500 nucleotides. In some embodiments, the length is at least 3000 nucleotides.
  • the poly(A) tail may be 80 nucleotides, 120 nucleotides, or 160 nucleotides in length. In some embodiments, the poly(A) tail may be 20, 40, 80, 100, 120, 140 or 160 nucleotides in length.
  • the poly(A) tail is designed relative to the length of the mRNA. This design may be based on the length of the coding region of the mRNA, the length of a particular feature or region of the mRNA, or based on the length of the ultimate product expressed from the RNA. When relative to any additional feature of the RNA (e.g., other than the mRNA portion which includes the poly(A) tail), poly(A) tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% greater in length than the additional feature.
  • the poly(A) tail may also be designed as a fraction of the mRNA to which it belongs.
  • the poly(A) tail may be 10, 20, 30, 40, 50, 60, 70, Attorney Docket No.: 45817-0156WO1 80, or 90% or more of the total length of the construct or the total length of the construct minus the poly(A) tail.
  • engineered binding sites and/or the conjugation of nucleic acids or mRNA for poly(A) binding protein may be used to enhance expression.
  • the engineered binding sites may be sensor sequences which can operate as binding sites for ligands of the local microenvironment of the nucleic acids and/or mRNA.
  • the nucleic acids and/or mRNA may include at least one engineered binding site to alter the binding affinity of poly(A) binding protein (PABP) and analogs thereof.
  • PABP poly(A) binding protein
  • the incorporation of at least one engineered binding site may increase the binding affinity of the PABP and analogs thereof.
  • multiple distinct nucleic acids or mRNA may be linked together to the PABP (poly(A) binding protein) through the 3′-end using nucleotides at the 3′- terminus of the poly(A) tail. Transfection experiments can be conducted in relevant cell lines and protein production can be assayed by ELISA at 12hr, 24hr, 48hr, 72hr, and day 7 post-transfection.
  • the transfection experiments may be used to evaluate the effect on PABP or analogs thereof binding affinity as a result of the addition of at least one engineered binding site.
  • a poly(A) tail may be used to modulate translation initiation. While not wishing to be bound by theory, the poly-A tail recruits PABP which in turn can interact with translation initiation complex and thus may be essential for protein synthesis.
  • a poly(A) tail may also be used in the present disclosure to protect against 3’-5’ exonuclease digestion.
  • the nucleic acids or mRNA of the present disclosure are designed to include a poly-A-G quartet.
  • the G-quartet is a cyclic hydrogen bonded array of four guanosine nucleotides that can be formed by G-rich sequences in both DNA and RNA.
  • the G-quartet is incorporated at the end of the poly-A tail.
  • the resultant nucleic acid or mRNA may be assayed for stability, Attorney Docket No.: 45817-0156WO1 protein production and other parameters including half-life at various time points. It has been discovered that the poly-A-G quartet results in protein production equivalent to at least 75% of that seen using a poly-A tail of 120 nucleotides alone (SEQ ID NO: 40).
  • the nucleic acids or mRNA of the present disclosure may include a poly(A) tail and may be stabilized by the addition of a chain terminating nucleoside.
  • the nucleic acids and/or mRNA with a poly(A) tail may further include a 5’cap structure.
  • the nucleic acids or mRNA of the present disclosure may include a poly-A-G quartet.
  • the nucleic acids and/or mRNA with a poly-A-G quartet may further include a 5’cap structure.
  • the chain terminating nucleoside which may be used to stabilize the nucleic acid or mRNA including a poly(A) tail or poly-A-G quartet may be, but is not limited to, those described in International Patent Publication No. WO2013103659, incorporated herein by reference in its entirety.
  • the chain terminating nucleosides which may be used with the present disclosure includes, but is not limited to, 3'-deoxyadenosine (cordycepin), 3'- deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymine, 2',3'- dideoxynucleosides, such as 2',3'- dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'- dideoxycytosine, 2',3'- dideoxyguanosine, 2',3'-dideoxythymine, a 2'- deoxynucleoside, or a -O- methylnucleoside.
  • 3'-deoxyadenosine cordycepin
  • 3'- deoxyuridine 3'-deoxycytosine
  • 3'-deoxyguanosine 3'-deoxythymine
  • the mRNA which includes a poly(A) tail or a poly-A-G quartet may be stabilized by an alteration to the 3’region of the nucleic acid that can prevent and/or inhibit the addition of oligo(U) (see, e.g., International Patent Publication No. WO2013/103659, incorporated herein by reference in its entirety).
  • the mRNA which includes a poly(A) tail or a poly-A-G quartet, may be stabilized by the addition of an oligonucleotide that terminates in a 3’-deoxynucleoside, 2’,3’-dideoxynucleoside 3'-O-methylnucleosides, Attorney Docket No.: 45817-0156WO1 3'-O-ethylnucleosides, 3'-arabinosides, and other alternative nucleosides known in the art and/or described herein.
  • the nucleic acids of the present disclosure may include a stem-loop such as, but not limited to, a histone stem-loop.
  • the stem-loop may be a nucleotide sequence that is about 25 or about 26 nucleotides in length such as, but not limited to, SEQ ID NOs: 7-17 as described in International Patent Publication No. WO2013/103659, incorporated herein by reference in its entirety.
  • the histone stem-loop may be located 3’ relative to the coding region (e.g., at the 3’ terminus of the coding region).
  • the stem-loop may be located at the 3’ end of a nucleic acid described herein.
  • the stem-loop may be located in the second terminal region.
  • the stem-loop may be located within an untranslated region (e.g., 3’-UTR) in the second terminal region.
  • the nucleic acid such as, but not limited to mRNA, which includes the histone stem-loop may be stabilized by the addition of at least one chain terminating nucleoside.
  • the addition of at least one chain terminating nucleoside may slow the degradation of a nucleic acid and thus can increase the half-life of the nucleic acid.
  • the chain terminating nucleoside may be, but is not limited to, those described in International Patent Publication No. WO2013/103659, incorporated herein by reference in its entirety.
  • the chain terminating nucleosides which may be used with the present disclosure includes, but is not limited to, 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymine, 2',3'-dideoxynucleosides, such as 2',3'- dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'- Attorney Docket No.: 45817-0156WO1 dideoxyguanosine, 2',3'-dideoxythymine, a 2'-deoxynucleoside, or a -O- methylnucleo
  • the nucleic acid such as, but not limited to mRNA, which includes the histone stem-loop may be stabilized by an alteration to the 3’region of the nucleic acid that can prevent and/or inhibit the addition of oligo(U) (see, e.g., International Patent Publication No. WO2013/103659, incorporated herein by reference in its entirety).
  • the nucleic acid such as, but not limited to, mRNA, which includes the histone stem-loop may be stabilized by the addition of an oligonucleotide that terminates in a 3’-deoxynucleoside, 2’,3’-dideoxynucleoside 3'- O-methylnucleosides, 3'-O-ethylnucleosides, 3'-arabinosides, and other alternative nucleosides known in the art and/or described herein.
  • the nucleic acids of the present disclosure may include a histone stem-loop, a poly(A) tail sequence, and/or a 5’-cap structure.
  • the histone stem-loop may be before and/or after the poly-A tail sequence.
  • the nucleic acids including the histone stem-loop and a poly(A) tail sequence may include a chain terminating nucleoside described herein.
  • the nucleic acids of the present disclosure may include a histone stem-loop and a 5’-cap structure.
  • the 5’-cap structure may include, but is not limited to, those described herein and/or known in the art.
  • the nucleic acids described herein may include at least one histone stem-loop and a poly(A) sequence or polyadenylation signal.
  • Non- limiting examples of nucleic acid sequences encoding for at least one histone stem- loop and a poly-A sequence or a polyadenylation signal are described in International Patent Publication Nos. WO2013/120497, WO2013/120629, WO2013/120500, WO2013/120627, WO2013/120498, WO2013/120626, WO2013/120499 and WO2013/120628, the contents of each of which are incorporated herein by reference in their entirety.
  • the nucleic acid encoding for a histone stem- Attorney Docket No.: 45817-0156WO1 loop and a poly(A) sequence or a polyadenylation signal may code for a pathogen antigen or fragment thereof such as the nucleic acid sequences described in International Patent Publication Nos. WO2013/120499 and WO2013/120628, the contents of both of which are incorporated herein by reference in their entirety.
  • the nucleic acid encoding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal may code for a therapeutic protein such as the nucleic acid sequences described in International Patent Publication Nos.
  • the nucleic acid encoding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal may code for a tumor antigen or fragment thereof such as the nucleic acid sequences described in International Patent Publication Nos. WO2013/120500 and WO2013/120627, the contents of both of which are incorporated herein by reference in their entirety.
  • nucleic acid encoding for a histone stem- loop and a poly(A) sequence or a polyadenylation signal may code for an autoimmune self-antigen such as the nucleic acid sequences described in International Patent Publication Nos. WO2013/120498 and WO2013/120626, the contents of both of which are incorporated herein by reference in their entirety.
  • mRNA Triple helices
  • nucleic acids of the present disclosure may include a triple helix on the 3’ end of the nucleic acid.
  • the 3’ end of the nucleic acids of the present disclosure may include a triple helix alone or in combination with a poly(A) tail.
  • the nucleic acid of the present disclosure may include at least a first and a second U-rich region, a conserved stem-loop region between the first and second region and an A-rich region.
  • the first and second U-rich region and the A-rich region may associate to form a triple helix on the 3’ end of the nucleic acid. This triple helix may stabilize the nucleic acid, enhance the translational efficiency of the nucleic acid and/or protect the 3’ end from degradation.
  • Triple helices include, but Attorney Docket No.: 45817-0156WO1 are not limited to, the triple helix sequence of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), MEN- ⁇ and polyadenylated nuclear (PAN) RNA (see, Wilusz et al., Genes & Development 26:2392-2407 (2012); herein incorporated by reference in its entirety).
  • the triple helix may be formed from the cleavage of a MALAT1 sequence prior to the cloverleaf structure.
  • MALAT1 is a long non-coding RNA which, when cleaved, forms a triple helix and a tRNA-like cloverleaf structure.
  • the MALAT1 transcript then localizes to nuclear speckles and the tRNA-like cloverleaf localizes to the cytoplasm (Wilusz et al., Cell.135(5): 919-932 (2008); incorporated herein by reference in its entirety).
  • the terminal end of the nucleic acid of the present disclosure including the MALAT1 sequence can then form a triple helix structure, after RNaseP cleavage from the cloverleaf structure, which stabilizes the nucleic acid (Peart et al., WIREs RNA.4(5):491-506 (2013); incorporated herein by reference in its entirety).
  • the nucleic acids or mRNA described herein include a MALAT1 sequence.
  • the nucleic acids or mRNA may be polyadenylated.
  • the nucleic acids or mRNA is not polyadenylated but has an increased resistance to degradation compared to unaltered nucleic acids or mRNA.
  • the nucleic acids of the present disclosure may include a MALAT1 sequence in the second flanking region (e.g., the 3’-UTR).
  • the MALAT1 sequence may be human or mouse.
  • mRNA Translation Enhancer Elements
  • TEEs translation Enhancer Elements
  • the 5’-UTR of the mRNA includes at least one TEE.
  • the TEE may be located between the transcription promoter and the start codon.
  • the mRNA with at least one TEE in the 5’-UTR may include a cap at the 5’-UTR. Further, at least one TEE may be located in the 5’-UTR of mRNA undergoing cap- dependent or cap-independent translation.
  • TEEs known may be in the 5′-leader of the Gtx homeodomain protein (Chappell et al., Proc. Natl. Acad. Sci. USA 101:9590-9594 (2004), incorporated herein by reference in their entirety).
  • TEEs are disclosed as SEQ ID NOs: 1-35 in US Patent Publication No. US20090226470, SEQ ID NOs: 1-35 in US Patent Publication No. US20130177581, SEQ ID NOs: 1-35 in International Patent Publication No. WO2009075886, SEQ ID NOs: 1-5, and 7-645 in International Patent Publication No. WO2012009644, SEQ ID NO: 1 in International Patent Publication No.
  • the TEE may be an internal ribosome entry site (IRES), HCV-IRES or an IRES element such as, but not limited to, those described in US Patent No. US7468275, US Patent Publication Nos. US20070048776 and US20110124100 and International Patent Publication Nos. WO2007025008 and WO2001055369, each of which is incorporated herein by reference in its entirety.
  • IRES internal ribosome entry site
  • HCV-IRES HCV-IRES
  • IRES element such as, but not limited to, those described in US Patent No. US7468275, US Patent Publication Nos. US20070048776 and US20110124100 and International Patent Publication Nos. WO2007025008 and WO2001055369, each of which is incorporated herein by reference in its entirety.
  • the IRES elements may include, but are not limited to, the Gtx sequences (e.g., Gtx9-nt, Gtx8-nt, Gtx7-nt) described by Chappell et al. (Proc. Natl. Acad. Sci. USA 101:9590-9594 (2004)) and Zhou et al. (PNAS 102:6273-6278 (2005)) and in US Patent Publication Nos. Attorney Docket No.: 45817-0156WO1 US20070048776 and US20110124100 and International Patent Publication No. WO2007025008, each of which is incorporated herein by reference in its entirety. Additional exemplary TEEs are disclosed in US Patent Nos.
  • the polynucleotides, primary constructs, alternative nucleic acids and/or mRNA may include at least one TEE that is described in International Patent Publication Nos.
  • WO1999024595 WO2012009644, WO2009075886, WO2007025008, WO1999024595, European Patent Publication Nos. EP2610341A1 and EP2610340A1, US Patent Nos. US6310197, US6849405, US7456273, US7183395, US Patent Publication No. US20090226470, US20110124100, US20070048776, US20090093049, and US20130177581 each of which is incorporated herein by reference in its entirety.
  • the TEE may be located in the 5’-UTR of the mRNA.
  • the polynucleotides, primary constructs, alternative nucleic acids and/or mmRNA may include at least one TEE that has 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 95% or at least 99% identity with the TEEs described in US Patent Publication Nos. US20090226470, US20070048776, US20130177581 and US20110124100, International Patent Publication Nos. WO1999024595, WO2012009644, WO2009075886 and WO2007025008, European Patent Publication No. EP2610341A1 and EP2610340A1, and US Patent Nos.
  • TEEs in the translational enhancer polynucleotides can be organized in one or more sequence segments.
  • a sequence segment can harbor one or more of the specific TEEs exemplified herein, with each TEE being present in one or more copies.
  • multiple sequence segments are present in a translational enhancer polynucleotide, they can be homogenous or heterogeneous.
  • the multiple sequence segments in a translational enhancer polynucleotide can harbor identical or different types of the specific TEEs exemplified herein, identical or different number of copies of each of the specific TEEs, and/or identical or different organization of the TEEs within each sequence segment.
  • the 5’-UTR of the mRNA may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55 or more than 60 TEE sequences.
  • the TEE sequences in the 5’-UTR of mRNA of the present disclosure may be the same or different TEE sequences.
  • the TEE sequences may be in a pattern such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times. In these patterns, each letter, A, B, or C represent a different TEE sequence at the nucleotide level.
  • the 5’-UTR may include a spacer to separate two TEE sequences. As a non-limiting example, the spacer may be a 15 nucleotide spacer and/or other spacers known in the art.
  • the 5’-UTR may include a TEE sequence-spacer module repeated at least once, at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times and at least 9 times, or more than 9 times in the 5’-UTR.
  • the TEE in the 5’-UTR of the mRNA of the present disclosure may include at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, Attorney Docket No.: 45817-0156WO1 at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more than 99% of the TEE sequences disclosed in US Patent Publication Nos. US20090226470, US20070048776, US20130177581 and US20110124100, International Patent Publication Nos.
  • the TEE in the 5’-UTR of the mRNA of the present disclosure may include a 5-30 nucleotide fragment, a 5-25 nucleotide fragment, a 5-20 nucleotide fragment, a 5-15 nucleotide fragment, a 5-10 nucleotide fragment of the TEE sequences disclosed in US Patent Publication Nos.
  • the TEE in the 5’-UTR of the mRNA of the present disclosure may include at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, 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 95%, at least 99% or more than 99% of the TEE sequences disclosed in Chappell et al. (Proc. Natl. Acad. Sci. USA 101:9590-9594 (2004)) and Zhou et al.
  • the TEE in the 5’-UTR of the polynucleotides, primary constructs, alternative nucleic acids and/or mmRNA of the present disclosure may include a 5-30 nucleotide fragment, a 5-25 nucleotide fragment, a 5-20 nucleotide fragment, a 5-15 nucleotide fragment, a 5-10 nucleotide fragment of the TEE sequences disclosed in Chappell et al. (Proc. Natl.
  • the TEE used in the 5’-UTR of the mRNA of the present disclosure is an IRES sequence such as, but not limited to, those described in US Patent No. US7468275 and International Patent Publication No. WO2001055369, each of which is incorporated herein by reference in its entirety.
  • the TEEs used in the 5’-UTR of the mRNA of the present disclosure may be identified by the methods described in US Patent Publication Nos. US20070048776 and US20110124100 and International Patent Publication Nos. WO2007025008 and WO2012009644, each of which is incorporated herein by reference in its entirety.
  • the TEEs used in the 5’-UTR of the mRNA of the present disclosure may be a transcription regulatory element described in US Patent Nos. US7456273 and US7183395, US Patent Publication No. US20090093049, and International Publication No. WO2001055371, each of which is incorporated herein by reference in its entirety.
  • the transcription regulatory elements may be identified by methods known in the art, such as, but not limited to, the methods described in US Patent Nos. US7456273 and US7183395, US Patent Publication No. US20090093049, and International Publication No. WO2001055371, each of which is incorporated herein by reference in its entirety.
  • the TEE used in the 5’-UTR of the mRNA of the present disclosure is an oligonucleotide or portion thereof as described in US Patent No. US7456273 and US7183395, US Patent Publication No. US20090093049, and International Publication No. WO2001055371, each of which is incorporated herein by reference in its entirety.
  • the 5’-UTR including at least one TEE described herein may be incorporated in a monocistronic sequence such as, but not limited to, a vector system or a nucleic Attorney Docket No.: 45817-0156WO1 acid vector.
  • a monocistronic sequence such as, but not limited to, a vector system or a nucleic Attorney Docket No.: 45817-0156WO1 acid vector.
  • the vector systems and nucleic acid vectors may include those described in US Patent Nos.7456273 and US7183395, US Patent Publication Nos. US20070048776, US20090093049, and US20110124100 and International Patent Publication Nos. WO2007025008 and WO2001055371, each of which is incorporated herein by reference in its entirety.
  • the TEEs described herein may be located in the 5’- UTR and/or the 3’-UTR of the mRNA.
  • the TEEs located in the 3’-UTR may be the same and/or different than the TEEs located in and/or described for incorporation in the 5’-UTR.
  • the 3’-UTR of the mRNA may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55 or more than 60 TEE sequences.
  • the TEE sequences in the 3’-UTR of the polynucleotides, primary constructs, alternative nucleic acids and/or mmRNA of the present disclosure may be the same or different TEE sequences.
  • the TEE sequences may be in a pattern such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times. In these patterns, each letter, A, B, or C represent a different TEE sequence at the nucleotide level.
  • the 3’-UTR may include a spacer to separate two TEE sequences. As a non-limiting example, the spacer may be a 15-nucleotide spacer and/or other spacers known in the art.
  • the 3’-UTR may include a TEE sequence-spacer module repeated at least once, at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times and at least 9 times or more than 9 times in the 3’-UTR.
  • mRNA: Heterologous 5’-UTRs Attorney Docket No.: 45817-0156WO1 5’-UTRs of an mRNA of the disclosure may be homologous or heterologous to the coding region found in the mRNA. Multiple 5′ UTRs may be included in mRNA and may be the same or of different sequences.
  • any portion of the mRNA, including none, may be codon optimized and any may independently contain one or more different structural or chemical alterations, before and/or after codon optimization.
  • each 5’-UTR (5’-UTR-005 to 5’-UTR 68511) is identified by its start and stop site relative to its native or wild type (homologous) transcript (ENST; the identifier used in the ENSEMBL database).
  • 5’-UTRs which are heterologous to the coding region of the mRNA are engineered into the mRNA.
  • the mRNA e.g., an mRNA in a composition described herein
  • the mRNA is administered to cells, tissue, or organisms, and outcomes such as protein level, localization, and/or half-life are measured to evaluate the beneficial effects the heterologous 5’-UTR may have on mRNA.
  • Variants of the 5’ UTRs may be utilized wherein one or more nucleotides are added or removed to the termini, including A, T, C or G.5’-UTRs may also be codon-optimized or altered in any manner described herein.
  • RNA binding proteins can regulate numerous aspects of co- and post- transcription gene expression, such as, but not limited to, RNA splicing, localization, translation, turnover, polyadenylation, capping, alteration, export, and localization.
  • RNA-binding domains such as, but not limited to, RNA recognition motif (RR) and hnRNP K-homology (KH) domains, typically regulate the sequence association between RBPs and their RNA targets (Ray et al., Nature.499:172-177 Attorney Docket No.: 45817-0156WO1 (2013); incorporated herein by reference in its entirety).
  • the canonical RBDs can bind short RNA sequences. In some embodiments, the canonical RBDs can recognize structure RNAs. In some embodiments, to increase the stability of the mRNA of interest, an mRNA encoding HuR is co-transfected or co-injected along with the mRNA of interest into the cells or into the tissue. These proteins can also be tethered to the mRNA of interest in vitro and then administered to the cells together. Poly A tail binding protein, PABP interacts with eukaryotic translation initiation factor eIF4G to stimulate translational initiation.
  • eIF4G eukaryotic translation initiation factor
  • the nucleic acids and/or mRNA may include at least one RNA-binding motif such as, but not limited to an RNA-binding domain (RBD).
  • RBP RNA-binding domain
  • the RBD may be any of the RBDs, fragments or variants thereof descried by Ray et al., (Nature.499:172-177 (2013); incorporated herein by reference in its entirety).
  • the nucleic acids or mRNA of the present disclosure may include a sequence for at least one RNA-binding domain (RBDs).
  • RBDs RNA-binding domain
  • the RBDs do not need to be from the same species or even the same structural class.
  • at least one flanking region e.g., the 5’-UTR and/or the 3’-UTR
  • the first flanking region and the second flanking region may both include at least one RBD.
  • the RBD may be the same or each of the RBDs may have at least 60% (e.g., at least 70%, 80%, Attorney Docket No.: 45817-0156WO1 or 90%) sequence identity to the other RBD.
  • at least on RBD may be located before, after and/or within the 3’-UTR of the nucleic acid or mRNA of the present disclosure.
  • at least one RBD may be located before or within the first 300 nucleosides of the 3’-UTR.
  • the nucleic acids and/or mRNA of the present disclosure may include at least one RBD in the first region of linked nucleosides.
  • the RBD may be located before, after, or within a coding region (e.g., the ORF).
  • the first region of linked nucleosides and/or at least one flanking region may include at least on RBD.
  • the first region of linked nucleosides may include a RBD related to splicing factors and at least one flanking region may include a RBD for stability and/or translation factors.
  • the nucleic acids and/or mRNA of the present disclosure may include at least one RBD located in a coding and/or non-coding region of the nucleic acids and/or mRNA.
  • at least one RBD may be incorporated into at least one flanking region to increase the stability of the nucleic acid and/or mRNA of the present disclosure.
  • an antisense locked nucleic acid (LNA) oligonucleotides and exon-junction complexes (EJCs) may be used in the RNA binding protein motif.
  • the LNA and EJCs may be used around a start codon (-4 to +37 where the A of the AUG codons is +1) in order to decrease the accessibility to the first start codon (AUG).
  • Nucleic acids as agents for delivering anti-CD16 antibodies or binding proteins The compositions of the disclosure can be administered not only as antibodies or antigen-binding fragments, but also in the form of nucleic acids.
  • the exemplary nucleic acids described herein may be used to deliver antibodies or antigen-binding Attorney Docket No.: 45817-0156WO1 fragments to a subject.
  • nucleic acids may be used as therapeutic agents to express antibodies or antigen-binding fragments of the disclosure as a therapy to treat a target disease.
  • Pharmaceutical Compositions Pharmaceutical compositions containing an anti-CD16 antibody, antigen- binding fragment, binding protein, or nucleic acid encoding the same, described herein can be prepared using methods known in the art. Pharmaceutical compositions described herein may contain an anti-CD16 antibody, antigen-binding fragment, binding protein, or a nucleic acid encoding the same, described herein in combination with one or more pharmaceutically acceptable excipients.
  • compositions described herein can be prepared using physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences (19th ed., 1995), incorporated herein by reference), and in a desired form, e.g., in the form of lyophilized formulations or aqueous solutions.
  • the compositions can also be prepared so as to contain the active agent (e.g., an anti-CD16 antibody, antigen- binding fragment, binding protein, or a nucleic acid encoding the same) at a desired concentration.
  • a pharmaceutical composition described herein may contain at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%) active agent by weight (w/w).
  • an active agent that can be incorporated into a pharmaceutical formulation can itself have a desired level of purity.
  • a polypeptide or nucleic acid described herein may be characterized by a certain degree of purity after isolating the antibody or binding protein from cell culture media or after chemical synthesis.
  • An antibody, antigen-biding fragment, binding protein, or nucleic acid described herein may be at least 10% pure prior to incorporating the antibody, antigen-biding fragment, binding protein or nucleic acid into a pharmaceutical composition (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or 100% pure).
  • compositions can be prepared for storage as lyophilized formulations or aqueous solutions by mixing the active agent having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers typically employed in the art, e.g., buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants, and other miscellaneous additives. See, e.g., Remington's Pharmaceutical Sciences (19th ed., 1995), incorporated herein by reference). Such additives must be nontoxic to the recipients at the dosages and concentrations employed. Buffering agents Buffering agents help to maintain the pH in the range which approximates physiological conditions.
  • Suitable buffering agents for use with the pharmaceutical compositions of the disclosure include both organic and inorganic acids and salts thereof, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g., succinic acid- monosodium succinate mixture, succinic acid- sodium hydroxide mixture, succinic acid- disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid- disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconate buffers (e.g.,
  • phosphate buffers histidine buffers, and trimethylamine salts such as Tris can be used.
  • Attorney Docket No.: 45817-0156WO1 Preservatives Preservatives can be added to a composition described herein, for example, to inhibit microbial growth.
  • Suitable preservatives for use with the pharmaceutical compositions of the disclosure include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalconium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.
  • Isotonifiers also known as “stabilizers,” can be added to ensure isotonicity of liquid compositions described herein and include polhydric sugar alcohols, for example trihydric or higher sugar alcohols, such as glycerin, arabitol, xylitol, sorbitol, and mannitol.
  • Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the therapeutic agent or helps to prevent denaturation or adherence to the container wall.
  • Typical stabilizers can be polyhydric sugar alcohols; amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L- leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a- monothioglycerol and sodium thio sulfate; low molecular weight polypeptide
  • non-ionic surfactants or detergents are added to the pharmaceutical composition, for example, to help Attorney Docket No.: 45817-0156WO1 solubilize the therapeutic agent as well as to protect the therapeutic agent against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stressed without causing denaturation of the protein.
  • Suitable non-ionic surfactants include, for example and without limitation, polysorbates (20, 80, etc.), polyoxamers (184, 188 etc.), Pluronic polyols, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.).
  • Alternative pharmaceutically acceptable carriers that can be incorporated into a pharmaceutical composition described herein may include dextrose, sucrose, sorbitol, mannitol, starch, rubber arable, potassium phosphate, arginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oils, but not limited to.
  • a pharmaceutical composition described herein may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative.
  • LNP Lipid Nanoparticle
  • the present disclosure provides LNP compositions with advantageous properties.
  • the lipid nanoparticle compositions described herein may be used for the delivery of therapeutic and/or prophylactic agents, e.g., mRNAs, to mammalian cells or organs.
  • the lipid nanoparticles described herein have little or no immunogenicity.
  • the lipid compounds disclosed herein have a lower immunogenicity as compared to a reference lipid (e.g., MC3, KC2, or DLinDMA).
  • a formulation comprising a lipid disclosed herein and a therapeutic or prophylactic agent, e.g., mRNA, has an increased therapeutic index as compared to a corresponding formulation which comprises a reference lipid (e.g., MC3, KC2, or DLinDMA) and the same therapeutic or prophylactic agent.
  • a reference lipid e.g., MC3, KC2, or DLinDMA
  • the present application provides pharmaceutical compositions comprising: (a) a delivery agent comprising a lipid nanoparticle; and (b) a polynucleotide encoding an antibody or antigen-binding fragment of the disclosure.
  • polynucleotides of the present disclosure are included in a lipid nanoparticle (LNP).
  • Lipid nanoparticles according to the present disclosure may comprise: (i) an ionizable lipid (e.g., an ionizable amino lipid); (ii) a sterol or other structural lipid; (iii) a non-cationic helper lipid or phospholipid; and (iv) a PEG-modified lipid.
  • lipid nanoparticles according to the present disclosure further comprise one or more polynucleotides of the present disclosure (e.g., mRNA).
  • the lipid nanoparticles according to the present disclosure can be generated using components, compositions, and methods as are generally known in the art, see, for example PCT/US2016/052352; PCT/US2016/068300; PCT/US2017/037551; PCT/US2015/027400; PCT/US2016/047406; PCT/US2016000129; PCT/US2016/014280; PCT/US2016/014280; PCT/US2017/038426; PCT/US2014/027077; PCT/US2014/055394; PCT/US2016/52117; PCT/US2012/069610; PCT/US2017/027492; PCT/US2016/059575 and PCT/US2016/069491 all of which are incorporated by reference herein in their entirety.
  • the lipid nanoparticle comprises: (i) 20 to 60 mol.% ionizable cationic lipid (e.g., ionizable amino lipid), (ii) 25 to 55 mol.% sterol or other structural lipid, (iii) 5 to 25 mol.% non-cationic lipid (e.g., phospholipid), and (iv) 0.5 to 15 mol.% PEG-modified lipid.
  • ionizable cationic lipid e.g., ionizable amino lipid
  • sterol or other structural lipid e.g., sterol or other structural lipid
  • non-cationic lipid e.g., phospholipid
  • iv 0.5 to 15 mol.% PEG-modified lipid.
  • the lipid nanoparticle comprises: (i) 40 to 50 mol.% ionizable cationic lipid (e.g., ionizable amino lipid), (ii) 30 to 45 mol.% sterol or other structural lipid, (iii) 5 to 15 mol.% non-cationic lipid (e.g., phospholipid), and (iv) 1 to 5 mol.% PEG-modified lipid.
  • ionizable cationic lipid e.g., ionizable amino lipid
  • sterol or other structural lipid e.g., sterol or other structural lipid
  • 5 to 15 mol.% non-cationic lipid e.g., phospholipid
  • 1 to 5 mol.% PEG-modified lipid e.g., PEG-modified lipid.
  • the lipid nanoparticle comprises: (i) 45 to 50 mol.% ionizable cationic lipid (e.g., ionizable amino lipid), (ii) 35 to 45 mol.% sterol or other structural lipid, (iii) 8 to 12 mol.% non-cationic lipid (e.g., phospholipid), and (iv) 1.5 to 3.5 mol.% PEG-modified lipid.
  • ionizable cationic lipid e.g., ionizable amino lipid
  • sterol or other structural lipid e.g., 8 to 12 mol.% non-cationic lipid (e.g., phospholipid)
  • iv 1.5 to 3.5 mol.% PEG-modified lipid.
  • the lipid nanoparticle of the present disclosure comprises an ionizable cationic lipid (e.g., an ionizable amino lipid) that is a compound of Formula (I): or its N-oxide, or a salt or isomer thereof, R’ branched denotes a point of attachment; wherein selected from the group consisting of H, C2-12 alkyl, and C2-12 alkenyl; R 2 and R 3 are each independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl; Attorney Docket No.: 45817-0156WO1 R 4 is selected from the group consisting of -(CH2)nOH, wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, , wherein denotes a point R 10 is N each R is independently selected from the group consisting of C 1-6 alkyl, C 2-3 alkenyl, and H; and n2 is selected from the group consisting of 1,
  • R’ a is R’ branched ;
  • R’ branched is denotes a point of attachment;
  • R a ⁇ , R a ⁇ , R a ⁇ , and R a ⁇ are each alkyl;
  • R 4 is -(CH ) OH;
  • n is 2;
  • each R is H;
  • M and M’ are each -C(O)O-;
  • R’ is a C1-12 alkyl; l is 5; and m is 7.
  • R’ a is R’ branched ;
  • R’ branched is denotes a point of attachment;
  • R a ⁇ , R a ⁇ , R a ⁇ , and R a ⁇ are C1-14 alkyl;
  • R 4 is -(CH2)nOH; n is 2;
  • each R 5 is H;
  • each R 6 is H;
  • M and M’ are each -C(O)O-;
  • R’ is a C 1-12 alkyl; l is 3; and m is 7.
  • R’ a is R’ branched ; of attachment; R a ⁇ is C 2-12 alkyl; alkyl; R 4 is ; R 10 is NH(C1-6 alkyl); n2 is 2; R 5 is H; each R 6 is H; M and M’ is a C 1-12 alkyl; l is 5; and m is 7.
  • R’ a is R’ branched ; a point of attachment; R a ⁇ , R a ⁇ , and each C 1-14 alkyl; R 4 is -(CH 2 ) n OH; n is 2; each R 5 is H; each R 6 is H; M and M’ are each -C(O)O-; R’ is a C1-12 alkyl; l is 5; and m is 7.
  • the compound of Formula (I) is selected from: Attorney Docket No.: 45817-0156WO1
  • the compound of Formula (I) is: (Compound I-1).
  • In (I) is: (Compound I-2).
  • the lipid composition of the lipid nanoparticle composition disclosed herein can comprise one or more phospholipids, for example, one or more saturated or (poly)unsaturated phospholipids or a combination thereof.
  • phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.
  • a phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.
  • a fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic Attorney Docket No.: 45817-0156WO1 acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.
  • Particular phospholipids can facilitate fusion to a membrane.
  • a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid- containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue.
  • elements e.g., a therapeutic agent
  • a lipid- containing composition e.g., LNPs
  • Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated.
  • a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond).
  • alkynes e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond.
  • an alkyne group can undergo a copper-catalyzed cycloaddition upon exposure to an azide.
  • Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye).
  • Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin.
  • a phospholipid of the present disclosure comprises 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero- 3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine
  • a phospholipid useful or potentially useful in the present disclosure is an analog or variant of DSPC.
  • Alternative Lipids In certain embodiments, a phospholipid useful or potentially useful in the present disclosure comprises a modified phosphocholine moiety, wherein the alkyl chain linking the quaternary amine to the phosphoryl group is not ethylene (e.g., n is not 2). Therefore, in certain embodiments, a phospholipid useful.
  • an alternative lipid is used in place of a phospholipid of the present disclosure.
  • an alternative lipid of the present disclosure is oleic acid.
  • the alternative lipid is one of the following: , Attorney Docket No.: 45817-0156WO1 , ,
  • the lipid composition of a pharmaceutical composition disclosed herein can comprise one or more structural lipids.
  • structural lipid refers to sterols and also to lipids containing sterol moieties.
  • Attorney Docket No.: 45817-0156WO1 Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle.
  • Structural lipids can be selected from the group including but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha- tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof.
  • the structural lipid is a sterol.
  • sterols are a subgroup of steroids consisting of steroid alcohols.
  • the structural lipid is a steroid.
  • the structural lipid is cholesterol.
  • the structural lipid is an analog of cholesterol.
  • the structural lipid is alpha-tocopherol. In some embodiments, the structural lipids may be one or more of the structural lipids described in U.S. Application No.62/520,530.
  • Polyethylene Glycol (PEG)-Lipids The lipid composition of a pharmaceutical composition disclosed herein can comprise one or more a polyethylene glycol (PEG) lipid. As used herein, the term “PEG-lipid” refers to polyethylene glycol (PEG)- modified lipids.
  • Non-limiting examples of PEG-lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2- diacyloxypropan-3-amines.
  • PEGylated lipids are also referred to as PEGylated lipids.
  • a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
  • the PEG-lipid includes, but not limited to 1,2- dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG- disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG- diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG- DPPE), or PEG-l,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA).
  • PEG-DMG 1,2- dimyristoyl-sn-glycerol methoxypolyethylene glycol
  • PEG-DSPE 1,2-distearoyl-sn- g
  • the PEG-lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG- modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.
  • the lipid moiety of the PEG-lipids includes those having lengths of from about C 14 to about C 22 , preferably from about C 14 to about C 16 .
  • a PEG moiety for example an mPEG-NH2 has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons.
  • the PEG- lipid is PEG2k-DMG.
  • the lipid nanoparticles described herein can comprise a PEG lipid which is a non-diffusible PEG.
  • Non-limiting examples of non-diffusible PEGs include PEG-DSG and PEG-DSPE.
  • PEG-lipids are known in the art, such as those described in U.S. Patent No. 8,158,601 and International Publ. No. WO 2015/130584 A2, which are incorporated herein by reference in their entirety.
  • lipid component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids.
  • a PEG lipid is a lipid modified with polyethylene glycol.
  • a PEG lipid may be selected from the non- limiting group including PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG- modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof.
  • a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
  • PEG lipids useful in the present disclosure can be PEGylated lipids described in International Publication No. WO2012099755, the contents of which is herein incorporated by reference in its entirety. Any of these exemplary PEG lipids described herein may be modified to comprise a hydroxyl group on the PEG chain.
  • the PEG lipid is a PEG-OH lipid.
  • a “PEG-OH lipid” (also referred to herein as “hydroxy- PEGylated lipid”) is a PEGylated lipid having one or more hydroxyl (–OH) groups on the lipid.
  • the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain.
  • a PEG-OH or hydroxy-PEGylated lipid comprises an —OH group at the terminus of the PEG chain.
  • the lipid composition of the pharmaceutical compositions disclosed herein does not comprise a PEG-lipid.
  • the PEG-lipids may be one or more of the PEG lipids described in U.S. Application No.62/520,530.
  • a PEG lipid of the present disclosure comprises a PEG- modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG- modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.
  • the PEG- modified lipid is PEG-DMG, PEG-c-DOMG (also referred to as PEG-DOMG), PEG- DSG and/or PEG-DPG.
  • a LNP of the present disclosure comprises an ionizable cationic lipid of Formula I, a phospholipid comprising DSPC, a structural lipid, and a PEG lipid comprising PEG-DMG.
  • a LNP of the present disclosure comprises an ionizable cationic lipid of Formula I, a phospholipid comprising DSPC, a structural lipid, and a PEG lipid.
  • a LNP of the present disclosure comprises an ionizable cationic lipid of .
