WO2024091979A1 - Cd276 (b7-h3) antibody-drug conjugates - Google Patents

Cd276 (b7-h3) antibody-drug conjugates Download PDF

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Publication number
WO2024091979A1
WO2024091979A1 PCT/US2023/077689 US2023077689W WO2024091979A1 WO 2024091979 A1 WO2024091979 A1 WO 2024091979A1 US 2023077689 W US2023077689 W US 2023077689W WO 2024091979 A1 WO2024091979 A1 WO 2024091979A1
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Prior art keywords
antibody
amino acid
seq
trna
unnatural amino
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PCT/US2023/077689
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French (fr)
Inventor
James Sebastian ITALIA
Colby Souders
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Brickbio, Inc.
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Publication of WO2024091979A1 publication Critical patent/WO2024091979A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68035—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a pyrrolobenzodiazepine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • 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
    • C07K16/2827—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 B7 molecules, e.g. CD80, CD86
    • 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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation

Definitions

  • ADCs antibody-drug conjugates
  • the disclosure described herein is based in part, upon the discovery of an anti-CD276 antibody containing one or more unnatural amino acids at one or more specific locations in an immunoglobulin heavy chain and/or immunoglobulin light chain of the anti-CD276 antibody that can be used to conjugate one or more payloads, e.g., one or more therapeutic molecules, to the antibody.
  • a payload can be linked directly to the unnatural amino acid or indirectly to the unnatural amino acid by a linker.
  • the resulting antibody conjugates (e.g., antibody-drug conjugate) can be used in methods for treating a disorder, e.g., cancer, associated with expression of elevated levels of CD276.
  • an engineered anti-CD276 antibody comprising: a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to one or more of: (i) A142 Attorney Docket No.
  • the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to one or more of: (i) A142 of SEQ ID NO: 61; (ii) T157 of SEQ ID NO: 61; (iii) T171 of SEQ ID NO: 61; (iv) A174 of SEQ ID NO: 61; (v) Y182 of SEQ ID NO: 61; (vi) T197 of SEQ ID NO: 61; (vii) D267 of SEQ ID NO: 61; or (viii) S241 of SEQ ID NO: 61.
  • the unnatural amino acid is incorporated at a position corresponding to T171 of SEQ ID NO: 61.
  • the antibody further comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 62.
  • the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 62.
  • the antibody further comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 62, with an unnatural amino acid incorporated at a position corresponding to Q162 of SEQ ID NO: 62.
  • the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to Q162 of SEQ ID NO: 62.
  • an engineered anti-CD276 antibody comprising: a light chain sequence, wherein the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 62, with an unnatural amino acid incorporated at a position corresponding to Q162 of SEQ ID NO: 62.
  • the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 62, with an unnatural amino acid incorporated at a position corresponding to Q162 of SEQ ID NO: 62.
  • the antibody further comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at Attorney Docket No.
  • BRI-023WO least 99%, or greater sequence identity to SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to one or more of: (i) A142 of SEQ ID NO: 61; (ii) T157 of SEQ ID NO: 61; (iii) T171 of SEQ ID NO: 61; (iv) A174 of SEQ ID NO: 61; (v) Y182 of SEQ ID NO: 61; (vi) T197 of SEQ ID NO: 61; (vii) D267 of SEQ ID NO: 61; or (viii) S241 of SEQ ID NO: 61.
  • the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to one or more of: (i) A142 of SEQ ID NO: 61; (ii) T157 of SEQ ID NO: 61; (iii) T171 of SEQ ID NO: 61; (iv) A174 of SEQ ID NO: 61; (v) Y182 of SEQ ID NO: 61; (vi) T197 of SEQ ID NO: 61; (vii) D267 of SEQ ID NO: 61; (viii) S241 of SEQ ID NO: 61.
  • the unnatural amino acid is incorporated at a position corresponding to T171 of SEQ ID NO: 61.
  • an engineered anti-CD276 antibody comprising: a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 137, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 138, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 139, and wherein the heavy chain and/or light chain comprises an
  • an engineered anti- CD276 antibody comprising: a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 137, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 138, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 139, and wherein the heavy chain comprises an unnatural amino acid incorporated at a position corresponding to one or more of: (i) A142 of SEQ ID NO: 61; (ii) T
  • an engineered anti-CD276 antibody comprising: a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 137, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 138, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 139, and wherein the heavy chain comprises an unnatural amino acid incorporated at a position corresponding to T171 of SEQ ID NO: 61.
  • the antibody comprises at least one, at least two, at least three, at least four, at least five, or more unnatural amino acids.
  • the unnatural amino acid can be a tryptophan analog, a leucine analog, a tyrosine analog, or a pyrrolysine analog.
  • the unnatural amino acid is a tryptophan analog (e.g., 5-HTP or 5-AzW).
  • the unnatural amino acid is a leucine analog (e.g., LCA or Cys-5-N3, or LCA).
  • the unnatural amino acid is a tyrosine analog (e.g., OmeY, AzF, or OpropY).
  • the unnatural amino acid is a pyrrolysine analog (e.g., BocK, CpK, or AzK).
  • the unnatural amino acid is chemically modified.
  • the chemical modification can comprise the unnatural amino acid conjugated to a molecule of interest, for example, a detectable label or a therapeutic molecule.
  • the therapeutic molecule is: AEB, AEVB, AFP, an amatoxin, an auristatin (e.g., auristatin E), a calicheamicin, CC-1065 or a CC-1065 analog, chalicheamicin, combretastatin, docetaxel, dolastatin-10, DUBA, a duocarmycin, echinomycin, FAM, maytansine, a maytansinoid (e.g.
  • DM1 or DM4 MMAD, MMAE, MMAF, a morpholino-doxorubicin (e.g., cyanomorpholino- doxorubicin), netropsin, an oligonucleotide (e.g., a DNA, RNA, or LNA oligonucleotide), paclitaxel, pyrrolobenzodiazepine dimer (PBD), a peptide (e.g., a therapeutic peptide), rhizoxin, a small molecule (e.g., a therapeutic small molecule), SN-38, topotecan, a topoisomerase inhibitor, or a toxoid.
  • oligonucleotide e.g., a DNA, RNA, or LNA oligonucleotide
  • PBD pyrrolobenzodiazepine dimer
  • a peptide e.g., a therapeutic peptide
  • the therapeutic molecule is PBD. Attorney Docket No. BRI-023WO [0016]
  • the molecule is conjugated to the antibody by a crosslinking agent.
  • crosslinking agents can include bissuflosuccinimidyl suberate (BS3), N-hydroxy-succinimide/1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (NHS/EDC), N- ⁇ -maleimidocaproyl-oxysulfosuccinimide ester (sulfoEMCS), N- ⁇ - maleimidocaproic acid hydrazide (EMCH), hydrazide, N-succinimidyl-S-acetylthioacetate (SATA), monofluorocyclooctyne (MFCO), bicyclo[6.1.0]nonyne (BCN), N succinimidyl-S- acetylthi
  • the molecule is conjugated to the antibody by a linker, which can contain a cleavable linker region or a non-cleavable linker region, and optionally, one or more spacer regions.
  • the cleavable or non-cleavable linker region is a peptide-linker region.
  • the peptide-linker region comprises a VA linker or a GGFG linker.
  • the spacer region comprises a PEG-based region (e.g., a PEG8 region).
  • the linker conjugates the molecule to the antibody via a cross- linking agent.
  • Exemplary cross-linking agents include bissuflosuccinimidyl suberate (BS3), N- hydroxy-succinimide/1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (NHS/EDC), N- ⁇ - maleimidocaproyl-oxysulfosuccinimide ester (sulfoEMCS), N- ⁇ -maleimidocaproic acid hydrazide (EMCH), hydrazide, N-succinimidyl-S-acetylthioacetate (SATA), monofluorocyclooctyne (MFCO), bicyclo[6.1.0]nonyne (BCN), N succinimidyl-S-acetylthiopropionate (SATP), a maleimido ester, a dibenzocyclooctyne (DBCO) ester, EDC, or any combination thereof.
  • BS3 bissuflosucc
  • the cross-linking agent or the linker conjugates the molecule to the antibody at the unnatural amino acid site.