  • a LNP of the present disclosure comprises an ionizable cationic lipid of , In some embodiments, a LNP of the present disclosure comprises an ionizable cationic lipid of , lipid comprising cholesterol, and a PEG lipid. In some embodiments, a LNP of the present disclosure comprises an ionizable cationic lipid of Attorney Docket No.: 45817-0156WO1 and a PEG lipid. In some embodiments, a LNP of the present disclosure comprises an ionizable cationic lipid of a phospholipid comprising DOPE, a structural lipid comprising cholesterol, and a PEG lipid. In some embodiments, a LNP of the present disclosure comprises an N:P ratio of from about 2:1 to about 30:1.
  • a LNP of the present disclosure comprises an N:P ratio of about 6:1. In some embodiments, a LNP of the present disclosure comprises an N:P ratio of about 3:1. In some embodiments, a LNP of the present disclosure comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of from about 10:1 to about 100:1. In some embodiments, a LNP of the present disclosure comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of about 20:1.
  • a LNP of the present disclosure comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of about 10:1.
  • a LNP of the present disclosure has a mean diameter from about 50nm to about 150nm.
  • a LNP of the present disclosure has a mean diameter from about 70nm to about 120nm.
  • Other Lipid Composition Components The lipid composition of a pharmaceutical composition disclosed herein can include one or more components in addition to those described above.
  • the lipid composition can include one or more permeability enhancer molecules, carbohydrates, polymers, surface altering agents (e.g., surfactants), or other components.
  • a permeability enhancer molecule can be a molecule described by U.S. Patent Application Publication No.2005/0222064.
  • Carbohydrates can include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives and analogs thereof).
  • a polymer can be included in and/or used to encapsulate or partially encapsulate a pharmaceutical composition disclosed herein (e.g., a pharmaceutical composition in lipid nanoparticle form).
  • a polymer can be biodegradable and/or biocompatible.
  • a polymer can be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.
  • the ratio between the lipid composition and the polynucleotide range can be from about 10:1 to about 60:1 (wt/wt).
  • the ratio between the lipid composition and the polynucleotide can be about 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, Attorney Docket No.: 45817-0156WO1 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1 or 60:1 (wt/wt).
  • the wt/wt ratio of the lipid composition to the polynucleotide encoding a therapeutic agent is about 20:1 or about 15:1.
  • the pharmaceutical composition disclosed herein can contain more than one polypeptides.
  • a pharmaceutical composition disclosed herein can contain two or more polynucleotides (e.g., RNA, e.g., mRNA).
  • the lipid nanoparticles described herein can comprise polynucleotides (e.g., mRNA) in a lipid:polynucleotide weight ratio of 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1 or 70:1, or a range or any of these ratios such as, but not limited to, 5:1 to about 10:1, from about 5:1 to about 15:1, from about 5:1 to about 20:1, from about 5:1 to about 25:1, from about 5:1 to about 30:1, from about 5:1 to about 35:1, from about 5:1 to about 40:1, from about 5:1 to about 45:1, from about 5:1 to about 50:1, from about 5:1 to about 55:1, from about 5:1 to about 60:1, from about 5:1 to about 70:1, from about 10:1 to about 15:1, from about 10:1 to about 20:1, from about 10:1 to about 25
  • the lipid nanoparticles described herein can comprise the polynucleotide in a concentration from approximately 0.1 mg/ml to 2 mg/ml such as, but not limited to, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3 Attorney Docket No.: 45817-0156WO1 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7 mg/ml, 1.8 mg/ml, 1.9 mg/ml, 2.0 mg/ml or greater than 2.0 mg/ml.
  • Nanoparticle Compositions are Formulated as lipid nanoparticles (LNP). Accordingly, the present disclosure also provides nanoparticle compositions comprising (i) a lipid composition comprising a delivery agent such as compound as described herein, and (ii) a polynucleotide encoding a polypeptide. In such nanoparticle composition, the lipid composition disclosed herein can encapsulate the polynucleotide encoding a polypeptide. Nanoparticle compositions are typically sized on the order of micrometers or smaller and can include a lipid bilayer.
  • Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes.
  • a nanoparticle composition can be a liposome having a lipid bilayer with a diameter of 500 nm or less.
  • Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, and lipoplexes.
  • nanoparticle compositions are vesicles including one or more lipid bilayers.
  • a nanoparticle composition includes two or more concentric bilayers separated by aqueous compartments. Lipid bilayers can be functionalized and/or crosslinked to one another.
  • Lipid bilayers can include one or more ligands, proteins, or channels.
  • a lipid nanoparticle comprises an ionizable amino lipid, a structural lipid, a phospholipid, and mRNA.
  • the LNP comprises an ionizable amino lipid, a PEG-modified lipid, a sterol and a structural lipid.
  • the LNP has a molar ratio of about 40-50% ionizable amino lipid; about 5-15% structural lipid; about 30-45% sterol; and about 1-5% PEG- modified lipid.
  • the lipid nanoparticle comprises 47-49 mol.% ionizable cationic lipid (e.g.
  • ionizable amino lipid e.g., Compound I-1, Compound I-2, or Attorney Docket No.: 45817-0156WO1 Compound I-3
  • 10-12 mol.% non-cationic lipid e.g., phospholipid, e.g., DSPC
  • 38- 40 mol.% sterol e.g., cholesterol
  • PEG- modified lipid e.g., PEG-DMG or Compound P-I
  • the lipid nanoparticle (“LNP-1”) may comprise the following components at the following molar ratios: (i) 45-50 mol.% Compound I-1 (ii) 35-45 mol.% sterol (e.g., cholesterol); (iii) 8-12 mol.% phospholipid (e.g., DSPC or DOPE); and (iv) 1.5-3.5 mol.% PEG-lipid.
  • the lipid nanoparticle (“LNP-1A”) may comprise the following components at the following molar ratios: (i) 45-50 mol.% Compound I-1 (ii) 35-45 mol.% Cholesterol; (iii) 8-12 mol.% DSPC; and (iv) 1.5-3.5 mol.% PEG-lipid.
  • the lipid nanoparticle (“LNP-1B”) may comprise the following components at the following molar ratios: (i) 45-50 mol.% Compound I-1 (ii) 35-45 mol.% Cholesterol; (iii) 8-12 mol.% DSPC; and (iv) 1.5-3.5 mol.% PEG-lipid.
  • the lipid nanoparticle (“LNP-2”) may comprise the following: (i) 45-50 mol.% Compound I-2; (ii) 35-45 mol.% sterol (e.g., Cholesterol); (iii) 8-12 mol.% phospholipid (e.g., DSPC or DOPE); and (iv) 1.5-3.5 mol.% PEG-lipid.
  • the lipid nanoparticle (“LNP-2A”) may comprise the following: (i) 45-50 mol.% Compound I-2; (ii) 35-45 mol.% Cholesterol; Attorney Docket No.: 45817-0156WO1 (iii) 8-12 mol.% DSPC; and (iv) 1.5-3.5 mol.% PEG-lipid.
  • the lipid nanoparticle (“LNP-2B”) may comprise the following components at the following molar ratios: (i) 45-50 mol.% Compound I-2; (ii) 35-45 mol.% Cholesterol; (iii) 8-12 mol.% DSPC; and (iv) 1.5-3.5 mol.% PEG-lipid.
  • the lipid nanoparticle (“LNP-3”) may comprise the following: (i) 45-50 mol.% Compound I-3; (ii) 35-45 mol.% sterol (e.g., Cholesterol); (iii) 8-12 mol.% phospholipid (e.g., DSPC or DOPE); and (iv) 1.5-3.5 mol.% PEG-lipid.
  • the lipid nanoparticle (“LNP-3A”) may comprise the following: (i) 45-50 mol.% Compound I-3; (ii) 35-45 mol.% Cholesterol; (iii) 8-12 mol.% DSPC; and (iv) 1.5-3.5 mol.% PEG-lipid.
  • the lipid nanoparticle (“LNP-3B”) may comprise the following: (i) 45-50 mol.% Compound I-3; (ii) 35-45 mol.% Cholesterol; (iii) 8-12 mol.% DSPC; and (iv) 1.5-3.5 mol.% PEG-lipid.
  • the LNP has a polydispersity value of less than 0.4.
  • the LNP has a net neutral charge at a neutral pH.
  • the LNP has a mean diameter of 50-150 nm. In some embodiments, the LNP has a mean diameter of 80-100 nm.
  • lipid refers to a small molecule that has hydrophobic or amphiphilic properties. Lipids may be naturally occurring or synthetic. Examples of classes of lipids include, but are not limited to, fats, waxes, sterol-containing metabolites, vitamins, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, and polyketides, and prenol lipids. In some instances, the amphiphilic properties of some lipids lead them to form liposomes, vesicles, or membranes in aqueous media.
  • a lipid nanoparticle may comprise an ionizable amino lipid.
  • the term “ionizable amino lipid” has its ordinary meaning in the art and may refer to a lipid comprising one or more charged moieties.
  • an ionizable amino lipid may be positively charged or negatively charged.
  • An ionizable amino lipid may be positively charged, in which case it can be referred to as “cationic lipid”.
  • an ionizable amino lipid molecule may comprise an amine group, and can be referred to as an ionizable amino lipid.
  • a “charged moiety” is a chemical moiety that carries a formal electronic charge, e.g., monovalent (+1, or -1), divalent (+2, or -2), trivalent (+3, or - 3), etc.
  • the charged moiety may be anionic (i.e., negatively charged) or cationic (i.e., positively charged).
  • positively-charged moieties include amine groups (e.g., primary, secondary, and/or tertiary amines), ammonium groups, pyridinium group, guanidine groups, and imidizolium groups.
  • the charged moieties comprise amine groups.
  • negatively- charged groups or precursors thereof include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, and the like.
  • the charge of the charged moiety may vary, in some cases, with the environmental conditions, for example, changes in pH may alter the charge of the moiety, and/or cause the moiety to become charged or uncharged. In general, the charge density of the molecule may be selected as desired. It should be understood that the terms “charged” or “charged moiety” does not refer to a “partial negative charge” or “partial positive charge” on a molecule.
  • partial negative charge and “partial positive charge” are given their ordinary Attorney Docket No.: 45817-0156WO1 meaning in the art.
  • a “partial negative charge” may result when a functional group comprises a bond that becomes polarized such that electron density is pulled toward one atom of the bond, creating a partial negative charge on the atom.
  • the ionizable lipid is sometimes referred to in the art as an “ionizable cationic lipid”.
  • the ionizable amino lipid may have a positively charged hydrophilic head and a hydrophobic tail that are connected via a linker structure.
  • an ionizable amino lipid may also be a lipid including a cyclic amine group.
  • the ionizable amino lipid may be selected from, but not limited to, an ionizable amino lipid described in International Publication Nos. WO2013086354 and WO2013116126; the contents of each of which are herein incorporated by reference in their entirety.
  • the ionizable amino lipid may be selected from, but not limited to, Formula CLI-CLXXXXII of US Patent No.7,404,969; each of which is herein incorporated by reference in their entirety.
  • the lipid may be a cleavable lipid such as those described in International Publication No.
  • the lipid may be synthesized by methods known in the art and/or as described in International Publication Nos. WO2013086354; the contents of each of which are herein incorporated by reference in their entirety.
  • Nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of a nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) can be used to measure zeta potentials.
  • Dynamic light Attorney Docket No.: 45817-0156WO1 scattering can also be utilized to determine particle sizes.
  • Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure multiple characteristics of a nanoparticle composition, such as particle size, polydispersity index, and zeta potential.
  • the size of the nanoparticles can help counter biological reactions such as, but not limited to, inflammation, or can increase the biological effect of the polynucleotide.
  • size or “mean size” in the context of nanoparticle compositions refers to the mean diameter of a nanoparticle composition.
  • the polynucleotide encoding a polypeptide are formulated in lipid nanoparticles having a diameter from about 10 to about 100 nm such as, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm,
  • the nanoparticles have a diameter from about 10 to 500 nm. In some embodiments, the nanoparticle has a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, Attorney Docket No.: 45817-0156WO1 greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm or greater than 1000 nm.
  • the largest dimension of a nanoparticle composition is 1 ⁇ m or shorter (e.g., 1 ⁇ m, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, or shorter).
  • a nanoparticle composition can be relatively homogenous.
  • a polydispersity index can be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle composition.
  • a small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution.
  • a nanoparticle composition can have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25.
  • the polydispersity index of a nanoparticle composition disclosed herein can be from about 0.10 to about 0.20.
  • the amount of a polynucleotide present in a pharmaceutical composition disclosed herein can depend on multiple factors such as the size of the polynucleotide, desired target and/or application, or other properties of the nanoparticle composition as well as on the properties of the polynucleotide.
  • the amount of an mRNA useful in a nanoparticle composition can depend on the size (expressed as length, or molecular mass), sequence, and other characteristics of the mRNA.
  • the relative amounts of a polynucleotide in a nanoparticle composition can also vary.
  • the relative amounts of the lipid composition and the polynucleotide present in a lipid nanoparticle composition of the present disclosure can be optimized according to considerations of efficacy and tolerability.
  • the N:P ratio can serve as a useful metric.
  • Attorney Docket No.: 45817-0156WO1 As the N:P ratio of a nanoparticle composition controls both expression and tolerability, nanoparticle compositions with low N:P ratios and strong expression are desirable. N:P ratios vary according to the ratio of lipids to RNA in a nanoparticle composition. In general, a lower N:P ratio is preferred.
  • the one or more RNA, lipids, and amounts thereof can be selected to provide an N:P ratio from about 2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1.
  • the N:P ratio can be from about 2:1 to about 8:1.
  • the N:P ratio is from about 5:1 to about 8:1.
  • the N:P ratio is between 5:1 and 6:1.
  • the N:P ratio is about is about 5.67:1.
  • the polynucleotides described herein can be Formulated for controlled release and/or targeted delivery.
  • controlled release refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome.
  • the polynucleotides can be encapsulated into a delivery agent described herein and/or known in the art for controlled release and/or targeted delivery.
  • encapsulate means to enclose, surround or encase. As it relates to the formulation of the compounds of the present disclosure, encapsulation can be substantial, complete or partial.
  • substantially encapsulated means that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, or greater than 99% of the pharmaceutical composition or compound of the present disclosure can be enclosed, surrounded or encased within the delivery agent.
  • partial encapsulation or “partially encapsulate” means that less than 10, 10, 20, 30, 4050 or less of the pharmaceutical composition or compound of the present disclosure can be enclosed, surrounded or encased within the delivery agent.
  • the therapeutic nanoparticle polynucleotide can be Formulated for sustained release.
  • sustained release refers to a pharmaceutical composition or compound that conforms to a release rate over a Attorney Docket No.: 45817-0156WO1 specific period of time.
  • the period of time can include, but is not limited to, hours, days, weeks, months and years.
  • the sustained release nanoparticle of the polynucleotides described herein can be Formulated as disclosed in Intl. Pub. No. WO2010075072 and U.S. Pub. Nos. US20100216804, US20110217377, US20120201859 and US20130150295, each of which is herein incorporated by reference in their entirety.
  • the therapeutic nanoparticle polynucleotide can be Formulated to be target specific, such as those described in Intl. Pub. Nos. WO2008121949, WO2010005726, WO2010005725, WO2011084521 and WO2011084518; and U.S. Pub. Nos. US20100069426, US20120004293 and US20100104655, each of which is herein incorporated by reference in its entirety.
  • the LNPs can be prepared using microfluidic mixers or micromixers.
  • Exemplary microfluidic mixers can include, but are not limited to, a slit interdigital micromixer including, but not limited to those manufactured by Microinnova (Allerheiligen bei Wildon, Austria) and/or a staggered herringbone micromixer (SHM) (see, Zhigaltsev et al., Langmuir.28:3633-40 (2012); Belliveau et al., Molecular Therapy-Nucleic Acids.1:e37 (2012); Chen et al., J. Am. Chem. Soc. 134(16):6948-51 (2012); each of which is herein incorporated by reference in its entirety).
  • SHM herringbone micromixer
  • Exemplary micromixers include Slit Interdigital Microstructured Mixer (SIMM-V2) or a Standard Slit Interdigital Micro Mixer (SSIMM) or Caterpillar (CPMM) or Impinging-jet (IJMM,) from the Institut für Mikrotechnik Mainz GmbH, Mainz Germany.
  • methods of making LNP using SHM further comprise mixing at least two input streams wherein mixing occurs by microstructure- induced chaotic advection (MICA).
  • MICA microstructure- induced chaotic advection
  • fluid streams flow through channels present in a herringbone pattern causing rotational flow and folding the fluids around each other.
  • This method can also comprise a surface for fluid mixing wherein the surface changes orientations during fluid cycling.
  • Methods of generating LNPs using SHM include those disclosed in U.S. Pub.
  • the polynucleotides described herein can be Formulated in lipid nanoparticles using microfluidic technology (see, Whitesides, George M., Nature 442: 368-373 (2006); and Abraham et al., Science 295: 647-651 (2002); each of which is herein incorporated by reference in its entirety).
  • the polynucleotides can be Formulated in lipid nanoparticles using a micromixer chip such as, but not limited to, those from Harvard Apparatus (Holliston, MA) or Dolomite Microfluidics (Royston, UK).
  • a micromixer chip can be used for rapid mixing of two or more fluid streams with a split and recombine mechanism.
  • the nanoparticles described herein are stealth nanoparticles or target-specific stealth nanoparticles such as, but not limited to, those described in U.S. Pub. No. US20130172406, herein incorporated by reference in its entirety.
  • the stealth or target-specific stealth nanoparticles can comprise a polymeric matrix, which can comprise two or more polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polyesters, polyanhydrides, polyethers, polyurethanes, polymethacrylates, polyacrylates, polycyanoacrylates, or combinations thereof.
  • polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyester
  • antibodies and/or binding proteins of the present disclosure are administered to a subject in need thereof.
  • the subject in need thereof is a subject with a disease, disorder, and/or condition that may be treated with technologies described herein. Specifically, a disease or disorder which would benefit from targeting cells expressing anti-CD16 for reduction or elimination.
  • the subject in need thereof is a subject with an infection, an inflammatory condition, or an autoimmune condition.
  • Attorney Docket No.: 45817-0156WO1 In some aspects, the subject in need thereof is a subject with cancer.
  • Certain cancers that may be treated in accordance with technologies of the present disclosure include, for example, adrenocortical carcinoma, astrocytoma, basal cell carcinoma, carcinoid, cardiac, cholangiocarcinoma, chordoma, chronic myeloproliferative neoplasms, craniopharyngioma, ductal carcinoma in situ, ependymoma, intraocular melanoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, glioma, histiocytosis, leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), hairy cell leukemia, myelogenous leukemia, and myeloid leukemia), lymphoma (e.g., Burkitt lymphoma
  • the subject in need thereof is a subject with multiple myeloma.
  • the subject in need thereof is a mammal.
  • a mammal includes, for example and without limitation, a household pet (e.g., a dog, a cat, a rabbit, a ferret, a hamster), a livestock or farm animal (e.g., a cow, a pig, a sheep, a goat, a pig, a chicken or another poultry), a horse, a monkey, a laboratory animal (e.g., a mouse, a rat, a rabbit) and a human.
  • the subject in need thereof is a human.
  • a subject in need thereof is a human.
  • the human is male.
  • the human is female.
  • the human is an adult (e.g., 18 or more years of age).
  • the adult is greater than 18 years old, greater than 25 years old, greater than 30 years old, greater than 40 years old, greater than 50 years old, greater than 55 years old, greater than 60 years old, greater than 65 years old, greater than 70 years old, greater than 75 years old, greater than 80 years old, greater than 85 years old, greater than 90 years old, greater than 95 years old, greater than 100 years old, or greater than 105 years old in age.
  • the human is a child.
  • the child is greater than 2 years old, greater than 3 years old, greater than 4 years old, greater than 5 years old, greater than 6 years old, greater than 7 years old, greater than 8 years old, greater than 9 years old, greater than 10 years old, greater than 11 years old, greater than 12 years old, greater than 13 years old, greater than 14 years old, greater than 15 years old, or greater than 16 years old in age.
  • a subject in need thereof is administered an antibody or binding protein of the present disclosure.
  • Routes of Administration and Dosing Anti-CD16 antibodies or binding molecules of the disclosure, and nucleic acids encoding the same can be administered to a subject (e.g., a mammalian subject, such as a human) by a variety of routes.
  • the antibody or nucleic acid is administered to the subject intravenously, subcutaneously, intramuscularly, parenterally, intrathecally, intracerebroventricularly, transdermally, or orally.
  • the most suitable route for administration in any given case will depend on the particular therapeutic agent administered, the patient, pharmaceutical formulation Attorney Docket No.: 45817-0156WO1 methods, and various patient-specific parameters, such as the patient's age, body weight, sex, severity of the diseases being treated, the patient’s diet, and the patient’s excretion rate.
  • an appropriate dosage of anti-CD16 antibodies or binding proteins, or nucleic acids encoding the same of the present disclosure will vary with the particular condition, disease and/or disease being treated, various subject-specific parameters (e.g., age, weight, physical condition of the subject), severity of the particular condition, disease, and/or disorder being treated, the nature of current or combination therapy (if any), the specific route of administration and other factors within the knowledge and expertise of a health practitioner.
  • a maximally tolerated dose of technologies described herein is to be used, e.g., the highest safe dose according to sound medical judgement.
  • technologies described herein are administered in an effective amount, e.g., a dose sufficient to provide one or more medically desirable results.
  • a therapeutic regiment for use in accordance with technologies described herein may include administration of such technologies or compositions comprising such technologies once a day, once every two days, once every three days, twice a week, once a week, once every two weeks, once every three weeks, once every month or four weeks, once every six weeks, once every two months or eight weeks, once every three months or twelve weeks.
  • a subject receives a single dose of a technology described herein.
  • a subject receives a plurality of doses of a technology described herein (e.g., at least two, at least three, at least four, at least five, at least six, at least eight, at least ten, or more doses).
  • technologies described herein are administered over a period of time, such as one week, two weeks, three weeks, four weeks, six weeks, two months, three months, four months, five months, six months, one year or more.
  • Appropriate therapeutic regimens are readily understood by medical practitioners and such regimens may be designed by a medical practitioner for a particular patient (e.g., a patient-specific regimen).
  • Attorney Docket No.: 45817-0156WO1 Kits Also included herein are kits that contain anti-CD16 antibodies, binding proteins, and/or nucleic acids encoding the same.
  • kits provided herein contain one or more cells engineered to express and secrete an anti- CD16 antibody, antigen-binding fragment, of binding protein of the disclosure, such as a cell containing a nucleic acid molecule of the disclosure.
  • a kit described herein may include reagents that can be used to produce a pharmaceutical composition of the disclosure.
  • kits described herein may include reagents that can induce the expression of anti-CD16 antibodies, antigen- binding fragments, or relate proteins of the present disclosure within cells (e.g., mammalian cells).
  • Other kits described herein may include tools for engineering a prokaryotic or eukaryotic cell (e.g., a CHO cell or a BL21(DE3) E.
  • kits may contain CHO cells stored in an appropriate media and optionally frozen according to methods known in the art.
  • the kit may also contain a nucleic acid encoding the desired antibody or binding protein as well as reagents for expressing the antibody or binding protein in the cell.
  • a kit described herein may also provide an a anti-CD16 antibody or binding protein of the disclosure, or a nucleic acid encoding the same in combination with a package insert describing how the antibody, binding protein, or nucleic acid may be administered to a subject, for example, for the treatment of a disease, disorder and/or condition (e.g., cancer).
  • a disease, disorder and/or condition e.g., cancer
  • Example 1 Antibody Discovery Immunization of Llamas: A Llama was immunized with a primary dose immunization of 200 ⁇ g of human CD16a (Acro CDA-H5220) in complete Freund’s Adjuvant (CFA), given subcutaneously. Following the primary immunization, 100 ⁇ g of human CD16a with incomplete Freund’s Adjuvant (IFA) was used for all following boosts weekly after 21 days.
  • CFA Complete Freund’s Adjuvant
  • a test bleed was taken on day 35 post immunization, to check for antibody titers against the immunized targets in the serum.
  • the production bleed was performed on day 42 after an IV boost without adjuvant, for a production bleed of 600 ml and was used to isolate and cryopreserve PBMCs.
  • FACS sorting of B-cells Antibody discovery was performed by Fluorescence Activated Cell Sorting (FACS) and culture of sorted Llama B cells, based on methods available on mammalian B cell culture (see, e.g., Kwakkenbos et al., Nat. Med.
  • B-cells were sorted at a concentration of 1 x 10 6 cells/mL while gating for double positive VHH and CD16a positive antibodies using biotinylated CD16a, streptavidin-APC and Rabbit anti- VHH-FITC (Rabbit anti-camelid VHH AF-647, GenScript).
  • B-cell culture B-cells were cultured at 2-4 cells/well and co-cultured with irradiated CD40L expressing feeder cells and the appropriate cytokines in 96 well plates.
  • the culture plates were incubated for 10-12 days in a CO 2 incubator at 37 o C and 5% CO2.
  • Proliferating B cell cultures were processed to harvest culture supernatants for screening the secreted antibodies against the target antigens, and the cell pellets were frozen for isolation of RNA and antibody sequencing.
  • Screening for Antibody Hits via ELISA 384 well ELISA plates were coated with 25 ⁇ l 1 ⁇ g/ml purified human CD16a. After blocking, 15 ⁇ l of primary B cell culture supernatants with 10 ⁇ l assay diluent was added into each well and incubated for 1 hr at room temperature.
  • VHH clones were PCR amplified and made into linear expression module LEM (or TAP: transcription activated PCR, (The rapid generation of recombinant functional monoclonal antibodies from individual, antigen-specific bone marrow-derived plasma cells isolated using a novel fluorescence-based method Clargo et al. MAbs, 6(1) 143- 159, 2014)), which was transfected into CHO cell line for VHH-Fc expression. The soup was used in ELISA for binding validation to CD16a. The positive clone VHH1 was sent for Sanger sequencing. The amino acid sequence of VHH1 is shown below. Table 4.
  • VHH1-His molecules were transiently expressed in ExpiCHO cells using the EpxiFectamine CHO (Thermofisher) transfection reagents.
  • ExpiCHO-S cells were cultured in ExpiCHO expression medium (ThermoFisher Scientific) in a shaker incubator set at 125 rpm, 37oC and 8% CO2. The day prior to transfection, ExpiCHO-S cells were seeded at 3 x 10 6 cells per ml in 30 ml of ExpiCHO Expression medium. On the day of transfection cells were split using pre-warmed (37oC) ExpiCHO expression media to a density of 6 x 10 6 cells per ml.
  • a total 20 ⁇ g of a single plasmid and 72 ⁇ L of ExpiFectamine CHO reagent were mixed in 2.4 ml of cold Opti-PRO SFM (Thermo Fisher Scientific), after incubating the mixture for 2 minutes it was then slowly added to the cells.
  • Opti-PRO SFM Thermo Fisher Scientific
  • 7.2 ml of ExpiCHO Feed and 180 ⁇ l of ExpiCHO Enhancer were added to the ExpiFectamine-transfected cultures.
  • Proteins were then dialyzed exhaustively using Slide-A-Lyzer® 10 or 3K as appropriate, (Dialysis cassette, Pierce) versus 1 x PBS (100 mM NaPO 4 pH 6.8, 200mM NaCl).
  • Expressed single domain antibody with a Fc C-terminal tag (VHH1-Fc) was purified from ExpiCHO media in the similar protocol except by capture on protein A resin and elution by 10 mM glycine pH 3.0 followed by immediate pH adjustment in 10% 1M Tris at pH 8.0.
  • Protein characterization SDS PAGE was run on each sample using gradient gels NuPAGE Bis-Tris 4-12% gradient gels using a MES running buffer (Thermo Fisher Scientific).
  • Samples were prepared with either reducing or non-reducing sample buffer and briefly heated to 95°C. To samples electrophoresed in non- reducing buffer N-ethyl maleimide was added to cap any free thiols and prevent unwanted disulfide scrambling as the samples cooled. Molecular weight standards (Blue Plus protein, Thermofisher) were included on the SDS-PAGE.
  • Non-denaturing protein electrophoresis was performed running 1 ⁇ g of each purified protein sample; reducing conditions were performed mixing each purified sample with 10 ⁇ l of Sample Reducing Agent (Invitrogen, Carlsbad, CA) and heating at 70°C for 10 min before electrophoresis on NuPAGE 4-12% Bis-Tris Mini Gels 1.0 mm (Invitrogen, Carlsbad, CA). The bands were visualized by SimplyBlueTM SafeStain (Invitrogen, Carlsbad, CA) staining, and the gel was dried using DryEase Mini-Gel Drying System (Invitrogen, Carlsbad, CA). All procedures were performed according to the manufacturer’s instructions.
  • Size Exclusion Chromatography with Light Scattering SEC- LS
  • Size exclusion chromatography SEC was carried out on a Zenix SEC 3004.6 x 300 mm (Sepax Technologies) in 20 mM sodium phosphate pH 7.2, 150 mM NaCl (PBS), 0.05% NaAzide at a flow rate of 0.35ml/min using an Agilent 1260 UPLC.
  • the eluent was monitored with a refractive index detector (Waters, Milford, MA).
  • Light scattering was monitored using a Wyatt Dawn 18 angle, coupled with an Optrex refractometer.
  • a buffer injection served as a negative control.
  • Channel 1 was blocked by ethanolamide and served as a reference channel.
  • surfaces were regenerated with 10 nM glycine pH 2.5 solution.
  • the association rates (ka), dissociation rate constants (kd), and affinity constants (KD) were calculated using Biacore T200 evaluation software. Each fit was evaluated by the agreement between experimental data and the calculated fits, where the Chi2 values were below 10% of Rmax. Surface densities of the molecule were optimized to minimize mass transfer while maintaining enough response. Binding and affinity results are shown in Table 6 below. Table 6.
  • SPR SPR
  • antibody binding was assessed against a number of purified CD16a and CD16b proteins with different alleles as well as to CD16a from varied species.
  • the antibody bound both alleles of human CD16a, V176 and F176, at 5nM and 13 nM, respectively (Table 6).
  • VHH1 was also cross reactive to cyno CD16 binding with a 9nM affinity and rat CD16 at 58nM (Table 6). Whereas no binding was seen to mouse CD16 (Table 6).
  • VHH1 did not bind to human CD16b NA1 variant and other human Fc receptor family members including all variants of CD32 and CD64. However, VHH1 did bind to human CD16b NA2 variant, indicating that antibody binding to neutrophils would be expected.
  • FACS Binding affinity CHO cell lines that stably express CD16a and CD16b were resuspended in FACS buffer (PBS pH7.4 + 2%FBS +1mM EDTA) at 1,000,000 cells/ml, and 100 ⁇ l of the cell suspension was mixed in a 96-well V bottom polypropylene plate (Nunc) with 100ul of VHH1-His or control-His fusion molecules in the same buffer with or without 33% human serum.
  • pelleted cells were washed 2x by ice cold FACS buffer and then resuspended in 100 ⁇ l anti-His-iFluor 647 from GenScript at 1:100 dilution in FACS buffer, incubated 15 min at 4°C, centrifuged at 1000 rpm for 3 min; cell pellets were washed, fixed with 200 ⁇ l/well 1% paraformaldehyde for 10 min at room temperature, centrifuged, the fix solution was decanted and cell pellets were resuspended in 200 ⁇ l FACS buffer for analysis. Cellular fluorescence was determined on Canto equipped with FlowJo software (Becton-Dickinson, Franklin Lakes, NJ).
  • the receptor EC50 values were determined by fitting the data with a single site total binding model using GraphPad Prism 6.0 (La Jolla, CA).
  • the llama anti-CD16 VHH antibody demonstrated 3.5 nM affinity to CHO-CD16a and micromolar affinity to CHO- CD16b.
  • Table 7 and FIG.1 The impact of VHH1 binding to CD16a in the presence and absence of human serum albumin was also evaluated. No significant effect was observed by presence of Attorney Docket No.: 45817-0156WO1 human serum (1:3 diluted) in contrast to a control antibody, indicating VHH1 does not compete with human IgG for binding to CD16a. (see, Table 7 and FIG.1) Table 7.
  • VHH1-His and VHH1-Fc show concentration dependent binding to both NK cells and neutrophils.
  • FIG.2. Attorney Docket No.: 45817-0156WO1
  • Example 3 Expression and in vitro Characterization of Humanized Antibody Sequence Humanization: The VHH antibody was humanized similar to the method of Hanf et al. (Methods.65(1):68-76 (2014)). Briefly sequences of the CDRs of VHH1 were annotated using the IMGT numbering scheme.
  • Each VHH nucleotide sequence is generated and used to identify the nearest human germline VH sequences by searching for similar sequences with the NCBI IgBLAST program. Common J and D gene sequences were attached to the VH as the acceptor. Next the most similar human VH sequences are identified using BLASTp and used to choose the nearest framework sequences into which the VHH1 CDR sequences are grafted replacing the human CDRs. Rosetta/Alpha fold was used to create the structural 3D homology model the of the appropriate CDRs that were grafted into the acceptor framework.
  • the framework residues that were critical for huVH/VL interactions are back mutated to llama sequence canonical llama residues, also potentially structural defects due to mismatches at the graft interface can be fixed by mutating some framework residues to llama, or by mutating some residues on the CDRs’ backside to human or to a de novo designed sequence. CDR stabilizing or overall fold stabilizing sequences were then back-mutated to the corresponding llama sequence to maintain the biophysical properties and target binding affinity. Sequences of 20 humanized variants of VHH1 are shown in Table 8 below. Table 8.
  • VHH1-H3 was selected for further characterization based on the humanness and close sequence analysis.
  • VHH1-H3 was produced with a His tag at the C-terminus in CHO cell line and purified by cobalt affinity column. The purified protein was analyzed with size exclusion chromatography (SEC). Parent llama antibody VHH1 was purified and analyzed the same way.

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Abstract

Described herein are CD16 singe domain antibodies and binding molecules including such single domain antibodies, as well as nucleic acids encoding the same. The disclosure also features methods of using such antibodies and binding molecules to treat various diseases such as cancer.