  • the antibody has an average drug-to-antibody ratio (DAR) of at least 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0, as measured by hydrophobic interaction chromatography (HIC);
  • HIC hydrophobic interaction chromatography
  • the antibody has an average DAR of about 2, as measured by HIC;
  • at least 40%, 50%, 60%, 70%, 80%, or 90% of the antibody remains following incubation in human plasma for 72 hours at 37 °C;
  • the antibody at least 40%, 50%, 60%, 70%, 80%, or 90% of the antibody remains following incubation with Cathepsin B for 240 minutes at 37 °C;
  • the antibody has a binding affinity for
  • the antibody is derived from an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the antibody is derived from an IgG1 antibody.
  • the disclosure provides a method of making an antibody as described herein.
  • the method comprising a step of culturing a cell comprising: (i) a first nucleotide sequence encoding a tRNA comprising an anticodon that hybridizes to a codon selected from UAG, UGA, and UAA, and is capable of being charged with an unnatural amino acid; (ii) a second nucleotide sequence encoding an aminoacyl-tRNA synthetase capable of charging the tRNA with the unnatural amino acid; and (iii) a third nucleotide sequence encoding the heavy chain sequence and/or a fourth nucleotide sequence encoding the light chain sequence, wherein the third nucleotide sequence and/or the fourth nucleotide sequence comprise a codon selected from UAG, UGA, and UAA; wherein the cell is cultured under conditions that permit the tRNA, when expressed in the cell and charged with the unnatural amino acid
  • the disclosure provides a method of making an engineered anti-CD276 antibody.
  • the method comprising a step of culturing a cell comprising: (i) a first nucleotide sequence encoding a tRNAs comprising an anticodon that hybridizes to a codon selected from UAG, UGA, and UAA, and is capable of being charged with an unnatural amino acid; (ii) a second nucleotide sequence encoding an aminoacyl-tRNA synthetase capable of charging the tRNA with the unnatural amino acid; and (iii) a third nucleotide sequence encoding a heavy chain sequence as set forth in SEQ ID NO: 61 and/or a fourth nucleotide sequence encoding a light chain sequence as set forth in SEQ ID NO: 62, wherein the third nucleotide sequence and/or the fourth nucleotide sequence comprise a codon selected from UAG, UGA, and UAA; where
  • the third nucleotide sequence and/or the fourth nucleotide sequence comprise a codon selected from UAG, UGA, and UAA at a site that corresponds to any site shown in FIG.5 or Table 1.
  • the amount of the antibody comprising the unnatural amino acid expressed by the cell is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the amount of a suitable reference antibody expressed by the same cell or a similar cell; and/or (b) following purification of the antibody, less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the antibody is aggregated, as measured by size exclusion chromatography (SEC).
  • the tRNA is an analog or derivative of a prokaryotic tryptophanyl-tRNA (e.g., E.
  • the nucleotide sequence encoding the tRNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 56-60.
  • the aminoacyl-tRNA synthetase is an analog or derivative of a prokaryotic tryptophanyl-tRNA synthetase (e.g., E. coli tryptophanyl- tRNA synthetase).
  • the aminoacyl-tRNA synthetase comprises an amino acid sequence selected from any one of SEQ ID NOs: 51-54.
  • the tRNA is an analog or derivative of a prokaryotic leucyl-tRNA (e.g., an E. coli leucyl-tRNA.
  • the nucleotide sequence encoding the tRNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 22-49.
  • the aminoacyl-tRNA synthetase is an analog or derivative of a prokaryotic leucyl-tRNA synthetase (e.g., an E. coli leucyl-tRNA synthetase).
  • the aminoacyl-tRNA synthetase comprises an amino acid sequence selected from any one of SEQ ID NOs: 8-21.
  • the tRNA is an analog or derivative of a prokaryotic tyrosyl-tRNA (e.g., an E. coli tyrosyl-tRNA).
  • the nucleotide sequence encoding the tRNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 140-144, 192, or 193.
  • the aminoacyl-tRNA synthetase is an analog or derivative of the prokaryotic tyrosyl-tRNA synthetase (e.g., an E. coli tyrosyl-tRNA synthetase).
  • the aminoacyl-tRNA synthetase comprises an amino acid sequence selected from any one of SEQ ID NOs: 145 or 147.
  • Attorney Docket No. BRI-023WO [0032]
  • the tRNA is an analog or derivative of an archael pyrrolysyl-tRNA (e.g., an M.
  • the nucleotide sequence encoding the tRNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 148-178, 181, or 182.
  • the aminoacyl-tRNA synthetase is an analog or derivative of the archael pyrrolysyl-tRNA synthetase (e.g., an M. barkeri pyrrolysyl- tRNA synthetase).
  • the aminoacyl-tRNA synthetase comprises an amino acid sequence selected from any one of SEQ ID NO: 179.
  • the reference antibody comprises a wild- type amino acid residue at the position corresponding to the unnatural amino acid.
  • the cell is a mammalian cell (e.g., a human cell).
  • the mammalian cell is a human embryonic kidney (HEK) cell or a Chinese hamster ovary (CHO) cell.
  • the method further comprises a step of purifying the antibody. Alternatively or in addition, the method further comprises a step of chemically modifying the unnatural amino acid.
  • the chemical modification can comprise conjugation to a molecule (e.g., a detectable label or a therapeutic molecule such as AEB, AEVB, AFP, an amatoxin, an auristatin (e.g., auristatin E), a calicheamicin, CC-1065 or a CC-1065 analog, chalicheamicin, combretastatin, docetaxel, dolastatin-10, DUBA, a duocarmycin, echinomycin, FAM, maytansine, a maytansinoid (e.g., DM1 or DM4), MMAD, MMAE, MMAF, a morpholino- doxorubicin (e.g., cyanomorpholino-doxorubicin), netropsin, an oligonucleotide (e.g., a DNA, RNA, or LNA oligonucleotide), paclitaxel, PBD, a
  • the therapeutic molecule is PBD.
  • the molecule is conjugated to the antibody by a crosslinking agent.
  • crosslinking agents include bissuflosuccinimidyl suberate (BS3), N-hydroxy- succinimide/1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (NHS/EDC), N- ⁇ -maleimidocaproyl- oxysulfosuccinimide ester (sulfoEMCS), N- ⁇ -maleimidocaproic acid hydrazide (EMCH), hydrazide, N-succinimidyl-S-acetylthioacetate (SATA), monofluorocyclooctyne (MFCO), bicyclo[6.1.0]nonyne (BCN), N succinimidyl-S-acetylthiopropionate (SATP), a maleimido ester, a di
  • the molecule is conjugated to the engineered antibody by a linker, which can comprise a cleavable or non-cleavable linker region, and optionally, one or more spacer regions.
  • the cleavable or non-cleavable linker region is a peptide-linker Attorney Docket No. BRI-023WO region.
  • the peptide-linker region comprises a VA linker or a GGFG linker.
  • the spacer region comprises a PEG-based domain (e.g., a PEG8 region).
  • the linker conjugates the molecule to the antibody via a cross- linking agent.
  • the cross-linking agent comprises bissuflosuccinimidyl suberate (BS3), N-hydroxy-succinimide/1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (NHS/EDC), N- ⁇ -maleimidocaproyl-oxysulfosuccinimide ester (sulfoEMCS), N- ⁇ - maleimidocaproic acid hydrazide (EMCH), hydrazide, N-succinimidyl-S-acetylthioacetate (SATA), monofluorocyclooctyne (MFCO), bicyclo[6.1.0]nonyne (BCN), N succinimidyl-S- acetylthiopropionate (SATP), a maleimido ester, a dibenzocyclooctyne (DBCO) ester, EDC, or any combination thereof.
  • BS3 bissuflosuccin
  • the cross-linking agent or the linker conjugates the molecule to the antibody at the unnatural amino acid site.
  • the disclosure provides an engineered anti-CD276 antibody comprising: a heavy chain sequence comprising an amino acid sequence as set forth in SEQ ID NO: 61, with an LCA incorporated at a position corresponding to T171 of SEQ ID NO: 61; a light chain sequence comprising an amino acid sequence as set forth in SEQ ID NO: 62; wherein a pyrrolobenzodiazepine dimer (PBD) is conjugated to the LCA via a linker and crosslinking agent, wherein the linker comprises a PEG8 spacer region and a VA linker region and the crosslinking agent is DBCO.