Description

Attorney Docket No.: 45817-0156WO1 CD16-BINDING ANTIBODIES AND USES THEREOF CROSS-REFERENCES TO RELATED APPLICATIONS This application claims the benefit of priority of U.S. Provisional Appl. No. 63/454,834, filed March 27, 2023, and U.S. Provisional Appl. No.63/601,308, filed November 21, 2023, the contents of both of which are incorporated by reference in their entirety herein. SEQUENCE LISTING [0001.1] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on February 29, 2024, is named 45817- 0156WO1_SL.xml and is 40,984 bytes in size. FIELD The present disclosure relates generally to antibodies and binding polypeptides that specifically bind CD16 and nucleic acids encoding the same. The present disclosure further relates to methods of producing the disclosed antibodies, binding domains, proteins (e.g., purified anti-CD16 binding proteins or chimeric molecules comprising such binding proteins), and nucleic acid molecules encoding such binding protein, as well as medical applications and treatments utilizing the disclosed antibodies, binding domains, proteins, and nucleic acid molecules. BACKGROUND The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology. CD16 (also known as FcyRIII), exists as two isoforms, CD16a and CD16b, that share 96% sequence identity in their extracellular immunoglobulin-binding regions. CD16a is expressed on macrophages, mast cells, and NK cells as a Attorney Docket No.: 45817-0156WO1 transmembrane receptor. CD16b is present on neutrophils as a glycosyl- phosphatidylinositol (GPI)-anchored receptor, as well as a soluble receptor in serum. CD16 is a low affinity receptor for the Fc portion of some IgGs known to be involved in antibody-dependent cellular cytotoxicity (ADCC), and has been associated with triggering of target cell lysis by NK cells (Mandelboim et al, 1999, PNAS 96:5640-5644). CD16 is thus an important therapeutic target for activation of NK cells and targeting tumor cells. Accordingly, there remains a need for antibodies, binding domains, and related proteins that bind CD16 and nucleic acids encoding the same. SUMMARY OF THE DISCLOSURE The present disclosure provides, among other things, antibodies, binding domains, and related proteins that bind CD16 and nucleic acids encoding the same. In some instances, the CD16 is human CD16a. In other cases, the CD16 is human CD16a and cynomolgus CD16. In certain cases, the antibodies, binding domains, and related proteins bind human, cynomolgus and rat CD16, but neither mouse CD16 nor human CD16b NA1. Such antibodies, binding domains, and binding proteins are useful for improved drug delivery and for extending the half-life of biologics and other therapeutics. In one aspect, the present disclosure provides a single-domain antibody the antibody specifically binds CD16 and comprises the following complementary determining regions (CDRs): CDR1 comprising the amino acid sequence GRTDSIYA (SEQ ID NO: 2), CDR2 comprising the amino acid sequence INSNTGRT (SEQ ID NO: 3), and CDR3 comprising the amino acid sequence AAGRGYGLLSISSNWYNY (SEQ ID NO: 4). In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 1 or 5-24. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence that is at least 95% identical to the Attorney Docket No.: 45817-0156WO1 amino acid sequence of any one of SEQ ID NOs: 1 or 5-24. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence of any one of SEQ ID NOs: 1 or 5-24. In some embodiments, the antibody binds CD16 with a KD of 20 nM or less. In some embodiments, the antibody binds CD16 with a KD of 10 nM or less, optionally wherein the antibody binds CD16 with a KD of 5 nM or less, optionally wherein the antibody binds CD16 with a KD of 1 nM or less, optionally wherein the antibody binds CD16 with a KD of 0.5 nM or less, optionally wherein the antibody binds CD16 with a KD of 0.1 nM or less, optionally wherein the antibody binds CD16 with a KD of 0.05 nM or less, optionally wherein the antibody binds CD16 with a KD of 0.01 nM or less. In some embodiments, the disclosed antibodies, antigen-binding fragments, and binding proteins do not compete with human IgG for binding to CD16a. In some embodiments, the present disclosure provides a conjugate comprising a biologic and a single-domain antibody described herein. In some embodiments, the biologic and the single-domain antibody of the conjugate are covalently bound to one another as part of a single polypeptide chain. In some embodiments, the biologic and the single-domain antibody of the conjugate are connected via a chemical linker. In some embodiments, the biologic of the conjugate is selected from an antibody, a cytokine, a growth factor, an enzyme, a polypeptide, a protein, a carbohydrate, and a nucleic acid. In some embodiments, the conjugate, when administered to a human subject, possesses a longer circulating half-life relative to the corresponding biologic that is not conjugated to the single-domain antibody. In one aspect, the present disclosure provides an antibody or binding protein that specifically binds CD16 and comprises a heavy chain comprising a CDR1 comprising the amino acid sequence GRTDSIYA (SEQ ID NO: 2), a CDR2 comprising the amino acid sequence INSNTGRT (SEQ ID NO: 3), and a CDR3 comprising the amino acid sequence AAGRGYGLLSISSNWYNY (SEQ ID NO: 4). Attorney Docket No.: 45817-0156WO1 In some embodiments, the antibody or binding protein comprises heavy chain comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 1 or 5-24. In some embodiments, the antibody or binding protein comprises a heavy chain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 1 or 5- 24. In some embodiments, the antibody or binding protein comprises heavy chain comprising an amino acid sequence of any one of SEQ ID NOs: 1 or 5-24. In some embodiments, the antibody or binding protein binds CD16 with a KD of 20 nM or less. In some embodiments, the antibody or binding protein binds CD16 with a KD of 10 nM or less, optionally the antibody or binding protein binds CD16 with a KD of 5 nM or less, optionally the antibody or binding protein binds CD16 with a KD of 1 nM or less, optionally the antibody or binding protein binds CD16 with a KD of 0.5 nM or less, optionally the antibody or binding protein binds CD16 with a KD of 0.1 nM or less, optionally the antibody or binding protein binds CD16 with a KD of 0.05 nM or less, optionally the antibody or binding protein binds CD16 with a KD of 0.01 nM or less. In some embodiments, the antibody or binding protein is a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen- binding fragment thereof, a human antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a primatized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a multi-specific antibody or antigen-binding fragment thereof, a dual-variable immunoglobulin domain, a monovalent antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a single-chain Fv molecule (scFv), a diabody, a triabody, an antibody-like protein scaffold, a domain antibody, a Fv fragment, a Fab fragment, a F(ab’)2 molecule, a tandem scFv (taFv), or a fusion protein. Attorney Docket No.: 45817-0156WO1 In some embodiments, the antibody or binding protein is a fusion protein comprising a biologic conjugated, either directly or indirectly, to the antibody or binding protein. In some embodiments, the antibody or binding protein is a multi-specific antibody comprising at least one binding domain that specifically binds to an antigen other than CD16. In some embodiments, the present disclosure provides a nucleic acid encoding a single-domain antibody, a conjugate, or an antibody or binding protein disclosed herein. In some embodiments, the nucleic acid is an mRNA. In some embodiments, the nucleic acid comprises, in the 5’-to-3’ direction: (a) a 5’ cap structure; (b) a 5’ untranslated region (UTR); (c) an open reading frame encoding the single-domain antibody, conjugate, antibody, antigen binging fragment, or binding protein, wherein the open reading frame consists of nucleosides is selected from the group consisting of (i) uridine or a modified uridine, (ii) cytidine or a modified cytidine, (iii) adenosine or a modified adenosine, and (iv) guanosine or a modified guanosine; (d) a 3’ UTR; and (e) a 3’ tailing sequence of linked nucleosides. In some embodiments, the nucleic acid comprises an open reading frame of nucleosides selected from the group consisting of (i) a modified uridine, (ii) cytidine, (iii) adenosine, and (iv) guanosine. In some embodiments, the nucleic acid comprises one or more modified uridines selected from 1-methylpseudouridine, pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4- thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine, 5- aminoallyl-uridine, 5-halo-uridine, 3-methyl-uridine, 5-methoxy-uridine, uridine 5- oxyacetic acid, uridine 5-oxyacetic acid methyl ester, 5-carboxymethyl-uridine, 1- carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine, 5- carboxyhydroxymethyl-uridine methyl ester, 5-methoxycarbonylmethyl-uridine, 5- Attorney Docket No.: 45817-0156WO1 methoxycarbonylmethyl-2-thio-uridine, 5-aminomethyl-2-thio-uridine, 5- methylaminomethyl-uridine, 5-methylaminomethyl-2-thio-uridine, 5- methylaminomethyl-2-seleno-uridine, 5-carbamoylmethyl-uridine, 5- carboxymethylaminomethyl-uridine, 5-carboxymethylaminomethyl-2-thio-uridine, 5- propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine, 1- taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio- pseudouridine, 5-methyl-uridine, 5-methyl-2-thio-uridine, 1-methyl-4-thio- pseudouridine, 4-thio-1-methyl-pseudouridine, 3-methylpseudouridine, 2-thio-1- methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza- pseudouridine, dihydrouridine, dihydropseudouridine, 5,6-dihydrouridine, 5-methyl- dihydrouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy- uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio- pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine, 1- methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 5- (isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thio-uridine, α-thio- uridine, 2′-O-methyl-uridine, 5,2′-O-dimethyl-uridine, 2′-O-methyl-pseudouridine, 2- thio-2′-O-methyl-uridine, 5-methoxycarbonylmethyl-2′-O-methyl-uridine, 5- carbamoylmethyl-2′-O-methyl-uridine, 5-carboxymethylaminomethyl-2′-O-methyl- uridine, 3,2′-O-dimethyl-uridine, 5-(isopentenylaminomethyl)-2′-O-methyl-uridine, 1- thio-uridine, deoxythymidine, 2’‐F‐ara‐uridine, 2’‐F‐uridine, 2’‐OH‐ara‐uridine, 5‐(2‐ carbomethoxyvinyl) uridine, or 5‐[3‐(1‐E‐propenylamino)uridine. In some embodiments, the nucleic acid comprises a modified uridine that is 1- methylpseudouridine. In some embodiments, the nucleic acid comprises one or more modified cytidines selected from 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl- cytidine, N4-acetyl-cytidine, 5-formyl-cytidine, N4-methyl-cytidine, 5-methyl- cytidine, 5-halo-cytidine, 5-hydroxymethyl-cytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1- deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza- Attorney Docket No.: 45817-0156WO1 zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2- methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4- methoxy-1-methyl-pseudoisocytidine, lysidine, α-thio-cytidine, 2′-O-methyl-cytidine, 5,2′-O-dimethyl-cytidine, N4-acetyl-2′-O-methyl-cytidine, N4,2′-O-dimethyl-cytidine, 5-formyl-2′-O-methyl-cytidine, N4,N4,2′-O-trimethyl-cytidine, 1-thio-cytidine, 2’‐F‐ ara‐cytidine, 2’‐F‐cytidine, or 2’‐OH‐ara‐cytidine. In some embodiments, the nucleic acid comprises one or more modified adenosines selected from 2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine, 6-halo-purine, 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7- deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7- deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine, 2- methyl-adenine, N6-methyl-adenosine, 2-methylthio-N6-methyl-adenosine, N6- isopentenyl-adenosine, 2-methylthio-N6-isopentenyl-adenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyl-adenosine, N6-threonylcarbamoyl-adenosine, N6-methyl-N6- threonylcarbamoyl-adenosine, 2-methylthio-N6-threonylcarbamoyl-adenosine, N6,N6-dimethyl-adenosine, N6-hydroxynorvalylcarbamoyl-adenosine, 2-methylthio- N6-hydroxynorvalylcarbamoyl-adenosine, N6-acetyl-adenosine, 7-methyl-adenine, 2- methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine, N6,2′-O-dimethyl-adenosine, N6,N6,2′-O-trimethyl-adenosine, 1,2′-O-dimethyl- adenosine, 2′-O-ribosyladenosine, 2-amino-N6-methyl-purine, 1-thio-adenosine, 8- azido-adenosine, 2’‐F‐ara‐adenosine, 2’‐F‐adenosine, 2’‐OH‐ara‐adenosine, or N6‐ (19‐amino‐pentaoxanonadecyl)-adenosine. In some embodiments, the nucleic acid comprises one or more modified guanosines selected from inosine, 1-methyl-inosine, wyosine, methylwyosine, 4- demethyl-wyosine, isowyosine, wybutosine, peroxywybutosine, hydroxywybutosine, 7-deaza-guanosine, queuosine, epoxyqueuosine, galactosyl-queuosine, mannosyl- queuosine, 7-cyano-7-deaza-guanosine, 7-aminomethyl-7-deaza-guanosine, archaeosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6- thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7- Attorney Docket No.: 45817-0156WO1 methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine, N2-methyl-guanosine, N2,N2-dimethyl-guanosine, N2,7-dimethyl-guanosine, N2, N2,7-dimethyl-guanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl- 6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2′-O-methyl- guanosine, N2-methyl-2′-O-methyl-guanosine, N2,N2-dimethyl-2′-O-methyl- guanosine, 1-methyl-2′-O-methyl-guanosine, N2,7-dimethyl-2′-O-methyl-guanosine, 2′-O-methyl-inosine, 1,2′-O-dimethyl-inosine, 2′-O-ribosylguanosine, 1-thio- guanosine, O6-methyl-guanosine, 2’‐F‐ara‐guanosine, or 2’‐F‐guanosine. In some embodiments, the nucleic acid comprises a 3’ tailing sequence of linked nucleosides, selected from a poly-adenylate (polyA) tail or a polyA-G quartet. In some embodiments, the 3’ tailing sequence of linked nucleosides is a polyA tail. In some embodiments, the nucleic acid comprises a 5’ cap structure selected from Cap0, Cap1, ARCA, inosine, 1-methyl-guanosine, 2′fluoroguanosine, 7-deaza- guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, or 2- azidoguanosine. In some embodiments, the present disclosure provides a pharmaceutical composition comprising a single domain antibody, a conjugate, an antibody or binding protein, or a nucleic acid disclosed herein. In some embodiments, the pharmaceutical composition additionally comprises one or more pharmaceutically acceptable carriers, diluents, excipients, or any combination thereof. In some embodiments, the pharmaceutical composition comprises a plurality of lipid nanoparticles encapsulating the nucleic acid. In some embodiments, the pharmaceutical composition comprises a plurality of lipid nanoparticles having a mean particle size of from 80 nm to 160 nm. In some embodiments, the pharmaceutical composition comprises a plurality of lipid nanoparticles having a polydispersity index (PDI) of from 0.02 to 0.2 and/or a lipid:nucleic acid ratio of from 10 to 20. In some embodiments, the pharmaceutical composition comprises a plurality Attorney Docket No.: 45817-0156WO1 of lipid nanoparticles comprising a neutral lipid, a cationic lipid, a polyethyleneglycol (PEG) lipid, and/or a sterol. In some embodiments, the pharmaceutical composition comprises a plurality of lipid nanoparticles comprising 1,2-distearoyl-sn-glycero-3- phosphocholine. In some embodiments, the pharmaceutical composition comprises a plurality of lipid nanoparticles comprising a compound of Formula (I). In some embodiments, the pharmaceutical composition comprises a plurality of lipid nanoparticles comprising PEG 2000 dimyristoyl glycerol. In some embodiments, the pharmaceutical composition comprises a sterol selected from cholesterol, adosterol, agosterol A, atheronals, avenasterol, azacosterol, blazein, cerevisterol, colestolone, cycloartenol, daucosterol, 7-dehydrocholesterol, 5- dehydroepisterol, 7-dehydrositosterol, 20α,22R-dihydroxycholesterol, dinosterol, epibrassicasterol, episterol, ergosterol, ergosterol, fecosterol, fucosterol, fungisterol, ganoderenic acid, ganoderic acid, ganoderiol, ganodermadiol, 7α-hydroxycholesterol, 22R-hydroxycholesterol, 27-hydroxycholesterol, inotodiol, lanosterol, lathosterol, lichesterol, lucidadiol, lumisterol, oxycholesterol, oxysterol, parkeol, saringosterol, spinasterol, sterol ester, trametenolic acid, zhankuic acid, or zymosterol. In some embodiments, the pharmaceutical composition comprises cholesterol. In some embodiments, the present disclosure provides a host cell comprising a single domain antibody, a conjugate, an antibody or binding protein, or a nucleic acid disclosed herein. In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a CHO cell or HEK cell. In some embodiments, the present disclosure provides a method of treating cancer comprising administering to a subject in need thereof a single domain antibody described herein or the antibody, a conjugate described herein, an antigen-binding fragment, or binding protein described herein, a nucleic acid described herein, a pharmaceutical composition described herein, or a host cell described herein. In some embodiments, the subject is human. Attorney Docket No.: 45817-0156WO1 In some embodiments, a VHH single domain antibody of the invention is incorporated into a binding molecule comprising at least one additional binding specificity (e.g., at least one binding moiety that binds to a molecule expressed on a cancer cell) and facilitates treatment of cancer. In some embodiments, the present disclosure provides a kit comprising (i) a single domain antibody, a conjugate, an antibody, antigen-binding fragment, binding protein, a nucleic acid, a pharmaceutical composition or host cell disclosed herein, and (ii) a package insert instructing a user of the kit to administer the single-domain antibody, conjugate, antibody, antigen-binding fragment, binding protein, nucleic acid, or pharmaceutical composition to a subject in need thereof. In another aspect, the disclosure features a binding molecule comprising a polypeptide that specifically binds CD16, wherein the polypeptide comprises the amino acid sequences of a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO:1 or SEQ ID NO:7. In some instances, the CD16 is human CD16a. In certain cases, the human CD16a is CD16aF176. In other cases, the human CD16a is CD16aV176. In some instances, the CD16 is human CD16b NA2. In some instances, the CD16 is cynomolgus CD16. In some instances, the CD16 is rat CD16. In some cases, the polypeptide binds to human and cynomolgus CD16. In other cases, the polypeptide binds to human, cynomolgus, and rat CD16. In some cases, the polypeptide binds human, cynomolgus, and rat CD16, but not mouse CD16 or human CD16b NA1 variant. In certain instances, the VHH-CDR1, VHH-CDR2, and VHH-CDR3 are based on Kabat, Chothia, enhanced Chothia, Contact, Aho, or IMGT CDR definitions. In certain instances, the binding molecule binds to human NK cells and/or human neutrophils. In one instance, the disclosure features a binding molecule comprising a polypeptide that specifically binds human CD16a, wherein the polypeptide comprises the amino acid sequences of a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of VHH1 based on any single CDR definition as set forth in Table 5. Attorney Docket No.: 45817-0156WO1 In some instances, the polypeptide is a VHH. In some cases, the VHH is humanized. In certain instances, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 8, 7, 6, 5, 43, 2, or 1 amino acids of the VHH are humanized. In certain cases, the humanization technique is resurfacing/veneering. In some cases, the humanization technique is CDR grafting. In some instances, one or more (1, 2, 3, 4, 5, 6) of positions 37, 44, 45, 47, 95, and 117 (numbering based on Kabat numbering) are humanized. In other instances, one or more (1, 2, 3, 4, 5, 6) of positions 37, 44, 45, 47, 95, and 117 (numbering based on Kabat numbering) are not humanized. In some cases, positions 37 and 47 (numbering based on Kabat numbering) are not humanized. In some cases, positions 44 and 45 (numbering based on Kabat numbering) are not humanized. In certain instances, one or more (1, 2, 3, 4, 5, 6) of positions 62, 65, 67, 72, 76, and 89 (numbering based on Kabat numbering) are not humanized. In certain instances, one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) of positions 1, 2, 37, 44, 45, 46, 47, 60, 61, 62, 65, and 117 (numbering based on Kabat numbering) are not humanized. In some instances, the binding molecule further comprises an agent selected from the group consisting of a purification tag, a fluorophore, a drug, a photosensitizer, a nanoparticle, a toxic agent, a radionuclide, a VHH, an Fab, a scFv, a multimerization module, a moiety that facilitates the polypeptide crossing the blood brain barrier, and a half-life extension moiety. In certain cases, the agent is a VHH, an Fab, or a scFv, and the agent is attached or linked to the N-terminus of the polypeptide. In other cases, the agent is a VHH, an Fab, or a scFv, and the agent is attached or linked to the C-terminus of the polypeptide. In another instance, the binding molecule further comprises a human Ig Fc domain. In some cases, the binding molecule further comprises a human Ig hinge domain. In some cases, the human Ig is a human IgG1, human IgG2, human IgG3, or human IgG4. In certain cases, the binding molecule comprises an amino acid of a human IgG4 PAA hinge and IgG4 Fc domain sequence. In some cases, the human IgG4PAA hinge + Fc sequence is at the N-terminus of the polypeptide. In certain Attorney Docket No.: 45817-0156WO1 cases, the Fc domain includes mutations that promote heterodimerization (e.g., knob- into-hole mutations, electrosteering mutations). In some instances, the polypeptide is a VHH and comprises the VHH-CDR1, VHH-CDR2, and VHH-CDR3 of any one of the CDR definitions set forth in Table 5. In some cases, the VHH comprises a sequence that is at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any amino acid sequence set forth in Table 8. In some cases, the VHH comprises a sequence that is identical to any amino acid sequence set forth in Table 8 except for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions. In some cases, the substitutions are conservative substitutions. In some cases, one or more (1, 2, 3, 4, 5, 6) of positions 37, 44, 45, 47, 95, and 117 (numbering based on Kabat numbering) are not substituted. In some cases, positions 37 and 47 (numbering based on Kabat numbering) are not substituted. In some cases, position 37 and/or 47 (numbering based on Kabat numbering) is/are substituted. In some cases, positions 44 and 45 (numbering based on Kabat numbering) are not substituted. In certain cases, one or more (1, 2, 3, 4, 5, 6) of positions 62, 65, 67, 72, 76, and 89 (numbering based on Kabat numbering) are not substituted. In certain cases, one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) of positions 1, 2, 37, 44, 45, 46, 47, 60, 61, 62, 65, and 117 (numbering based on Kabat numbering) are not substituted. In another aspect, the disclosure features a bispecific antibody comprising (i) a binding molecule described above; and (ii) a second binding molecule that binds to a different epitope of human CD16 than the binding molecule, or that binds to a different antigen. In some cases, the different antigen is an antigen on a T cell, a neutrophil, an NK cell, or a tumor cell. In some cases, the second binding molecule is a VHH. In other cases, the second binding molecule is a scFv. In certain cases, the second binding molecule is a Fab. In other cases, the second binding molecule is a F(ab)2. In certain instances, the binding molecule and the second binding molecule are connected via a linker. In some cases, the linker is a polypeptide linker. In some cases, the polypeptide linker comprises an IgG Fc domain and optionally a human Attorney Docket No.: 45817-0156WO1 IgG hinge region (which may be mutated – e.g., IgG4PAA hinge). In certain cases, the IgG is a human IgG1, human IgG2, human IgG3, or human IgG4. In one cases, the polypeptide linker comprises an IgG4PAA hinge+ Fc region. In some cases, the linker is a glycine linker, a serine linker, or a glycine-serine linker. In one case, the linker is (G4S)n , wherein n= 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO:35). In another aspect the disclosure provides a bispecific antibody comprising a means for binding CD16 and a second binding molecule that binds to a different antigen. In some instances, the CD16 is human CD16a. In certain cases, the human CD16a is CD16aF176. In other cases, the human CD16a is CD16aV176. In some instances, the CD16 is human CD16b NA2. In some instances, the CD16 is cynomolgus CD16. In some instances, the CD16 is rat CD16. In some cases, the polypeptide binds to human and cynomolgus CD16. In other cases, the polypeptide binds to human, cynomolgus, and rat CD16. In some cases, the polypeptide binds human, cynomolgus, and rat CD16, but not mouse CD16 or human CD16b NA1 variant. In some cases, the different antigen is an antigen on a T cell or NK cell. The means for binding CD16 is a VHH comprising the three VHH CDRs (according to any CDR definition such as Kabat, Chothia, enhanced Chothia, Aho, contact, IMGT) of any one clone listed in Table 8, or a VHH comprising the amino acid sequence of any clone of Table 8. In another aspect, the disclosure provides a human CD16a binding chimeric antigen receptor (CAR) comprising a binding molecule described above. In some cases, the human CD16a binding CAR further comprises a transmembrane domain, a costimulatory domain, and an intracellular signaling domain. In some cases, the transmembrane domain is derived from an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9. In certain cases, the costimulatory domain comprises a costimulatory region of CD3, CD4, CD8, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-l, ICOS, Attorney Docket No.: 45817-0156WO1 lymphocyte function-associated antigen-l (LFA-l), CD2, CD7, LIGHT, NKG2C, B7- H3, or a fragment thereof. In some cases, the intracellular signaling domain comprises a fragment or domain from one or more molecules selected from the group consisting of a T cell receptor (TCR), CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma Rlla, DAP 10, DAP 12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, 0X40, CD30, CD40, PD-l, ICOS, a KIR family protein, lymphocyte function-associated antigen-l (LFA-l), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-l, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD 160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CD 11 a, LFA-l, ITGAM, CD lib, ITGAX, CD l lc, ITGB1, CD29, ITGB2, CD 18, LFA- 1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD 96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD 100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13. In some cases, the human CD16a binding CAR further comprises CD28 and CD137 signaling domains and CD3ζ (CD16-28-137-3ζ). In some instances, the disclosure features a T cell or a NK cell expressing a human CD16a binding CAR described above. In a further aspect, the disclosure provides a human CD16a binding CAR comprising a means for binding human CD16a as well as a transmembrane domain, a costimulatory domain, and an intracellular signaling domain. The means for binding human CD16a is a VHH comprising the three VHH CDRs (according to any CDR definition such as Kabat, Chothia, enhanced Chothia, Aho, contact, IMGT) of any one clone listed in Table 8, or a VHH comprising the amino acid sequence of any clone of Table 8. Attorney Docket No.: 45817-0156WO1 In another aspect, the disclosure relates to a nucleic acid or nucleic acids encoding a binding molecule, a bispecific antibody, or the human CD16a binding CAR described above. In some cases, also provided are a vector or vectors comprising the nucleic acid or nucleic acids. Also provided are a host cell comprising the nucleic acid or nucleic acids, or vector or vectors described herein. In some cases, the host cell is a COS cell, a CHO cell, a 293 cell, or a NIH3T3 cell. In some cases, the host cell is a yeast cell. The disclosure also provides a method of making a binding molecule or a bispecific antibody. The method comprises culturing the host cell described herein under conditions that facilitate expression of the binding molecule or the bispecific antibody, and isolating the binding molecule or the bispecific antibody. In some cases, the method further comprises formulating the binding molecule or the bispecific antibody as a sterile pharmaceutical composition. In another aspect, the disclosure provides a pharmaceutical composition comprising a binding molecule, a bispecific antibody, or a T cell or NK cell expressing the human CD16a binding CAR described herein, and a pharmaceutically acceptable carrier. In yet another aspect, the disclosure features a pharmaceutical composition comprising a means for binding human CD16a and a pharmaceutically acceptable carrier. The means for binding human CD16a is a VHH comprising the three VHH CDRs (according to any CDR definition such as Kabat, Chothia, enhanced Chothia, Aho, contact, IMGT) of any one clone listed in Table 8, or a VHH comprising the amino acid sequence of any clone of Table 8. In another aspect, the disclosure relates to a method of treating a cancer in a human subject in need thereof, killing a tumor cell in a human subject in need thereof, or decreasing the rate of tumor growth in a human subject in need thereof. The method comprises administering to the human subject a therapeutically effective amount of a binding molecule, a bispecific antibody, a T cell or NK cell expressing the human CD16a binding CAR, or a pharmaceutical composition described herein. Attorney Docket No.: 45817-0156WO1 In some instances, the cancer is one which benefits from NK cell targeting and killing of a tumor. In another aspect, the disclosure features polynucleotide comprising an mRNA comprising: (i) a 5' UTR; (ii) an open reading frame (ORF) encoding a binding molecule, a bispecific antibody, or a human CD16a binding CAR described herein; (iii) a stop codon; and (iv) a 3' UTR. In some instances, the mRNA comprises a microRNA (miR) binding site. In some cases, the microRNA is expressed in an immune cell of hematopoietic lineage or a cell that expresses TLR7 and/or TLR8 and secretes pro-inflammatory cytokines and/or chemokines. In certain cases, the microRNA binding site is for a microRNA selected from miR-126, miR-142, miR-144, miR-146, miR-150, miR-155, miR-16, miR-21, miR-223, miR-24, miR-27, miR-26a, or any combination thereof. In some cases, the microRNA binding site is for a microRNA selected from miR126-3p, miR- 142-3p, miR-142-5p, miR-155, or any combination thereof. In certain cases, the microRNA binding site is located in the 3' UTR of the mRNA. In some instances, the mRNA comprises a 5' terminal cap. In certain cases, the 5' terminal cap comprises a Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA- guanosine, 2-azidoguanosine, Cap2, Cap4, 5' methylG cap, or an analog thereof. In some instances, the mRNA comprises a poly-A region. In some cases, the poly-A region is at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90 nucleotides in length, or at least about 100 nucleotides in length. In certain cases, the poly-A region is about 10 to about 200, about 20 to about 180, about 50 to about 160, about 70 to about 140, or about 80 to about 120 nucleotides in length. In some instances, the mRNA comprises at least one chemically modified nucleobase, sugar, backbone, or any combination thereof. In some cases, the at least one chemically modified nucleobase is selected from the group consisting of Attorney Docket No.: 45817-0156WO1 pseudouracil (ψ), N1-methylpseudouracil (m1ψ), 1-ethylpseudouracil, 2-thiouracil (s2U), 4’-thiouracil, 5-methylcytosine, 5-methyluracil, 5-methoxyuracil, and any combination thereof. In certain cases, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or 100% of the uracils are N1-methylpseudouracils. In some instances, the open reading frame consists of nucleosides selected from the group consisting of (i) uridine or a modified uridine, (ii) cytidine or a modified cytidine, (iii) adenosine or a modified adenosine, and (iv) guanosine or a modified guanosine. In some cases, the modified uridine is 1-methylpseudouridine. In certain instances, the mRNA comprises a 5’terminal cap comprising Cap1 and a poly-A region 100 nucleotides in length (SEQ ID NO: 37). In some instances, all uracils of the polynucleotide are N1- methylpseudouracils. In another aspect, the disclosure provides a pharmaceutical composition comprising a polynucleotide described above, and a delivery agent. In some cases, the delivery agent comprises a lipid nanoparticle. In certain cases, the lipid nanoparticle has a mean particle size of from 80 nm to 160 nm. In some cases, the lipid nanoparticle has a polydispersity index (PDI) of from 0.02 to 0.2 and/or a lipid:nucleic acid ratio of from 10 to 20. In some cases, the lipid nanoparticle comprises a neutral lipid, an ionizable amino lipid, a polyethyleneglycol (PEG) lipid, and/or a sterol. In some cases, the neutral lipid is 1,2-distearoyl-sn-glycero-3- phosphocholine. In some cases, the ionizable amino lipid is a compound of Formula (I). In certain cases, the PEG lipid is PEG 2000 dimyristoyl glycerol or 134-hydroxy- 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87 ,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132- tetratetracontaoxatetratriacontahectyl stearate or a salt thereof. In some cases, the sterol is cholesterol, adosterol, agosterol A, atheronals, avenasterol, azacosterol, blazein, cerevisterol, colestolone, cycloartenol, daucosterol, 7-dehydrocholesterol, 5- Attorney Docket No.: 45817-0156WO1 dehydroepisterol, 7-dehydrositosterol, 20α,22R-dihydroxycholesterol, dinosterol, epibrassicasterol, episterol, ergosterol, ergosterol, fecosterol, fucosterol, fungisterol, ganoderenic acid, ganoderic acid, ganoderiol, ganodermadiol, 7α-hydroxycholesterol, 22R-hydroxycholesterol, 27-hydroxycholesterol, inotodiol, lanosterol, lathosterol, lichesterol, lucidadiol, lumisterol, oxycholesterol, oxysterol, parkeol, saringosterol, spinasterol, sterol ester, trametenolic acid, zhankuic acid, or zymosterol. In one case, the sterol is cholesterol. In another aspect, the disclosure features a method of treating a cancer in a human subject in need thereof, killing a tumor cell in a human subject in need thereof, or decreasing the rate of tumor growth in a human subject in need thereof. The method comprises administering to the human subject a therapeutically effective amount of a polynucleotide, or a pharmaceutical composition described above. In some instances, the cancer is one that benefits from NK cell targeting to kill a tumor. In another aspect, the disclosure relates to a kit comprising a binding molecule, bispecific antibody, human CD16a binding CAR, polynucleotide, or a pharmaceutical composition described above and a package insert instructing a user of the kit to administer the binding molecule, bispecific antibody, human CD16a binding CAR, polynucleotide, or a pharmaceutical composition to a human subject in need thereof. In another aspect the disclosure features a lipid nanoparticle comprising an mRNA that encodes a binding molecule comprising a polypeptide that specifically binds CD16, wherein the polypeptide is a VHH and comprises the amino acid sequences of a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO: 1 or 7, optionally wherein the polypeptide binds human, cynomolgus and rat CD16, but not mouse CD16, or human CD16b NA1 variant, and further optionally wherein the binding molecule binds to human NK cells and/or human neutrophils. In some instances, the VHH-CDR1, VHH-CDR2, and VHH-CDR3 comprise the sequences set forth in SEQ ID NOs: (i) 2, 3, 4, respectively; (ii) 25, 26, 27, respectively; (iii) 28, 29, 27, respectively; (iv) 28, 26, 27, respectively; (v) 30, 31, Attorney Docket No.: 45817-0156WO1 32, respectively; (vi) 30, 36, 32, respectively; or (vii) 33, 34, 27, respectively. In certain instances, the VHH comprises a sequence that is 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%, at least 99%, or 100% identical to any amino acid sequence set forth in Table 8; or wherein the VHH comprises a sequence with 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 8, 7, 6, 5, 4, 3, 2, or 1 substitution relative to any amino acid sequence set forth in Table 8. In some cases, the polypeptide further comprises an agent selected from the group consisting of a purification tag, a fluorophore, a drug, a photosensitizer, a nanoparticle, a toxic agent, a radionuclide, a VHH, an Fab, a scFv, a multimerization module, a moiety that facilitates the polypeptide crossing the blood brain barrier, and a half-life extension moiety, optionally wherein when the agent is a VHH, an Fab, or a scFv, it is attached or linked to the N-terminus of the polypeptide. In certain cases, the polypeptide further comprises a human Ig Fc domain, optionally further comprising a human Ig hinge domain, and further optionally wherein the human Ig is a human IgG1, human IgG2, human IgG3, or human IgG4. In some cases, the polypeptide comprises an amino acid of an human IgG4 PAA hinge and Fc domain sequence, optionally wherein the human IgG4PAA sequence is at the N-terminus of the polypeptide, optionally wherein the Fc domain includes mutations that promote heterodimerization. In certain instances, the lipid nanoparticle comprises an ionizable amino lipid, a PEG-lipid, a structural lipid, and a phospholipid. In certain cases the lipid nanoparticle comprises: about 47.5 mol % of ionizable amino lipid; about 39 mol % of cholesterol; about 10.5 mol % of DSPC; and about 3 mol % of PEG-lipid. In certain cases, the lipid nanoparticle comprises: 47.5 mol % of an ionizable amino lipid; 39 mol % of cholesterol; 10.5 mol % of DSPC; and 3 mol % of a PEG-lipid. In some cases, the ionizable amino acid lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(8- (nonyloxy)-8-oxooctyl)amino)octanoate which has the formula Attorney Docket No.: 45817-0156WO1 (Compound I-1), or a salt thereof. is heptadecan-9-yl 8-((2-
Figure imgf000021_0001
oxo- amino)octanoate or a salt thereof. In some cases, the PEG lipid is 134-hydroxy- 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,8 4,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132- tetratetracontaoxatetratriacontahectyl stearate which has the formula
Figure imgf000021_0002
cases, all uridines of the mRNA are 1-methylpseudouridine. In certain cases, the mRNA comprises a 5’ terminal cap, a 5’UTR, a 3’UTR, and a poly A tail. In another aspect, the disclosure features a method of treating a cancer in a human subject in need thereof; killing a tumor cell in a human subject in need thereof; or decreasing the rate of tumor growth in a human subject in need thereof. The method comprises administering to the human subject a therapeutically effective amount of a LNP described above. In some instances, the cancer is one that benefits from NK cell targeting to kill a tumor. In some cases, the LNP is administered subcutaneously. In other cases, the LNP is administered intravenously. In yet other cases, the LNP is administered intramuscularly. In yet another aspect, the disclosure relates to a kit comprising LNP described above and a package insert instructing a user of the kit to administer the LNP to a human subject in need thereof. In some instances, the human subject has a cancer. The following drawings and detailed description are exemplary and explanatory, but it is not intended to be limiting. Attorney Docket No.: 45817-0156WO1 BRIEF DESCRIPTION OF THE DRAWINGS FIG.1 shows results of FACS binding affinity assay of His labelled anti- CD16 sdAb in CHO cell lines stably expressing CD16a and CD16b compared to control antibody, in the presence and absence of HSA. FIG.2 shows results of FACS binding affinity assay of His labelled anti- CD16 sdAb on neutrophils and NK cells. Anti-His or anti-VHH antibodies were used to detect CD16 binding on each cell type. DETAILED DESCRIPTION The compositions and methods of the disclosure feature CD16 heavy chain variable domains (anti-CD16 VHH domains) and complementarity determining regions (CDRs) thereof, as well as antibodies, antigen-binding fragments, and other related binding proteins that comprise the disclosed VHH domains or CDRs. The disclosure also provides nucleic acids encoding the disclosed proteins, and methods of using such antibodies, antigen-binding fragments, binding proteins, and nucleic acids. In some embodiments, the antibodies are single domain antibodies (e.g., a VHH). In some embodiments, the antibodies are full-length antibodies that include a pair of heavy chains and a pair of light chains, each containing a variable domain and a constant region. In some embodiments, the antibodies are single-domain antibodies (sdAbs) or single chain Fv (scFv) molecules, among other antigen-binding fragments described herein. In some embodiments, the antibodies are bispecific antibodies (i.e., engagers) that bind to CD16 and another antigenic target. In some cases, these binding molecules bind human CD16a. In other cases, these binding molecules bind human and cynomolgus CD16. In certain cases, these binding molecules bind human, cynomolgus, and rat CD16. In some cases, these binding molecules bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1. The compositions and methods of the disclosure exhibit a series of beneficial biochemical properties. For example, VHH domains described herein and antibodies and binding polypeptides are capable of binding CD16 with high affinity. More Attorney Docket No.: 45817-0156WO1 specifically, the anti-CD16 VHH domains described herein is not affected by the presence of human serum. Because the binding of the instant VHH domains does not compete with human IgG for binding to CD16a, these VHH domains are particularly suited for therapies where binding to CD16a in vivo is desired. Definitions As used herein, the term “about” refers to a stated numerical term and a value that is no more than 10% above or below the value being described. For example, the term “about 5 nM” indicates disclosure of both the stated value of 5 nM and a range of from 4.5 nM to 5.5 nM. As used herein, the term “CD16 antibody” or “CD16-antibody” refers to an antibody or fragment thereof that specifically binds to, or is immunologically reactive with, CD16. Similarly, a “CD16 binding protein” or “anti-CD16 binding protein” refers to any protein comprising at least one domain (such as a VHH domain disclosed herein) that specifically binds to or is immunologically reactive with CD16. Accordingly, a “CD16 binding protein” or “anti-CD16 binding protein” includes, for example, anti-CD16 antibodies (both monospecific and bispecific), and other constructs that bind to CD16. As used herein, the term “antibody” (Ab) refers to an immunoglobulin molecule, or a molecule having an immunoglobulin-like scaffold, that specifically binds to, or is immunologically reactive with, a particular antigen. The term “antibody” includes polyclonal, monoclonal, genetically engineered, and otherwise modified forms of antibodies, including, but not limited to, chimeric antibodies, humanized antibodies, and heteroconjugate antibodies (e.g., bi- tri-, quad-, and multispecific antibodies, diabodies, triabodies, and tetrabodies). The term “antigen-binding fragment,” as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be performed by fragments of a full- length antibody. The antibody fragments can be, e.g., a single-domain antibody Attorney Docket No.: 45817-0156WO1 (sdAb), Fab, F(ab’)2, Fab Fv, VHH, scFv, SMIP, diabody, a triabody, an affibody, an aptamer, or recombinant fragments thereof. Examples of binding fragments encompassed by the term “antigen-binding fragment” of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb including VH and VL domains; (vi) a dAb fragment (Ward et al., Nature 341:544-546, 1989), which consists of a VH domain; (vii) a dAb which consists of a VH or a VL domain; (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426 (1988), and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). These antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments can be screened for utility in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in some embodiments, by chemical peptide synthesis procedures known in the art. As used herein, term “biologic” refers to a medicinal preparation that is created by biological processes rather than chemical synthesis. Exemplary biologics include certain vaccines, antibodies, cell preparations, tissue preparations, recombinant proteins, nucleic acids, cytokines, growth factors, enzymes, peptides, proteins, carbohydrates, or combinations thereof. Biologics also include biosimilar molecules (or biosimilars), which are molecular entities that are structurally similar to and have no clinically meaningful differences in terms of safety, purity, and potency from known biologics. Attorney Docket No.: 45817-0156WO1 As used herein, the term “bispecific antibodies” refers to monoclonal, often human or humanized antibodies that have binding specificities for at least two different antigens. Bispecific CD16 antibodies of the disclosure may have binding specificities that are directed towards CD16 and any other antigen, e.g., for a cell- surface protein, receptor, receptor subunit, or tissue-specific antigen. A bispecific antibody may also be an antibody or antigen-binding fragment thereof that includes two separate antigen-binding domains (e.g., two scFvs joined by a linker). The scFvs may bind the same antigen or different antigens. For the purposes of the present disclosure, the term “engager” may be used interchangeably with “bispecific antibody.” As used herein, the term “multispecific antibodies” refers to monoclonal, often human or humanized antibodies that have binding specificities for at least two different antigens, e.g., bispecific antibodies. Multispecific CD16 antibodies of the disclosure may have binding specificities that are directed towards CD16 and any other antigen(s), e.g., for a cell-surface protein, receptor, receptor subunit, or tissue- specific antigen. A multispecific antibody may also be an antibody or antigen-binding fragment thereof that includes multiple separate antigen-binding domains (e.g., two scFvs joined by a linker). The scFvs may bind the same antigen or different antigens. Such structures can include, but not limited to, IgG-Fv, IgG-(scFv)2, DVD-Ig, (scFv)2-(scFv)2-Fc and (scFv)2-Fc-(scFv)2. In case of IgG-(scFv)2, the scFv can be attached to either the N-terminal or the C- terminal end of either the heavy chain or the light chain. Exemplary multi-specific molecules that include Fc regions and into which CD16 antibodies or antigen-binding fragments thereof are disclosed in Kontermann, 2012, mAbs 4(2):182-197, Yazaki et al., Protein Engineering, Design & Selection 26(3):187- 193 (2013), and Grote et al., in Proetzel & Ebersbach (eds.), Antibody Methods and Protocols, Methods in Molecular Biology vol.901, chapter 16:247-263 (2012); incorporated herein by reference. In some embodiments, antibody fragments can be components of multi-specific molecules without Fc regions, based on fragments of IgG or DVD or scFv. Exemplary multi-specific molecules that lack Fc regions and into which antibodies or antibody fragments can be incorporated include scFv dimers (diabodies), trimers (triabodies) and tetramers (tetrabodies), Fab Attorney Docket No.: 45817-0156WO1 dimers (conjugates by adhesive polypeptide or protein domains) and Fab trimers (chemically conjugated), are described by Hudson and Souriau, 2003, Nature Medicine 9:129-134; incorporated herein by reference. Multi-specific antibodies also include tetravalent bispecific antibody. As used herein, the term “chimeric” antibody refers to an antibody having portions of its sequence derived from at least two different sources, such as variable domain sequences (e.g., CDR sequences) derived from an immunoglobulin of one source organism, such as rat or mouse, and constant regions derived from an immunoglobulin of a different organism (e.g., a human, another primate, pig, goat, rabbit, hamster, cat, dog, guinea pig, member of the bovidae family (such as cattle, bison, buffalo, elk, and yaks, among others), cow, sheep, horse, bison, llama, camel, or shark among others). Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, Science 229(4719):1202-7 (1985); Oi et al., BioTechniques. 4:214-221 (1986); Gillies et al., J. Immunol. Methods 125:191-202 (1985); U.S. Pat. Nos.5,807,715; 4,816,567; and 4,816,397; incorporated herein by reference. As used herein, the term “complementarity determining region” or “CDR” refers to a hypervariable region found in the light chain and/or the heavy chain variable domains of an antibody. The more highly conserved portions of variable domains are called the framework regions (FRs). As is appreciated in the art, the amino acid positions that delineate a hypervariable region of an antibody can vary, depending on the context and the various definitions known in the art. Some positions within a variable domain may be viewed as hybrid hypervariable positions in that these positions can be deemed to be within a hypervariable region under one set of criteria while being deemed to be outside a hypervariable region under a different set of criteria. One or more of these positions can also be found in extended hypervariable regions. The disclosure includes antibodies comprising modifications in these hybrid hypervariable positions. The variable domains of native heavy and light chains each comprise four framework regions that primarily adopt a β-sheet configuration, connected by three CDRs, which form loops that connect, and in some cases form part of, the β-sheet structure. The CDRs in each chain are held together in Attorney Docket No.: 45817-0156WO1 close proximity by the FR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3- FR4 and, with the CDRs from the other antibody chains, contribute to the formation of the target binding site of antibodies (see, Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. (1987); incorporated herein by reference). As used herein, numbering of immunoglobulin amino acid residues is performed according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated. As used herein, the terms “conservative mutation,” “conservative substitution,” “conservative amino acid substitution,” and the like refer to a substitution of one or more amino acids for one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and/or steric volume. These properties are summarized for each of the twenty naturally-occurring amino acids in Table 1 below. Table 1 – Representative physicochemical properties of naturally-occurring amino acids Electrostatic 3 1 Side- e e e e e