  • PBD pyrrolobenzodiazepine dimer
  • Such an engineered anti-CD276 antibody can be produced by a method comprising a step of culturing a cell comprising: (i) a first nucleotide sequence encoding a tRNAs comprising an anticodon that hybridizes to a codon selected from UAG, UGA, and UAA, and is capable of being charged with an LCA; (ii) a second nucleotide sequence encoding an aminoacyl- tRNA synthetase capable of charging the tRNA with the LCA; and (iii) a third nucleotide sequence encoding the heavy chain sequence and a fourth nucleotide sequence encoding the light chain sequence, wherein the third nucleotide sequence comprises a codon selected from UAG, UGA, and UAA at a site corresponding to T171 of SEQ ID NO: 61; wherein the cell is cultured under conditions that permit the tRNA, when expressed in the cell and charged with the LCA, to hybridize to the codon
  • an engineered anti-CD276 antibody comprising: an immunoglobulin heavy chain sequence comprising an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to one or more sites selected from TABLE 1 or FIG.5; and an immunoglobulin light chain sequence comprising an amino acid sequence with at least 75%, at Attorney Docket No.
  • BRI-023WO least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 62, with an unnatural amino acid incorporated at a position corresponding to one or more sites selected from TABLE 1 or FIG.5.
  • the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to one or more sites selected from Table 1 or FIG.5; and the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 62, with an unnatural amino acid incorporated at a position corresponding to one or more sites selected from TABLE 1 or FIG.5.
  • the at least one unnatural amino acid is: (a) a leucine analog, a tyrosine analog, a tryptophan analog, or a pyrrololysine analog; (b) 5-HTP or 5-AzW; (c) LCA or Cys-5-N43; (d) LCA; (e) OmeY, AzF, or OpropY; and/or (f) BocK, CpK, or AzK.
  • the unnatural amino acid is chemically modified, wherein the chemical modification comprises conjugation of the unnatural amino acid to a molecule: (a) wherein the molecule is a detectable label; (b) wherein the molecule is a therapeutic molecule; (c) wherein the molecule is conjugated to the antibody by a crosslinking reagent; (d) wherein the molecule is conjugated to the antibody by a linker; (e) wherein the molecule is conjugated to the antibody by a linker forming a linker-payload structure of U’-X1-L-X2-P, where U’ is DBCO, X1 is PEG8, L is VA, X2 is PAB, and P is PBD, and DBCO crosslinks the linker-payload to the unnatural amino acid; and/or (f) wherein the molecule is conjugated to the antibody by a linker forming a linker-payload structure of U’-X1-L-P, where U’ is a detectable label; (b)
  • the disclosure provides a pharmaceutical composition comprising an engineered antibody as described herein, and optionally comprising one or more pharmaceutically acceptable excipients or carriers.
  • the disclosure provides a method of treating (a) a subject with a disease or a disorder, the method comprising administering to the subject an effective amount of an engineered antibody as described herein, a pharmaceutical composition as described herein, or an antibody made by a method described herein, (b) a subject with a cancer that overexpresses CD276, the method comprising administering to the subject an effective amount of an engineered antibody as described herein, a pharmaceutical composition as described herein, or an antibody made by a method as described herein, or (c) a subject with CD276 positive tumor vasculature, the method comprising administering to the subject an effective amount of an engineered antibody as described Attorney Docket No.
  • FIG.1 depicts a sequence alignment between an exemplary anti-CD276 antibody heavy chain amino acid sequence (SEQ ID NO: 61) that may be used in accordance with the present disclosure, and, for sequence comparison purposes, a Trastuzumab heavy chain amino acid sequence (SEQ ID NO: 183).
  • FIG.2 depicts a sequence alignment between an exemplary anti-CD276 antibody light chain amino acid sequence (SEQ ID NO: 62) that may be used in accordance with the present disclosure, and, for sequence comparison purposes, a Trastuzumab light chain amino acid sequence (SEQ ID NO: 184).
  • FIGs.3A-3E depict exemplary unnatural amino acids (UAAs) that may be used in accordance with the present disclosure.
  • FIG.4 depict graphs showing % wild-type expression for certain UAA-containing antibodies generated in accordance with the present disclosure.
  • FIG.5 depicts a list of exemplary ADCs generated in accordance with the present disclosure.
  • FIG.6 depict exemplary QC profiles using biophysical assays for certain ADCs generated in accordance with the present disclosure.
  • FIGs.7A-7J depict exemplary QC profiles using stability, cathepsin cleavage assays, and LCMS assays for certain ADCs generated in accordance with the present disclosure.
  • FIG.8 depicts an analysis of drug-to-antibody ratio (DAR) in certain ADCs via LCMS.
  • FIG.9 depicts an exemplary broad site-select screening panel for certain ADCs generated in accordance with the present disclosure.
  • DAR drug-to-antibody ratio
  • FIG.10 depicts an exemplary full site-panel of certain ADCs generated in accordance with the present disclosure.
  • Panel assays included HIC, SDS-PAGE, SEC, DAR assessment, yield, human plasma stability, mouse plasma stability, and cathepsin cleavage.
  • FIGs.11A-11B depict graphs showing cytotoxicity of certain ADCs in CD276+ cells and CD276- cells. Attorney Docket No.
  • FIG.12 depicts graphs showing cytotoxicity of certain ADCs in lung cancer cells, breast cancer cells, and ovarian cancer cells.
  • FIG.13 depicts a graph showing cytotoxicity of certain ADCs in CD276+ cells and CD276- cells.
  • FIG.14 depicts a schematic overview of genetic code expansion and unnatural amino acid (UAA) incorporation.
  • UAA unnatural amino acid
  • FIG.15 depicts exemplary auristatin payloads that may be used in accordance with the present disclosure.
  • FIG.16 depicts exemplary pyrrolobenzodiazepine (PBD) payloads that may be used in accordance with the present disclosure.
  • PBD pyrrolobenzodiazepine
  • FIG.17 depicts exemplary camptothecin payloads that may be used in accordance with the present disclosure.
  • FIG.18 depicts exemplary branched payload adapters that may be used in accordance with the present disclosure.
  • FIGs.19A-19C depict exemplary PBD structures that may be used in accordance with the present disclosure. Structures depicted are adapted from Angew. Chem. Int. Ed.2017, 56, 462-488.
  • FIG.20 depicts exemplary PBD cores that may be used in accordance with the present disclosure.
  • FIG.21 depicts an exemplary PBD based ADC connectivity that may be used in accordance with the present disclosure.
  • FIGS.22A-B depict an exemplary pro-PBD based ADC connectivity that may be used in accordance with the present disclosure.
  • FIGS.23A-23B depict an exemplary connectivity to ADCs via click chemistry that may be used in accordance with the present disclosure.
  • FIG.24 depicts a table and graphs showing efficacy of certain ADCs in a HCT116 CDX in vivo mouse model.
  • FIG.25 depicts graphs showing tumor regression in a HCT116 CDX in vivo mouse model.
  • FIG.26 depicts a graph showing body weight of mice treated with certain ADCs at different time points. Attorney Docket No.
  • FIG.27 depicts a graph showing tumor volume reduction efficacy of certain interchain cysteine conjugate ADCs in a HCT116 CDX in vivo mouse model.
  • FIG.28 depicts a graph showing differential cathepsin B cleavage rate as a function of site of conjugation in certain ADCs
  • FIGs.29A-29B depict exemplary an connectivity between an exemplary antibody, e.g., an anti-CD276 antibody provided herein, containing an unnatural amino acid (A-U) modified with a linker-payload as described herein (e.g., U’-X1-L-X2-P).
  • A-U unnatural amino acid
  • FIG.29A represents a schematic representation of an antibody with an exemplary payload attached
  • FIG 29B represents an unnatural amino acid (with peptide bonds connecting the unnatural amino acid to the adjacent amino acids in remainder of the immunoglobulin chain, e.g., the immunoglobulin heavy chain, shown with wavy lines) with the payload attached.