Figure imgf000027_0001
Attorney Docket No.: 45817-0156WO1 equilibrium at pH 7.4 e e e
Figure imgf000028_0001
From this table it is appreciated that the conservative amino acid families include, e.g., (i) G, A, V, L, I, P, and M; (ii) D and E; (iii) C, S and T; (iv) H, K and R; (v) N and Q; and (vi) F, Y and W. A conservative mutation or substitution is therefore one that substitutes one amino acid for a member of the same amino acid family (e.g., a substitution of Ser for Thr or Lys for Arg). As used herein, the term “conjugate” refers to a compound formed by the chemical bonding of a reactive functional group of one molecule with an appropriately reactive functional group of another molecule. Conjugates may additionally be produced, e.g., as two polypeptide domains covalently bound to one another as part of a single polypeptide chain that is synthesized by the translation of a single RNA transcript encoding both polypeptides in frame with one another. Attorney Docket No.: 45817-0156WO1 As used herein in the context of a CD16-binding protein, the term “construct” refers to a fusion protein containing a first polypeptide domain bound to a second polypeptide domain. The polypeptide domains may each independently be anti-CD16 single chain polypeptides, for instance, as described herein. The first polypeptide domain may be covalently bound to the second polypeptide domain, for instance, by way of a linker, such as a peptide linker or a disulfide bridge, among others. Exemplary linkers that may be used to join the polypeptide domains of a CD16 construct include, without limitation, those that are described in Leriche et al., Bioorg. Med. Chem., 20:571-582 (2012), the disclosure of which is incorporated herein by reference in its entirety. As used herein, the term “derivatized antibodies” refers to antibodies that are modified by a chemical reaction so as to cleave residues or add chemical moieties not native to an isolated antibody. Derivatized antibodies can be obtained by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by addition of known chemical protecting/blocking groups, proteolytic cleavage, and/or linkage to a cellular ligand or other protein. Any of a variety of chemical modifications can be carried out by known techniques, including, without limitation, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. using established procedures. Additionally, the derivative can contain one or more non-natural amino acids, e.g., using amber suppression technology (see, e.g., US Patent No.6,964,859; incorporated herein by reference). As used herein, the term “diabodies” refers to bivalent antibodies comprising two polypeptide chains, in which each polypeptide chain includes VH and VL domains joined by a linker that is too short (e.g., a linker composed of five amino acids) to allow for intramolecular association of VH and VL domains on the same peptide chain. This configuration forces each domain to pair with a complementary domain on another polypeptide chain so as to form a homodimeric structure. Accordingly, the term “triabodies” refers to trivalent antibodies comprising three peptide chains, each of which contains one VH domain and one VL domain joined by a linker that is exceedingly short (e.g., a linker composed of 1-2 amino acids) to permit Attorney Docket No.: 45817-0156WO1 intramolecular association of VH and VL domains within the same peptide chain. In order to fold into their native structure, peptides configured in this way typically trimerize so as to position the VH and VL domains of neighboring peptide chains spatially proximal to one another to permit proper folding (see, Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993); incorporated herein by reference). As used herein, a “tetravalent bispecific antibody” or “tetravalent bsAB” refers to an antibody comprising two peptide chains. Each of the peptide chains contains two sdAB sequences. For example, each peptide chain may include an anti- CD16 sdAB and a second sdAB that binds to a different target than the first sdAB. The peptide chains can dimerize at the hinge region of their respective Fc domains, thus resulting in a tetravalent construct. As used herein, the term “endogenous” describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell). As used herein, the term “exogenous” describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is not found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell). Exogenous materials include those that are provided from an external source to an organism or to cultured matter extracted there from. As used herein, the term “framework region” or “FW region” includes amino acid residues that are adjacent to the CDRs. FW region residues may be present in, for example, human antibodies, rodent-derived antibodies (e.g., murine antibodies), humanized antibodies, primatized antibodies, chimeric antibodies, antibody fragments (e.g., Fab fragments), single-chain antibody fragments (e.g., scFv fragments), antibody domains, and bispecific antibodies, among others. Attorney Docket No.: 45817-0156WO1 As used herein, the term “fusion protein” refers to a protein that is joined via a covalent bond to another molecule. A fusion protein can be chemically synthesized by, e.g., an amide-bond forming reaction between the N-terminus of one protein to the C-terminus of another protein. Alternatively, a fusion protein containing one protein covalently bound to another protein can be expressed recombinantly in a cell (e.g., a eukaryotic cell or prokaryotic cell) by expression of a polynucleotide encoding the fusion protein, for example, from a vector or the genome of the cell. A fusion protein may contain one protein that is covalently bound to a linker, which in turn is covalently bound to another molecule. Examples of linkers that can be used for the formation of a fusion protein include peptide-containing linkers, such as those that contain naturally occurring or non-naturally occurring amino acids. In some embodiments, it may be desirable to include D-amino acids in the linker, as these residues are not present in naturally-occurring proteins and are thus more resistant to degradation by endogenous proteases. Linkers can be prepared using a variety of strategies that are well known in the art, and depending on the reactive components of the linker, can be cleaved by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (Leriche et al., Bioorg. Med. Chem., 20:571-582 (2012)). As used herein, the term “heterospecific antibodies” refers to monoclonal (e.g., human or humanized) antibodies that have binding specificities for at least two different antigens. Traditionally, the recombinant production of heterospecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Milstein et al., Nature 305:537 (1983)). Similar procedures are disclosed, e.g., in WO 93/08829, U.S. Pat. Nos.6,210,668; 6,193,967; 6,132,992; 6,106,833; 6,060,285; 6,037,453; 6,010,902; 5,989,530; 5,959,084; 5,959,083; 5,932,448; 5,833,985; 5,821,333; 5,807,706; 5,643,759, 5,601,819; 5,582,996, 5,496,549, 4,676,980, WO 91/00360, WO 92/00373, EP 03089, Traunecker et al., EMBO J.10:3655 (1991), Suresh et al., Methods in Enzymology 121:210 (1986); incorporated herein by reference. Heterospecific antibodies can include Fc mutations that enforce correct chain Attorney Docket No.: 45817-0156WO1 association in multi-specific antibodies, as described by Klein et al., mAbs 4(6):653- 663 (2012); incorporated herein by reference. As used herein, the term “human antibody” refers to an antibody in which substantially every part of the protein (e.g., CDR, framework, CL, CH domains (e.g., CH1, CH2, CH3), hinge, (VL, VH)) is substantially non-immunogenic in humans, with only minor sequence changes or variations. A human antibody can be produced in a human cell (e.g., by recombinant expression), or by a non-human animal or a prokaryotic or eukaryotic cell that is capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and/or light chain) genes. Further, when a human antibody is a single-chain antibody, it can include a linker peptide that is not found in native human antibodies. For example, an Fv can comprise a linker peptide, such as two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain. Human antibodies can be made by a variety of methods known in the art including phage display methods using antibody libraries derived from human immunoglobulin sequences. See, U.S. Patent Nos.4,444,887 and 4,716,111; and PCT publications WO 1998/46645; WO 1998/50433; WO 1998/24893; WO 1998/16654; WO 1996/34096; WO 1996/33735; and WO 1991/10741; incorporated herein by reference. Human antibodies can also be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. See, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; U.S. Patent Nos.5,413,923; 5,625, 126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598; incorporated by reference herein. As used herein, the term “humanized” antibodies refers to forms of non- human (e.g., murine) antibodies that are chimeric immunoglobulins, or immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other target-binding subdomains of antibodies), which contain minimal sequences derived from non-human immunoglobulin. In general, a humanized antibody will contain substantially all of at least one, and typically two, variable domains, in which all or Attorney Docket No.: 45817-0156WO1 substantially all of the CDRs correspond to those of a non-human immunoglobulin. All or substantially all of the FRs may also be those of a human immunoglobulin sequence. The humanized antibody may also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence. Methods of antibody humanization are known in the art. See, e.g., Riechmann et al., Nature 332:323-7 (1988); U.S. Patent Nos: 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370 to Queen et al; EP239400; PCT publication WO 91/09967; U.S. Patent No.5,225,539; EP592106; and EP519596; the disclosure of each of which is incorporated herein by reference. As used herein, the term “lipid nanoparticle” refers to a transfer vehicle including one or more lipids (e.g., cationic lipids, non-cationic lipids, and PEG- modified lipids). Exemplary lipid nanoparticles are formulated to deliver one or more mRNA to one or more target cells. Examples of suitable lipids include, for example, the phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides). Lipid nanoparticles may contain a cationic lipid, or a lipid species with a net positive charge at a selected pH (e.g., physiological pH), to encapsulate and/or enhance the delivery of mRNA into the target cells. As used herein, the terms “messenger RNA” or “mRNA” refer to any polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ, or ex vivo. Traditionally, the basic components of an mRNA molecule include a coding region, a 5’UTR, a 3’UTR, a 5’ cap, and a poly-A tail. As used herein, the terms “modified messenger RNA” or “modified mRNA” refer to mRNA polynucleotides that include naturally occurring and/or non-naturally occurring modifications, for example, of a sugar, a nucleobase, or an internucleoside linkage (e.g., to a linking phosphate, to a phosphodiester linkage, or to the phosphodiester backbone). Non-natural modified nucleotides may be introduced during synthesis of post-synthesis of the polynucleotides to achieve desired functions Attorney Docket No.: 45817-0156WO1 or properties. The modifications may be present on an internucleoside linkage, purine or pyrimidine base, or sugar. The modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a polynucleotide may be chemically modified. As used herein, the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. As used herein, the term “nucleic acid” includes any compound containing a continuous segment of nucleosides joined by way of one or more internucleoside linkages (e.g., polymers of nucleosides linked by way of phosphodiester bonds). Exemplary nucleic acids include ribonucleic acids (RNA, in particular mRNA), deoxyribonucleic acids (DNA), threose nucleic acids (TNA), glycol nucleic acids (GNA), peptide nucleic acids (PNA), locked nucleic acids (LNA), or hybrids thereof. Nucleic acids also include RNAi inducers, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNAs, tRNAs, RNAs that induce triple spiral formation, aptamers, vectors, and the like. In a preferred embodiment, the nucleic acid is one or more modified messenger RNAs (modified mRNAs). As used herein, the terms “percent (%) sequence identity,” “percent (%) identity,” and the like, with respect to a reference polynucleotide or polypeptide sequence, is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence Attorney Docket No.: 45817-0156WO1 identity values may be generated using the sequence comparison computer program BLAST. As an illustration, the percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which can alternatively be phrased as a given nucleic acid or amino acid sequence, A that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as: 100 multiplied by (the fraction X/Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program’s alignment of A and B, and where Y is the total number of nucleic acids in B. It will be appreciated that where the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A. As used herein, the term “primatized antibody” refers to an antibody comprising framework regions from primate-derived antibodies and other regions, such as CDRs and/or constant regions, from antibodies of a non-primate source. Methods for producing primatized antibodies are known in the art. See, e.g., U.S. Patent Nos.5,658,570; 5,681,722; and 5,693,780; incorporated herein by reference. For instance, a primatized antibody or antigen-binding fragment thereof described herein can be produced by inserting the CDRs of a non-primate antibody or antigen- binding fragment thereof into an antibody or antigen-binding fragment thereof that contains one or more framework regions of a primate. As used herein, the term “operatively linked” in the context of a polynucleotide fragment is intended to mean that the two polynucleotide fragments are joined such that the amino acid sequences encoded by the two polynucleotide fragments remain in-frame. As used herein, the term “pharmacokinetic profile” refers to the absorption, distribution, metabolism, and clearance of a therapeutic agent (e.g., a polypeptide, such as an anti-CD16 antibody, antigen-binding fragment thereof, single-chain Attorney Docket No.: 45817-0156WO1 polypeptide, or construct of the disclosure) over time following administration of the drug to a patient. As used herein, the term “regulatory sequence” includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation, e.g., of antibody chain genes. Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, CA, 1990); incorporated herein by reference. As used herein, the term “scFv” refers to a single-chain Fv antibody in which the variable domains of the heavy chain and the light chain from an antibody have been joined to form one chain. ScFv fragments contain a single polypeptide chain that includes the variable region of an antibody light chain (VL) (e.g., CDR-L1, CDR-L2, and/or CDR-L3) and the variable region of an antibody heavy chain (VH) (e.g., CDR- H1, CDR-H2, and/or CDR-H3) separated by a linker. The linker that joins the VL and VH regions of a scFv fragment can be a peptide linker composed of proteinogenic amino acids. Alternative linkers can be used to so as to increase the resistance of the scFv fragment to proteolytic degradation (e.g., linkers containing D-amino acids), in order to enhance the solubility of the scFv fragment (e.g., hydrophilic linkers such as polyethylene glycol-containing linkers or polypeptides containing repeating glycine and serine residues), to improve the biophysical stability of the molecule (e.g., a linker containing cysteine residues that form intramolecular or intermolecular disulfide bonds), or to attenuate the immunogenicity of the scFv fragment (e.g., linkers containing glycosylation sites). ScFv molecules are known in the art and are described, e.g., in US patent 5,892,019, Flo et al., Gene 77:51 (1989); Bird et al., Science 242:423 (1988); Pantoliano et al., Biochemistry 30:10117 (1991); Milenic et al., Cancer Research 51:6363 (1991); and Takkinen et al., Protein Engineering 4:837 (1991). The VL and VH domains of a scFv molecule can be derived from one or more antibody molecules. It will also be understood by one of ordinary skill in the art that the variable regions of the scFv molecules described herein can be modified such that they vary in amino acid sequence from the antibody molecule from which they were Attorney Docket No.: 45817-0156WO1 derived. For example, in one embodiment, nucleotide or amino acid substitutions leading to conservative substitutions or changes at amino acid residues can be made (e.g., in CDR and/or framework residues). Alternatively or in addition, mutations are made to CDR amino acid residues to optimize antigen binding using art recognized techniques. ScFv fragments are described, for example, in WO 2011/084714; incorporated herein by reference. As used herein, the terms “single-domain antibody,” “sdAb,” “nanobody,” and “VHH antibody” are used interchangeably to refer to a single-chain antibody fragment that contains only a single heavy-chain variable domain. Unlike a traditional, full-length antibody, which includes heavy chains and light chains, each containing a corresponding variable domain (i.e., a heavy chain variable domain, VH, and a light chain variable domain, VL) having three CDRs, a single-domain antibody only includes one heavy-chain variable domain having a total of three CDRs (referred to herein as CDR-H1, CDR-H2, and CDR-H3). As used herein, the phrase “specifically binds” refers to a binding reaction which is determinative of the presence of an antigen in a heterogeneous population of proteins and other biological molecules that is recognized, e.g., by an antibody or antigen-binding fragment thereof, with particularity. An antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of less than 100 nM. For example, an antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen via the antigen binding domain with a KD of up to 100 nM (e.g., between 1 pM and 100 nM). An antibody or antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or epitope thereof will exhibit a KD of greater than 100 nM (e.g., greater than 500 nm, 1 µM, 100 µM, 500 µM, or 1 mM) for that particular antigen or epitope thereof. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein or carbohydrate. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein or carbohydrate. See, Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Attorney Docket No.: 45817-0156WO1 Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. As used herein, the terms “subject” and “patient” refer to an organism that receives treatment (e.g., by administration of a CD16 polypeptide, such as an antibody, antigen-binding fragment thereof, single-chain polypeptide, or construct described herein) for a particular disease or condition, such as a cancer or an immunological disorder (e.g., an autoimmune disease). Examples of subjects and patients include mammals, such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the bovidae family (such as cattle, bison, buffalo, and yaks, among others), sheep, and horses, among others, receiving treatment for a cancer, immunological diseases or conditions, such as autoimmune disorders (e.g., allograft rejection) and graft-versus-host disease, among others. A patient that may be treated using the compositions and methods described herein may have an established disease, in which case the patient has been diagnosed as having the disease and has shown symptoms of the disease for a prolonged period of time (e.g., over the course of days, weeks, months, or years). Alternatively, a patient may be symptomatic for a particular disease, but has yet to be diagnosed with the disease by a physician. Other patients that may be treated using the compositions and methods described herein include those that have been diagnosed as having a disease or disorder, and may or may not be showing symptoms of the disease as of yet. As used herein, the term “transfection” refers to any of a wide variety of techniques commonly used for the introduction of a nucleic acid molecule, e.g., exogenous DNA or RNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, lipofection, calcium- phosphate precipitation, DEAE- dextran transfection and the like. As used herein, the terms “treat” or “treatment” refer to therapeutic treatment, in which the object is to inhibit or slow down (lessen) an undesired physiological change or disorder, such as a cancer or an immunological disorder (e.g., autoimmune Attorney Docket No.: 45817-0156WO1 disorders (e.g., allograft rejection) and graft-versus-host disease, among others). Beneficial or desired clinical results of treatment include, without limitation, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Those in need of treatment include those already having the condition or disorder, as well as those prone to have the condition or disorder or those in which the condition or disorder is to be inhibited. As used herein the term “variable region CDR” includes amino acids in a CDR or complementarity determining region as identified using sequence or structure-based methods. As used herein, the term “CDR” or “complementarity determining region” refers to the noncontiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem.252:6609-6616 (1977) and Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.91 -3242 (1991); by Chothia et al., J. Mol. Biol.196:901-917 (1987), and by MacCallum et al., J. Mol. Biol.262:732-745 (1996) where the definitions include overlapping or subsets of amino acid residues when compared against each other. In certain embodiments, the term “CDR” is a CDR as defined by Kabat based on sequence comparisons. As used herein, the term “vector” includes a nucleic acid vector, e.g., a DNA vector, such as a plasmid, an RNA vector, virus or other suitable replicon (e.g., viral vector). A variety of vectors have been developed for the delivery of polynucleotides encoding exogenous proteins into a prokaryotic or eukaryotic cell. Examples of such expression vectors are disclosed in, e.g., WO 1994/11026; incorporated herein by reference. Expression vectors described herein contain a polynucleotide sequence as well as, e.g., additional sequence elements used for the expression of proteins and/or the integration of these polynucleotide sequences into the genome of a mammalian cell. Certain vectors that can be used for the expression of antibodies, antibody fragments, and/or binding proteins described herein include plasmids that contain Attorney Docket No.: 45817-0156WO1 regulatory sequences, such as promoter and enhancer regions, which direct gene transcription. Other useful vectors for expression of antibodies, antibody fragments, and/or binding proteins contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements include, e.g., 5’ and 3’ untranslated regions, an internal ribosomal entry site (IRES), and polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector. The expression vectors described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker include genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, or nourseothricin. As used herein, the term “VH” refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, or Fab. References to “VL” refer to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv or Fab. Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules which lack target specificity. Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain of a native antibody has at the amino terminus a variable domain (VH) followed by a number of constant domains. Each light chain of a native antibody has a variable domain at the amino terminus (VL) and a constant domain at the carboxy terminus. Structural characteristics of exemplary anti-CD16 antibodies Among the molecular features of anti-CD16 antibodies and binding proteins comprising anti-CD16 VHH domains described herein, it will be appreciated by one of skill in the art that the CDRs are those regions that predominantly dictate the CD16-binding properties of the molecule. This selection provides amino acid Attorney Docket No.: 45817-0156WO1 sequence information for the CDRs of anti-CD16 VHH domains and antibodies comprising such anti-CD16 VHH domains of the disclosure. In some embodiments, the disclosure provides an anti-CD16 antibody or binding protein having one, two, or three of the CDRs described in Table 4, below. In one instance, the disclosure features an anti-CD16 binding molecule comprising a VHH that binds to human CD16a, the VHH comprising a VHH-CDR1, a VHH- CDR2, and a VHH-CDR3 of any one of SEQ ID NO: 1 or 5-24. The VHH-CDRs can be based on any known definition in the art. For example, the anti-CD16 binding molecule comprises the three VHH-CDRs of SEQ ID NO:1 based on any single definition as shown in Table 5. Exemplary CDRs of SEQ ID NO:7 (VHH1-H3) are shown in Table A below. Table A: Exemplary CDR Definitions of anti-CD16 VHH, VHH1-H3 CDR VHH-CDR1 VHH-CDR2 VHH-CDR3 Definition
Figure imgf000041_0001
In one instance, the binding molecule comprises the three VHH-CDRs of SEQ ID NO:7 based on any single CDR definition as shown in Table A. For example, in some embodiments, an anti-CD16 antibody or binding protein of the disclosure is an antibody (e.g., a VHH or bispecific antibody) or antigen- binding fragment thereof (e.g., a scFv) having one or more of the following CDRs: Attorney Docket No.: 45817-0156WO1 (a) a CDR1 having the amino acid sequence GRTDSIYA (SEQ ID NO: 2) or an amino acid sequence having up to two amino acid substitutions (e.g., conservative amino acid substitutions) relative to SEQ ID NO: 2; (b) a CDR2 having the amino acid sequence INSNTGRT (SEQ ID NO: 3), or an amino acid sequence having up to two amino acid substitutions (e.g., conservative amino acid substitutions) relative to SEQ ID NO: 3; and (c) a CDR3 having the amino acid sequence AAGRGYGLLSISSNWYNY (SEQ ID NO: 4), or an amino acid sequence having up to two amino acid substitutions (e.g., conservative amino acid substitutions) relative to SEQ ID NO: 4. In certain cases, the anti-CD16 antibody or binding protein comprises the three CDRs based on any single definition as shown in Table A or Table 5. In some cases, the anti-CD16 antibody or binding protein binds human CD16a. In other cases, the anti- CD16 antibody or binding protein binds cynomolgus CD16. In certain cases, the anti- CD16 antibody or binding protein binds human CD16a and cynomolgus CD16. In some cases, the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1. In some embodiments, the anti-CD16 antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH1, shown below (CDR sequences shown in bold): QVQLVESGGGLVQAGGSLRLSCAASGRTDSIYAMGWFRQAPGKERDFVAAI NSNTGRTYHADSVKGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAAGRG YGLLSISSNWYNYWGQGTQVTVSS (VHH1, SEQ ID NO: 1). In some embodiments, the anti-CD16 antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the anti-CD16 Attorney Docket No.: 45817-0156WO1 antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the anti-CD16 antibody or binding protein contains a VHH domain having the amino acid sequence of SEQ ID NO: 1. In some cases, the anti-CD16 antibody or binding protein binds human CD16a. In other cases, the anti-CD16 antibody or binding protein binds cynomolgus CD16. In certain cases, the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16. In some cases, the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H1, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKELDFVSAIN SNTGRTYHADSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCAAGRGY GLLSISSNWYNYWGQGTLVTVSS (VHH1-H1, SEQ ID NO: 5). In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 5. In some cases, the anti- CD16 antibody or binding protein binds human CD16a. In other cases, the anti-CD16 antibody or binding protein binds cynomolgus CD16. In certain cases, the anti-CD16 Attorney Docket No.: 45817-0156WO1 antibody or binding protein binds human CD16a and cynomolgus CD16. In some cases, the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H2, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKELEFVAAI NSNTGRTYHADSVKGRFTISRDNAKNTLYLQMNSLKAEDTAVYYCAAGRG YGLLSISSNWYNYWGQGTLVTVSS (VHH1-H2, SEQ ID NO: 6). In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H3, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKEREFVSAIN SNTGRTYHADSVKGRFTISRDNAKNMVYLQMNSLRAEDTAVYYCAAGRGY GLLSISSNWYNYWGQGTLVTVSS (VH1-H3, SEQ ID NO: 7). Attorney Docket No.: 45817-0156WO1 In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 7. In some cases, the anti- CD16 antibody or binding protein binds human CD16a. In other cases, the anti-CD16 antibody or binding protein binds cynomolgus CD16. In certain cases, the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16. In some cases, the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H4, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKELEFVSAIN SNTGRTYHADSVKGRFTISRDNAKNMVYLQMNSLKAEDTAVYYCAAGRGY GLLSISSNWYNYWGQGTLVTVSS (VHH1-H4, SEQ ID NO: 8). In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the aanti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 8. In some Attorney Docket No.: 45817-0156WO1 embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 8. In some cases, the anti- CD16 antibody or binding protein binds human CD16a. In other cases, the anti-CD16 antibody or binding protein binds cynomolgus CD16. In certain cases, the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16. In some cases, the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H5, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKELEFVSAIN SNTGRTYHADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAAGRGY GLLSISSNWYNYWGQGTLVTVSS (VHH1-H5, SEQ ID NO: 9). In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 9. In some cases, the anti- CD16 antibody or binding protein binds human CD16a. In other cases, the anti-CD16 antibody or binding protein binds cynomolgus CD16. In certain cases, the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16. In some cases, the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1. Attorney Docket No.: 45817-0156WO1 In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H6, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKEREFVSAIN SNTGRTYHADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAGRGY GLLSISSNWYNYWGQGTLVTVSS (VHH1-H6, SEQ ID NO: 10). In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 10. In some cases, the anti- CD16 antibody or binding protein binds human CD16a. In other cases, the anti-CD16 antibody or binding protein binds cynomolgus CD16. In certain cases, the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16. In some cases, the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H7, shown below (CDR sequences shown in bold): Attorney Docket No.: 45817-0156WO1 EVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKEREFVSAIN SNTGRTYHADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAAGRGY GLLSISSNWYNYWGQGTLVTVSS (VHH1-H7, SEQ ID NO: 11). In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-CD1`6 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 11. In some cases, the anti- CD16 antibody or binding protein binds human CD16a. In other cases, the anti-CD16 antibody or binding protein binds cynomolgus CD16. In certain cases, the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16. In some cases, the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H8, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKELEFVSAIN SNTGRTYHADSVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCAAGRGY GLLSISSNWYNYWGQGTLVTVSS (VHH1-H8, SEQ ID NO: 12). In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the Attorney Docket No.: 45817-0156WO1 anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 12. In some cases, the anti- CD16 antibody or binding protein binds human CD16a. In other cases, the anti-CD16 antibody or binding protein binds cynomolgus CD16. In certain cases, the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16. In some cases, the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H9, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKERDFVSAI NSNTGRTYHADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAGRG YGLLSISSNWYNYWGQGTLVTVSS (VHH1-H9, SEQ ID NO: 13). In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 13. In some cases, the anti- CD16 antibody or binding protein binds human CD16a. In other cases, the anti-CD16 antibody or binding protein binds cynomolgus CD16. In certain cases, the anti-CD16 Attorney Docket No.: 45817-0156WO1 antibody or binding protein binds human CD16a and cynomolgus CD16. In some cases, the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H10, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKERDFVSAIN SNTGRTYHADSVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCAAGRGY GLLSISSNWYNYWGQGTLVTVSS (VHH1-H10, SEQ ID NO: 14). In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 14. In some cases, the anti- CD16 antibody or binding protein binds human CD16a. In other cases, the anti-CD16 antibody or binding protein binds cynomolgus CD16. In certain cases, the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16. In some cases, the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Attorney Docket No.: 45817-0156WO1 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H11, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKELDFVSAIN SNTGRTYHADSVKGRFTISRDNAKNMVYLQMNSLRPEDTAVYYCAAGRGY GLLSISSNWYNYWGQGTLVTVSS (VHH1-H11, SEQ ID NO: 15). In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 15. In some cases, the anti- CD16 antibody or binding protein binds human CD16a. In other cases, the anti-CD16 antibody or binding protein binds cynomolgus CD16. In certain cases, the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16. In some cases, the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H12, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKERDFVSAI NSNTGRTYHADSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCAAGRG YGLLSISSNWYNYWGQGTLVTVSS (VHH1-H12, SEQ ID NO: 16). Attorney Docket No.: 45817-0156WO1 In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 16. In some cases, the anti- CD16 antibody or binding protein binds human CD16a. In other cases, the anti-CD16 antibody or binding protein binds cynomolgus CD16. In certain cases, the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16. In some cases, the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H13, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKERDFVAAI NSNTGRTYHADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAAGRG YGLLSISSNWYNYWGQGTLVTVSS (VHH1-H13, SEQ ID NO: 17). In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 17. In some Attorney Docket No.: 45817-0156WO1 embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 17. In some cases, the anti- CD16 antibody or binding protein binds human CD16a. In other cases, the anti-CD16 antibody or binding protein binds cynomolgus CD16. In certain cases, the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16. In some cases, the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H14, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKEREFVSAIN SNTGRTYHADSVKGRFTISRDNAKNMLYLQMNSLKAEDTAVYYCAAGRGY GLLSISSNWYNYWGQGTLVTVSS (VHH1-H14, SEQ ID NO: 18). In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 18. In some cases, the anti- CD16 antibody or binding protein binds human CD16a. In other cases, the anti-CD16 antibody or binding protein binds cynomolgus CD16. In certain cases, the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16. In some cases, the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1. Attorney Docket No.: 45817-0156WO1 In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H15, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKEREFVAAI NSNTGRTYHADSVKGRFTISRDNAKNTLYLQMNSLKAEDTAVYYCAAGRG YGLLSISSNWYNYWGQGTLVTVSS (VHH1-H15, SEQ ID NO: 19). In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 19. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 19. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 19. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H16, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKERDFVSAI NSNTGRTYHADSVKGRFTISRDNAKNTLYLQMNSLKAEDTAVYYCAAGRG YGLLSISSNWYNYWGQGTLVTVSS (VHH1-H16, SEQ ID NO: 20). In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% Attorney Docket No.: 45817-0156WO1 identical) to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 20. In some cases, the anti- CD16 antibody or binding protein binds human CD16a. In other cases, the anti-CD16 antibody or binding protein binds cynomolgus CD16. In certain cases, the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16. In some cases, the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H17, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKELDFVAAI NSNTGRTYHADSVKGRFTISRDNAKNMLYLQMNSLRAEDTAVYYCAAGRG YGLLSISSNWYNYWGQGTLVTVSS (VHH1-H17, SEQ ID NO: 21). In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 21. In some cases, the anti- CD16 antibody or binding protein binds human CD16a. In other cases, the anti-CD16 Attorney Docket No.: 45817-0156WO1 antibody or binding protein binds cynomolgus CD16. In certain cases, the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16. In some cases, the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H18, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKERDFVSAI NSNTGRTYHADSVKGRFTISRDNAKNMLYLQMNSLKAEDTAVYYCAAGRG YGLLSISSNWYNYWGQGTLVTVSS (VHH1-H18, SEQ ID NO: 22). In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 22. In some cases, the anti- CD16 antibody or binding protein binds human CD16a. In other cases, the anti-CD16 antibody or binding protein binds cynomolgus CD16. In certain cases, the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16. In some cases, the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Attorney Docket No.: 45817-0156WO1 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH1-H19, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKERDFVAAI NSNTGRTYHADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAGRG YGLLSISSNWYNYWGQGTLVTVSS (VHH1-H19, SEQ ID NO: 23). In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 23. In some cases, the anti- CD16 antibody or binding protein binds human CD16a. In other cases, the anti-CD16 antibody or binding protein binds cynomolgus CD16. In certain cases, the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16. In some cases, the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VH, VHH1-H20, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGKERDFVAAI NSNTGRTYHADSVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCAAGRG YGLLSISSNWYNYWGQGTLVTVSS (VHH1-H20, SEQ ID NO: 24). Attorney Docket No.: 45817-0156WO1 In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 24. In some embodiments, the anti-CD16 antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 24. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 24. In some cases, the anti-CD16 antibody or binding protein binds human CD16a. In other cases, the anti-CD16 antibody or binding protein binds cynomolgus CD16. In certain cases, the anti-CD16 antibody or binding protein binds human CD16a and cynomolgus CD16. In some cases, the anti-CD16 antibody or binding protein bind human, cynomolgus, and rat CD16, but neither mouse CD16 nor human CD16b NA1. In some embodiments, an antibody or antigen-binding fragment is a murine- specific antibody or antigen-binding fragment, e.g., the antibody or binding protein specifically binds the murine antigen. In some embodiments, an antibody or antigen- binding fragment is a rat-specific antibody or antigen-binding fragment, e.g., the antibody or binding protein specifically binds the rat antigen. In some embodiments, an antibody or antigen-binding fragment is a llama-specific antibody or antigen- binding fragment, e.g., the antibody or binding protein specifically binds the llama antigen. In some embodiments, an antibody or antigen-binding fragment is a human- specific antibody or antigen-binding fragment, e.g., the antibody or binding protein specifically binds the human antigen. In some embodiments, an antibody or antigen- binding fragment is human-specific even if the antibody or binding protein is not human or humanized. Multispecific antibodies In another aspect, the present disclosure provides multispecific antibodies, for example, bispecific antibodies (BsAbs), that may have binding specificities that are Attorney Docket No.: 45817-0156WO1 directed towards CD16 and any other antigen, e.g., for a cell-surface protein, receptor, receptor subunit, or tissue-specific antigen, or other non-CD16 antigen. Multispecific antibodies typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen (i.e., CD16 and any other antigen). Each antigen-binding domain of a bispecific antibody can comprise a heavy chain variable domain (VH), a light chain variable domain (VL), or a VHH and a VL. In the context of a bispecific antigen-binding fragment comprising a first and a second antigen-binding domain (e.g., a bispecific antibody), each antigen binding domain comprises at least one CDR that alone, or in combination with one or more additional CDRs and/or framework regions, specifically binds to a particular antigen (i.e., CD16, any other antigen). The first antigen-binding domain and the second antigen-binding domain may be directly or indirectly connected to one another to form a bispecific antigen-binding fragment (i.e., bispecific scFv) further bound to an Fc domain. Alternatively, the first antigen-binding domain and the second antigen-binding domain may each be connected to a separate Fc domain. Bispecific antigen-binding fragments of the present disclosure may comprise two Fc domains that are each individually part of a separate antibody heavy chain. The first and second Fc domains may be of the same sequence, or the Fc domains may have a mutation in the CH3 domain intended for the facilitation or ease of purification of heterodimeric (i.e., bispecific) molecules. A multispecific antibody may also be an antibody or antigen-binding fragment thereof that includes at least two separate antigen-binding domains (e.g., two scFvs joined by a linker). The scFvs may bind the same antigen or different antigens. A bispecific antibody can also comprise multiple chains. A bispecific antibody may be an antibody or antigen-binding fragment thereof that includes a F(ab) with binding specificity directed towards a first antigen and a VHH domain with binding specificity directed towards a second antigen (e.g., CD16) joined by a linker. In some embodiments, a bispecific antibody may be a tetrameric bispecific antibody. Attorney Docket No.: 45817-0156WO1 In some embodiments, multispecific antibodies of the present disclosure are secreted (e.g., released from a cell, for example, into the extracellular milieu). Multispecific antibodies of the present disclosure can include any anti-CD16 CDRs, or VHH domains described herein. Multispecific antibodies of the present disclosure can comprise binding specificities that are directed towards CD16 and any other antigen. Any other antigen may be or comprise, for example, a cancer cell antigen/marker, an immune cell antigen (e.g., a T cell activation marker), a pathogenic antigen, or any other non- CD16 antigen. The disclosed multispecific antibodies may be produced by any means known in the art for producing multispecific antibodies, so long as the resulting multispecific antibody retains the functional characteristic of being able to specifically bind CD16 and at least one other antigen. In some embodiments, the BsAbs may be created using the methods described in Labrijin et al., Proc. Natl. Acad. Sci. USA, 110(13):5145-50 (2013). Briefly, the two parental Abs, each containing single matched point mutations in the CH3 domains, are separately expressed and then mixed under reducing conditions in vitro. This separates the Abs into half-molecules, followed by reassembly, to form bispecific antibodies, and is compatible with large-scale manufacturing of bispecific antibodies. However, this is simply one exemplary method for making a multispecific antibody. Those of skill in the art will be aware that other methods of producing multispecific antibodies are available, and the present disclosure is not intended to be limited solely to the methods of making and type of multispecific antibodies disclosed herein. Other multispecific antibody formats or technologies may be used to make the multispecific antigen-binding molecules of the present disclosure. For example, an antibody or fragment thereof having a first antigen binding specificity can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity to produce Attorney Docket No.: 45817-0156WO1 a bispecific antigen-binding molecule. Specific exemplary bispecific formats that can be used in the context of the present disclosure include, without limitation, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into- holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgGl/IgG2, dual acting Fab (DAF)-IgG, and Mab2 bispecific formats (see, e.g., Klein et al.2012, mAbs 4:6, 1-11 , and references cited therein, for a review of the foregoing formats). The disclosed multispecific antibodies can be made from or incorporate the CDRs or variable regions from polyclonal, monoclonal, chimeric, human, partially or fully humanized, and/or recombinant antibodies. Thus, the “parent” antibodies for the disclosed multispecific antibodies are not particularly limited; however, they are preferably fully human or humanized. In some embodiments, the parent antibody can be a polyclonal antibody. In some embodiments, the parent antibody can be a monoclonal. In some embodiments, the parent antibody can be a human antibody. Affinity of antibodies, antigen-binding fragments, or binding proteins of the disclosure Thermodynamic properties of anti-CD16 antibodies, antigen-binding fragments, or binding proteins Antibodies, antigen-binding fragments, or binding proteins of the disclosure may have an affinity for CD16 of, for example, from 1 nM to 100 nM (e.g., from 10 nM to 90 nM, from 20 nM to 80 nM, from 30 nM to 70 nM, from 40 nM to 60 nM, or about 50 nM). In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of from about 1 nM to about 100 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of from about 1 nM to about 90 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity CD16 of from about 1 nM to about 80 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of from about 1 nM to 60 nM. In Attorney Docket No.: 45817-0156WO1 some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of from about 1 nM to 40 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of from about 1 nM to 20 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 100 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 95 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 90 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 85 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 80 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 75 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 70 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 65 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 60 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 55 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 50 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 45 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 40 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 35 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 30 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 25 Attorney Docket No.: 45817-0156WO1 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 20 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 15 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 10 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 5 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for CD16 of about 1 nM. The specific binding of an antibody, antigen-binding fragments, or binding proteins described herein to CD16 can be determined by any of a variety of established methods. The affinity can be represented quantitatively by various measurements, including the concentration of antibody or binding protein needed to achieve the equilibrium constant (KD) of the antibody- , antigen-binding fragment-, or binding proteins - antigen complex dissociation. The equilibrium constant, KD, which describes the interaction of CD16 with an antibody or binding proteins described herein is the chemical equilibrium constant for the dissociation reaction of an antigen- antibody, –antigen-binding fragment, or – binding protein complex into solvent- separated antigen and antibody or binding proteins that do not interact with one another. Antibodies, antigen-binding fragments, or binding proteins described herein include those that specifically bind to CD16 with a KD value of less than 100 nM (e.g., less than 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM). In some embodiments, the antibodies, antigen-binding fragments, or binding proteins described herein specifically bind to CD16 with a KD value of less than 10 nM (e.g., less than 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM). Attorney Docket No.: 45817-0156WO1 The antibodies, antigen-binding fragments, and binding proteins described herein may also not compete with human IgG for binding to CD16a. This can be shown by observing that the disclosed antibodies, antigen-binding fragments, and binding proteins are not significantly affected by the addition of 33% human serum in binding assays (see, e.g., Example 5). Antibodies, antigen-binding fragments, or binding proteins described herein can also be characterized by a variety of in vitro binding assays. Examples of experiments that can be used to determine the KD or EC50 of an anti-CD16 antibody or binding protein include, e.g., surface plasmon resonance, isothermal titration calorimetry, fluorescence anisotropy, ELISA-based assays, gene expression assays, and protein expression assays, among others. ELISA represents a particularly useful method for analyzing antibody or binding protein activity, as such assays typically require minimal concentrations of binding domains (e.g., antibodies, antigen-binding fragments, binding proteins). A common signal that is analyzed in a typical ELISA assay is luminescence, which is typically the result of the activity of a peroxidase conjugated to a secondary antibody that specifically binds a primary antibody (e.g., an anti-CD16 antibody, antigen-binding fragment described herein). Antibodies, antigen- binding fragments, or binding proteins described herein may bind CD16 and fragments thereof. Antibodies, antigen-binding fragments, or binding proteins described herein may additionally bind isolated peptides derived from CD16 that structurally pre-organize various residues in a manner that simulates the conformation of the above fragments in the native protein. In a direct ELISA experiment, this binding can be quantified, e.g., by analyzing the luminescence that occurs upon incubation of an HRP substrate (e.g., 2,2’-azino-di-3- ethylbenzthiazoline sulfonate) with an antigen-antibody, antigen-antigen-binding fragment, or antigen-binding protein complex bound to a HRP-conjugated secondary antibody. Attorney Docket No.: 45817-0156WO1 Kinetic properties of anti-CD16 antibodies, antigen-binding fragments, or binding proteins In addition to the thermodynamic parameters of a CD16-antibody, –antigen- binding fragment, or -binding protein interaction, it is also possible to quantitatively characterize the kinetic association and dissociation of an antibody or binding proteins described herein with CD16. This can be done, e.g., by monitoring the rate of antibody-, antigen-binding fragment-, or binding protein-antigen complex formation according to established procedures. For example, one can use surface plasmon resonance (SPR) to determine the rate constants for the formation (kon) and dissociation (koff) of an antibody-, antigen-binding fragment-, or binding protein- CD16 complex. These data also enable calculation of the equilibrium constant of (KD) of antibody-, antigen-binding fragment-, or binding protein-CD16 complex dissociation, since the equilibrium constant of this unimolecular dissociation can be expressed as the ratio of the koff to kon values. SPR is a technique that is particularly advantageous for determining kinetic and thermodynamic parameters of antigen- antibody, -antigen-binding fragment, or -binding protein interactions since the experiment does not require that one component be modified by attachment of a chemical label. Rather, the antigen is typically immobilized on a solid metallic surface which is treated in pulses with solutions of increasing concentrations of antibody or binding proteins. Antibody-, antigen-binding fragment-, or binding protein-antigen binding induces distortion in the angle of reflection of incident light at the metallic surface, and this change in refractive index over time as antibody or binding protein is introduced to the system can be fit to established regression models in order to calculate the association and dissociation rate constants of an antibody- or antigen- binding-fragment- or binding protein- antigen interaction. Antibodies, antigen-binding fragments, or binding proteins described herein may exhibit high kon and low koff values upon interaction with CD16. For example, antibodies, antigen-binding fragments, or binding proteins described herein may exhibit kon values in the presence of CD16 of greater than 104 M-1s-1 (e.g., 1.0 x 104 M-1s-1, 1.5 x 104 M-1s-1, 2.0 x 104 M-1s-1, 2.5 x 104 M-1s-1, 3.0 x 104 M-1s-1, 3.5 x 104 Attorney Docket No.: 45817-0156WO1 M-1s-1, 4.0 x 104 M-1s-1, 4.5 x 104 M-1s-1, 5.0 x 104 M-1s-1, 5.5 x 104 M-1s-1, 6.0 x 104 M- 1s-1, 6.5 x 104 M-1s-1, 7.0 x 104 M-1s-1, 7.5 x 104 M-1s-1, 8.0 x 104 M-1s-1, 8.5 x 104 M-1s- 1, 9.0 x 104 M-1s-1, 9.5 x 104 M-1s-1, 1.0 x 105 M-1s-1, 1.5 x 105 M-1s-1, 2.0 x 105 M-1s-1, x 105 M-1s-1, 3.0 x 105 M-1s-1, 3.5 x 105 M-1s-1, 4.0 x 105 M-1s-1, 4.5 x 105 M-1s-1, 5.0 x 105 M-1s-1, 5.5 x 105 M-1s-1, 6.0 x 105 M-1s-1, 6.5 x 105 M-1s-1, 7.0 x 105 M-1s-1, 7.5 x 105 M-1s-1, 8.0 x 105 M-1s-1, 8.5 x 105 M-1s-1, 9.0 x 105 M-1s-1, 9.5 x 105 M-1s-1, or 1.0 x 106 M-1s- . Antibodies, antigen-binding fragments, or binding proteins
Figure imgf000066_0001
may exhibit low koff values when bound to CD16. For instance, antibodies, antigen-binding fragments, or binding proteins described herein may exhibit koff values of less than 10-3 s-1 when complexed to CD16 (e.g., 1.0 x 10-3 s-1, 9.5 x 10-4 s-1, 9.0 x 10-4 s-1, 8.5 x 10-4 s-1, 8.0 x 10 -4 s-1, 7.5 x 10-4 s-1, 7.0 x 10-4 s-1, 6.5 x 10-4 s-1, 6.0 x 10-4 s-1, 5.5 x 10-4 s-1, 5.0 x 10-4 s-1, 4.5 x 10-4 s-1, 4.0 x 10-4 s-1, 3.5 x 10-4 s-1, 3.0 x 10-4 s-1, 2.5 x 10-4 s-1, 2.0 x 10-4 s-1, 1.5 x 10-4 s-1, 1.0 x 10-4 s-1, 9.5 x 10-5 s-1, 9.0 x 10-5 s-1, 8.5 x 10-5 s-1, 8.0 x 10-5 s-1, 7.5 x 10-5 s-1, 7.0 x 10-5 s-1, 6.5 x 10-5 s-1, 6.0 x 10-5 s-1, 5.5 x 10-5 s-1, 5.0 x 10-5 s-1, 4.5 x 10-5 s-1, 4.0 x 10-5 s-1, 3.5 x 10-5 s-1, 3.0 x 10-5 s-1, 2.5 x 10-5 s-1, 2.0 x 10-5 s-1, 1.5 x 10-5 s-1, or 1.0 x 10-5 s-1). Methods for Humanization Antibodies, antigen-binding fragments, or binding proteins described herein can include fully human, humanized, primatized, and chimeric antibodies that contain one or more of the CDR sequences shown in Table 4, below. As an example, one strategy that can be used to design humanized antibodies, antigen-binding fragments, or binding proteins described herein is to align the sequences of the VH and/or VL of an antibody or binding protein (e.g., of the present disclosure) with the VH and/or VL of a consensus human antibody. Consensus human antibody heavy chain and light chain sequences are known in the art (see, e.g., the “VBASE” human germline sequence database; see also Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.91 -3242 (1991); Tomlinson et al., J. Mol. Biol.227:776-98 (1992); and Cox et al., Eur. J. Immunol.24:827-836 (1994); the disclosure of which is incorporated herein by reference). In this way, the variable domain framework Attorney Docket No.: 45817-0156WO1 residues and CDRs can be identified by sequence alignment (see, Kabat, supra). One can then substitute, for example, one or more of the CDRs of the consensus human antibody with the corresponding CDR(s) of an antibody or antigen-binding fragment or binding protein of the disclosure, thereby producing a humanized antibody or binding protein. Similarly, this strategy can also be used to produce primatized anti-CD16 antibodies, antigen-binding fragments, or binding proteins, as one can substitute, for example, one or more, or all, of the CDRs of a primate antibody consensus sequence with, for example, one or more, or all, of the CDRs of an antibody or binding protein of the disclosure. Consensus primate antibody sequences known in the art (see, e.g., U.S. Patent Nos.5,658,570; 5,681,722; and 5,693,780; the disclosures of each of which are incorporated herein by reference). In some embodiments, it may be desirable to import particular framework residues in addition to CDR sequences from an anti-CD16 antibody or binding protein into the VH and/or VL of a human antibody. For instance, US Patent No.6,054,297 identifies several instances when it may be advantageous to retain certain framework residues from a particular antibody heavy chain or light chain variable region in the resulting humanized antibody or binding proteins. In some embodiments, framework residues may engage in non-covalent interactions with the antigen and thus contribute to the affinity of the antibody or binding proteins for the target antigen. In some embodiments, individual framework residues may modulate the conformation of a CDR, and thus indirectly influence the interaction of the antibody, antigen-binding domain, or binding proteins with the antigen. Certain framework residues may form the interface between VH and VL domains, and may therefore contribute to the global antibody, antigen-binding domain, or binding protein structure. In some cases, framework residues may constitute functional glycosylation sites (e.g., Asn-X- Ser/Thr) which may dictate antibody, antigen-binding domain, or binding protein structure and antigen affinity upon attachment to carbohydrate moieties. In cases such as those described above, it may be beneficial to retain certain framework residues of an anti-CD16 antibody or binding protein in, e.g., a humanized or primatized antibody Attorney Docket No.: 45817-0156WO1 or antigen-binding fragment or binding protein thereof, as various framework residues may promote high epitope affinity and improved biochemical activity of the antibody or antigen-binding fragment or binding protein thereof. Examples of the humanized variant sequences of the antibodies, antigen- binding fragments, or binding proteins described herein can be found in Table 7 below. Antibodies described herein also include antibody fragments, Fab domains, F(ab’) molecules, F(ab’)2 molecules, single-chain variable fragments (scFvs), tandem scFv fragments, diabodies, triabodies, dual variable domain immunoglobulins, multi- specific antibodies, bispecific antibodies, VHH, and heterospecific antibodies that contain one or more of the CDRs in Table 4, below, or a CDR having at least 85% sequence identity thereto (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto). These molecules can be expressed recombinantly, e.g., by incorporating polynucleotides encoding these proteins into expression vectors for transfection in a eukaryotic or prokaryotic cell using techniques described herein or known in the art, or synthesized chemically, e.g., by solid phase peptide synthesis methods described herein or known in the art. Nucleic Acids and Expression systems Anti-CD16 antibodies, antigen-binding fragments, or binding proteins described herein can be prepared by any of a variety of established techniques. For instance, an anti- CD16 antibody or antigen-binding fragment described herein can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell. To express an antibody or antigen-binding fragment or binding protein recombinantly, a host cell can be transfected with one or more recombinant expression vectors carrying DNA fragments encoding the desired antibody chain(s), antigen-binding fragments, and/or additional binding protein domains (e.g., transmembrane domains, hinge domains). For example, the light and/or heavy chains of an antibody or an antigen-binding fragment can be expressed in the host cell and, Attorney Docket No.: 45817-0156WO1 optionally, secreted into the medium in which the host cells are cultured, from which medium the antibodies can be recovered. Standard recombinant DNA methodologies are used to obtain antibody heavy chain genes, light chain genes, and binding protein domains and to incorporate these genes into recombinant expression vectors and introduce the vectors into host cells, such as those described in Molecular Cloning; A Laboratory Manual, Second Edition (Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, N. Y., 1989), Current Protocols in Molecular Biology (Ausubel et al., eds., Greene Publishing Associates (1989), and in U.S. Patent No.4,816,397; the disclosures of each of which are incorporated herein by reference. Suitable vectors include, but are not limited to, viral vectors and non-viral vectors. Viral vectors can include retrovirus, adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., Measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses useful for delivering polynucleotides encoding antibody light and heavy chains or antibody fragments or binding proteins described herein include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Non-viral vectors can include plasmids. In addition to viral vectors, a variety of additional methods have been developed for the incorporation of genes, e.g., those encoding antibody light and heavy chains, single-domain antibodies, single-chain variable fragments (scFvs), tandem scFvs, Fab domains, F(ab’)2 domains, diabodies, and triabodies, among others, into the genomes of target cells for antibody, antigen-binding fragment, and/or binding protein expression. One such method that can be used for incorporating polynucleotides encoding anti-CD16 antibodies, antigen-binding fragments, or binding proteins into prokaryotic or eukaryotic cells includes the use of transposons. Attorney Docket No.: 45817-0156WO1 Another useful method for the integration of nucleic acid molecules encoding anti-CD16 antibodies, antigen-binding fragments, or binding proteins into the genome of a prokaryotic or eukaryotic cell is the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system, which is a system that originally evolved as an adaptive defense mechanism in bacteria and archaea against infection by viruses. Alternative methods for site-specifically cleaving genomic DNA prior to the incorporation of a polynucleotide encoding an anti-CD16 antibody or binding protein described herein include the use of zinc finger nucleases and transcription activator- like effector nucleases (TALENs). Additional genome editing techniques that can be used to incorporate polynucleotides encoding antibodies, antigen-binding fragments, or binding proteins described herein into the genome of a prokaryotic or eukaryotic cell include the use of ARCUSTM meganucleases that can be rationally designed so as to site-specifically cleave genomic DNA. To express an anti-CD16 antibodies, antigen-binding fragments, or binding proteins described herein, polynucleotides encoding partial or full-length light and heavy chains, e.g., polynucleotides that encode a one or more of the CDR sequences of an antibody or binding protein described herein, can be inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. Polynucleotides encoding the light chain gene and the heavy chain of an anti-CD16 antibody or binding protein can be inserted into separate vectors, or, optionally, both polynucleotides can be incorporated into the same expression vector using established techniques described herein or known in the art. In addition to polynucleotides encoding the heavy and light chains of an antibody, or a polynucleotide encoding a single-chain antibody, an antibody fragment, such as a scFv molecule, or a construct described herein, or a binding protein, the recombinant expression vectors described herein may carry regulatory sequences that Attorney Docket No.: 45817-0156WO1 control the expression of the antibody chain genes or binding protein domains in a host cell. The design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed or the level of expression of protein desired. For instance, suitable regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma. Viral regulatory elements, and sequences thereof, are described in detail, for instance, in U.S. Patent No.5, 168,062, U.S. Patent No.4,510,245, and U.S. Patent No.4,968,615, the disclosures of each of which are incorporated herein by reference. In addition to, for example, the antibody chain genes and regulatory sequences, the recombinant expression vectors described herein can carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. A selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Patents Nos.4,399,216, 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers resistance to cytotoxic drugs, such as G418, puromycin, blasticidin, hygromycin or methotrexate, to a host cell into which the vector has been introduced. Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in DHFR” host cells with methotrexate selection/amplification) and the neo gene (for G418 selection). In order to express the light and heavy chains of an anti-CD16 antibody, anti-CD16 antibody fragment, or binding protein, the expression vector(s) containing polynucleotides encoding the heavy and light chains can be transfected into a host cell by standard techniques. Host cells for expression of anti-CD16 antibodies, antigen-binding fragments, or binding proteins It is possible to express the antibodies, antigen-binding fragments, or binding proteins described herein in either prokaryotic or eukaryotic host cells. In some Attorney Docket No.: 45817-0156WO1 embodiments, expression of antibodies, antigen-binding fragments, or binding proteins is performed in eukaryotic cells, e.g., mammalian host cells, for high secretion of a properly folded and immunologically active antibody or antigen- binding fragments. Exemplary mammalian host cells for expressing the recombinant antibodies, antigen-binding fragments, or binding proteins described herein include Chinese Hamster Ovary (CHO cells) (including DHFR CHO cells, described in Urlaub and Chasin (1980, Proc. Natl. Acad. Sci. USA 77:4216-4220), used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982, Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, 293 cells (e.g., expi293), and SP2/0 cells. Additional cell types that may be useful for the expression of antibodies, antigen-binding fragments, or binding proteins include bacterial cells, such as BL- 21(DE3) E. coli cells, which can be transformed with vectors containing foreign DNA according to established protocols. Additional eukaryotic cells that may be useful for expression of antibodies, antigen-binding fragments, or binding proteins include yeast cells, such as auxotrophic strains of S. cerevisiae, which can be transformed and selectively grown in incomplete media according to established procedures known in the art. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are grown. Half-life Extension of anti-CD16 Antibodies In some embodiments, an anti-CD16 antibody or antigen-binding fragment of the disclosure is conjugated to a second molecule, e g., to extend the half-life of the anti-CD16 antibody or antigen-binding fragment in vivo. Such molecules that can extend half-life of the anti-CD16 antibody or antigen-binding fragment are described below, and include polyethylene glycol (PEG), among others. Anti-CD16 antibodies and fragments thereof can be conjugated to these half-life extending molecules at, e.g., the N-terminus or C-terminus of a light and/or heavy chain of the antibody using any one of a variety of conjugation strategies known in the art. Examples of pairs of reactive functional groups that can be used to covalently tether an anti-CD16 antibody Attorney Docket No.: 45817-0156WO1 or fragment thereof to a half-life extending or other molecule include, without limitation, thiol pairs, carboxylic acids and amino groups, ketones and amino groups, aldehydes and amino groups, thiols and alpha,beta-unsaturated moieties (such as maleimides or dehydroalanine), thiols and alpha-halo amides, carboxylic acids and hydrazides, aldehydes and hydrazides, and ketones and hydrazides. Anti-CD16 antibodies can be conjugated to various molecules for the purpose of improving the half-life, solubility, and stability of the protein in aqueous solution. Examples of such molecules include polyethylene glycol (PEG), murine serum albumin (MSA), bovine serum albumin (BSA), and human serum albumin (HSA), among others. For instance, one can conjugate an anti-CD16 antibody or antigen- binding fragment to carbohydrate moieties in order to evade detection of the antibody antigen-binding fragment by the immune system of the patient receiving treatment. This process of hyperglycosylation reduces the immunogenicity of therapeutic proteins by sterically inhibiting the interaction of the protein with B cell receptors in circulation. Additionally, anti-CD16 antibodies, antigen-binding fragments, or binding proteins can be conjugated to molecules that prevent clearance from human serum and improve the pharmacokinetic profile of the antibodies, antigen-binding fragments, or binding proteins. Anti-CD16 antibodies, antigen-binding fragments, or binding proteins can be covalently appended directly to a half-life extending or other molecule by chemical conjugation as described. Alternatively, fusion proteins containing anti-CD16 antibodies, antigen-binding fragments, or binding proteins can be expressed recombinantly from a cell (e.g., a eukaryotic cell or prokaryotic cell). This can be accomplished, for example, by incorporating a polynucleotide encoding the fusion protein into the genome of a cell (e.g., using techniques described herein or known in the art). Optionally, antibodies and fragments thereof described herein can be joined to a half-life extending molecule by forming a covalent bond between the antibody and a linker. This linker can then be subsequently conjugated to another molecule, or the linker can be conjugated to another molecule prior to ligation to the anti-CD16 antibody or antigen-binding fragment. Examples of linkers that can be used for the Attorney Docket No.: 45817-0156WO1 formation of a conjugate include polypeptide linkers, such as those that contain naturally occurring or non-naturally occurring amino acids. In some embodiments, it may be desirable to include D-amino acids in the linker, as these residues are not present in naturally-occurring proteins and are thus more resistant to degradation by endogenous proteases. Fusion proteins containing polypeptide linkers can be made using chemical synthesis techniques, such as those described herein, or through recombinant expression of a polynucleotide encoding the fusion protein in a cell (e.g., a prokaryotic or eukaryotic cell). Linkers can be prepared using a variety of strategies that are well known in the art, and depending on the reactive components of the linker, can be cleaved by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (Leriche et al., Bioorg. Med. Chem., 20:571-582 (2012)). Nucleic Acids Encoding Anti-CD16 Antibodies or Binding Proteins This section provides exemplary nucleic acids that may be used to encode antibodies or antigen-binding fragments of the disclosure. The nucleic acid molecules of the disclosure may include one or more alterations. Herein, in a nucleotide, nucleoside, or polynucleotide (such as the nucleic acids of the disclosure (e.g., an mRNA or an oligonucleotide)), the terms “alteration” or, as appropriate, “alternative” refer to alteration with respect to A, G, U or C ribonucleotides. The alterations may be various distinct alterations. In some embodiments, where the nucleic acid is an mRNA, the coding region, the flanking regions, and/or the terminal regions may contain one, two, or more (optionally different) nucleoside or nucleotide alterations. In some embodiments, an alternative polynucleotide introduced to a cell may exhibit reduced degradation in the cell, as compared to an unaltered polynucleotide. The polynucleotides can include any useful alteration, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g., to a linking phosphate, to a phosphodiester linkage, or to the phosphodiester backbone). In certain embodiments, Attorney Docket No.: 45817-0156WO1 alterations (e.g., one or more alterations) are present in each of the sugar and the internucleoside linkage. Alterations according to the present disclosure may be alterations of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs) (e.g., the substitution of the 2’OH of the ribofuranosyl ring to 2’H), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof. Additional alterations are described herein. In certain embodiments, it may be desirable for a nucleic acid molecule introduced into the cell to be degraded intracellularly. For example, degradation of a nucleic acid molecule may be preferable if precise timing of protein production is desired. Thus, in some embodiments, the disclosure provides an alternative nucleic acid molecule containing a degradation domain, which is capable of being acted on in a directed manner within a cell. The polynucleotides can optionally include other agents (e.g., RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamers, vectors, etc.). In some embodiments, the polynucleotides may include one or more messenger RNAs (mRNAs) having one or more alternative nucleoside or nucleotides (i.e., mRNA molecules). In some embodiments, the polynucleotides may include one or more oligonucleotides having one or more alternative nucleoside or nucleotides. In some embodiments, a composition of the disclosure includes an mRNA and/or one or more oligonucleotides having one or more alternative nucleoside or nucleotides. Modified nucleic acids According to Aduri et al., (Aduri, R. et al., Journal of Chemical Theory and Computation.3(4):1464-75(2006)), there are 107 naturally occurring nucleosides, including 1-methyladenosine, 2-methylthio-N6-hydroxynorvalyl carbamoyladenosine, 2-methyladenosine, 2-O-ribosylphosphate adenosine, N6-methyl-N6- threonylcarbamoyladenosine, N6-acetyladenosine, N6-glycinylcarbamoyladenosine, N6-isopentenyladenosine, N6-methyladenosine, N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, N6- Attorney Docket No.: 45817-0156WO1 hydroxynorvalylcarbamoyladenosine, 1,2-O-dimethyladenosine, N6,2-O- dimethyladenosine, 2-O-methyladenosine, N6,N6,O-2-trimethyladenosine, 2- methylthio-N6-(cis-hydroxyisopentenyl) adenosine, 2-methylthio-N6- methyladenosine, 2-methylthio-N6-isopentenyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, 2-thiocytidine, 3-methylcytidine , N4-acetylcytidine, 5- formylcytidine, N4-methylcytidine, 5-methylcytidine, 5-hydroxymethylcytidine, lysidine, N4-acetyl-2-O-methylcytidine, 5-formyl-2-O-methylcytidine, 5,2-O- dimethylcytidine, 2-O-methylcytidine, N4,2-O-dimethylcytidine, N4,N4,2-O- trimethylcytidine, 1-methylguanosine, N2,7-dimethylguanosine, N2-methylguanosine, 2-O-ribosylphosphate guanosine, 7-methylguanosine, under modified hydroxywybutosine, 7-aminomethyl-7-deazaguanosine, 7-cyano-7-deazaguanosine, N2,N2-dimethylguanosine, 4-demethylwyosine, epoxyqueuosine, hydroxywybutosine, isowyosine, N2,7,2-O-trimethylguanosine, N2,2-O- dimethylguanosine, 1,2-O-dimethylguanosine, 2-O-methylguanosine, N2,N2,2-O- trimethylguanosine, N2,N2,7-trimethylguanosine, peroxywybutosine, galactosyl- queuosine, mannosyl-queuosine, queuosine, archaeosine, wybutosine, methylwyosine, wyosine, 2-thiouridine, 3-(3-amino-3-carboxypropyl)uridine, 3-methyluridine, 4- thiouridine, 5-methyl-2-thiouridine, 5-methylaminomethyluridine, 5- carboxymethyluridine, 5-carboxymethylaminomethyluridine, 5-hydroxyuridine, 5- methyluridine, 5-taurinomethyluridine, 5-carbamoylmethyluridine, 5- (carboxyhydroxymethyl)uridine methyl ester, dihydrouridine, 5- methyldihydrouridine, 5-methylaminomethyl-2-thiouridine, 5- (carboxyhydroxymethyl)uridine, 5-(isopentenylaminomethyl)uridine, 5- (isopentenylaminomethyl)-2-thiouridine, 3,2-O-dimethyluridine, 5- carboxymethylaminomethyl-2-O-methyluridine, 5-carbamoylmethyl-2-O- methyluridine, 5-methoxycarbonylmethyl-2-O-methyluridine, 5- (isopentenylaminomethyl)-2-O-methyluridine, 5,2-O-dimethyluridine, 2-O- methyluridine, 2-thio-2-O-methyluridine, uridine 5-oxyacetic acid, 5- methoxycarbonylmethyluridine, uridine 5-oxyacetic acid methyl ester, 5- methoxyuridine, 5-aminomethyl-2-thiouridine, 5-carboxymethylaminomethyl-2- thiouridine, 5-methylaminomethyl-2-selenouridine, 5-methoxycarbonylmethyl-2- Attorney Docket No.: 45817-0156WO1 thiouridine, 5-taurinomethyl-2-thiouridine, pseudouridine, 1-methyl-3-(3-amino-3- carboxypropyl)pseudouridine, 1-methylpseudouridine, 3-methylpseudouridine, 2-O- methylpseudouridine, inosine, 1-methylinosine, 1,2-O-dimethylinosine, and 2-O- methylinosine. Each of these may be components of nucleic acids of the present disclosure. Nucleosides containing modified sugars The alternative nucleosides and nucleotides (e.g., building block molecules), which may be incorporated into a polynucleotide (e.g., RNA or mRNA, as described herein), can be altered on the sugar of the ribonucleic acid. For example, the 2′ hydroxyl group (OH) can be modified or replaced with a number of different substituents. Exemplary substitutions at the 2′-position include, but are not limited to, H, halo, optionally substituted C1-6 alkyl; optionally substituted C1-6 alkoxy; optionally substituted C6-10 aryloxy; optionally substituted C3-8 cycloalkyl; optionally substituted C3-8 cycloalkoxy; optionally substituted C6-10 aryloxy; optionally substituted C6-10 aryl- C1-6 alkoxy, optionally substituted C1-12 (heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any described herein); a polyethyleneglycol (PEG), - O(CH2CH2O)nCH2CH2OR, where R is H or optionally substituted alkyl, and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20); “locked” nucleic acids (LNA) in which the 2′-hydroxyl is connected by a C1-6 alkylene or C1-6 heteroalkylene bridge to the 4’-carbon of the same ribose sugar, where exemplary bridges included methylene, propylene, ether, or amino bridges; aminoalkyl, as defined herein; aminoalkoxy, as defined herein; amino as defined herein; and amino acid, as defined herein Generally, RNA includes the sugar group ribose, which is a 5-membered ring having an oxygen. Exemplary, non-limiting alternative nucleotides include replacement of the oxygen in ribose (e.g., with S, Se, or alkylene, such as methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or Attorney Docket No.: 45817-0156WO1 cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone); multicyclic forms (e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), threose nucleic acid (TNA, where ribose is replace with α-L-threofuranosyl-(3′→2′)), and peptide nucleic acid (PNA, where 2-amino-ethyl-glycine linkages replace the ribose and phosphodiester backbone). The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a polynucleotide molecule can include nucleotides containing, e.g., arabinose, as the sugar. Alterations on the nucleobase The present disclosure provides for alternative nucleosides and nucleotides. As described herein “nucleoside” is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). As described herein, “nucleotide” is defined as a nucleoside including a phosphate group. Exemplary non-limiting alterations include an amino group, a thiol group, an alkyl group, a halo group, or any described herein. The alternative nucleotides may by synthesized by any useful method, as described herein (e.g., chemically, enzymatically, or recombinantly to include one or more alternative or alternative nucleosides). In some embodiments, a nucleic acid of the disclosure (e.g., an mRNA or an oligonucleotide) includes one or more 2’-OMe nucleotides, 2’-methoxyethyl nucleotides (2’-MOE nucleotides), 2’-F nucleotide, 2’-NH2 nucleotide, Attorney Docket No.: 45817-0156WO1 2’fluoroarabino nucleotides (FANA nucleotides), locked nucleic acid nucleotides (LNA nucleotides), or 4’-S nucleotides. The alternative nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, and guanosine-cytosine base pairs, but also base pairs formed between nucleotides and/or alternative nucleotides including non- standard or alternative bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures. One example of such non-standard base pairing is the base pairing between the alternative nucleotide inosine and adenine, cytosine, or uracil. The alternative nucleosides and nucleotides can include an alternative nucleobase. Examples of nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil. Examples of nucleobase found in DNA include, but are not limited to, adenine, guanine, cytosine, and thymine. These nucleobases can be altered or wholly replaced to provide polynucleotide molecules having enhanced properties (e.g., resistance to nucleases and stability), and these properties may manifest through disruption of the binding of a major groove binding partner. In some embodiments, the alternative nucleobase is an alternative uracil. Exemplary nucleobases and nucleosides having an alternative uracil include pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio- 5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio- pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridineor 5-bromo-uridine), 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5- carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5- methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine Attorney Docket No.: 45817-0156WO1 (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio- uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5- carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1- propynyl-pseudouridine, 5-taurinomethyl-uridine (τm5U), 1-taurinomethyl- pseudouridine, 5-taurinomethyl-2-thio-uridine(τm5s2U), 1-taurinomethyl-4-thio- pseudouridine, 5-methyl-uridine (m5U, i.e., having the nucleobase deoxythymine), 1- methyl-pseudouridine (m1ψ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio- pseudouridine 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine 3
Figure imgf000080_0001
(m ψ), 2-thio-1- 1-methyl-1-deaza-pseudouridine, 2-thio-1- methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6- dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio- dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy- pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3- amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3- carboxypropyl)pseudouridine (acp3 ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2- (inm5s2U), α-thio-uridine, 2′-O-methyl-
Figure imgf000080_0002