  • DETAILED DESCRIPTION [0073] The disclosure described herein is based in part, upon the discovery of an anti-CD276 antibody containing one or more unnatural amino acids at one or more specific locations in an immunoglobulin heavy chain and/or immunoglobulin light chain of the anti-CD276 antibody that can be used to conjugate one or more payloads, e.g., one or more therapeutic molecules, to the antibody.
  • a payload can be linked directly to the unnatural amino acid or indirectly to the unnatural amino acid by a linker.
  • the resulting antibody conjugates e.g., antibody-drug conjugate
  • a disorder e.g., cancer
  • the approach described herein involves a method of modifying proteins via the site- specific incorporation of one or more unnatural amino acids (also referred to as UAAs) into a protein of interest in vivo.
  • the ability to site-specifically incorporate unnatural amino acids e.g., non-natural amino acids, non-canonical amino acids, nonstandard amino acids, etc.
  • unnatural amino acids e.g., non-natural amino acids, non-canonical amino acids, nonstandard amino acids, etc.
  • the core elements used to implement this technology include: an engineered tRNA/aminoacyl-tRNA synthetase (aaRS) pair and a unique codon directing the incorporation of the unnatural amino acid.
  • aaRS engineered tRNA/aminoacyl-tRNA synthetase
  • An engineered tRNA/aaRS pair can derived from a tRNA/aaRS pair obtained from a different organism than that used as an expression host (to maintain orthogonality, and minimize cross-reactivity) that are imported into the desired expression host (e.g., a cell, such as a eukaryotic cell).
  • a tRNA/aaRS pair obtained from a different organism than that used as an expression host (to maintain orthogonality, and minimize cross-reactivity) that are imported into the desired expression host (e.g., a cell, such as a eukaryotic cell).
  • the tRNA/aaRS pairs can be engineered to incorporate an unnatural amino acid to a site- specific locus in a protein of interest at the direction of the charged tRNA-unnatural amino acid complex towards a unique codon, such as stop codons (e.g., TAG, TGA, TAA, etc.), four-base frameshift codons, sense codons, or non-canonical codons.
  • a unique codon such as stop codons (e.g., TAG, TGA, TAA, etc.), four-base frameshift codons, sense codons, or non-canonical codons.
  • This technology has been established for a variety of tRNA/aaRS pairs for some of the 20 natural amino acids (e.g., E. coli TrpRS/tRNA, E. coli LeuRS/tRNA, E. coli TyrRS/tRNA, M. barkeri PylRS/tRNA, M.
  • ADCs antibody-drug conjugates
  • mAbs bi-specific monoclonal antibodies
  • nanobodies chemokines
  • vaccines coagulation factors
  • hormones hormones
  • enzyme therapies etc.
  • payloads e.g., drugs, oligos, half-life extenders, and other molecules
  • biologics such as antibodies, cytokines, peptides, and other protein biologics
  • the present disclosure is based, in part, upon the recognition that the site of conjugation can make drastic differences to molecular characteristics such as biophysical properties, PK/PD, efficacy, as well as the logistical challenges such as yield, conjugation efficiency, and modularity.
  • the site-specific conjugation of cytotoxic payloads to targeting antibodies has been shown to increase the therapeutic window of these molecules.
  • the site and location of conjugation can have a major impact on the overall characteristics of an ADC. For example, site and location of conjugation have been found to impact properties that prevent non-specific uptake (Mahalingaiah, PK. et al., (2019) PHARMACOL.
  • CD276 is overexpressed in many solid tumors, but has minimal to no expression in healthy tissues.
  • CD276 also known as 4Ig-B7-H3, B7 homolog 3 costimulatory molecule, and B7-H3
  • B7-H3 is a transmembrane protein of the B7 family (e.g., PD-L1, etc.) which Attorney Docket No. BRI-023WO includes many immune checkpoint molecules.
  • mRNA expression is broadly observed, but protein expression is low in healthy tissues and organs.
  • the physiological function of CD276 is still under investigation but the receptor has implications towards stimulation of T-cells, as well as influencing tumor-infiltration, cancer progression, migration, and other tumorigenic functions.
  • CD276 is an attractive target as a cancer therapy for which 50+ clinical trials have commenced, some progressing to Phase III.
  • the broad expression profile of CD276 in malignant tissue has prompted investigation into therapeutic modalities across many different solid cancers, such as lung, esophageal, sarcomas, endometrial, prostate, breast, and colon cancers (Michelakos, T.
  • ADCs provide an attractive strategy due to their high specificity and ability to selectively deliver highly potent payloads to the site of interest. Regardless of payload used, there is a balance between increasing and improving the efficacy of the ADC conjugate versus maintaining a high safety profile.
  • the present disclosure appreciates that modular, tunable site- specific conjugation enabled by unnatural amino acid conjugation handles can provide best-in-class safety profiles while allowing for easy control over drug to antibody ratio (DAR), conjugation site location, and linker chemistry variation to enhance the desired properties of an ADC (e.g., any desired properties described herein).
  • DAR drug to antibody ratio
  • conjugation site location e.g., any desired properties described herein.
  • linker chemistry variation e.g., any desired properties described herein.
  • the one or more UAAs can be incorporated into an engineered anti-CD276 antibody using one or more orthogonal tRNA/aminoacyl-tRNA synthetase pairs that are present in a desired expression system (e.g., in an in vitro system, in a host cell, etc.).
  • the incorporated UAAs can subsequently be used to conjugate a payload (e.g., a cytotoxic payload, an immune modulatory payload, or any other payload described herein) to generate a therapeutic ADC for the treatment of disorders associated with elevated levels of CD276 expression, e.g., CD276 positive oncological malignancies.
  • a payload e.g., a cytotoxic payload, an immune modulatory payload, or any other payload described herein
  • the resulting anti-CD276 antibody conjugate (e.g., an anti-CD276 antibody drug conjugate described herein) can be used to deliver one or more payload moieties to a target cell or tissue (e.g., a cancerous target cell, or cancerous tissue) expressing CD276.
  • a target cell or tissue e.g., a cancerous target cell, or cancerous tissue
  • CD276 e.g., a cancerous target cell, or cancerous tissue
  • an antibody includes a plurality of antibodies and reference to “an antibody” in some embodiments includes multiple antibodies, and so forth.
  • all numerical values or numerical ranges include whole integers within or encompassing such ranges and fractions of the values or the integers within or encompassing ranges unless the context clearly indicates otherwise.
  • reference to a range of 90-100% includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth.
  • reference to a range of 1-5,000 fold includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 fold, etc., as well as 1.1, 1.2, 1.3, 1.4, 1.5 fold, etc., 2.1, 2.2, 2.3, 2.4, 2.5 fold, etc., and so forth.
  • the term “about” refers to a ⁇ 10% variation from the nominal value unless otherwise indicated or inferred.
  • percent identity refers to the extent to which two sequences e.g., two polypeptides or two nucleic acids have the same respective amino acid or nucleotide at the same positions in an alignment.
  • percent identity between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
  • percent identity between a nucleic acid sequence and a reference sequence is defined as the percentage of nucleotides in the nucleic acid sequence that are identical to the nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity (e.g., nucleic acid sequence identity or amino acid sequence identity) can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST (Basic Local Alignment Search Tool), BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software.
  • BLAST Basic Local Alignment Search Tool
  • BLAST-2 Basic Local Alignment Search Tool
  • ALIGN ALIGN
  • MEGALIGN MEGALIGN
  • CLUSTALW CLUSTAL OMEGA
  • MUSCLE software MUSCLE software
  • the BLOSUM-62 scoring matrix assigns positive, zero, or negative scores between each pair or standard amino acid residues (see Henikoff and Henikoff (1992) PROC. NATL. ACAD. SCI. USA, 89: 10915–19 at FIG.2). A positive score between two amino acid residues indicates that substitution of these amino acid residues for each other is conservative.
  • similarity between a subject amino acid sequence and a reference amino acid sequence refers to the percentage of amino acid residues in the subject amino acid sequence that are identical or have a conservative substitution according to the BLOSUM-62 scoring matrix, relative to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum sequence alignment score.
  • the terms “subject” and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably include humans.
  • the term “effective amount” refers to the amount of an active agent sufficient to effect beneficial or desired results (e.g., a desired prophylactic or therapeutic effect). An effective amount can be administered in one or more administration(s), application(s) or dosage(s) and is not intended to be limited to a particular formulation or administration route.