uridine (Um), 5,2′-O-dimethyl-uridine (m5Um), 2′-O-methyl-pseudouridine (ψm), 2- thio-2′-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2′-O-methyl-uridine (ncm5Um), 5- carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm5Um), 3,2′-O-dimethyl- uridine (m3Um), and 5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm5Um), 1- thio-uridine, deoxythymidine, 2’‐F‐ara‐uridine, 2’‐F‐uridine, 2’‐OH‐ara‐uridine, 5‐(2‐ carbomethoxyvinyl) uridine, and 5‐[3‐(1‐E‐propenylamino)uridine. In preferred embodiments, the nucleic acid is modified to contain 1- methylpseudouridine (m1ψ) in lieu of uridine at each instance. In some embodiments, the alternative nucleobase is an alternative cytosine. Exemplary nucleobases and nucleosides having an alternative cytosine include 5-aza- cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl- cytidine (ac4C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl- Attorney Docket No.: 45817-0156WO1 cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2- thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1- methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1- deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2- thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm), 5,2′-O-dimethyl-cytidine (m5Cm), N4- acetyl-2′-O-methyl-cytidine (ac4Cm), N4,2′-O-dimethyl-cytidine (m4Cm), 5-formyl- 2′-O-methyl-cytidine (f5Cm), N4,N4,2′-O-trimethyl-cytidine (m42Cm), 1-thio- cytidine, 2’‐F‐ara‐cytidine, 2’‐F‐cytidine, and 2’‐OH‐ara‐cytidine. In some embodiments, the alternative nucleobase is an alternative adenine. Exemplary nucleobases and nucleosides having an alternative adenine include 2- amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro- purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido- adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7- deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6- diaminopurine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl- adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl- adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis- hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis- hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl- adenosine (m6 2A), N6-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio- N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7- methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2′-O- methyl-adenosine (Am), N6,2′-O-dimethyl-adenosine (m6Am), N6,N6,2′-O-trimethyl- adenosine (m6 2Am), 1,2′-O-dimethyl-adenosine (m1Am), 2′-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, Attorney Docket No.: 45817-0156WO1 2’‐F‐ara‐adenosine, 2’‐F‐adenosine, 2’‐OH‐ara‐adenosine, and N6‐(19‐amino‐ pentaoxanonadecyl)-adenosine. In some embodiments, the alternative nucleobase is an alternative guanine. Exemplary nucleobases and nucleosides having an alternative guanine include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 4-demethyl- wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OhyW), undermodified hydroxywybutosine (OhyW*), 7-deaza- guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7- deaza-guanosine (preQ1), archaeosine (G+), 7-deaza-8-aza-guanosine, 6-thio- guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl- guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy- guanosine, 1-methyl-guanosine (m1G), N2-methyl-guanosine (m2G), N2,N2- dimethyl-guanosine (m2 2G), N2,7-dimethyl-guanosine (m2,7G), N2, N2,7-dimethyl- guanosine (m2,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio- guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio- guanosine, 2′-O-methyl-guanosine (Gm), N2-methyl-2′-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2′-O-methyl-guanosine (m22Gm), 1-methyl-2′-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2′-O-methyl-guanosine (m2,7Gm), 2′-O-methyl-inosine (Im), 1,2′-O-dimethyl-inosine (m1Im), 2′-O-ribosylguanosine (phosphate) (Gr(p)) , 1-thio- guanosine, O6-methyl-guanosine, 2’‐F‐ara‐guanosine, and 2’‐F‐guanosine. The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine, or pyrimidine analog. For example, the nucleobase can each be independently selected from adenine, cytosine, guanine, uracil, or hypoxanthine. In some embodiments, the nucleobase can also include, for example, naturally-occurring and synthetic derivatives of a base, including pyrazolo[3,4-d]pyrimidines, 5- methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl, and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5- Attorney Docket No.: 45817-0156WO1 uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3- deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5 triazinones, 9- deazapurines, imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones, 1,2,4-triazine, pyridazine; and 1,3,5 triazine. When the nucleotides are depicted using the shorthand A, G, C, T or U, each letter refers to the representative base and/or derivatives thereof (e.g., A includes adenine or adenine analogs (e.g., 7-deaza adenine)). In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uracil, uracil, 5-methyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uracil, uracil, 5-trifluoromethyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uracil, uracil, 5-hydroxymethyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uracil, uracil, 5-bromo-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uracil, uracil, 5-iodo-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 5-methoxy- uracil, uracil, 5-methoxy-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-ethyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-phenyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-ethnyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, N4-methyl-cytosine, and cytosine as the only uracils and cytosines. In some Attorney Docket No.: 45817-0156WO1 embodiments, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-fluoro-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, N4-acetyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, pseudoisocytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-formyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-aminoallyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-carboxy-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl- pseudouracil, uracil, 5-methyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl- pseudouracil, uracil, 5-trifluoromethyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 1- methyl-pseudouracil, uracil, 5-hydroxymethyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-bromo-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-iodo-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-methoxy-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-ethyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-phenyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-ethnyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure Attorney Docket No.: 45817-0156WO1 contain 1-methyl-pseudouracil, uracil, N4-methyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-fluoro-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, N4-acetyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, pseudoisocytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-formyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-aminoallyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-carboxy-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, 5-methyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, 5-trifluoromethyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, 5-hydroxymethyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, 5-bromo-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, 5-iodo-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, 5-methoxy-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, 5-ethyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, 5-phenyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 5- Attorney Docket No.: 45817-0156WO1 methoxy-uridine, uridine, 5-ethnyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, N4-methyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, 5-fluoro-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, N4-acetyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, pseudoisocytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, 5-formyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, 5-aminoallyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, 5-carboxy-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl- pseudouridine, uridine, 5-methyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 1- methyl-pseudouridine, uridine, 5-trifluoromethyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-hydroxymethyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-bromo-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-iodo-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-methoxy-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-ethyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the Attorney Docket No.: 45817-0156WO1 disclosure contain 1-methyl-pseudouridine, uridine, 5-phenyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-ethnyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, N4-methyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-fluoro-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, N4-acetyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, pseudoisocytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-formyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-aminoallyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-carboxy-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the disclosure contain the uracil of one of the nucleosides of Table 2 and uracil as the only uracils. In other embodiments, the polynucleotides of the disclosure contain a uridine of Table 2 and uridine as the only uridines. Table 2 – Exemplary uracil-containing nucleosides Nucleoside Name
Figure imgf000087_0001
Attorney Docket No.: 45817-0156WO1 Nucleoside Name 5-methyl-2-thiouridine '
Figure imgf000088_0001
Attorney Docket No.: 45817-0156WO1 Nucleoside Name 1-benzyl-pseudo-uridine
Figure imgf000089_0001
Attorney Docket No.: 45817-0156WO1 Nucleoside Name 1-(2-amino-ethyl)pseudo-uridine
Figure imgf000090_0001
Attorney Docket No.: 45817-0156WO1 Nucleoside Name 1-methyl-6-iodo-pseudo-uridine
Figure imgf000091_0001
Attorney Docket No.: 45817-0156WO1 Nucleoside Name 1-Propargylpseudouridine
Figure imgf000092_0001
Attorney Docket No.: 45817-0156WO1 Nucleoside Name 5-methoxy-carbonyl-methyl-2’-OMe-uridine
Figure imgf000093_0001
In some embodiments, the polynucleotides of the disclosure contain the cytosine of one of the nucleosides of Table 3 and cytosine as the only cytosines. In other embodiments, the polynucleotides of the disclosure contain a cytidine of Table 3 and cytidine as the only cytidines. Attorney Docket No.: 45817-0156WO1 Table 3 – Exemplary cytosine containing nucleosides Nucleoside Name α-thio-cytidine
Figure imgf000094_0001
Attorney Docket No.: 45817-0156WO1 Nucleoside Name 2'-Deoxy-2'-b-mercaptocytidine ' '
Figure imgf000095_0001
Alterations on the internucleoside linkage The alternative nucleotides, which may be incorporated into a polynucleotide molecule, can be altered on the internucleoside linkage (e.g., phosphate backbone). Herein, in the context of the polynucleotide backbone, the phrases “phosphate” and “phosphodiester” are used interchangeably. Backbone phosphate groups can be altered by replacing one or more of the oxygen atoms with a different substituent. The alternative nucleosides and nucleotides can include the wholesale replacement of an unaltered phosphate moiety with another internucleoside linkage as described herein. Examples of alternative phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be altered by the replacement of a linking oxygen with Attorney Docket No.: 45817-0156WO1 nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates). The alternative nucleosides and nucleotides can include the replacement of one or more of the non-bridging oxygens with a borane moiety (BH3), sulfur (thio), methyl, ethyl and/or methoxy. As a non-limiting example, two non-bridging oxygens at the same position (e.g., the alpha (α), beta (β) or gamma (γ) position) can be replaced with a sulfur (thio) and a methoxy. The replacement of one or more of the oxygen atoms at the α position of the phosphate moiety (e.g., α-thio phosphate) is provided to confer stability (such as against exonucleases and endonucleases) to RNA and DNA through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment. While not wishing to be bound by theory, phosphorothioate linked polynucleotide molecules are expected to also reduce the innate immune response through weaker binding/activation of cellular innate immune molecules. In specific embodiments, an alternative nucleoside includes an alpha-thio- nucleoside (e.g., 5′-O-(1-thiophosphate)-adenosine, 5′-O-(1-thiophosphate)-cytidine (α-thio-cytidine), 5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine, or 5′-O-(1-thiophosphate)-pseudouridine). Other internucleoside linkages that may be employed according to the present disclosure, including internucleoside linkages which do not contain a phosphorous atom, are described herein below. Combinations of alternative sugars, nucleobases, and internucleoside linkages The polynucleotides of the disclosure can include a combination of alterations to the sugar, the nucleobase, and/or the internucleoside linkage. These combinations can include any one or more alterations described herein. Attorney Docket No.: 45817-0156WO1 Synthesis of polynucleotides The polynucleotide molecules for use in accordance with the disclosure may be prepared according to any useful technique, as described herein. The alternative nucleosides and nucleotides used in the synthesis of polynucleotide molecules disclosed herein can be prepared from readily available starting materials using the following general methods and procedures. Where typical or preferred process conditions (e.g., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are provided, a skilled artisan would be able to optimize and develop additional process conditions. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures. The processes described herein can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1H or 13C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography (e.g., high performance liquid chromatography (HPLC) or thin layer chromatography). Preparation of polynucleotide molecules of the present disclosure can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Greene, et al., Protective Groups in Organic Synthesis, 2d. Ed., Wiley & Sons, (1991), which is incorporated herein by reference in its entirety. The reactions of the processes described herein can be carried out in suitable solvents, which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out (i.e., temperatures which can range from the solvent’s freezing temperature to the solvent’s boiling temperature). A given reaction can be carried out Attorney Docket No.: 45817-0156WO1 in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected. Resolution of racemic mixtures of alternative polynucleotides or nucleic acids (e.g., polynucleotides or mRNA molecules) can be carried out by any of numerous methods known in the art. An example method includes fractional recrystallization using a “chiral resolving acid” which is an optically active, salt-forming organic acid. Suitable resolving agents for fractional recrystallization methods are, for example, optically active acids, such as the D and L forms of tartaric acid, acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or the various optically active camphorsulfonic acids. Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elution solvent composition can be determined by one skilled in the art. Alternative nucleosides and nucleotides (e.g., building block molecules) can be prepared according to the synthetic methods described in Ogata et al., J. Org. Chem.74:2585-2588 (2009); Purmal et al., Nucl. Acids Res.22(1): 72-78 (1994); Fukuhara et al., Biochemistry, 1(4): 563-568 (1962); and Xu et al., Tetrahedron, 48(9): 1729-1740 (1992), each of which are incorporated by reference in their entirety. If the polynucleotide includes one or more alternative nucleosides or nucleotides, the polynucleotides of the disclosure may or may not be uniformly altered along the entire length of the molecule. For example, one or more or all types of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may or may not be uniformly altered in a polynucleotide of the disclosure, or in a given predetermined sequence region thereof. In some embodiments, all nucleotides X in a polynucleotide of the disclosure (or in a given sequence region thereof) are altered, wherein X may any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C. Attorney Docket No.: 45817-0156WO1 Different sugar alterations, nucleotide alterations, and/or internucleoside linkages (e.g., backbone structures) may exist at various positions in the polynucleotide. One of ordinary skill in the art will appreciate that the nucleotide analogs or other alteration(s) may be located at any position(s) of a polynucleotide such that the function of the polynucleotide is not substantially decreased. An alteration may also be a 5′ or 3′ terminal alteration. The polynucleotide may contain from 1% to 100% alternative nucleosides, nucleotides, or internucleoside linkages (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e. any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100. In some embodiments, the remaining percentage is accounted for by the presence of A, G, U, or C. When referring to percentage incorporation by an alternative nucleoside, nucleotide, or internucleoside linkage, in some embodiments the remaining percentage necessary to total 100% is accounted for by the corresponding natural nucleoside, nucleotide, or internucleoside linkage. In other embodiments, the remaining percentage necessary to total 100% is accounted for by a second alternative nucleoside, nucleotide, or internucleoside linkage. Messenger RNA The present disclosure features compositions including one or more mRNAs, where each mRNA encodes a polypeptide (e.g., an anti-CD16 antibody or antigen- Attorney Docket No.: 45817-0156WO1 binding fragment described herein). Exemplary mRNAs of the disclosure each include (i) a 5’-cap structure; (ii) a 5’-UTR; (iii) an open reading frame encoding the polypeptide; (iv) a 3’-untranslated region (3’-UTR); and (v) a poly-A region. In some embodiments, the mRNA includes from about 30 to about 3,000 (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to 3,000, from 2,000 to 3,000, from 2,000 to 2,500, or from 2,500 to 3,000) nucleotides. mRNA: 5’-cap The 5′-cap structure of an mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5′ proximal introns removal during mRNA splicing. Endogenous mRNA molecules may be 5′-end capped generating a 5′-ppp-5′- triphosphate linkage between a terminal guanosine cap residue and the 5′-terminal transcribed sense nucleotide of the mRNA. This 5′-guanylate cap may then be methylated to generate an N7-methyl-guanylate residue. The ribose sugars of the terminal and/or anteterminal transcribed nucleotides of the 5′ end of the mRNA may optionally also be 2′-O-methylated.5′-decapping through hydrolysis and cleavage of the guanylate cap structure may target a nucleic acid molecule, such as an mRNA molecule, for degradation. Attorney Docket No.: 45817-0156WO1 Alterations to the nucleic acids of the present disclosure may generate a non- hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5′-ppp-5′ phosphorodiester linkages, alternative nucleotides may be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) may be used with α-thio-guanosine nucleotides according to the manufacturer’s instructions to create a phosphorothioate linkage in the 5′-ppp-5′ cap. Additional alternative guanosine nucleotides may be used such as α-methyl-phosphonate and seleno-phosphate nucleotides. Additional alterations include, but are not limited to, 2′-O-methylation of the ribose sugars of 5′-terminal and/or 5′-anteterminal nucleotides of the mRNA (as mentioned above) on the 2′-hydroxyl group of the sugar ring. Multiple distinct 5′-cap structures can be used to generate the 5′-cap of a nucleic acid molecule, such as an mRNA molecule. 5’-cap structures include those described in International Patent Publication Nos. WO2008/127688, WO2008/016473, and WO2011/015347, each of which is incorporated herein by reference in its entirety. Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e., endogenous, wild-type or physiological) 5′-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e., non- enzymatically) or enzymatically synthesized and/linked to a nucleic acid molecule. For example, the Anti-Reverse Cap Analog (ARCA) cap contains two guanosines linked by a 5′-5′-triphosphate group, wherein one guanosine contains an N7 methyl group as well as a 3′-O-methyl group (i.e., N7,3′-O-dimethyl-guanosine-5′- triphosphate-5′-guanosine (m7G-3′mppp-G; which may equivalently be designated 3′ O-Me-m7G(5')ppp(5')G)). The 3′-O atom of the other, unaltered, guanosine becomes linked to the 5′-terminal nucleotide of the capped nucleic acid molecule (e.g., an Attorney Docket No.: 45817-0156WO1 mRNA or mmRNA). The N7- and 3′-O-methlyated guanosine provides the terminal moiety of the capped nucleic acid molecule (e.g., mRNA or mmRNA). Another exemplary cap is mCAP, which is similar to ARCA but has a 2′-O- methyl group on guanosine (i.e., N7,2′-O-dimethyl-guanosine-5′-triphosphate-5′- guanosine, m7Gm-ppp-G). In some embodiments, the cap is a dinucleotide cap analog. As a non-limiting example, the dinucleotide cap analog may be modified at different phosphate positions with a boranophosphate group or a phophoroselenoate group such as the dinucleotide cap analogs described in US Patent No. US 8,519,110, the contents of which are herein incorporated by reference in its entirety. In some embodiments, the cap analog is a N7-(4-chlorophenoxyethyl) substituted dicnucleotide form of a cap analog known in the art and/or described herein. Non-limiting examples of a N7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analog include a N7-(4-chlorophenoxyethyl)- G(5’)ppp(5’)G and a N7-(4-chlorophenoxyethyl)-m3’-OG(5’)ppp(5’)G cap analog (see, e.g., the various cap analogs and the methods of synthesizing cap analogs described in Kore et al. Bioorganic & Medicinal Chemistry 21:4570-4574 (2013); the contents of which are herein incorporated by reference in its entirety). In some embodiments, a cap analog of the present disclosure is a 4-chloro/bromophenoxyethyl analog. While cap analogs allow for the concomitant capping of a nucleic acid molecule in an in vitro transcription reaction, up to 20% of transcripts remain uncapped. This, as well as the structural differences of a cap analog from endogenous 5′-cap structures of nucleic acids produced by the endogenous, cellular transcription machinery, may lead to reduced translational competency and reduced cellular stability. Nucleic acids of the disclosure (e.g., mRNAs of the disclosure) may also be capped post-transcriptionally, using enzymes.5’ cap structures produced by enzymatic capping may enhance binding of cap binding proteins, increase half-life, Attorney Docket No.: 45817-0156WO1 reduce susceptibility to 5′ endonucleases and/or reduce 5′ decapping, as compared to synthetic 5′-cap structures known in the art (or to a wild-type, natural or physiological 5′-cap structure). For example, recombinant Vaccinia Virus Capping Enzyme and recombinant 2′-O-methyltransferase enzyme can create a canonical 5′-5′-triphosphate linkage between the 5′-terminal nucleotide of an mRNA and a guanosine cap nucleotide wherein the cap guanosine contains an N7 methylation and the 5′-terminal nucleotide of the mRNA contains a 2′-O-methyl. Such a structure is termed the Cap1 structure. This cap results in a higher translational-competency and cellular stability and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5′cap analog structures known in the art. Cap structures include 7mG(5')ppp(5')N,pN2p (cap 0), 7mG(5')ppp(5')NlmpNp (cap 1), 7mG(5')- ppp(5')NlmpN2mp (cap 2), and m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up (cap 4). According to the present disclosure, 5′ terminal caps may include endogenous caps or cap analogs. According to the present disclosure, a 5′ terminal cap may include a guanosine analog. Useful guanosine analogs include inosine, N1-methyl- guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino- guanosine, LNA-guanosine, and 2-azido-guanosine. In some embodiments, the nucleic acids described herein may contain a modified 5’-cap. A modification on the 5’-cap may increase the stability of mRNA, increase the half-life of the mRNA, and could increase the mRNA translational efficiency. The modified 5’-cap may include, but is not limited to, one or more of the following modifications: modification at the 2’ and/or 3’ position of a capped guanosine triphosphate (GTP), a replacement of the sugar ring oxygen (that produced the carbocyclic ring) with a methylene moiety (CH2), a modification at the triphosphate bridge moiety of the cap structure, or a modification at the nucleobase (G) moiety. mRNA: Coding region Attorney Docket No.: 45817-0156WO1 Provided are nucleic acids that encode antibodies or antigen-binding fragments of the disclosure. As recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of this present disclosure. For example, provided herein is any protein fragment of a reference protein (meaning a polypeptide sequence at least one amino acid residue shorter than a reference polypeptide sequence but otherwise identical) 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or greater than 100 amino acids in length. In another example, any protein that includes a stretch of about 20, about 30, about 40, about 50, or about 100 amino acids which are about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% identical to any of the sequences described herein can be utilized in accordance with the present disclosure. In certain embodiments, a protein sequence to be utilized in accordance with the present disclosure includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided or referenced herein. mRNA: Poly-A tail During RNA processing, a long chain of adenosine nucleotides (poly(A) tail) is normally added to mRNA molecules to increase the stability of the mRNA. Immediately after transcription, the 3' end of the transcript is cleaved to free a 3' hydroxyl. Then poly(A) polymerase adds a chain of adenosine nucleotides to the RNA. The process, called polyadenylation, adds a poly-A tail that is between 100 and 250 residues long (SEQ ID NO: 38). Methods for the stabilization of RNA by incorporation of chain-terminating nucleosides at the 3’-terminus include those described in International Patent Publication No. WO2013/103659, incorporated herein in its entirety. Poly(A) tail deadenylation by 3′ exonucleases is a key step in cellular mRNA degradation in eukaryotes. By blocking 3' exonucleases, the functional half-life of mRNA can be increased, resulting in increased protein expression. Chemical and enzymatic ligation strategies to modify the 3' end of mRNA with reverse chirality adenosine (LA10) and/or inverted deoxythymidine (IdT) are known to those of skill in Attorney Docket No.: 45817-0156WO1 the art and have been demonstrated to extend mRNA half-life in cellular and in vivo studies. In some embodiments, the poly(A)tail of the mRNA includes a 3’ LA10 or IdT modification. For example, as described in International Patent Publication No. WO2017/049275, the tail modifications of which are incorporated by reference in their entirety. Additional strategies have been explored to further stabilize mRNA, including: chemical modification of the 3’ nucleotide (e.g., conjugation of a morpholino to the 3’ end of the poly(A)tail); incorporation of stabilizing sequences after the poly(A) tail (e.g., a co-polymer, a stem-loop, or a triple helix); and/or annealing of structured oligos to the 3' end of an mRNA, as described, for example, in International Patent Publication No. WO2017/049286, the stabilized linkages of which are incorporated by reference in their entirety. Annealing an oligonucleotide (e.g., an oligonucleotide conjugate) with a complex secondary structure (e.g., a triple-helix structure or a stem-loop structure) at the 3’end may provide nuclease resistance and increase half-life of mRNA. Unique poly(A) tail lengths may provide certain advantages to the RNAs of the present disclosure. Generally, the length of a poly(A) tail of the present disclosure is greater than 30 nucleotides in length (SEQ ID NO: 39). In some embodiments, the poly(A) tail is greater than 35 nucleotides in length. In some embodiments, the length is at least 40 nucleotides. In another embodiment, the length is at least 45 nucleotides. In some embodiments, the length is at least 50 nucleotides. In some embodiments, the length is at least 55 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 65 nucleotides. In another embodiment, the length is at least 70 nucleotides. In some embodiments, the length is at least 80 nucleotides. In some embodiments, the length is at least 90 nucleotides. In some embodiments, the length is at least 100 nucleotides. In some embodiments, the length is at least 120 nucleotides. In some embodiments, the length is at least 140 nucleotides. In some embodiments, the length is at least 160 nucleotides. In some embodiments, the length is at least 180 nucleotides. In some embodiments, the length Attorney Docket No.: 45817-0156WO1 is at least 200 nucleotides. In some embodiments, the length is at least 250 nucleotides. In some embodiments, the length is at least 300 nucleotides. In some embodiments, the length is at least 350 nucleotides. In some embodiments, the length is at least 400 nucleotides. In some embodiments, the length is at least 450 nucleotides. In some embodiments, the length is at least 500 nucleotides. In some embodiments, the length is at least 600 nucleotides. In some embodiments, the length is at least 700 nucleotides. In some embodiments, the length is at least 800 nucleotides. In some embodiments, the length is at least 900 nucleotides. In some embodiments, the length is at least 1000 nucleotides. In some embodiments, the length is at least 1100 nucleotides. In some embodiments, the length is at least 1200 nucleotides. In some embodiments, the length is at least 1300 nucleotides. In some embodiments, the length is at least 1400 nucleotides. In some embodiments, the length is at least 1500 nucleotides. In some embodiments, the length is at least 1600 nucleotides. In some embodiments, the length is at least 1700 nucleotides. In some embodiments, the length is at least 1800 nucleotides. In some embodiments, the length is at least 1900 nucleotides. In some embodiments, the length is at least 2000 nucleotides. In some embodiments, the length is at least 2500 nucleotides. In some embodiments, the length is at least 3000 nucleotides. In some embodiments, the poly(A) tail may be 80 nucleotides, 120 nucleotides, or 160 nucleotides in length. In some embodiments, the poly(A) tail may be 20, 40, 80, 100, 120, 140 or 160 nucleotides in length. In some embodiments, the poly(A) tail is designed relative to the length of the mRNA. This design may be based on the length of the coding region of the mRNA, the length of a particular feature or region of the mRNA, or based on the length of the ultimate product expressed from the RNA. When relative to any additional feature of the RNA (e.g., other than the mRNA portion which includes the poly(A) tail), poly(A) tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% greater in length than the additional feature. The poly(A) tail may also be designed as a fraction of the mRNA to which it belongs. In this context, the poly(A) tail may be 10, 20, 30, 40, 50, 60, 70, Attorney Docket No.: 45817-0156WO1 80, or 90% or more of the total length of the construct or the total length of the construct minus the poly(A) tail. In some embodiments, engineered binding sites and/or the conjugation of nucleic acids or mRNA for poly(A) binding protein may be used to enhance expression. The engineered binding sites may be sensor sequences which can operate as binding sites for ligands of the local microenvironment of the nucleic acids and/or mRNA. As a non-limiting example, the nucleic acids and/or mRNA may include at least one engineered binding site to alter the binding affinity of poly(A) binding protein (PABP) and analogs thereof. The incorporation of at least one engineered binding site may increase the binding affinity of the PABP and analogs thereof. Additionally, multiple distinct nucleic acids or mRNA may be linked together to the PABP (poly(A) binding protein) through the 3′-end using nucleotides at the 3′- terminus of the poly(A) tail. Transfection experiments can be conducted in relevant cell lines and protein production can be assayed by ELISA at 12hr, 24hr, 48hr, 72hr, and day 7 post-transfection. As a non-limiting example, the transfection experiments may be used to evaluate the effect on PABP or analogs thereof binding affinity as a result of the addition of at least one engineered binding site. In some embodiments, a poly(A) tail may be used to modulate translation initiation. While not wishing to be bound by theory, the poly-A tail recruits PABP which in turn can interact with translation initiation complex and thus may be essential for protein synthesis. In some embodiments, a poly(A) tail may also be used in the present disclosure to protect against 3’-5’ exonuclease digestion. In some embodiments, the nucleic acids or mRNA of the present disclosure are designed to include a poly-A-G Quartet. The G-quartet is a cyclic hydrogen bonded array of four guanosine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of the poly-A tail. The resultant nucleic acid or mRNA may be assayed for stability, Attorney Docket No.: 45817-0156WO1 protein production and other parameters including half-life at various time points. It has been discovered that the poly-A-G quartet results in protein production equivalent to at least 75% of that seen using a poly-A tail of 120 nucleotides alone (SEQ ID NO: 40). In some embodiments, the nucleic acids or mRNA of the present disclosure may include a poly(A) tail and may be stabilized by the addition of a chain terminating nucleoside. The nucleic acids and/or mRNA with a poly(A) tail may further include a 5’cap structure. In some embodiments, the nucleic acids or mRNA of the present disclosure may include a poly-A-G Quartet. The nucleic acids and/or mRNA with a poly-A-G Quartet may further include a 5’cap structure. In some embodiments, the chain terminating nucleoside which may be used to stabilize the nucleic acid or mRNA including a poly(A) tail or poly-A-G Quartet may be, but is not limited to, those described in International Patent Publication No. WO2013103659, incorporated herein by reference in its entirety. In some embodiments, the chain terminating nucleosides which may be used with the present disclosure includes, but is not limited to, 3'-deoxyadenosine (cordycepin), 3'- deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymine, 2',3'- dideoxynucleosides, such as 2',3'- dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'- dideoxycytosine, 2',3'- dideoxyguanosine, 2',3'-dideoxythymine, a 2'- deoxynucleoside, or a -O- methylnucleoside. In some embodiments, the mRNA which includes a poly(A) tail or a poly-A-G Quartet may be stabilized by an alteration to the 3’region of the nucleic acid that can prevent and/or inhibit the addition of oligo(U) (see, e.g., International Patent Publication No. WO2013/103659, incorporated herein by reference in its entirety). In yet another embodiment, the mRNA, which includes a poly(A) tail or a poly-A-G Quartet, may be stabilized by the addition of an oligonucleotide that terminates in a 3’-deoxynucleoside, 2’,3’-dideoxynucleoside 3'-O-methylnucleosides, Attorney Docket No.: 45817-0156WO1 3'-O-ethylnucleosides, 3'-arabinosides, and other alternative nucleosides known in the art and/or described herein. mRNA: Stem-loops In some embodiments, the nucleic acids of the present disclosure (e.g., the mRNA of the present disclosure) may include a stem-loop such as, but not limited to, a histone stem-loop. The stem-loop may be a nucleotide sequence that is about 25 or about 26 nucleotides in length such as, but not limited to, SEQ ID NOs: 7-17 as described in International Patent Publication No. WO2013/103659, incorporated herein by reference in its entirety. The histone stem-loop may be located 3’ relative to the coding region (e.g., at the 3’ terminus of the coding region). As a non-limiting example, the stem-loop may be located at the 3’ end of a nucleic acid described herein. In some embodiments, the stem-loop may be located in the second terminal region. As a non-limiting example, the stem-loop may be located within an untranslated region (e.g., 3’-UTR) in the second terminal region. In some embodiments, the nucleic acid such as, but not limited to mRNA, which includes the histone stem-loop may be stabilized by the addition of at least one chain terminating nucleoside. Not wishing to be bound by theory, the addition of at least one chain terminating nucleoside may slow the degradation of a nucleic acid and thus can increase the half-life of the nucleic acid. In some embodiments, the chain terminating nucleoside may be, but is not limited to, those described in International Patent Publication No. WO2013/103659, incorporated herein by reference in its entirety. In some embodiments, the chain terminating nucleosides which may be used with the present disclosure includes, but is not limited to, 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymine, 2',3'-dideoxynucleosides, such as 2',3'- dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'- Attorney Docket No.: 45817-0156WO1 dideoxyguanosine, 2',3'-dideoxythymine, a 2'-deoxynucleoside, or a -O- methylnucleoside. In some embodiments, the nucleic acid such as, but not limited to mRNA, which includes the histone stem-loop may be stabilized by an alteration to the 3’region of the nucleic acid that can prevent and/or inhibit the addition of oligo(U) (see, e.g., International Patent Publication No. WO2013/103659, incorporated herein by reference in its entirety). In yet another embodiment, the nucleic acid such as, but not limited to, mRNA, which includes the histone stem-loop may be stabilized by the addition of an oligonucleotide that terminates in a 3’-deoxynucleoside, 2’,3’-dideoxynucleoside 3'- O-methylnucleosides, 3'-O-ethylnucleosides, 3'-arabinosides, and other alternative nucleosides known in the art and/or described herein. In some embodiments, the nucleic acids of the present disclosure may include a histone stem-loop, a poly(A) tail sequence, and/or a 5’-cap structure. The histone stem-loop may be before and/or after the poly-A tail sequence. The nucleic acids including the histone stem-loop and a poly(A) tail sequence may include a chain terminating nucleoside described herein. In some embodiments, the nucleic acids of the present disclosure may include a histone stem-loop and a 5’-cap structure. The 5’-cap structure may include, but is not limited to, those described herein and/or known in the art. In some embodiments, the nucleic acids described herein may include at least one histone stem-loop and a poly(A) sequence or polyadenylation signal. Non- limiting examples of nucleic acid sequences encoding for at least one histone stem- loop and a poly-A sequence or a polyadenylation signal are described in International Patent Publication Nos. WO2013/120497, WO2013/120629, WO2013/120500, WO2013/120627, WO2013/120498, WO2013/120626, WO2013/120499 and WO2013/120628, the contents of each of which are incorporated herein by reference in their entirety. In some embodiments, the nucleic acid encoding for a histone stem- Attorney Docket No.: 45817-0156WO1 loop and a poly(A) sequence or a polyadenylation signal may code for a pathogen antigen or fragment thereof such as the nucleic acid sequences described in International Patent Publication Nos. WO2013/120499 and WO2013/120628, the contents of both of which are incorporated herein by reference in their entirety. In some embodiments, the nucleic acid encoding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal may code for a therapeutic protein such as the nucleic acid sequences described in International Patent Publication Nos. WO2013/120497 and WO2013/120629, the contents of both of which are incorporated herein by reference in their entirety. In some embodiments, the nucleic acid encoding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal may code for a tumor antigen or fragment thereof such as the nucleic acid sequences described in International Patent Publication Nos. WO2013/120500 and WO2013/120627, the contents of both of which are incorporated herein by reference in their entirety. In some embodiments, the nucleic acid encoding for a histone stem- loop and a poly(A) sequence or a polyadenylation signal may code for an autoimmune self-antigen such as the nucleic acid sequences described in International Patent Publication Nos. WO2013/120498 and WO2013/120626, the contents of both of which are incorporated herein by reference in their entirety. mRNA: Triple helices In some embodiments, nucleic acids of the present disclosure (e.g., the mRNA of the present disclosure) may include a triple helix on the 3’ end of the nucleic acid. The 3’ end of the nucleic acids of the present disclosure may include a triple helix alone or in combination with a poly(A) tail. In some embodiments, the nucleic acid of the present disclosure may include at least a first and a second U-rich region, a conserved stem-loop region between the first and second region and an A-rich region. The first and second U-rich region and the A-rich region may associate to form a triple helix on the 3’ end of the nucleic acid. This triple helix may stabilize the nucleic acid, enhance the translational efficiency of the nucleic acid and/or protect the 3’ end from degradation. Triple helices include, but Attorney Docket No.: 45817-0156WO1 are not limited to, the triple helix sequence of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), MEN-β and polyadenylated nuclear (PAN) RNA (see, Wilusz et al., Genes & Development 26:2392-2407 (2012); herein incorporated by reference in its entirety). In some embodiments, the triple helix may be formed from the cleavage of a MALAT1 sequence prior to the cloverleaf structure. While not meaning to be bound by theory, MALAT1 is a long non-coding RNA which, when cleaved, forms a triple helix and a tRNA-like cloverleaf structure. The MALAT1 transcript then localizes to nuclear speckles and the tRNA-like cloverleaf localizes to the cytoplasm (Wilusz et al., Cell.135(5): 919-932 (2008); incorporated herein by reference in its entirety). As a non-limiting example, the terminal end of the nucleic acid of the present disclosure including the MALAT1 sequence can then form a triple helix structure, after RNaseP cleavage from the cloverleaf structure, which stabilizes the nucleic acid (Peart et al., WIREs RNA.4(5):491-506 (2013); incorporated herein by reference in its entirety). In some embodiments, the nucleic acids or mRNA described herein include a MALAT1 sequence. In some embodiments, the nucleic acids or mRNA may be polyadenylated. In yet another embodiment, the nucleic acids or mRNA is not polyadenylated but has an increased resistance to degradation compared to unaltered nucleic acids or mRNA. In some embodiments, the nucleic acids of the present disclosure may include a MALAT1 sequence in the second flanking region (e.g., the 3’-UTR). As a non- limiting example, the MALAT1 sequence may be human or mouse. mRNA: Translation Enhancer Elements (TEEs) The term “translational enhancer element” or “translation enhancer element” (herein collectively referred to as “TEE”) refers to sequences that increase the amount of polypeptide or protein produced from an mRNA. TEEs are conserved elements in the UTR which can promote translational activity of a nucleic acid such as, but not Attorney Docket No.: 45817-0156WO1 limited to, cap-dependent or cap-independent translation. The conservation of these sequences has been previously shown by Pánek et al., Nucleic Acids Research. 