  • the term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vivo or ex vivo.
  • the terms “treat,” “treating,” or “treatment,” and other grammatical equivalents as used in this disclosure, include alleviating, abating, ameliorating, or preventing a disease, condition or symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition, and are intended to include prophylaxis.
  • the terms further include achieving a therapeutic benefit and/or a prophylactic benefit.
  • therapeutic benefit refers to eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder.
  • the term “combination” in the context of therapies means that two or more different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, such that the effects of the treatments on the patient overlap at a point in time.
  • the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.”
  • the delivery of one treatment ends before the delivery of the other treatment begins.
  • the treatment is more effective because of combined administration.
  • the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second Attorney Docket No. BRI-023WO treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment.
  • delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other.
  • the effect of the two treatments can be partially additive, wholly additive, or greater than additive.
  • the delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.
  • compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited steps.
  • elements and/or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present invention, whether explicit or implicit herein.
  • that compound can be used in various embodiments of compositions of the present invention and/or in methods of the present invention, unless otherwise understood from the context.
  • the present disclosure provides, among other things, engineered antibodies (e.g., engineered anti-CD276 antibodies) that comprise at least one unnatural amino acid that functions as conjugation site (e.g., as a bioconjugation handle) for at least one payload molecule (e.g., a therapeutic payload, or any payload described herein), and methods of making and using the same.
  • the present disclosure provides engineered anti-CD276 antibodies, or anti- CD276 antibody drug conjugates (ADCs), that utilize unnatural amino acid conjugation sites to attach one or more payload molecules (e.g., any payload described herein, e.g., PBD or PBD dimers).
  • the conjugated antibodies can have enhanced efficacy compared to similar antibodies that do not comprise a conjugated payload.
  • the present disclosure further provides, among other things, methods of producing and methods of using the conjugated antibodies.
  • specific amino acid residue locations e.g., sites or positions
  • an antibody e.g., an anti-CD276 antibody, such as an m276 antibody
  • payload molecules e.g., cytotoxic linker-payloads, or any other payload described herein.
  • engineered antibodies of the present disclosure may provide, among other things, enhanced release kinetics for therapeutic use, increased cytotoxic windows between cancerous and healthy cells, improved toxicity profiles, lower uptake in healthy tissues, increased efficacy at lower dosages, increased therapeutic window (efficacy to toxicity dosing range), and increased maximum tolerated dose (MTD) while maintaining effective concentrations for therapeutic dosages.
  • ADCs such as anti-CD276 ADCs
  • DAR drug-to-antibody ratio
  • engineered antibodies of the present disclosure may provide, among other things, enhanced release kinetics for therapeutic use, increased cytotoxic windows between cancerous and healthy cells, improved toxicity profiles, lower uptake in healthy tissues, increased efficacy at lower dosages, increased therapeutic window (efficacy to toxicity dosing range), and increased maximum tolerated dose (MTD) while maintaining effective concentrations for therapeutic dosages.
  • MTD maximum tolerated dose
  • an antibody provided by the present disclosure (e.g., an engineered antibody, such as a conjugated antibody or ADC) comprises a heavy chain amino acid sequence as shown in TABLE 2.
  • an antibody provided by the present disclosure comprises a heavy chain amino acid sequence as shown in TABLE 2, with an unnatural amino acid incorporated at one or more positions in the heavy chain amino acid sequence.
  • an antibody provided by the present disclosure comprises a heavy chain amino acid sequence as shown in TABLE 2, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1.
  • an antibody provided by the present disclosure comprises a light chain amino acid sequence as shown in TABLE 2. In some embodiments, an antibody provided by the present disclosure comprises a light chain amino acid sequence as shown in TABLE 2, with an unnatural amino acid incorporated at one or more positions in the light chain amino acid sequence. In some embodiments, an antibody provided by the present disclosure comprises a light chain amino acid sequence as shown in TABLE 2, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1. [0099] In some embodiments, an antibody provided by the present disclosure comprises a heavy chain sequence as shown in TABLE 2, and a light chain sequence as shown in TABLE 2.
  • an antibody provided by the present disclosure comprises a heavy chain sequence as shown in TABLE 2, with an unnatural amino acid incorporated at one or more positions in the heavy chain, and a light chain sequence as shown in TABLE 2, with an unnatural amino acid incorporated at one or more positions in the light chain.
  • an antibody provided by the present disclosure comprises a heavy chain sequence as shown in TABLE 2, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1, and a light chain sequence as shown in TABLE 2, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1.
  • an antibody or engineered antibody provided by the present disclosure comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 1.
  • an antibody or engineered antibody comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 1.
  • an antibody or engineered antibody provided by the present disclosure comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at Attorney Docket No. BRI-023WO least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 1, with an unnatural amino acid incorporated at one or more amino acid positions of the heavy chain.
  • an antibody or engineered antibody comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 1, with an unnatural amino acid incorporated at one or more amino acid positions of the heavy chain.
  • an antibody or engineered antibody provided by the present disclosure comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 1, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1.
  • an antibody or engineered antibody comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 1, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1.
  • an antibody or engineered antibody provided by the present disclosure comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 61.
  • an antibody or engineered antibody comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 61.
  • an antibody or engineered antibody provided by the present disclosure comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 61, with an unnatural amino acid incorporated at one or more amino acid positions of the heavy chain.
  • An exemplary payload can be attached to the unnatural amino acid, for example, as shown in FIGs. 29A-29B.
  • an antibody or engineered antibody comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 61, with an unnatural amino acid incorporated at one or more amino acid positions of the heavy chain.
  • An exemplary payload can be attached to the unnatural amino acid, for example, as shown in FIGs.29A-29B.
  • an antibody or engineered antibody provided by the present disclosure comprises a heavy chain sequence, wherein the heavy chain sequence comprises an Attorney Docket No.
  • an antibody or engineered antibody comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 61, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1.
  • an antibody or engineered antibody provided by the present disclosure comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to one or more of: (i) A142 of SEQ ID NO: 61; (ii) T157 of SEQ ID NO: 61; (iii) T171 of SEQ ID NO: 61; (iv) A174 of SEQ ID NO: 61; (v) Y182 of SEQ ID NO: 61; (vi) T197 of SEQ ID NO: 61; (vii) D267 of SEQ ID NO: 61; or (viii) S241 of SEQ ID NO: 61.
  • an exemplary payload can be attached to the unnatural amino acid, for example, as shown in FIGs.29A-29B.
  • an antibody or engineered antibody comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to one or more of: (i) A142 of SEQ ID NO: 61; (ii) T157 of SEQ ID NO: 61; (iii) T171 of SEQ ID NO: 61; (iv) A174 of SEQ ID NO: 61; (v) Y182 of SEQ ID NO: 61; (vi) T197 of SEQ ID NO: 61; (vii) D267 of SEQ ID NO: 61; or (viii) S241 of SEQ ID NO: 61.
  • an exemplary payload can be attached to the unnatural amino acid, for example, as shown in FIGs.29A-29B.
  • an antibody or engineered antibody provided by the present disclosure comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to T171 of SEQ ID NO: 61.
  • An exemplary payload can be attached to T171, for example, as shown in FIG.29A and FIG.29B.
  • an antibody or engineered antibody comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to T171 of SEQ ID NO: 61.
  • An exemplary payload can be attached to T171, for example, as shown in FIG.29A and FIG. 29B.
  • an antibody or engineered antibody provided by the present disclosure comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 3.
  • an antibody or engineered antibody comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 3.
  • an antibody or engineered antibody provided by the present disclosure comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 3, with an unnatural amino acid incorporated at one or more amino acid positions of the light chain.
  • an antibody or engineered antibody comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 3, with an unnatural amino acid incorporated at one or more amino acid positions of the light chain.
  • an antibody or engineered antibody provided by the present disclosure comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 3, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1.
  • an antibody or engineered antibody comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 3, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1.
  • an antibody or engineered antibody provided by the present disclosure comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 62.
  • an antibody or engineered antibody comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 62.
  • an antibody or engineered antibody provided by the present disclosure comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 62, with an unnatural amino acid incorporated at one or more amino acid positions of the light chain.
  • an antibody or engineered antibody comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 62, with an unnatural amino acid incorporated at one or more amino acid positions of the light chain.
  • an antibody or engineered antibody provided by the present disclosure comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 62, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1.
  • an antibody or engineered antibody comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 62, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1.
  • an antibody or engineered antibody provided by the present disclosure comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 62, with an unnatural amino acid incorporated at a position corresponding to Q162 of SEQ ID NO: 62.
  • an antibody or engineered antibody comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 62, with an unnatural amino acid incorporated at a position corresponding to Q162 of SEQ ID NO: 62.
  • an antibody or engineered antibody provided by the present disclosure may comprises a combination of any heavy chain amino acid sequence and any light chain amino acid sequence described herein.
  • an antibody or engineered antibody provided by the present disclosure comprises a heavy chain sequence and a light chain sequence, wherein the heavy chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 1 or SEQ ID NO: 61, and the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 3 or SEQ ID NO: 62.
  • an antibody or engineered antibody comprises a heavy chain sequence and a light chain sequence, wherein the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 61, and the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 62.
  • the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 61
  • the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 62.
  • an antibody or engineered antibody provided by the present disclosure comprises a heavy chain sequence and a light chain sequence, wherein the heavy chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 1 or SEQ ID NO: 61, with an unnatural amino acid incorporated at one or more positions in the heavy chain, and the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 3 or SEQ ID NO: 62, with an unnatural amino acid incorporated at one or more positions in the light chain.
  • an exemplary payload can be attached to an unnatural amino acid, for example, as shown in FIGs.29A-29B.
  • an antibody or engineered antibody comprises a heavy chain sequence and a light chain sequence, wherein the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 61, with an unnatural amino acid incorporated at one or more positions in the heavy chain, and the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 62, with an unnatural amino acid incorporated at one or more positions in the light chain.
  • An exemplary payload can be attached to an unnatural amino acid, for example, as shown in FIGs.29A-29B.
  • an antibody or engineered antibody provided by the present disclosure comprises a heavy chain sequence and a light chain sequence, wherein the heavy chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 1 or SEQ ID NO: 61, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1, and the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 3 or SEQ ID NO: 62, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1.
  • an antibody or engineered antibody comprises a heavy chain sequence and a light chain sequence, wherein the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 61, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1, and the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 62, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1.
  • an engineered anti-CD276 antibody may comprise or more unnatural amino acids at positions selected from TABLE 1.
  • an engineered antibody comprises a modification at one or more unnatural amino acid residues.
  • the modification can comprise a payload molecule or moiety (e.g., any payload molecule or moiety described herein) that is conjugated using any crosslinker agent and/or linker described herein to yield an antibody conjugate, or an antibody-drug conjugate (ADC).
  • an antibody provided herein comprises a heavy chain amino acid sequence as shown in TABLE 2.
  • an antibody provided herein comprises a heavy chain amino acid sequence as shown in TABLE 2, with an unnatural amino acid incorporated at one or more positions in the heavy chain amino acid sequence.
  • an antibody provided by the present disclosure comprises a heavy chain amino acid sequence as shown in TABLE 2, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1.
  • an antibody provided herein comprises a light chain amino acid sequence as shown in TABLE 2.
  • an antibody provided herein comprises a light chain amino acid sequence as shown in TABLE 2, with an unnatural amino acid incorporated at one or more positions in the light chain amino acid sequence.
  • an antibody provided by the present disclosure comprises a light chain amino acid sequence as shown in TABLE 2, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1.
  • an antibody or engineered antibody exhibits enhanced stability compared to a similar antibody that does not comprise a modification such as conjugation to a payload molecule.
  • stability of an antibody can be assessed using any suitable method in the field.
  • antibody stability is assessed using a plasma stability assay.
  • antibody stability is assessed in vivo after administration in animals such as mice, rats or non-human primates.
  • a conjugated antibody exhibits enhanced payload release characteristics compared to a similar antibody where the payload is conjugated to different site.
  • Release characteristics of an antibody can be assessed using any suitable method in the field. For example, payload release can be assessed using a Cathepsin B assay.
  • at least 40%, 50%, 60%, 70%, 80%, or 90% of an antibody provided herein remains following incubation in human plasma for 72 hours at 37 °C.
  • at least 40%, 50%, 60%, 70%, 80%, or 90% of the conjugated payload of an antibody provided herein is released from the antibody following incubation with Cathepsin B for 240 minutes at 37 °C.
  • the antibody has a binding affinity for a target antigen of 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.75 nM, 0.5 nM, 0.1 nM, 0.075 nM, or 0.05 nM or lower, as measured by enzyme-linked immunosorbent assay (ELISA) or any other suitable method.
  • ELISA enzyme-linked immunosorbent assay
  • the antibody has a binding affinity for a target antigen that is within 0.1 fold, 0.2 fold, 0.3 fold, 0.4 fold, 0.5 fold, 1.0 fold, 1.5 fold, 2.0 fold, 3.0 fold, 4.0 fold, 5.0 fold, 6.0 fold, 8.0 fold, or 10.0 fold of the binding affinity for the target antigen of a reference antibody, wherein the reference antibody is an otherwise identical antibody that does not comprise the unnatural amino acid, as measured by ELISA or any other suitable method.
  • an antibody provided herein has improved biophysical characteristics, such as enhanced or tuned half-life (increased or decreased) and correlated to a desired improved efficacy and toxicity profile. Furthermore it is contemplated that an antibody Attorney Docket No.
  • BRI-023WO conjugate provided herein has improved efficacy compared to other antibodies using different conjugation strategies.
  • the site-specifically conjugated drug substance can have reduced off target toxicity or non-specific uptake (see, e.g., FIG.26).
  • the antibodies provided herein have reduced platform toxicity of the payload with an increased maximum tolerated dose as compared to a similar antibody or therapeutic modality. The resulting antibody can exhibit a difference in activity, toxicity, and overall therapeutic window based on the site of conjugation and DAR that results in greater therapeutic potential.
  • an antibody provided herein which may include an Antibody-UAA-Linker-PBD structure, has enhanced activity in cell lines where other payloads (e.g, camptothecins, auristatins, etc.) are less active or inactive, or visa versa. Ideally the antibody has optimal activity as an ADC.
  • UAAs unnatural amino acids
  • an unnatural amino acid refers to any amino acid, modified amino acid, or amino acid analogue other than the following twenty genetically encoded alpha-amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine. See, e.g., Biochemistry by L. Stryer, 3rd ed. 1988, Freeman and Company, New York, for structures of the twenty natural amino acids.
  • unnatural amino acid also includes amino acids that occur by modification (e.g., post- translational modifications) of a natural amino acid, but are not themselves naturally incorporated into a growing polypeptide chain by the translation complex. Any unnatural amino acid described herein (e.g., those depicted in FIGs.3A-3E), or any suitable unnatural amino acid may be used in accordance with the present disclosure.
  • unnatural amino acids typically differ from natural amino acids only in the structure of the side chain, unnatural amino acids may, for example, form amide bonds with other amino acids in the same manner in which they are formed in naturally occurring proteins. However, unnatural amino acids may have side chain groups that distinguish them from the natural amino acids.
  • an unnatural amino acid side chain may comprise an alkyl-, aryl-, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkyl, ether, thiol, seleno-, sulfonyl-, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amine, and the like, or any combination thereof.
  • BRI-023WO naturally occurring amino acids include, but are not limited to, amino acids comprising a photoactivatable cross-linker, spin-labeled amino acids, fluorescent amino acids, metal binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids with novel functional groups, amino acids that covalently or noncovalently interact with other molecules, photocaged and/or photoisomerizable amino acids, amino acids comprising biotin or a biotin analogue, glycosylated amino acids such as a sugar substituted serine, other carbohydrate modified amino acids, keto-containing amino acids, amino acids comprising polyethylene glycol or polyether, heavy atom substituted amino acids, chemically cleavable and/or photocleavable amino acids, amino acids with an elongated side chains as compared to natural amino acids, including but not limited to, polyethers or long chain hydrocarbons, including but not limited to, greater than about 5 or greater than about 10 carbons, carbon-linked sugar-containing amino acids, redox-active amino acids, amino
  • unnatural amino acids In addition to unnatural amino acids that contain novel side chains, unnatural amino acids also optionally comprise modified backbone structures. [0130] Many unnatural amino acids are based on natural amino acids, such as tyrosine, glutamine, phenylalanine, and the like.