41(16): 7625-7634 (2013); incorporated herein by reference in its entirety) across 14 species including humans. In some embodiments, the 5’-UTR of the mRNA includes at least one TEE. The TEE may be located between the transcription promoter and the start codon. The mRNA with at least one TEE in the 5’-UTR may include a cap at the 5’-UTR. Further, at least one TEE may be located in the 5’-UTR of mRNA undergoing cap- dependent or cap-independent translation. The TEEs known may be in the 5′-leader of the Gtx homeodomain protein (Chappell et al., Proc. Natl. Acad. Sci. USA 101:9590-9594 (2004), incorporated herein by reference in their entirety). In another non-limiting example, TEEs are disclosed as SEQ ID NOs: 1-35 in US Patent Publication No. US20090226470, SEQ ID NOs: 1-35 in US Patent Publication No. US20130177581, SEQ ID NOs: 1-35 in International Patent Publication No. WO2009075886, SEQ ID NOs: 1-5, and 7-645 in International Patent Publication No. WO2012009644, SEQ ID NO: 1 in International Patent Publication No. WO1999024595, SEQ ID NO: 1 in US Patent No. US6310197, and SEQ ID NO: 1 in US Patent No. US6849405, each of which is incorporated herein by reference in its entirety. The TEE may be an internal ribosome entry site (IRES), HCV-IRES or an IRES element such as, but not limited to, those described in US Patent No. US7468275, US Patent Publication Nos. US20070048776 and US20110124100 and International Patent Publication Nos. WO2007025008 and WO2001055369, each of which is incorporated herein by reference in its entirety. The IRES elements may include, but are not limited to, the Gtx sequences (e.g., Gtx9-nt, Gtx8-nt, Gtx7-nt) described by Chappell et al. (Proc. Natl. Acad. Sci. USA 101:9590-9594 (2004)) and Zhou et al. (PNAS 102:6273-6278 (2005)) and in US Patent Publication Nos. Attorney Docket No.: 45817-0156WO1 US20070048776 and US20110124100 and International Patent Publication No. WO2007025008, each of which is incorporated herein by reference in its entirety. Additional exemplary TEEs are disclosed in US Patent Nos. US6310197, US6849405, US7456273, US7183395; US Patent Publication Nos. US20090226470, US20070048776, US20110124100, US20090093049, US20130177581; International Patent Publication Nos. WO2009075886, WO2007025008, WO2012009644, WO2001055371 WO1999024595; and European Patent Publications Nos. EP2610341A1 and EP2610340A1; each of which is incorporated herein by reference in its entirety. In some embodiments, the polynucleotides, primary constructs, alternative nucleic acids and/or mRNA may include at least one TEE that is described in International Patent Publication Nos. WO1999024595, WO2012009644, WO2009075886, WO2007025008, WO1999024595, European Patent Publication Nos. EP2610341A1 and EP2610340A1, US Patent Nos. US6310197, US6849405, US7456273, US7183395, US Patent Publication No. US20090226470, US20110124100, US20070048776, US20090093049, and US20130177581 each of which is incorporated herein by reference in its entirety. The TEE may be located in the 5’-UTR of the mRNA. In some embodiments, the polynucleotides, primary constructs, alternative nucleic acids and/or mmRNA may include at least one TEE that has 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 95% or at least 99% identity with the TEEs described in US Patent Publication Nos. US20090226470, US20070048776, US20130177581 and US20110124100, International Patent Publication Nos. WO1999024595, WO2012009644, WO2009075886 and WO2007025008, European Patent Publication No. EP2610341A1 and EP2610340A1, and US Patent Nos. US6310197, US6849405, US7456273, and US7183395, each of which is incorporated herein by reference in its entirety. Attorney Docket No.: 45817-0156WO1 Multiple copies of a specific TEE can be present in mRNA. The TEEs in the translational enhancer polynucleotides can be organized in one or more sequence segments. A sequence segment can harbor one or more of the specific TEEs exemplified herein, with each TEE being present in one or more copies. When multiple sequence segments are present in a translational enhancer polynucleotide, they can be homogenous or heterogeneous. Thus, the multiple sequence segments in a translational enhancer polynucleotide can harbor identical or different types of the specific TEEs exemplified herein, identical or different number of copies of each of the specific TEEs, and/or identical or different organization of the TEEs within each sequence segment. In some embodiments, the 5’-UTR of the mRNA may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55 or more than 60 TEE sequences. The TEE sequences in the 5’-UTR of mRNA of the present disclosure may be the same or different TEE sequences. The TEE sequences may be in a pattern such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times. In these patterns, each letter, A, B, or C represent a different TEE sequence at the nucleotide level. In some embodiments, the 5’-UTR may include a spacer to separate two TEE sequences. As a non-limiting example, the spacer may be a 15 nucleotide spacer and/or other spacers known in the art. As another non-limiting example, the 5’-UTR may include a TEE sequence-spacer module repeated at least once, at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times and at least 9 times, or more than 9 times in the 5’-UTR. In some embodiments, the TEE in the 5’-UTR of the mRNA of the present disclosure may include at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, Attorney Docket No.: 45817-0156WO1 at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more than 99% of the TEE sequences disclosed in US Patent Publication Nos. US20090226470, US20070048776, US20130177581 and US20110124100, International Patent Publication Nos. WO1999024595, WO2012009644, WO2009075886 and WO2007025008, European Patent Publication Nos. EP2610341A1 and EP2610340A1, and US Patent No. US6310197, US6849405, US7456273, and US7183395 each of which is incorporated herein by reference in its entirety. In some embodiments, the TEE in the 5’-UTR of the mRNA of the present disclosure may include a 5-30 nucleotide fragment, a 5-25 nucleotide fragment, a 5-20 nucleotide fragment, a 5-15 nucleotide fragment, a 5-10 nucleotide fragment of the TEE sequences disclosed in US Patent Publication Nos. US20090226470, US20070048776, US20130177581, and US20110124100, International Patent Publication No. WO1999024595, WO2012009644, WO2009075886, and WO2007025008, European Patent Publication No. EP2610341A1 and EP2610340A1, and US Patent Nos. US6310197, US6849405, US7456273, and US7183395; each of which is incorporated herein by reference in its entirety. In some embodiments, the TEE in the 5’-UTR of the mRNA of the present disclosure may include at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, 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 95%, at least 99% or more than 99% of the TEE sequences disclosed in Chappell et al. (Proc. Natl. Acad. Sci. USA 101:9590-9594 (2004)) and Zhou et al. (PNAS 102:6273-6278 (2005)), in Supplemental Table 1 and in Supplemental Table 2 disclosed by Wellensiek et al. (Nature Methods.10(8):747-750 (2013)); each of which is herein incorporated by reference in its entirety. In some embodiments, the TEE in the 5’-UTR of the polynucleotides, primary constructs, alternative nucleic acids and/or mmRNA of the present disclosure may include a 5-30 nucleotide fragment, a 5-25 nucleotide fragment, a 5-20 nucleotide fragment, a 5-15 nucleotide fragment, a 5-10 nucleotide fragment of the TEE sequences disclosed in Chappell et al. (Proc. Natl. Acad. Sci. USA 101:9590-9594 (2004)) and Zhou et al. Attorney Docket No.: 45817-0156WO1 (PNAS 102:6273-6278 (2005)), in Supplemental Table 1 and in Supplemental Table 2 disclosed by Wellensiek et al. (Nature Methods.10(8):747-750 (2013)); each of which is incorporated herein by reference in its entirety. In some embodiments, the TEE used in the 5’-UTR of the mRNA of the present disclosure is an IRES sequence such as, but not limited to, those described in US Patent No. US7468275 and International Patent Publication No. WO2001055369, each of which is incorporated herein by reference in its entirety. In some embodiments, the TEEs used in the 5’-UTR of the mRNA of the present disclosure may be identified by the methods described in US Patent Publication Nos. US20070048776 and US20110124100 and International Patent Publication Nos. WO2007025008 and WO2012009644, each of which is incorporated herein by reference in its entirety. In some embodiments, the TEEs used in the 5’-UTR of the mRNA of the present disclosure may be a transcription regulatory element described in US Patent Nos. US7456273 and US7183395, US Patent Publication No. US20090093049, and International Publication No. WO2001055371, each of which is incorporated herein by reference in its entirety. The transcription regulatory elements may be identified by methods known in the art, such as, but not limited to, the methods described in US Patent Nos. US7456273 and US7183395, US Patent Publication No. US20090093049, and International Publication No. WO2001055371, each of which is incorporated herein by reference in its entirety. In yet another embodiment, the TEE used in the 5’-UTR of the mRNA of the present disclosure is an oligonucleotide or portion thereof as described in US Patent No. US7456273 and US7183395, US Patent Publication No. US20090093049, and International Publication No. WO2001055371, each of which is incorporated herein by reference in its entirety. The 5’-UTR including at least one TEE described herein may be incorporated in a monocistronic sequence such as, but not limited to, a vector system or a nucleic Attorney Docket No.: 45817-0156WO1 acid vector. As a non-limiting example, the vector systems and nucleic acid vectors may include those described in US Patent Nos.7456273 and US7183395, US Patent Publication Nos. US20070048776, US20090093049, and US20110124100 and International Patent Publication Nos. WO2007025008 and WO2001055371, each of which is incorporated herein by reference in its entirety. In some embodiments, the TEEs described herein may be located in the 5’- UTR and/or the 3’-UTR of the mRNA. The TEEs located in the 3’-UTR may be the same and/or different than the TEEs located in and/or described for incorporation in the 5’-UTR. In some embodiments, the 3’-UTR of the mRNA may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55 or more than 60 TEE sequences. The TEE sequences in the 3’-UTR of the polynucleotides, primary constructs, alternative nucleic acids and/or mmRNA of the present disclosure may be the same or different TEE sequences. The TEE sequences may be in a pattern such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times. In these patterns, each letter, A, B, or C represent a different TEE sequence at the nucleotide level. In some embodiments, the 3’-UTR may include a spacer to separate two TEE sequences. As a non-limiting example, the spacer may be a 15-nucleotide spacer and/or other spacers known in the art. As another non-limiting example, the 3’-UTR may include a TEE sequence-spacer module repeated at least once, at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times and at least 9 times or more than 9 times in the 3’-UTR. mRNA: Heterologous 5’-UTRs Attorney Docket No.: 45817-0156WO1 5’-UTRs of an mRNA of the disclosure may be homologous or heterologous to the coding region found in the mRNA. Multiple 5′ UTRs may be included in mRNA and may be the same or of different sequences. Any portion of the mRNA, including none, may be codon optimized and any may independently contain one or more different structural or chemical alterations, before and/or after codon optimization. Shown in Lengthy Table 21 in International Patent Publication No. WO 2014/081507, and in Lengthy Table 21 and in Table 22 in International Patent Publication No. WO 2014/081507, the contents of each of which are incorporated herein by reference in their entirety, is a listing of the start and stop site of mRNAs. In Table 21 each 5’-UTR (5’-UTR-005 to 5’-UTR 68511) is identified by its start and stop site relative to its native or wild type (homologous) transcript (ENST; the identifier used in the ENSEMBL database). To alter one or more properties of the mRNA of the disclosure, 5’-UTRs which are heterologous to the coding region of the mRNA are engineered into the mRNA. The mRNA (e.g., an mRNA in a composition described herein) is administered to cells, tissue, or organisms, and outcomes such as protein level, localization, and/or half-life are measured to evaluate the beneficial effects the heterologous 5’-UTR may have on mRNA. Variants of the 5’ UTRs may be utilized wherein one or more nucleotides are added or removed to the termini, including A, T, C or G.5’-UTRs may also be codon-optimized or altered in any manner described herein. mRNA: RNA motifs for RNA binding proteins RNA binding proteins (RBPs) can regulate numerous aspects of co- and post- transcription gene expression, such as, but not limited to, RNA splicing, localization, translation, turnover, polyadenylation, capping, alteration, export, and localization. RNA-binding domains (RBDs), such as, but not limited to, RNA recognition motif (RR) and hnRNP K-homology (KH) domains, typically regulate the sequence association between RBPs and their RNA targets (Ray et al., Nature.499:172-177 Attorney Docket No.: 45817-0156WO1 (2013); incorporated herein by reference in its entirety). In some embodiments, the canonical RBDs can bind short RNA sequences. In some embodiments, the canonical RBDs can recognize structure RNAs. In some embodiments, to increase the stability of the mRNA of interest, an mRNA encoding HuR is co-transfected or co-injected along with the mRNA of interest into the cells or into the tissue. These proteins can also be tethered to the mRNA of interest in vitro and then administered to the cells together. Poly A tail binding protein, PABP interacts with eukaryotic translation initiation factor eIF4G to stimulate translational initiation. Co-administration of mRNAs encoding these RBPs along with the mRNA drug and/or tethering these proteins to the mRNA drug in vitro and administering the protein-bound mRNA into the cells can increase the translational efficiency of the mRNA. The same concept can be extended to co- administration of mRNA along with mRNAs encoding various translation factors and facilitators as well as with the proteins themselves to influence RNA stability and/or translational efficiency. In some embodiments, the nucleic acids and/or mRNA may include at least one RNA-binding motif such as, but not limited to an RNA-binding domain (RBD). In some embodiments, the RBD may be any of the RBDs, fragments or variants thereof descried by Ray et al., (Nature.499:172-177 (2013); incorporated herein by reference in its entirety). In some embodiments, the nucleic acids or mRNA of the present disclosure may include a sequence for at least one RNA-binding domain (RBDs). When the nucleic acids or mRNA of the present disclosure include more than one RBD, the RBDs do not need to be from the same species or even the same structural class. In some embodiments, at least one flanking region (e.g., the 5’-UTR and/or the 3’-UTR) may include at least one RBD. In some embodiments, the first flanking region and the second flanking region may both include at least one RBD. The RBD may be the same or each of the RBDs may have at least 60% (e.g., at least 70%, 80%, Attorney Docket No.: 45817-0156WO1 or 90%) sequence identity to the other RBD. As a non-limiting example, at least on RBD may be located before, after and/or within the 3’-UTR of the nucleic acid or mRNA of the present disclosure. As another non-limiting example, at least one RBD may be located before or within the first 300 nucleosides of the 3’-UTR. In some embodiments, the nucleic acids and/or mRNA of the present disclosure may include at least one RBD in the first region of linked nucleosides. The RBD may be located before, after, or within a coding region (e.g., the ORF). In another embodiment, the first region of linked nucleosides and/or at least one flanking region may include at least on RBD. As a non-limiting example, the first region of linked nucleosides may include a RBD related to splicing factors and at least one flanking region may include a RBD for stability and/or translation factors. In some embodiments, the nucleic acids and/or mRNA of the present disclosure may include at least one RBD located in a coding and/or non-coding region of the nucleic acids and/or mRNA. In some embodiments, at least one RBD may be incorporated into at least one flanking region to increase the stability of the nucleic acid and/or mRNA of the present disclosure. In some embodiments, an antisense locked nucleic acid (LNA) oligonucleotides and exon-junction complexes (EJCs) may be used in the RNA binding protein motif. The LNA and EJCs may be used around a start codon (-4 to +37 where the A of the AUG codons is +1) in order to decrease the accessibility to the first start codon (AUG). Nucleic acids as agents for delivering anti-CD16 antibodies or binding proteins The compositions of the disclosure can be administered not only as antibodies or antigen-binding fragments, but also in the form of nucleic acids. The exemplary nucleic acids described herein may be used to deliver antibodies or antigen-binding Attorney Docket No.: 45817-0156WO1 fragments to a subject. These nucleic acids (e.g., RNAs, such as mRNAs) may be used as therapeutic agents to express antibodies or antigen-binding fragments of the disclosure as a therapy to treat a target disease. Pharmaceutical Compositions Pharmaceutical compositions containing an anti-CD16 antibody, antigen- binding fragment, binding protein, or nucleic acid encoding the same, described herein can be prepared using methods known in the art. Pharmaceutical compositions described herein may contain an anti-CD16 antibody, antigen-binding fragment, binding protein, or a nucleic acid encoding the same, described herein in combination with one or more pharmaceutically acceptable excipients. For instance, pharmaceutical compositions described herein can be prepared using physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences (19th ed., 1995), incorporated herein by reference), and in a desired form, e.g., in the form of lyophilized formulations or aqueous solutions. The compositions can also be prepared so as to contain the active agent (e.g., an anti-CD16 antibody, antigen- binding fragment, binding protein, or a nucleic acid encoding the same) at a desired concentration. For example, a pharmaceutical composition described herein may contain at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%) active agent by weight (w/w). Additionally, an active agent that can be incorporated into a pharmaceutical formulation can itself have a desired level of purity. For example, a polypeptide or nucleic acid described herein may be characterized by a certain degree of purity after isolating the antibody or binding protein from cell culture media or after chemical synthesis. An antibody, antigen-biding fragment, binding protein, or nucleic acid described herein may be at least 10% pure prior to incorporating the antibody, antigen-biding fragment, binding protein or nucleic acid into a pharmaceutical composition (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or 100% pure). Attorney Docket No.: 45817-0156WO1 Pharmaceutical compositions can be prepared for storage as lyophilized formulations or aqueous solutions by mixing the active agent having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers typically employed in the art, e.g., buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants, and other miscellaneous additives. See, e.g., Remington's Pharmaceutical Sciences (19th ed., 1995), incorporated herein by reference). Such additives must be nontoxic to the recipients at the dosages and concentrations employed. Buffering agents Buffering agents help to maintain the pH in the range which approximates physiological conditions. Suitable buffering agents for use with the pharmaceutical compositions of the disclosure include both organic and inorganic acids and salts thereof, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g., succinic acid- monosodium succinate mixture, succinic acid- sodium hydroxide mixture, succinic acid- disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid- disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodium gluconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium gluconate mixture, etc.), oxalate buffer (e.g., oxalic acid- sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.), and acetate buffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.). Additionally, phosphate buffers, histidine buffers, and trimethylamine salts such as Tris can be used. Attorney Docket No.: 45817-0156WO1 Preservatives Preservatives can be added to a composition described herein, for example, to inhibit microbial growth. Suitable preservatives for use with the pharmaceutical compositions of the disclosure include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalconium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol. Isotonifiers, also known as “stabilizers,” can be added to ensure isotonicity of liquid compositions described herein and include polhydric sugar alcohols, for example trihydric or higher sugar alcohols, such as glycerin, arabitol, xylitol, sorbitol, and mannitol. Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the therapeutic agent or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can be polyhydric sugar alcohols; amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L- leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a- monothioglycerol and sodium thio sulfate; low molecular weight polypeptides (e.g., peptides of 10 residues or fewer); proteins such as HSA, BSA, MSA, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose, fructose, glucose; disaccharides such as lactose, maltose, sucrose and trisaccharides such as raffinose; and polysaccharides such as dextran. Detergents In some embodiments, non-ionic surfactants or detergents (also known as “wetting agents”) are added to the pharmaceutical composition, for example, to help Attorney Docket No.: 45817-0156WO1 solubilize the therapeutic agent as well as to protect the therapeutic agent against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stressed without causing denaturation of the protein. Suitable non-ionic surfactants include, for example and without limitation, polysorbates (20, 80, etc.), polyoxamers (184, 188 etc.), Pluronic polyols, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.). Other pharmaceutical carriers Alternative pharmaceutically acceptable carriers that can be incorporated into a pharmaceutical composition described herein may include dextrose, sucrose, sorbitol, mannitol, starch, rubber arable, potassium phosphate, arginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oils, but not limited to. A pharmaceutical composition described herein may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative. Details of suitable pharmaceutically acceptable carriers and formulations can be found in Remington's Pharmaceutical Sciences (19th ed., 1995), which is incorporated herein by reference. Lipid Nanoparticle (LNP) Compositions The present disclosure provides LNP compositions with advantageous properties. The lipid nanoparticle compositions described herein may be used for the delivery of therapeutic and/or prophylactic agents, e.g., mRNAs, to mammalian cells or organs. For example, the lipid nanoparticles described herein have little or no immunogenicity. For example, the lipid compounds disclosed herein have a lower immunogenicity as compared to a reference lipid (e.g., MC3, KC2, or DLinDMA). For example, a formulation comprising a lipid disclosed herein and a therapeutic or prophylactic agent, e.g., mRNA, has an increased therapeutic index as compared to a corresponding formulation which comprises a reference lipid (e.g., MC3, KC2, or DLinDMA) and the same therapeutic or prophylactic agent. Attorney Docket No.: 45817-0156WO1 In some embodiments, the present application provides pharmaceutical compositions comprising: (a) a delivery agent comprising a lipid nanoparticle; and (b) a polynucleotide encoding an antibody or antigen-binding fragment of the disclosure. Lipid Nanoparticles In some embodiments, polynucleotides of the present disclosure (e.g., mRNA) are included in a lipid nanoparticle (LNP). Lipid nanoparticles according to the present disclosure may comprise: (i) an ionizable lipid (e.g., an ionizable amino lipid); (ii) a sterol or other structural lipid; (iii) a non-cationic helper lipid or phospholipid; and (iv) a PEG-modified lipid. In some embodiments, lipid nanoparticles according to the present disclosure further comprise one or more polynucleotides of the present disclosure (e.g., mRNA). The lipid nanoparticles according to the present disclosure can be generated using components, compositions, and methods as are generally known in the art, see, for example PCT/US2016/052352; PCT/US2016/068300; PCT/US2017/037551; PCT/US2015/027400; PCT/US2016/047406; PCT/US2016000129; PCT/US2016/014280; PCT/US2016/014280; PCT/US2017/038426; PCT/US2014/027077; PCT/US2014/055394; PCT/US2016/52117; PCT/US2012/069610; PCT/US2017/027492; PCT/US2016/059575 and PCT/US2016/069491 all of which are incorporated by reference herein in their entirety. In some embodiments, the lipid nanoparticle comprises: (i) 20 to 60 mol.% ionizable cationic lipid (e.g., ionizable amino lipid), (ii) 25 to 55 mol.% sterol or other structural lipid, (iii) 5 to 25 mol.% non-cationic lipid (e.g., phospholipid), and (iv) 0.5 to 15 mol.% PEG-modified lipid. In some embodiments, the lipid nanoparticle comprises: (i) 40 to 50 mol.% ionizable cationic lipid (e.g., ionizable amino lipid), (ii) 30 to 45 mol.% sterol or other structural lipid, (iii) 5 to 15 mol.% non-cationic lipid (e.g., phospholipid), and (iv) 1 to 5 mol.% PEG-modified lipid. Attorney Docket No.: 45817-0156WO1 In some embodiments, the lipid nanoparticle comprises: (i) 45 to 50 mol.% ionizable cationic lipid (e.g., ionizable amino lipid), (ii) 35 to 45 mol.% sterol or other structural lipid, (iii) 8 to 12 mol.% non-cationic lipid (e.g., phospholipid), and (iv) 1.5 to 3.5 mol.% PEG-modified lipid. In the following sections, “Compounds” numbered with an “I-” prefix (e.g., “Compound I-1,” “Compound I-2,” “Compound I-3,” “Compound I-VI,” etc., indicate specific ionizable amino lipid compounds. Likewise, compounds numbered with a “P-” prefix (e.g., “Compound P-I,” etc.) indicate a specific PEG-modified lipid compound. Ionizable amino lipids In some embodiments, the lipid nanoparticle of the present disclosure comprises an ionizable cationic lipid (e.g., an ionizable amino lipid) that is a compound of Formula (I): or its N-oxide, or a salt or isomer thereof,
Figure imgf000127_0001
R’branched denotes a point of attachment; wherein
Figure imgf000127_0002
selected from the group consisting of H, C2-12 alkyl, and C2-12 alkenyl; R2 and R3 are each independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl; Attorney Docket No.: 45817-0156WO1 R4 is selected from the group consisting of -(CH2)nOH, wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, , wherein denotes a point
Figure imgf000128_0001
R10 is N each R is independently selected from the group
Figure imgf000128_0002
consisting of C1-6 alkyl, C2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; M and M’ are each independently selected from the group consisting of - C(O)O- and -OC(O)-; R’ is a C1-12 alkyl or C2-12 alkenyl; l is selected from the group consisting of 1, 2, 3, 4, and 5; and m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13. In some embodiments, in Formula (I), R’a is R’branched; R’branched is
Figure imgf000128_0003
denotes a point of attachment; Raα, Raβ, Raγ, and Raδ are each alkyl; R4 is -(CH ) OH; n is 2; each R5 i 6
Figure imgf000128_0004
14 2 n s H; each R is H; M and M’ are each -C(O)O-; R’ is a C1-12 alkyl; l is 5; and m is 7. In some embodiments, in Formula (I), R’a is R’branched; R’branched is denotes a point of attachment; Raα, Raβ, Raγ, and Raδ are
Figure imgf000128_0005
C1-14 alkyl; R4 is -(CH2)nOH; n is 2; each R5 is H; each R6 is H; M and M’ are each -C(O)O-; R’ is a C1-12 alkyl; l is 3; and m is 7. Attorney Docket No.: 45817-0156WO1 In some embodiments of the compounds of Formula (I), R’a is R’branched; of attachment; Raα is C2-12 alkyl; alkyl; R4 is
Figure imgf000129_0001
; R10 is NH(C1-6 alkyl); n2 is 2; R5 is H; each R6 is H; M and M’ is a C1-12 alkyl; l is 5; and m is 7.
Figure imgf000129_0002
In some embodiments of the compounds of Formula (I), R’a is R’branched; a point of attachment; Raα, Raβ, and each C1-14 alkyl; R4 is -(CH2)nOH;
Figure imgf000129_0003
n is 2; each R5 is H; each R6 is H; M and M’ are each -C(O)O-; R’ is a C1-12 alkyl; l is 5; and m is 7. In some embodiments, the compound of Formula (I) is selected from:
Figure imgf000129_0004
Attorney Docket No.: 45817-0156WO1 In some embodiments, the compound of Formula (I) is: (Compound I-1). In (I) is:
Figure imgf000130_0001
(Compound I-2). In (I) is:
Figure imgf000130_0002
(Compound I-3).
Figure imgf000130_0003
Phospholipids The lipid composition of the lipid nanoparticle composition disclosed herein can comprise one or more phospholipids, for example, one or more saturated or (poly)unsaturated phospholipids or a combination thereof. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties. A phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin. A fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic Attorney Docket No.: 45817-0156WO1 acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Particular phospholipids can facilitate fusion to a membrane. For example, a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid- containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue. Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond). Under appropriate reaction conditions, an alkyne group can undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye). Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin. In some embodiments, a phospholipid of the present disclosure comprises 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero- 3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Attorney Docket No.: 45817-0156WO1 Diether PC), 1-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3-phosphocholine,1,2-diarachidonoyl-sn-glycero-3- phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2- diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn- glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero- 3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof. In certain embodiments, a phospholipid useful or potentially useful in the present disclosure is an analog or variant of DSPC. Alternative Lipids In certain embodiments, a phospholipid useful or potentially useful in the present disclosure comprises a modified phosphocholine moiety, wherein the alkyl chain linking the quaternary amine to the phosphoryl group is not ethylene (e.g., n is not 2). Therefore, in certain embodiments, a phospholipid useful. In certain embodiments, an alternative lipid is used in place of a phospholipid of the present disclosure. In certain embodiments, an alternative lipid of the present disclosure is oleic acid. In certain embodiments, the alternative lipid is one of the following: ,
Figure imgf000132_0001
Attorney Docket No.: 45817-0156WO1 , ,
Figure imgf000133_0001
The lipid composition of a pharmaceutical composition disclosed herein can comprise one or more structural lipids. As used herein, the term "structural lipid" refers to sterols and also to lipids containing sterol moieties. Attorney Docket No.: 45817-0156WO1 Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle. Structural lipids can be selected from the group including but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha- tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a sterol. As defined herein, "sterols" are a subgroup of steroids consisting of steroid alcohols. In certain embodiments, the structural lipid is a steroid. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol. In certain embodiments, the structural lipid is alpha-tocopherol. In some embodiments, the structural lipids may be one or more of the structural lipids described in U.S. Application No.62/520,530. Polyethylene Glycol (PEG)-Lipids The lipid composition of a pharmaceutical composition disclosed herein can comprise one or more a polyethylene glycol (PEG) lipid. As used herein, the term “PEG-lipid” refers to polyethylene glycol (PEG)- modified lipids. Non-limiting examples of PEG-lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2- diacyloxypropan-3-amines. Such lipids are also referred to as PEGylated lipids. For example, a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid. In some embodiments, the PEG-lipid includes, but not limited to 1,2- dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG- disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG- diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG- DPPE), or PEG-l,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). Attorney Docket No.: 45817-0156WO1 In some embodiments, the PEG-lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG- modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the lipid moiety of the PEG-lipids includes those having lengths of from about C14 to about C22, preferably from about C14 to about C16. In some embodiments, a PEG moiety, for example an mPEG-NH2, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons. In some embodiments, the PEG- lipid is PEG2k-DMG. In some embodiments, the lipid nanoparticles described herein can comprise a PEG lipid which is a non-diffusible PEG. Non-limiting examples of non-diffusible PEGs include PEG-DSG and PEG-DSPE. PEG-lipids are known in the art, such as those described in U.S. Patent No. 8,158,601 and International Publ. No. WO 2015/130584 A2, which are incorporated herein by reference in their entirety. In general, some of the other lipid components (e.g., PEG lipids) of various Formulae, described herein may be synthesized as described International Patent Application No. PCT/US2016/000129, filed December 10, 2016, entitled “Compositions and Methods for Delivery of Therapeutic Agents,” which is incorporated by reference in its entirety. The lipid component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids. A PEG lipid is a lipid modified with polyethylene glycol. A PEG lipid may be selected from the non- limiting group including PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG- modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For Attorney Docket No.: 45817-0156WO1 example, a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid. In some embodiments, PEG lipids useful in the present disclosure can be PEGylated lipids described in International Publication No. WO2012099755, the contents of which is herein incorporated by reference in its entirety. Any of these exemplary PEG lipids described herein may be modified to comprise a hydroxyl group on the PEG chain. In certain embodiments, the PEG lipid is a PEG-OH lipid. As generally defined herein, a “PEG-OH lipid” (also referred to herein as “hydroxy- PEGylated lipid”) is a PEGylated lipid having one or more hydroxyl (–OH) groups on the lipid. In certain embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In certain embodiments, a PEG-OH or hydroxy-PEGylated lipid comprises an –OH group at the terminus of the PEG chain. Each possibility represents a separate embodiment of the present disclosure. In some aspects, the lipid composition of the pharmaceutical compositions disclosed herein does not comprise a PEG-lipid. In some embodiments, the PEG-lipids may be one or more of the PEG lipids described in U.S. Application No.62/520,530. In some embodiments, a PEG lipid of the present disclosure comprises a PEG- modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG- modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the PEG- modified lipid is PEG-DMG, PEG-c-DOMG (also referred to as PEG-DOMG), PEG- DSG and/or PEG-DPG. In some embodiments, a LNP of the present disclosure comprises an ionizable cationic lipid of Formula I, a phospholipid comprising DSPC, a structural lipid, and a PEG lipid comprising PEG-DMG. Attorney Docket No.: 45817-0156WO1 In some embodiments, a LNP of the present disclosure comprises an ionizable cationic lipid of Formula I, a phospholipid comprising DSPC, a structural lipid, and a PEG lipid. In some embodiments, a LNP of the present disclosure comprises an ionizable cationic lipid of . comprises an ionizable
Figure imgf000137_0001
cationic lipid of ,
Figure imgf000137_0002
In some embodiments, a LNP of the present disclosure comprises an ionizable cationic lipid of ,
Figure imgf000137_0003
lipid comprising cholesterol, and a PEG lipid. In some embodiments, a LNP of the present disclosure comprises an ionizable cationic lipid of Attorney Docket No.: 45817-0156WO1 and a
Figure imgf000138_0001
PEG lipid. In some embodiments, a LNP of the present disclosure comprises an ionizable cationic lipid of
Figure imgf000138_0002
a phospholipid comprising DOPE, a structural lipid comprising cholesterol, and a PEG lipid. In some embodiments, a LNP of the present disclosure comprises an N:P ratio of from about 2:1 to about 30:1. In some embodiments, a LNP of the present disclosure comprises an N:P ratio of about 6:1. In some embodiments, a LNP of the present disclosure comprises an N:P ratio of about 3:1. In some embodiments, a LNP of the present disclosure comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of from about 10:1 to about 100:1. In some embodiments, a LNP of the present disclosure comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of about 20:1. Attorney Docket No.: 45817-0156WO1 In some embodiments, a LNP of the present disclosure comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of about 10:1. In some embodiments, a LNP of the present disclosure has a mean diameter from about 50nm to about 150nm. In some embodiments, a LNP of the present disclosure has a mean diameter from about 70nm to about 120nm. Other Lipid Composition Components The lipid composition of a pharmaceutical composition disclosed herein can include one or more components in addition to those described above. For example, the lipid composition can include one or more permeability enhancer molecules, carbohydrates, polymers, surface altering agents (e.g., surfactants), or other components. For example, a permeability enhancer molecule can be a molecule described by U.S. Patent Application Publication No.2005/0222064. Carbohydrates can include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives and analogs thereof). A polymer can be included in and/or used to encapsulate or partially encapsulate a pharmaceutical composition disclosed herein (e.g., a pharmaceutical composition in lipid nanoparticle form). A polymer can be biodegradable and/or biocompatible. A polymer can be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. The ratio between the lipid composition and the polynucleotide range can be from about 10:1 to about 60:1 (wt/wt). In some embodiments, the ratio between the lipid composition and the polynucleotide can be about 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, Attorney Docket No.: 45817-0156WO1 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1 or 60:1 (wt/wt). In some embodiments, the wt/wt ratio of the lipid composition to the polynucleotide encoding a therapeutic agent is about 20:1 or about 15:1. In some embodiments, the pharmaceutical composition disclosed herein can contain more than one polypeptides. For example, a pharmaceutical composition disclosed herein can contain two or more polynucleotides (e.g., RNA, e.g., mRNA). In some embodiments, the lipid nanoparticles described herein can comprise polynucleotides (e.g., mRNA) in a lipid:polynucleotide weight ratio of 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1 or 70:1, or a range or any of these ratios such as, but not limited to, 5:1 to about 10:1, from about 5:1 to about 15:1, from about 5:1 to about 20:1, from about 5:1 to about 25:1, from about 5:1 to about 30:1, from about 5:1 to about 35:1, from about 5:1 to about 40:1, from about 5:1 to about 45:1, from about 5:1 to about 50:1, from about 5:1 to about 55:1, from about 5:1 to about 60:1, from about 5:1 to about 70:1, from about 10:1 to about 15:1, from about 10:1 to about 20:1, from about 10:1 to about 25:1, from about 10:1 to about 30:1, from about 10:1 to about 35:1, from about 10:1 to about 40:1, from about 10:1 to about 45:1, from about 10:1 to about 50:1, from about 10:1 to about 55:1, from about 10:1 to about 60:1, from about 10:1 to about 70:1, from about 15:1 to about 20:1, from about 15:1 to about 25:1,from about 15:1 to about 30:1, from about 15:1 to about 35:1, from about 15:1 to about 40:1, from about 15:1 to about 45:1, from about 15:1 to about 50:1, from about 15:1 to about 55:1, from about 15:1 to about 60:1 or from about 15:1 to about 70:1. In some embodiments, the lipid nanoparticles described herein can comprise the polynucleotide in a concentration from approximately 0.1 mg/ml to 2 mg/ml such as, but not limited to, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3 Attorney Docket No.: 45817-0156WO1 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7 mg/ml, 1.8 mg/ml, 1.9 mg/ml, 2.0 mg/ml or greater than 2.0 mg/ml. Nanoparticle Compositions In some embodiments, the pharmaceutical compositions disclosed herein are Formulated as lipid nanoparticles (LNP). Accordingly, the present disclosure also provides nanoparticle compositions comprising (i) a lipid composition comprising a delivery agent such as compound as described herein, and (ii) a polynucleotide encoding a polypeptide. In such nanoparticle composition, the lipid composition disclosed herein can encapsulate the polynucleotide encoding a polypeptide. Nanoparticle compositions are typically sized on the order of micrometers or smaller and can include a lipid bilayer. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, a nanoparticle composition can be a liposome having a lipid bilayer with a diameter of 500 nm or less. Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, and lipoplexes. In some embodiments, nanoparticle compositions are vesicles including one or more lipid bilayers. In certain embodiments, a nanoparticle composition includes two or more concentric bilayers separated by aqueous compartments. Lipid bilayers can be functionalized and/or crosslinked to one another. Lipid bilayers can include one or more ligands, proteins, or channels. In some embodiments, a lipid nanoparticle comprises an ionizable amino lipid, a structural lipid, a phospholipid, and mRNA. In some embodiments, the LNP comprises an ionizable amino lipid, a PEG-modified lipid, a sterol and a structural lipid. In some embodiments, the LNP has a molar ratio of about 40-50% ionizable amino lipid; about 5-15% structural lipid; about 30-45% sterol; and about 1-5% PEG- modified lipid. In some embodiments, the lipid nanoparticle comprises 47-49 mol.% ionizable cationic lipid (e.g. ionizable amino lipid, e.g., Compound I-1, Compound I-2, or Attorney Docket No.: 45817-0156WO1 Compound I-3), 10-12 mol.% non-cationic lipid (e.g., phospholipid, e.g., DSPC), 38- 40 mol.% sterol (e.g., cholesterol) or other structural lipid, and 1-3 mol.% PEG- modified lipid (e.g., PEG-DMG or Compound P-I). For instance, in some embodiments, the lipid nanoparticle (“LNP-1”) may comprise the following components at the following molar ratios: (i) 45-50 mol.% Compound I-1 (ii) 35-45 mol.% sterol (e.g., cholesterol); (iii) 8-12 mol.% phospholipid (e.g., DSPC or DOPE); and (iv) 1.5-3.5 mol.% PEG-lipid. For instance, in some embodiments, the lipid nanoparticle (“LNP-1A”) may comprise the following components at the following molar ratios: (i) 45-50 mol.% Compound I-1 (ii) 35-45 mol.% Cholesterol; (iii) 8-12 mol.% DSPC; and (iv) 1.5-3.5 mol.% PEG-lipid. For instance, in some embodiments, the lipid nanoparticle (“LNP-1B”) may comprise the following components at the following molar ratios: (i) 45-50 mol.% Compound I-1 (ii) 35-45 mol.% Cholesterol; (iii) 8-12 mol.% DSPC; and (iv) 1.5-3.5 mol.% PEG-lipid. In some embodiments, the lipid nanoparticle (“LNP-2”) may comprise the following: (i) 45-50 mol.% Compound I-2; (ii) 35-45 mol.% sterol (e.g., Cholesterol); (iii) 8-12 mol.% phospholipid (e.g., DSPC or DOPE); and (iv) 1.5-3.5 mol.% PEG-lipid. In some embodiments, the lipid nanoparticle (“LNP-2A”) may comprise the following: (i) 45-50 mol.% Compound I-2; (ii) 35-45 mol.% Cholesterol; Attorney Docket No.: 45817-0156WO1 (iii) 8-12 mol.% DSPC; and (iv) 1.5-3.5 mol.% PEG-lipid. For instance, in some embodiments, the lipid nanoparticle (“LNP-2B”) may comprise the following components at the following molar ratios: (i) 45-50 mol.% Compound I-2; (ii) 35-45 mol.% Cholesterol; (iii) 8-12 mol.% DSPC; and (iv) 1.5-3.5 mol.% PEG-lipid. In some embodiments, the lipid nanoparticle (“LNP-3”) may comprise the following: (i) 45-50 mol.% Compound I-3; (ii) 35-45 mol.% sterol (e.g., Cholesterol); (iii) 8-12 mol.% phospholipid (e.g., DSPC or DOPE); and (iv) 1.5-3.5 mol.% PEG-lipid. In some embodiments, the lipid nanoparticle (“LNP-3A”) may comprise the following: (i) 45-50 mol.% Compound I-3; (ii) 35-45 mol.% Cholesterol; (iii) 8-12 mol.% DSPC; and (iv) 1.5-3.5 mol.% PEG-lipid. In some embodiments, the lipid nanoparticle (“LNP-3B”) may comprise the following: (i) 45-50 mol.% Compound I-3; (ii) 35-45 mol.% Cholesterol; (iii) 8-12 mol.% DSPC; and (iv) 1.5-3.5 mol.% PEG-lipid. In some embodiments, the LNP has a polydispersity value of less than 0.4. In some embodiments, the LNP has a net neutral charge at a neutral pH. In some embodiments, the LNP has a mean diameter of 50-150 nm. In some embodiments, the LNP has a mean diameter of 80-100 nm. Attorney Docket No.: 45817-0156WO1 As generally defined herein, the term “lipid” refers to a small molecule that has hydrophobic or amphiphilic properties. Lipids may be naturally occurring or synthetic. Examples of classes of lipids include, but are not limited to, fats, waxes, sterol-containing metabolites, vitamins, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, and polyketides, and prenol lipids. In some instances, the amphiphilic properties of some lipids lead them to form liposomes, vesicles, or membranes in aqueous media. In some embodiments, a lipid nanoparticle (LNP) may comprise an ionizable amino lipid. As used herein, the term “ionizable amino lipid” has its ordinary meaning in the art and may refer to a lipid comprising one or more charged moieties. In some embodiments, an ionizable amino lipid may be positively charged or negatively charged. An ionizable amino lipid may be positively charged, in which case it can be referred to as “cationic lipid”. In certain embodiments, an ionizable amino lipid molecule may comprise an amine group, and can be referred to as an ionizable amino lipid. As used herein, a “charged moiety” is a chemical moiety that carries a formal electronic charge, e.g., monovalent (+1, or -1), divalent (+2, or -2), trivalent (+3, or - 3), etc. The charged moiety may be anionic (i.e., negatively charged) or cationic (i.e., positively charged). Examples of positively-charged moieties include amine groups (e.g., primary, secondary, and/or tertiary amines), ammonium groups, pyridinium group, guanidine groups, and imidizolium groups. In a particular embodiment, the charged moieties comprise amine groups. Examples of negatively- charged groups or precursors thereof, include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, and the like. The charge of the charged moiety may vary, in some cases, with the environmental conditions, for example, changes in pH may alter the charge of the moiety, and/or cause the moiety to become charged or uncharged. In general, the charge density of the molecule may be selected as desired. It should be understood that the terms “charged” or “charged moiety” does not refer to a “partial negative charge" or “partial positive charge" on a molecule. The terms “partial negative charge" and “partial positive charge" are given their ordinary Attorney Docket No.: 45817-0156WO1 meaning in the art. A “partial negative charge" may result when a functional group comprises a bond that becomes polarized such that electron density is pulled toward one atom of the bond, creating a partial negative charge on the atom. Those of ordinary skill in the art will, in general, recognize bonds that can become polarized in this way. The ionizable lipid is sometimes referred to in the art as an “ionizable cationic lipid”. In some embodiments, the ionizable amino lipid may have a positively charged hydrophilic head and a hydrophobic tail that are connected via a linker structure. In addition to these, an ionizable amino lipid may also be a lipid including a cyclic amine group. In some embodiments, the ionizable amino lipid may be selected from, but not limited to, an ionizable amino lipid described in International Publication Nos. WO2013086354 and WO2013116126; the contents of each of which are herein incorporated by reference in their entirety. In yet another embodiment, the ionizable amino lipid may be selected from, but not limited to, Formula CLI-CLXXXXII of US Patent No.7,404,969; each of which is herein incorporated by reference in their entirety. In some embodiments, the lipid may be a cleavable lipid such as those described in International Publication No. WO2012170889, herein incorporated by reference in its entirety. In some embodiments, the lipid may be synthesized by methods known in the art and/or as described in International Publication Nos. WO2013086354; the contents of each of which are herein incorporated by reference in their entirety. Nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of a nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) can be used to measure zeta potentials. Dynamic light Attorney Docket No.: 45817-0156WO1 scattering can also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure multiple characteristics of a nanoparticle composition, such as particle size, polydispersity index, and zeta potential. The size of the nanoparticles can help counter biological reactions such as, but not limited to, inflammation, or can increase the biological effect of the polynucleotide. As used herein, “size” or “mean size” in the context of nanoparticle compositions refers to the mean diameter of a nanoparticle composition. In some embodiments, the polynucleotide encoding a polypeptide are formulated in lipid nanoparticles having a diameter from about 10 to about 100 nm such as, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 to about 60 nm, about 50 to about 70 nm, about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nm and/or about 90 to about 100 nm. In some embodiments, the nanoparticles have a diameter from about 10 to 500 nm. In some embodiments, the nanoparticle has a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, Attorney Docket No.: 45817-0156WO1 greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm or greater than 1000 nm. In some embodiments, the largest dimension of a nanoparticle composition is 1 µm or shorter (e.g., 1 µm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, or shorter). A nanoparticle composition can be relatively homogenous. A polydispersity index can be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle composition. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A nanoparticle composition can have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of a nanoparticle composition disclosed herein can be from about 0.10 to about 0.20. The amount of a polynucleotide present in a pharmaceutical composition disclosed herein can depend on multiple factors such as the size of the polynucleotide, desired target and/or application, or other properties of the nanoparticle composition as well as on the properties of the polynucleotide. For example, the amount of an mRNA useful in a nanoparticle composition can depend on the size (expressed as length, or molecular mass), sequence, and other characteristics of the mRNA. The relative amounts of a polynucleotide in a nanoparticle composition can also vary. The relative amounts of the lipid composition and the polynucleotide present in a lipid nanoparticle composition of the present disclosure can be optimized according to considerations of efficacy and tolerability. For compositions including an mRNA as a polynucleotide, the N:P ratio can serve as a useful metric. Attorney Docket No.: 45817-0156WO1 As the N:P ratio of a nanoparticle composition controls both expression and tolerability, nanoparticle compositions with low N:P ratios and strong expression are desirable. N:P ratios vary according to the ratio of lipids to RNA in a nanoparticle composition. In general, a lower N:P ratio is preferred. The one or more RNA, lipids, and amounts thereof can be selected to provide an N:P ratio from about 2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain embodiments, the N:P ratio can be from about 2:1 to about 8:1. In other embodiments, the N:P ratio is from about 5:1 to about 8:1. In certain embodiments, the N:P ratio is between 5:1 and 6:1. In one specific aspect, the N:P ratio is about is about 5.67:1. In some embodiments, the polynucleotides described herein can be Formulated for controlled release and/or targeted delivery. As used herein, "controlled release" refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome. In some embodiments, the polynucleotides can be encapsulated into a delivery agent described herein and/or known in the art for controlled release and/or targeted delivery. As used herein, the term "encapsulate" means to enclose, surround or encase. As it relates to the formulation of the compounds of the present disclosure, encapsulation can be substantial, complete or partial. The term "substantially encapsulated" means that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, or greater than 99% of the pharmaceutical composition or compound of the present disclosure can be enclosed, surrounded or encased within the delivery agent. "Partial encapsulation" or “partially encapsulate” means that less than 10, 10, 20, 30, 4050 or less of the pharmaceutical composition or compound of the present disclosure can be enclosed, surrounded or encased within the delivery agent. In some embodiments, the therapeutic nanoparticle polynucleotide can be Formulated for sustained release. As used herein, "sustained release" refers to a pharmaceutical composition or compound that conforms to a release rate over a Attorney Docket No.: 45817-0156WO1 specific period of time. The period of time can include, but is not limited to, hours, days, weeks, months and years. As a non-limiting example, the sustained release nanoparticle of the polynucleotides described herein can be Formulated as disclosed in Intl. Pub. No. WO2010075072 and U.S. Pub. Nos. US20100216804, US20110217377, US20120201859 and US20130150295, each of which is herein incorporated by reference in their entirety. In some embodiments, the therapeutic nanoparticle polynucleotide can be Formulated to be target specific, such as those described in Intl. Pub. Nos. WO2008121949, WO2010005726, WO2010005725, WO2011084521 and WO2011084518; and U.S. Pub. Nos. US20100069426, US20120004293 and US20100104655, each of which is herein incorporated by reference in its entirety. The LNPs can be prepared using microfluidic mixers or micromixers. Exemplary microfluidic mixers can include, but are not limited to, a slit interdigital micromixer including, but not limited to those manufactured by Microinnova (Allerheiligen bei Wildon, Austria) and/or a staggered herringbone micromixer (SHM) (see, Zhigaltsev et al., Langmuir.28:3633-40 (2012); Belliveau et al., Molecular Therapy-Nucleic Acids.1:e37 (2012); Chen et al., J. Am. Chem. Soc. 134(16):6948-51 (2012); each of which is herein incorporated by reference in its entirety). Exemplary micromixers include Slit Interdigital Microstructured Mixer (SIMM-V2) or a Standard Slit Interdigital Micro Mixer (SSIMM) or Caterpillar (CPMM) or Impinging-jet (IJMM,) from the Institut für Mikrotechnik Mainz GmbH, Mainz Germany. In some embodiments, methods of making LNP using SHM further comprise mixing at least two input streams wherein mixing occurs by microstructure- induced chaotic advection (MICA). According to this method, fluid streams flow through channels present in a herringbone pattern causing rotational flow and folding the fluids around each other. This method can also comprise a surface for fluid mixing wherein the surface changes orientations during fluid cycling. Methods of generating LNPs using SHM include those disclosed in U.S. Pub. Nos. US20040262223 and US20120276209, each of which is incorporated herein by reference in their entirety. Attorney Docket No.: 45817-0156WO1 In some embodiments, the polynucleotides described herein can be Formulated in lipid nanoparticles using microfluidic technology (see, Whitesides, George M., Nature 442: 368-373 (2006); and Abraham et al., Science 295: 647-651 (2002); each of which is herein incorporated by reference in its entirety). In some embodiments, the polynucleotides can be Formulated in lipid nanoparticles using a micromixer chip such as, but not limited to, those from Harvard Apparatus (Holliston, MA) or Dolomite Microfluidics (Royston, UK). A micromixer chip can be used for rapid mixing of two or more fluid streams with a split and recombine mechanism. In some embodiment, the nanoparticles described herein are stealth nanoparticles or target-specific stealth nanoparticles such as, but not limited to, those described in U.S. Pub. No. US20130172406, herein incorporated by reference in its entirety. The stealth or target-specific stealth nanoparticles can comprise a polymeric matrix, which can comprise two or more polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polyesters, polyanhydrides, polyethers, polyurethanes, polymethacrylates, polyacrylates, polycyanoacrylates, or combinations thereof. Methods of Using Anti-CD16 Antibodies and Binding Proteins In some aspects, antibodies and/or binding proteins of the present disclosure are administered to a subject in need thereof. In some aspects, the subject in need thereof is a subject with a disease, disorder, and/or condition that may be treated with technologies described herein. Specifically, a disease or disorder which would benefit from targeting cells expressing anti-CD16 for reduction or elimination. In some aspects, the subject in need thereof is a subject with an infection, an inflammatory condition, or an autoimmune condition. Attorney Docket No.: 45817-0156WO1 In some aspects, the subject in need thereof is a subject with cancer. Certain cancers that may be treated in accordance with technologies of the present disclosure include, for example, adrenocortical carcinoma, astrocytoma, basal cell carcinoma, carcinoid, cardiac, cholangiocarcinoma, chordoma, chronic myeloproliferative neoplasms, craniopharyngioma, ductal carcinoma in situ, ependymoma, intraocular melanoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, glioma, histiocytosis, leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), hairy cell leukemia, myelogenous leukemia, and myeloid leukemia), lymphoma (e.g., Burkitt lymphoma (non-Hodgkin lymphoma), cutaneous T-cell lymphoma, Hodgkin lymphoma, mycosis fungoides, Sezary syndrome, AIDS-related lymphoma, follicular lymphoma, diffuse large B-cell lymphoma), melanoma, merkel cell carcinoma, mesothelioma, myeloma (e.g., multiple myeloma), myelodysplastic syndrome, papillomatosis, paraganglioma, pheochromocytoma, pleuropulmonary blastoma, retinoblastoma, sarcoma (e.g., Ewing sarcoma, Kaposi sarcoma, osteosarcoma, rhabdomyosarcoma, uterine sarcoma, vascular sarcoma), Wilms' tumor, and/or cancer of the adrenal cortex, anus, appendix, bile duct, bladder, bone, brain, breast, bronchus, central nervous system, cervix, colon, endometrium, esophagus, eye, fallopian tube, gall bladder, gastrointestinal tract, germ cell, head and neck, heart, intestine, kidney (e.g., Wilms' tumor), larynx, liver, lung (e.g., non-small cell lung cancer, small cell lung cancer), mouth, nasal cavity, oral cavity, ovary, pancreas, rectum, skin, stomach, testes, throat, thyroid, penis, pharynx, peritoneum, pituitary, prostate, rectum, salivary gland, ureter, urethra, uterus, vagina, or vulva. In some aspects, the subject in need thereof is a subject with multiple myeloma. In some aspects, the subject in need thereof is a mammal. In some embodiments, a mammal includes, for example and without limitation, a household pet (e.g., a dog, a cat, a rabbit, a ferret, a hamster), a livestock or farm animal (e.g., a cow, a pig, a sheep, a goat, a pig, a chicken or another poultry), a horse, a monkey, a laboratory animal (e.g., a mouse, a rat, a rabbit) and a human. In a preferred embodiment, the subject in need thereof is a human. Technologies of the present Attorney Docket No.: 45817-0156WO1 disclosure can be practiced in any subject in need thereof that is likely to benefit from administration of technologies of the present disclosure (e.g., a subject with cancer). In some embodiments, a subject in need thereof is a human. In some embodiments, the human is male. In some embodiments, the human is female. In some embodiments, the human is an adult (e.g., 18 or more years of age). In some embodiments, the adult is greater than 18 years old, greater than 25 years old, greater than 30 years old, greater than 40 years old, greater than 50 years old, greater than 55 years old, greater than 60 years old, greater than 65 years old, greater than 70 years old, greater than 75 years old, greater than 80 years old, greater than 85 years old, greater than 90 years old, greater than 95 years old, greater than 100 years old, or greater than 105 years old in age. In some embodiments, the human is a child. In some embodiments, the child is greater than 2 years old, greater than 3 years old, greater than 4 years old, greater than 5 years old, greater than 6 years old, greater than 7 years old, greater than 8 years old, greater than 9 years old, greater than 10 years old, greater than 11 years old, greater than 12 years old, greater than 13 years old, greater than 14 years old, greater than 15 years old, or greater than 16 years old in age. In some aspects, a subject in need thereof is administered an antibody or binding protein of the present disclosure. Routes of Administration and Dosing Anti-CD16 antibodies or binding molecules of the disclosure, and nucleic acids encoding the same, can be administered to a subject (e.g., a mammalian subject, such as a human) by a variety of routes. In some embodiments, the antibody or nucleic acid is administered to the subject intravenously, subcutaneously, intramuscularly, parenterally, intrathecally, intracerebroventricularly, transdermally, or orally. The most suitable route for administration in any given case will depend on the particular therapeutic agent administered, the patient, pharmaceutical formulation Attorney Docket No.: 45817-0156WO1 methods, and various patient-specific parameters, such as the patient's age, body weight, sex, severity of the diseases being treated, the patient’s diet, and the patient’s excretion rate. An appropriate dosage of anti-CD16 antibodies or binding proteins, or nucleic acids encoding the same of the present disclosure will vary with the particular condition, disease and/or disease being treated, various subject-specific parameters (e.g., age, weight, physical condition of the subject), severity of the particular condition, disease, and/or disorder being treated, the nature of current or combination therapy (if any), the specific route of administration and other factors within the knowledge and expertise of a health practitioner. In some embodiments, a maximally tolerated dose of technologies described herein is to be used, e.g., the highest safe dose according to sound medical judgement. In some embodiments, technologies described herein are administered in an effective amount, e.g., a dose sufficient to provide one or more medically desirable results. A therapeutic regiment for use in accordance with technologies described herein may include administration of such technologies or compositions comprising such technologies once a day, once every two days, once every three days, twice a week, once a week, once every two weeks, once every three weeks, once every month or four weeks, once every six weeks, once every two months or eight weeks, once every three months or twelve weeks. In some certain embodiments, a subject receives a single dose of a technology described herein. In certain embodiments, a subject receives a plurality of doses of a technology described herein (e.g., at least two, at least three, at least four, at least five, at least six, at least eight, at least ten, or more doses). In some embodiments, technologies described herein are administered over a period of time, such as one week, two weeks, three weeks, four weeks, six weeks, two months, three months, four months, five months, six months, one year or more. Appropriate therapeutic regimens are readily understood by medical practitioners and such regimens may be designed by a medical practitioner for a particular patient (e.g., a patient-specific regimen). Attorney Docket No.: 45817-0156WO1 Kits Also included herein are kits that contain anti-CD16 antibodies, binding proteins, and/or nucleic acids encoding the same. In some embodiments, the kits provided herein contain one or more cells engineered to express and secrete an anti- CD16 antibody, antigen-binding fragment, of binding protein of the disclosure, such as a cell containing a nucleic acid molecule of the disclosure. A kit described herein may include reagents that can be used to produce a pharmaceutical composition of the disclosure. Optionally, kits described herein may include reagents that can induce the expression of anti-CD16 antibodies, antigen- binding fragments, or relate proteins of the present disclosure within cells (e.g., mammalian cells). Other kits described herein may include tools for engineering a prokaryotic or eukaryotic cell (e.g., a CHO cell or a BL21(DE3) E. Coli cell or an immune cell) so as to express and secrete an anti-CD16 antibody or binding protein described herein. For example, a kit may contain CHO cells stored in an appropriate media and optionally frozen according to methods known in the art. The kit may also contain a nucleic acid encoding the desired antibody or binding protein as well as reagents for expressing the antibody or binding protein in the cell. A kit described herein may also provide an a anti-CD16 antibody or binding protein of the disclosure, or a nucleic acid encoding the same in combination with a package insert describing how the antibody, binding protein, or nucleic acid may be administered to a subject, for example, for the treatment of a disease, disorder and/or condition (e.g., cancer). Examples The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods claimed herein can be performed, made, and evaluated, and are intended to be purely exemplary and are not intended to limit the scope of the present disclosure. Attorney Docket No.: 45817-0156WO1 Example 1: Antibody Discovery Immunization of Llamas: A Llama was immunized with a primary dose immunization of 200 µg of human CD16a (Acro CDA-H5220) in complete Freund’s Adjuvant (CFA), given subcutaneously. Following the primary immunization, 100 µg of human CD16a with incomplete Freund’s Adjuvant (IFA) was used for all following boosts weekly after 21 days. A test bleed was taken on day 35 post immunization, to check for antibody titers against the immunized targets in the serum. The production bleed was performed on day 42 after an IV boost without adjuvant, for a production bleed of 600 ml and was used to isolate and cryopreserve PBMCs. FACS sorting of B-cells: Antibody discovery was performed by Fluorescence Activated Cell Sorting (FACS) and culture of sorted Llama B cells, based on methods available on mammalian B cell culture (see, e.g., Kwakkenbos et al., Nat. Med. 16(1):123-128 (2010); WO2013076139 A1; Carbonetti et al., JIM 448: 66-73 (2017), herein incorporated by reference in their entirety). B-cells were sorted at a concentration of 1 x 106 cells/mL while gating for double positive VHH and CD16a positive antibodies using biotinylated CD16a, streptavidin-APC and Rabbit anti- VHH-FITC (Rabbit anti-camelid VHH AF-647, GenScript). B-cell culture: B-cells were cultured at 2-4 cells/well and co-cultured with irradiated CD40L expressing feeder cells and the appropriate cytokines in 96 well plates. The culture plates were incubated for 10-12 days in a CO2 incubator at 37oC and 5% CO2. Proliferating B cell cultures were processed to harvest culture supernatants for screening the secreted antibodies against the target antigens, and the cell pellets were frozen for isolation of RNA and antibody sequencing. Screening for Antibody Hits via ELISA: 384 well ELISA plates were coated with 25 µl 1 µg/ml purified human CD16a. After blocking, 15 µl of primary B cell culture supernatants with 10 µl assay diluent was added into each well and incubated for 1 hr at room temperature. The plate was detected by Rabbit Anti‐Camelid VHH Antibody at 1:5000 dilution (GenScript A01860) and goat anti rabbit‐HRP. All wells with a reading above 0.3 were deemed positive. Attorney Docket No.: 45817-0156WO1 Total RNA was extracted from the positive clones (Thermo Fisher 61012, Dynabeads™ mRNA DIRECT™ Purification Kit) and cDNA is made with SuperScript® IV Reverse Transcriptase (Thermo Fisher 18090050). VHH clones were PCR amplified and made into linear expression module LEM (or TAP: transcription activated PCR, (The rapid generation of recombinant functional monoclonal antibodies from individual, antigen-specific bone marrow-derived plasma cells isolated using a novel fluorescence-based method Clargo et al. MAbs, 6(1) 143- 159, 2014)), which was transfected into CHO cell line for VHH-Fc expression. The soup was used in ELISA for binding validation to CD16a. The positive clone VHH1 was sent for Sanger sequencing. The amino acid sequence of VHH1 is shown below. Table 4. Amino acid sequences of anti-CD16 VHH domain (CDRs are underlined) Clone Amino acid sequence SEQ ID NO
Figure imgf000156_0001
Table 5. Exemplary CDR definitions of the anti-CD16 VHH, VHH1 CDR Def VHH-CDR1 VHH-CDR2 VHH-CDR3
Figure imgf000156_0002
Attorney Docket No.: 45817-0156WO1 VHH expression and purification: The VHH1-His molecules were transiently expressed in ExpiCHO cells using the EpxiFectamine CHO (Thermofisher) transfection reagents. The ExpiCHO-S cells were cultured in ExpiCHO expression medium (ThermoFisher Scientific) in a shaker incubator set at 125 rpm, 37ºC and 8% CO2. The day prior to transfection, ExpiCHO-S cells were seeded at 3 x 106 cells per ml in 30 ml of ExpiCHO Expression medium. On the day of transfection cells were split using pre-warmed (37ºC) ExpiCHO expression media to a density of 6 x 106 cells per ml. Using the manufacturer’s recommended protocol, a total 20 µg of a single plasmid and 72 µL of ExpiFectamine CHO reagent were mixed in 2.4 ml of cold Opti-PRO SFM (Thermo Fisher Scientific), after incubating the mixture for 2 minutes it was then slowly added to the cells. One day (~20h) post-transfection, 7.2 ml of ExpiCHO Feed and 180 µl of ExpiCHO Enhancer were added to the ExpiFectamine-transfected cultures. Culture supernatants were harvested when cell viability dropped below 60% (~day 8), clarified by centrifugation at 3,000 rpm for 30 min and filtered using a 0.2 μm filter (Thermo Fisher Scientific). Expressed single domain antibody with a 6xHis (SEQ ID NO: 41) C-terminal tag (VHH1-His) was purified from ExpiCHO media by capture on TALON (TAKRA) cobalt immobilized metal affinity chromatography (IMAC) resin. Resin was prewashed in 50 mM sodium phosphate buffer pH 7.4 (wash buffer) with 15x the volume of resin, then centrifuged for 2 min @ 700 x g to pellet the resin. The supernatant discarded and the resin wash repeated. To the filtered media containing the expressed protein was added 3 mL of IMAC resin, then allowed to bind overnight with gentle shaking. The media resin mixture was gravity loaded into a 25 ml column. Columns containing IMAC resin were then washed with 10 column volumes (~30 ml) of wash buffer (PBS pH 7.4 (Life Tech cat# 10010-023)., 2 mM imidazole) then 2x with 10 ml of wash buffer. Protein was then eluted using a total of 7.5 mL 150 mM imidazole /PBS pH 7 in three 2.5 mL aliquots. Proteins were then dialyzed exhaustively using Slide-A-Lyzer® 10 or 3K as appropriate, (Dialysis cassette, Pierce) versus 1 x PBS (100 mM NaPO4 pH 6.8, 200mM NaCl). Attorney Docket No.: 45817-0156WO1 Expressed single domain antibody with a Fc C-terminal tag (VHH1-Fc) was purified from ExpiCHO media in the similar protocol except by capture on protein A resin and elution by 10 mM glycine pH 3.0 followed by immediate pH adjustment in 10% 1M Tris at pH 8.0. Protein characterization: SDS PAGE was run on each sample using gradient gels NuPAGE Bis-Tris 4-12% gradient gels using a MES running buffer (Thermo Fisher Scientific). Samples were prepared with either reducing or non-reducing sample buffer and briefly heated to 95°C.   To samples electrophoresed in non- reducing buffer N-ethyl maleimide was added to cap any free thiols and prevent unwanted disulfide scrambling as the samples cooled.  Molecular weight standards (Blue Plus protein, Thermofisher) were included on the SDS-PAGE. Non-denaturing protein electrophoresis was performed running 1 μg of each purified protein sample; reducing conditions were performed mixing each purified sample with 10 μl of Sample Reducing Agent (Invitrogen, Carlsbad, CA) and heating at 70°C for 10 min before electrophoresis on NuPAGE 4-12% Bis-Tris Mini Gels 1.0 mm (Invitrogen, Carlsbad, CA). The bands were visualized by SimplyBlue™ SafeStain (Invitrogen, Carlsbad, CA) staining, and the gel was dried using DryEase Mini-Gel Drying System (Invitrogen, Carlsbad, CA). All procedures were performed according to the manufacturer’s instructions.  Analysis of native molecule homogeneity and determination of molecular weight was done using Size Exclusion Chromatography with Light Scattering (SEC- LS) when indicated. Size exclusion chromatography (SEC) was carried out on a Zenix SEC 3004.6 x 300 mm (Sepax Technologies) in 20 mM sodium phosphate pH 7.2, 150 mM NaCl (PBS), 0.05% NaAzide at a flow rate of 0.35ml/min using an Agilent 1260 UPLC.  In addition to UV detection, the eluent was monitored with a refractive index detector (Waters, Milford, MA).  Light scattering was monitored using a Wyatt Dawn 18 angle, coupled with an Optrex refractometer. Intact mass of molecules was determined mass spectrometry (Merrigen, Lowell MA).  Attorney Docket No.: 45817-0156WO1 Example 2: In vitro characterization of candidate antibody sequence VHH1 SPR binding affinity determination: Briefly, binding of an anti-CD16 VHH antibody molecule (VHH1) to various CD16 molecules was analyzed on a Biacore T200 (Cytiva) at 25°C. VHH1-His was covalently coated to CM5 sensor chip by amine-coupling using the coupling kit (Cytiva). Various Fc receptor molecules were injected over each flow cell at the flow rate of 30 µl /min in HBS-EP+ buffer at concentrations ranging from 3 nM to 200 nM. A buffer injection served as a negative control. Channel 1 was blocked by ethanolamide and served as a reference channel. Upon completion of each association and dissociation cycle, surfaces were regenerated with 10 nM glycine pH 2.5 solution. The association rates (ka), dissociation rate constants (kd), and affinity constants (KD) were calculated using Biacore T200 evaluation software. Each fit was evaluated by the agreement between experimental data and the calculated fits, where the Chi2 values were below 10% of Rmax. Surface densities of the molecule were optimized to minimize mass transfer while maintaining enough response. Binding and affinity results are shown in Table 6 below. Table 6. Binding profile and cross-reactivity of the anti-CD16 antibody, VHH1, to purified CD16 Binding Affinity
Figure imgf000159_0001
Attorney Docket No.: 45817-0156WO1 Using SPR, antibody binding was assessed against a number of purified CD16a and CD16b proteins with different alleles as well as to CD16a from varied species. The antibody bound both alleles of human CD16a, V176 and F176, at 5nM and 13 nM, respectively (Table 6). VHH1 was also cross reactive to cyno CD16 binding with a 9nM affinity and rat CD16 at 58nM (Table 6). Whereas no binding was seen to mouse CD16 (Table 6). Additionally, VHH1 did not bind to human CD16b NA1 variant and other human Fc receptor family members including all variants of CD32 and CD64. However, VHH1 did bind to human CD16b NA2 variant, indicating that antibody binding to neutrophils would be expected. FACS Binding affinity: CHO cell lines that stably express CD16a and CD16b were resuspended in FACS buffer (PBS pH7.4 + 2%FBS +1mM EDTA) at 1,000,000 cells/ml, and 100 µl of the cell suspension was mixed in a 96-well V bottom polypropylene plate (Nunc) with 100ul of VHH1-His or control-His fusion molecules in the same buffer with or without 33% human serum. After one-hour incubation at 4°C, pelleted cells were washed 2x by ice cold FACS buffer and then resuspended in 100 µl anti-His-iFluor 647 from GenScript at 1:100 dilution in FACS buffer, incubated 15 min at 4°C, centrifuged at 1000 rpm for 3 min; cell pellets were washed, fixed with 200 µl/well 1% paraformaldehyde for 10 min at room temperature, centrifuged, the fix solution was decanted and cell pellets were resuspended in 200 µl FACS buffer for analysis. Cellular fluorescence was determined on Canto equipped with FlowJo software (Becton-Dickinson, Franklin Lakes, NJ). The receptor EC50 values were determined by fitting the data with a single site total binding model using GraphPad Prism 6.0 (La Jolla, CA).  The llama anti-CD16 VHH antibody demonstrated 3.5 nM affinity to CHO-CD16a and micromolar affinity to CHO- CD16b. (see, Table 7 and FIG.1) The impact of VHH1 binding to CD16a in the presence and absence of human serum albumin was also evaluated. No significant effect was observed by presence of Attorney Docket No.: 45817-0156WO1 human serum (1:3 diluted) in contrast to a control antibody, indicating VHH1 does not compete with human IgG for binding to CD16a. (see, Table 7 and FIG.1) Table 7. Binding profile of the anti-CD16 antibody, VHH1, to CHO-CD16a and CHO-CD16b (NA1) Antibody Cell line Serum Kd (nM) VHH1-His CHO-CD16a No 3.5
Figure imgf000161_0001
FACS Binding affinity on neutrophils and NK cells: Whole blood containing neutrophils and NK from the same donor was ordered from Research Blood Components. The red cells were lysed with Ammonium Chloride Solution at 9:1 ratio on ice for 10 minutes and then 500K lysed whole blood cells were plated into each well of 96 round bottom plate. Diluted VHH1 was added to each well at starting concentration of 1000 nM with 12 step 1:3 dilutions and incubated for 30 minutes at 4ºC. Cells were washed and then a flow cytometry panel including markers for neutrophils and NK cells plus anti-His or anti-VHH was used to detect CD16 binding on each cell type. Penta-His AF488 (Qiagen 35310) was used to detect VHH1-His and anti-VHH (Genscript A01862) was used for the VHH1-Fc version. Cells were incubated with this panel for 30 minutes at 4C. Cells were washed and then ran on Fortessa. Penta-His (SEQ ID NO: 42) was used at 1:100 so a final concentration of 2 ug/mL. MonoRab Rabbit anti-Camelid VHH Antibody (iFluor488) mAb was used at 1:100. TruStain blocking was used to prevent non-specific binding of staining antibodies. Both VHH1-His and VHH1-Fc show concentration dependent binding to both NK cells and neutrophils. (FIG.2.) Attorney Docket No.: 45817-0156WO1 Example 3. Expression and in vitro Characterization of Humanized Antibody Sequence Humanization: The VHH antibody was humanized similar to the method of Hanf et al. (Methods.65(1):68-76 (2014)).  Briefly sequences of the CDRs of VHH1 were annotated using the IMGT numbering scheme. Each VHH nucleotide sequence is generated and used to identify the nearest human germline VH sequences by searching for similar sequences with the NCBI IgBLAST program. Common J and D gene sequences were attached to the VH as the acceptor. Next the most similar human VH sequences are identified using BLASTp and used to choose the nearest framework sequences into which the VHH1 CDR sequences are grafted replacing the human CDRs.   Rosetta/Alpha fold was used to create the structural 3D homology model the of the appropriate CDRs that were grafted into the acceptor framework. The framework residues that were critical for huVH/VL interactions are back mutated to llama sequence canonical llama residues, also potentially structural defects due to mismatches at the graft interface can be fixed by mutating some framework residues to llama, or by mutating some residues on the CDRs’ backside to human or to a de novo designed sequence. CDR stabilizing or overall fold stabilizing sequences were then back-mutated to the corresponding llama sequence to maintain the biophysical properties and target binding affinity.  Sequences of 20 humanized variants of VHH1 are shown in Table 8 below. Table 8. Sequences of humanized VHH1 variants (CDRs are underlined) Clone Amino acid sequence SEQ O
Figure imgf000162_0001
Attorney Docket No.: 45817-0156WO1 VHH1- EVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGK 5 H1 ELDFVSAINSNTGRTYHADSVKGRFTISRDNAKNTVYLQMNSLR AEDTA YY AA R Y LL I Y Y TL T
Figure imgf000163_0001
Attorney Docket No.: 45817-0156WO1 VHH1- QVQLVESGGGLVQPGGSLRLSCAASGRTDSIYAMGWFRQAPGK 20 H16 ERDFVSAINSNTGRTYHADSVKGRFTISRDNAKNTLYLQMNSLK AEDTA YY AA R Y LL I Y Y TL T
Figure imgf000164_0001
The 20 humanized variants of VHH1 were generated as SASA fusion (anti- BSA VHH) and produced as secreted protein by CHO cell line. The supernatant was then injected into a BSA preloaded CM5 Chip in a BiaCore 8K.200 nM CD16a was injected as analyte for 120 seconds and dissociation was carried out in 360 seconds. All the data were processed using the Biacore 8K Evaluation software version 1.1. Flow cell 1 and blank injection of buffer in each cycle were used as double reference for Response Units subtraction. Three negative controls were included in the assay: 3H01 is an irrelevant VHH, NC is an irrelevant antibody, and blank is buffer. (Table 9) Table 9. Binding kinetics of supernatant to human CD16a Capture Ligand Chi2 Ka Kd KD Rmax
Figure imgf000164_0002
Attorney Docket No.: 45817-0156WO1 195.3 VHH1-H4 3.23E-01 1.20E+05 2.13E-03 1.78E-08 129.3 174.9 VHH1-H5 4.98E-01 1.32E+05 3.48E-03 2.64E-08 113.3
Figure imgf000165_0001
Attorney Docket No.: 45817-0156WO1 The humanized version, VHH1-H3, was selected for further characterization based on the humanness and close sequence analysis. VHH1-H3 was produced with a His tag at the C-terminus in CHO cell line and purified by cobalt affinity column. The purified protein was analyzed with size exclusion chromatography (SEC). Parent llama antibody VHH1 was purified and analyzed the same way. 

Claims

Attorney Docket No.: 45817-0156WO1 WHAT IS CLAIMED IS: 1. A binding molecule comprising a polypeptide that specifically binds CD16, wherein the polypeptide is a VHH and comprises the amino acid sequences of a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of the VHH set forth in SEQ ID NO: 1 or 7, optionally wherein the polypeptide binds human, cynomolgus and rat CD16, but not mouse CD16, or human CD16b NA1 variant, and further optionally wherein the binding molecule binds to human NK cells and/or human neutrophils. 2. The binding molecule of claim 1, wherein the VHH-CDR1, VHH-CDR2, and VHH-CDR3 comprise the sequences set forth in SEQ ID NOs: (i) 2, 3, 4, respectively; (ii) 25, 26, 27, respectively; (iii) 28, 29, 27, respectively; (iv) 28, 26, 27, respectively; (v) 30, 31, 32, respectively; (vi) 30, 36, 32, respectively; or (vii) 33, 34, 27, respectively. 3. The binding molecule of claim 1 or 2, wherein the VHH is humanized, optionally wherein 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid(s) of the polypeptide of SEQ ID NO:1 are humanized. 4. The binding molecule of claim 3, wherein: (i) one or more of positions 42, 49, 50, and 52 (numbering based on IMGT numbering) of the polypeptide of SEQ ID NO:1 are humanized; (ii) one or more of positions 42, 49, 50, and 52 (numbering based on IMGT numbering) of the polypeptide of SEQ ID NO:1 are not humanized, optionally wherein all of these positions are not humanized; Attorney Docket No.: 45817-0156WO1 (iii) positions 42 and/or 52 (numbering based on IMGT numbering) of the polypeptide of SEQ ID NO:1 are not humanized; or (iv) positions 49 and/or 50 (numbering based on IMGT numbering) of the polypeptide of SEQ ID NO:1 are humanized. 5. The binding molecule of any one of claims 1 to 4, wherein the VHH comprises a sequence that is 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%, at least 99%, or 100% identical to any amino acid sequence set forth in Table 8; or wherein the VHH comprises a sequence with 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 8, 7, 6, 5, 4, 3, 2, or 1 substitution relative to any amino acid sequence set forth in Table 8. 6. The binding molecule of any one of claims 1 to 5, further comprising an agent selected from the group consisting of a purification tag, a fluorophore, a drug, a photosensitizer, a nanoparticle, a toxic agent, a radionuclide, a VHH, an Fab, a scFv, a multimerization module, a moiety that facilitates the polypeptide crossing the blood brain barrier, and a half-life extension moiety, optionally wherein when the agent is a VHH, an Fab, or a scFv, it is attached or linked to the N-terminus of the polypeptide. 7. The binding molecule of any one of claims 1 to 6, further comprising a human Ig Fc domain, optionally further comprising a human Ig hinge domain, and further optionally wherein the human Ig is a human IgG1, human IgG2, human IgG3, or human IgG4. 8. The binding molecule of any one of claims 1 to 7, comprising an amino acid of an human IgG4 PAA hinge and Fc domain sequence, optionally wherein the human IgG4PAA sequence is at the N-terminus of the polypeptide, optionally wherein the Fc domain includes mutations that promote heterodimerization. 9. A bispecific antibody comprising (i) the binding molecule of any one of claims 1 to 8; and (ii) a second binding molecule that binds to a different epitope of human CD16 than the polypeptide or binds to a different antigen, optionally wherein the different Attorney Docket No.: 45817-0156WO1 antigen is an antigen on a T cell, a neutrophil, an NK cell, or a tumor cell; and further optionally wherein the second binding molecule is a VHH, a scFv, a Fab or a F(ab)2. 10. A human CD16a binding chimeric antigen receptor (CAR) comprising the binding molecule of any one of claims 1 to 8 and further comprising two or more of a transmembrane domain, a costimulatory domain, and an intracellular signaling domain; optionally wherein the transmembrane domain is derived from an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9; further optionally wherein the costimulatory domain comprises a costimulatory region of CD3, CD4, CD8, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-l, ICOS, lymphocyte function-associated antigen-l (LFA-l), CD2, CD7, LIGHT, NKG2C, B7-H3, or a fragment thereof; and further optionally wherein the intracellular signaling domain comprises a fragment or domain from one or more molecules selected from the group consisting of a T cell receptor (TCR), CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma Rlla, DAP 10, DAP 12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, 0X40, CD30, CD40, PD-l, ICOS, a KIR family protein, lymphocyte function-associated antigen-l (LFA-l), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-l, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD 160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CD 11 a, LFA-l, ITGAM, CD lib, ITGAX, CD l lc, ITGB1, CD29, ITGB2, CD 18, LFA- 1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD 96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD 100 (SEMA4D), CD69, SLAMF6 (NTB- A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, Attorney Docket No.: 45817-0156WO1 Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13. 11. A T or NK cell expressing the human CD16a binding CAR of claim 10. 12. A nucleic acid or nucleic acids encoding the binding molecule of any one of claims 1 to 8, the bispecific antibody of claim 9, or the human CD16a binding CAR of claim 10. 13. A vector or vectors comprising the nucleic acid or nucleic acids of claim 12. 14. A host cell comprising the vector or vectors of claim 13. 15. A method of making a binding molecule or a bispecific antibody, the method comprising culturing the host cell of claim 14 under conditions that facilitate expression of the binding molecule or the bispecific antibody, and isolating the binding molecule or the bispecific antibody, optionally further comprising formulating the binding molecule or the bispecific antibody as a sterile pharmaceutical composition. 16. A pharmaceutical composition comprising the binding molecule of any one of claims 1 to 8, the bispecific antibody of claim 9, or a T cell or NK cell expressing the human CD16a binding CAR of claim 10, and a pharmaceutically acceptable carrier. 17. A messenger RNA (mRNA) or mRNAs comprising an open reading frame (ORF) encoding the binding molecule of any one of claims 1 to 8, the bispecific antibody of claim 9, or the human CD16a binding CAR of claim 10. 18. A polynucleotide comprising an mRNA comprising: (i) a 5' UTR; (ii) an open reading frame (ORF) encoding the binding molecule of any one of claims 1 to 8, the bispecific antibody of claim 9, or the human CD16a binding CAR of claim 10; (iii) a stop codon; and Attorney Docket No.: 45817-0156WO1 (iv) a 3' UTR, optionally wherein the mRNA comprises a microRNA (miR) binding site; optionally wherein the miR is expressed in an immune cell of hematopoietic lineage or a cell that expresses TLR7 and/or TLR8 and secretes pro- inflammatory cytokines and/or chemokines; and further optionally wherein the miR binding site is for a miR selected from miR-126, miR126-3p, miR-142, miR-142-3p, miR-142-5p, miR-144, miR-146, miR-150, miR-155, miR-16, miR-21, miR-223, miR-24, miR-27, miR-26a, or any combination thereof; and further optionally wherein the microRNA binding site is located in the 3' UTR of the mRNA. 19. The polynucleotide of claim 18, wherein the mRNA comprises a 5' terminal cap, optionally wherein the 5' terminal cap comprises a Cap0, Cap1, ARCA, inosine, N1- methyl-guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2- amino-guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5' methylG cap, or an analog thereof. 20. The polynucleotide of any one of claims 18 or 19, wherein the mRNA comprises a poly-A region, optionally wherein the poly-A region is at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90 nucleotides in length, or at least about 100 nucleotides in length; and further optionally wherein the poly-A region is about 10 to about 200, about 20 to about 180, about 50 to about 160, about 70 to about 140, or about 80 to about 120 nucleotides in length. 21. The polynucleotide of any one of claims 18 to 20, wherein the mRNA comprises at least one chemically modified nucleobase, sugar, backbone, or any combination thereof. 22. The polynucleotide of claim 21, wherein the mRNA comprises at least one chemically modified nucleobase and the at least one chemically modified nucleobase is selected from the group consisting of pseudouracil (ψ), N1-methylpseudouracil (m1ψ), 1-ethylpseudouracil, 2-thiouracil (s2U), 4’-thiouracil, 5-methylcytosine, 5- methyluracil, 5-methoxyuracil, and any combination thereof; optionally wherein at Attorney Docket No.: 45817-0156WO1 least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or 100% of the uracils are N1-methylpseudouracils. 23. The polynucleotide of any one of claims 18 to 22, wherein the mRNA comprises a 5’terminal cap comprising Cap1 and a poly-A region 100 nucleotides in length (SEQ ID NO: 37), and optionally wherein all uracils of the polynucleotide are N1- methylpseudouracils. 24. A pharmaceutical composition comprising the polynucleotide of any one of claims 13 to 18 and a pharmaceutically acceptable excipient. 25. A lipid nanoparticle comprising the mRNA or mRNAs of claim 17 or the polynucleotide of any one of claims 18 to 23. 26. The lipid nanoparticle of claim 25, wherein the lipid nanoparticle comprises an ionizable lipid, a structural lipid, a phospholipid, and a polyethylene glycol (PEG)- modified lipid, optionally wherein (i) the ionizable amino lipid is a compound of Formula (I); the structural lipid is cholesterol; the phospholipid is 1,2-distearoyl-sn- glycero-3-phosphocholine or 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); and the PEG lipid is PEG 2000 dimyristoyl glycerol or 134-hydroxy- 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87 ,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132- tetratetracontaoxatetratriacontahectyl stearate or a salt thereof; or (ii) the ionizable amino lipid is a compound of Formula I-1 or a salt thereof; the structural lipid is cholesterol; the phospholipid is DSPC; and the PEG-lipid is 134-hydroxy- 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87 ,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132- tetratetracontaoxatetratriacontahectyl stearate or PEG-DMG, or a salt thereof. Attorney Docket No.: 45817-0156WO1 27. The lipid nanoparticle of claim 26, wherein the lipid nanoparticle comprises about 47.5 mol % of the ionizable amino lipid; about 39 mol % of cholesterol; about 10.5 mol % of DSPC; and about 3 mol % of the PEG-lipid; optionally wherein the lipid nanoparticle comprises 47.5 mol % of the ionizable amino lipid; 39 mol % of cholesterol; 10.5 mol % of DSPC; and 3 mol % of the PEG-lipid. 28. A method of treating a cancer in a human subject in need thereof, killing a tumor cell in a human subject in need thereof, or decreasing the rate of tumor growth in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of the polynucleotide of any one of claims 18 to 23, or the pharmaceutical composition of claim 24, or the lipid nanoparticle of claim 25, 26, or 27.
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