  • Tyrosine analogs include para-substituted tyrosines, ortho- substituted tyrosines, and meta substituted tyrosines, wherein the substituted tyrosine comprises a keto group (including but not limited to, an acetyl group), a benzoyl group, an amino group, a hydrazine, an hydroxyamine, a thiol group, a carboxy group, an isopropyl group, a methyl group, a C 6 -C 20 straight chain or branched hydrocarbon, a saturated or unsaturated hydrocarbon, an O- methyl group, a polyether group, a nitro group, or the like.
  • multiply substituted aryl rings are also contemplated.
  • Glutamine analogs include, but are not limited to, ⁇ -hydroxy derivatives, ⁇ -substituted derivatives, cyclic derivatives, and amide substituted glutamine derivatives.
  • Exemplary phenylalanine analogs include, but are not limited to, para-substituted phenylalanines, ortho-substituted phenylalanines, and meta-substituted phenylalanines, wherein the substituent comprises a hydroxy group, a methoxy group, a methyl group, an allyl group, an aldehyde, an azido, an iodo, a bromo, a keto group (including but not limited to, an acetyl group), or the like.
  • unnatural amino acids include, but are not limited to, a p-acetyl-L- phenylalanine, a p-propargyl-phenylalanine, O-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine, a 3- methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-GlcNAc ⁇ - serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L- phenylalanine, a p-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a
  • BRI-023WO amino-L-phenylalanine an isopropyl-L-phenylalanine, and a p-propargyloxy-phenylalanine, and the like.
  • Examples of structures of a variety of unnatural amino acids are provided in U.S. Patent Application Publication Nos.2003/0082575 and 2003/0108885, PCT Publication No. WO 2002/085923, and Kiick et al. (2002) PROC. NATL. ACAD. SCI. USA 99:19-24.
  • Any suitable unnatural amino acid can be used in the methods and compositions of the present disclosure for incorporation into a protein of interest (e.g., an antibody).
  • an unnatural amino acid used in accordance with the present disclosure may be a leucine analog (also referred to herein as a derivative), for example, a non- naturally occurring leucine analog.
  • methods and compositions of the present disclosure utilize a leucine analog depicted in FIG.3A, or a composition comprising the leucine analog.
  • Formula A in FIG.3A depicts an amino acid analog containing a side chain including a carbon containing chain n units (0-20 units) long.
  • An O, S, CH 2 , or NH is present in at position X, and another carbon containing chain of n units (0-20 units) long can follow.
  • a functional group Y is attached to the terminal carbon of second carbon containing chain (e.g., functional groups 1-12 as depicted in FIG.3A, where R represents a linkage to the terminal carbon atom the second carbon containing side chain).
  • these functional groups can be used for bioconjugation of any amenable ligand to any protein of interest that is amenable to site-specific unnatural amino acid incorporation.
  • Formula B in FIG.3A depicts a similar amino acid analog containing a side chain denoted as either Z-Y 2 or Z-Y 3 attached to the second carbon containing chain or the first carbon containing chain, respectively.
  • Z represents a carbon chain comprising (CH 2 )n units, where n is any integer from 0-20.
  • Y 2 or Y 3 independently, can be the same or different groups as those of Y 1 .
  • methods and compositions of the present disclosure utilize a leucine analog depicted in FIG.3B (e.g., LCA, LKET, or ACA), or a composition comprising the leucine analog depicted in FIG.3B.
  • Additional exemplary leucine analogs include those selected from linear alkyl halides and linear aliphatic chains comprising a functional group, for example, an alkyne, azide, cyclopropene, alkene, ketone, aldehyde, diazirine, or tetrazine functional group, as well as structures 1-6 shown in FIG.3B.
  • the amino and carboxylate groups both attached to the first carbon of any amino acid shown in FIGs.3A-3B would constitute portions of peptide bonds when the leucine analog is incorporated into a protein or polypeptide chain.
  • Attorney Docket No. BRI-023WO [0135] the leucine analogs set forth in FIG.3C, referred to as C5AzMe and LCA can be used in the practice of the invention. Methods for preparing leucine analogs, e.g., C5AzMe or LCA, are described in International (PCT) Publication No. WO2021026506.
  • an unnatural amino acid used in accordance with the present disclosure is a tryptophan analog (also referred to herein as a derivative).
  • the tryptophan analog is a non-naturally occurring tryptophan analog.
  • Exemplary tryptophan analogs include 5-azidotryptophan, 5-propargyloxytryptophan, 5-aminotryptophan, 5- methoxytryptophan, 5-O-allyltryptophan or 5-bromotryptophan. Additional exemplary tryptophan analogs are depicted in FIG.3A (right side).
  • an unnatural amino acid used in accordance with the present disclosure is a tyrosine analog (also referred to herein as a derivative).
  • the tyrosine analog is a non-naturally occurring tyrosine analog.
  • Exemplary tyrosine analogs include o-methyltyrosine (OmeY), p-azidophenylalanine (AzF), o-propargyltyrosine (OpropY or PrY), and p-acetylphenylalanine (AcF).
  • Exemplary tryptophan analogs are depicted in FIG.3D.
  • an unnatural amino acid used in accordance with the present disclosure is a pyrrolysine analog (also referred to herein as a derivative).
  • the pyrrolysine analog is a non-naturally occurring pyrrolysine analog.
  • Exemplary pyrrolysine analogs include aminocaprylic acid (Cap), H-Lys(Boc)-OH (Boc-Lysine, BocK), azidolysine (AzK), H-propargyl- lysine (hPrK), and cyclopropenelysine (CpK).
  • Exemplary pyrrolysine analogs are depicted in FIG. 3E.
  • Any commercially available unnatural amino acid may be used in accordance with the present disclosure. Many unnatural amino acids are commercially available, e.g., from Sigma- Aldrich (St. Louis, Mo., USA), Novabiochem (Darmstadt, Germany), or Peptech (Burlington, Mass., USA).
  • Patent Application Publication No.2004/0198637 Matsoukas et al. (1995) J. MED. CHEM., 38:4660-4669, King et al. (1949) J. CHEM. SOC., 3315- 3319, Friedman et al. (1959) J. AM. CHEM. SOC., 81: 3750-3752, Craig et al. (1988) J. ORG. CHEM., 53: 1167-1170, Azoulay et al. (1991) EUR. J. MED. CHEM., 26: 201-5, Koskinen et al. (1989) J. ORG. CHEM., 54: 1859-1866, Christie et al.
  • an antibody provided herein comprises an unnatural amino acid that is chemically modified, e.g., conjugated to a payload molecule (e.g., a therapeutic molecule, or any other payload).
  • a payload molecule e.g., a therapeutic molecule, or any other payload.
  • a polypeptide may comprise one or more unnatural amino acids (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more unnatural amino acids, each of which may be the same or different), and similarly, may be conjugated to one or more molecules (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more than ten payload molecules, each of which may be the same or different).
  • unnatural amino acids e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more unnatural amino acids, each of which may be the same or different
  • molecules e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more than ten payload molecules, each of which may be the same or different.
  • a polypeptide may comprise one or more unnatural amino acids (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more unnatural amino acids, each of which may be the same or different) at positions selected from TABLE 1, and similarly, may be conjugated to one or more molecules (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more than ten payload molecules, each of which may be the same or different), e.g., selected from any molecules or payloads described herein.
  • unnatural amino acids e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more unnatural amino acids, each of which may be the same or different
  • molecules e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more than ten payload molecules, each of which may be the same or different
  • Exemplary molecules that can be conjugated to a polypeptide containing an unnatural amino acid include, for example, a label, a dye, a polymer, a water-soluble polymer, a stabilizing agent (e.g., a derivative of polyethylene glycol), a photoactivatable crosslinker, a radionuclide, a cytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound, a resin, a second protein or polypeptide or polypeptide analog (e.g., a therapeutic peptide or polypeptide), an antibody or antibody fragment (e.g., an anti-CD276 antibody or antibody fragment), a metal chelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide, a DNA (e.g., a DNA Attorney Docket No.
  • a stabilizing agent e.g., a derivative of polyethylene glycol
  • a photoactivatable crosslinker e
  • RNA e.g., a RNA oligonucleotide
  • LNA e.g., a LNA oligonucleotide
  • an antisense polynucleotide e.g., a saccharide, a water-soluble dendrimer, a cyclodextrin, an inhibitory ribonucleic acid (e.g., a small interfering RNA (siRNA), a small nuclear RNA (snRNA), or a non- coding RNA), a biomaterial, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, a radioactive moiety, a novel functional group, a group that covalently or noncovalently interacts with other molecules, a photocaged moiety, an actinic radiation excitable moiety, a photoisomerizable moiety, biotin, a derivative of biotin, a
  • Additional exemplary molecules or payloads for conjugation to a protein of interest include any cytotoxic, cytostatic or immunomodulatory drug.
  • useful classes of cytotoxic or immunomodulatory agents include, for example, anti-tubulin agents, auristatins (e.g., vedotin or MMAE, mafodotin or MMAF, etc.), maytansinoids (e.g., Emtansine, soravtansine, etc.), calicheamicin (e.g., ozogamicin, etc.), anthramycin, SG2000, pyrrolobenzodiazapines (e.g., PBD, talirine (SGD-1910), tesirine (SG3249), etc.), indolobenzodiazapines (e.g., IGNs), pyrrolobenzodiazepine (PBD) and derivatives thereof (Lai et al.
  • auristatins e.g., vedot
  • DNA minor groove binders DNA replication inhibitors
  • alkylating agents e.g., platinum complexes such as cis-platin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin
  • anthracyclines antibiotics, antifolates, antimetabolites, calmodulin inhibitors, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, pore-forming compounds, purine antimetabolites, puromycins, radiation sensitizers, rapamycins, steroids, taxanes, topoisomerase inhibitors (e.g., camptothecin based topopisomerase inhibitors (e.g., deruxtecan, SN-38, etc.), duocarmycins, amanitins, vin
  • cytotoxic or immunomodulatory agents that may be used in accordance with the present disclosure include, for example, an androgen, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan, buthionine sulfoximine, calicheamicin, calicheamicin derivatives, camptothecin, carboplatin, carmustine (BSNU), CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin (formerly actinomycin), daunorubicin, decarbazine, DM1, DM4, docetaxel, doxorubicin, etoposide, an estrogen, 5-fluordeoxyuridine, 5-fluorour
  • suitable cytotoxic agents for use in accordance with the present disclosure include, for example, DNA minor groove binders (e.g., enediynes and lexitropsins, a CBI compound), duocarmycins, taxanes (e.g., paclitaxel and docetaxel), puromycins, vinca alkaloids, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino- doxorubicin, echinomycin, combretastatin, netropsin, epothilone A and B, estramustine, cryptophycins, cemadotin, maytansinoids, discodermolide, eleutherobin, and mitoxantrone.
  • DNA minor groove binders e.g., enediynes and lexitropsins, a CBI compound
  • duocarmycins e.g.,
  • a molecule or payload used in accordance with the present disclosure is an anti-tubulin agent.
  • anti-tubulin agents include taxanes (e.g., Taxol ® (paclitaxel), Taxotere ® (docetaxel)), T67 (Tularik) and vinca alkyloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine).
  • antitubulin agents include, for example, baccatin derivatives, taxane analogs, epothilones (e.g., epothilone A and B), nocodazole, colchicine and colcimid, estramustine, cryptophycins, cemadotin, maytansinoids, combretastatins, discodermolide, and eleutherobin.
  • the cytotoxic agent is a maytansinoid (e.g., maytansine or DM1).
  • a molecule or payload used in accordance with the present disclosure is an auristatin, such as auristatin E or a derivative thereof.
  • the auristatin E derivative can be an ester formed between auristatin E and a keto acid.
  • auristatin E can be reacted with paraacetyl benzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively.
  • Other typical auristatin derivatives include AFP, MMAF, and MMAE.
  • the antimetabolite can be, for example, a purine antagonist (e.g., azothioprine or mycophenolate mofetil), a dihydrofolate reductase inhibitor (e.g., methotrexate), acyclovir, ganciclovir, zidovudine, vidarabine, ribavarin, azidothymidine, cytidine arabinoside, amantadine, dideoxyuridine, iododeoxyuridine, poscarnet, or trifluridine.
  • a molecule or payload used in accordance with the present disclosure is tacrolimus, cyclosporine, FU506 or rapamycin.
  • the molecule or payload is aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, bexarotene, bexarotene, calusterone, capecitabine, celecoxib, cladribine, Darbepoetin alfa, Denileukin diftitox, dexrazoxane, dromostanolone propionate, epirubicin, Epoetin alfa, estramustine, exemestane, Filgrastim, floxuridine, fludarabine, fulvestrant, gemcitabine, gemtuzumab ozogamicin (MYLOTARG), goserelin, idarubicin, ifosfamide, imatinib mesylate, Interferon alfa-2a, irinotecan, letroz
  • a molecule or payload used in accordance with the present disclosure is an immunomodulatory agent.
  • the immunomodulatory agent can be, for example, ganciclovir, etanercept, tacrolimus, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil or methotrexate.
  • the immunomodulatory agent can be, for example, a glucocorticoid (e.g., cortisol or aldosterone) or a glucocorticoid analogue (e.g., prednisone or dexamethasone).
  • the immunomodulatory agent can be, for example, a Toll-like receptor (TLR) agonist, e.g., a TLR7 or TLR8 agonist, e.g., imiquimod, 852A, hiltonol, resiquimod, 3M-052, CpG oligodeoxynucleotides (CpG ODN), 1V270, or SD-101.
  • TLR Toll-like receptor
  • the immunomodulatory agent is an anti-inflammatory agent, such as arylcarboxylic derivatives, pyrazole-containing derivatives, oxicam derivatives and nicotinic acid derivatives.
  • Classes of anti-inflammatory agents include, for example, cyclooxygenase inhibitors, 5-lipoxygenase inhibitors, and leukotriene receptor antagonists.
  • Suitable cyclooxygenase inhibitors may include meclofenamic acid, mefenamic acid, carprofen, diclofenac, diflunisal, fenbufen, fenoprofen, indomethacin, ketoprofen, nabumetone, Attorney Docket No. BRI-023WO sulindac, tenoxicam and tolmetin.
  • Leukotriene receptor antagonists may include calcitriol, and ontazolast.
  • Suitable lipoxygenase inhibitors include redox inhibitors (e.g., catechol butane derivatives, nordihydroguaiaretic acid (NDGA), masoprocol, phenidone, Ianopalen, indazolinones, naphazatrom, benzofuranol, alkylhydroxylamine), and non-redox inhibitors (e.g., hydroxythiazoles, methoxyalkylthiazoles, benzopyrans and derivatives thereof, methoxytetrahydropyran, boswellic acids and acetylated derivatives of boswellic acids, and quinolinemethoxyphenylacetic acids substituted with cycloalkyl radicals), and precursors of redox inhibitors.
  • redox inhibitors e.g., catechol butane derivatives, nordihydroguaiaretic acid (NDGA), masoprocol, phenidone, Ianopalen, indazolinones, naphaz
  • lipoxygenase inhibitors include antioxidants (e.g., phenols, propyl gallate, flavonoids and/or naturally occurring substrates containing flavonoids, hydroxylated derivatives of the flavones, flavonol, dihydroquercetin, luteolin, galangin, orobol, derivatives of chalcone, 4,2',4'- trihydroxychalcone, ortho-aminophenols, N-hydroxyureas, benzofuranols, ebselen and species that increase the activity of the reducing selenoenzymes), iron chelating agents (e.g., hydroxamic acids and derivatives thereof, N-hydroxyureas, 2-benzyl-1-naphthol, catechols, hydroxylamines, carnosol trolox C, catechol, naphthol, sulfasalazine, zyleuton, 5-hydroxyanthranilic acid and 4-(omega- arylal
  • lipoxygenase inhibitors include inhibitors of eicosanoids (e.g., octadecatetraenoic, eicosatetraenoic, docosapentaenoic, eicosahexaenoic and docosahexaenoic acids and esters thereof, PGE1 (prostaglandin E1), PGA2 (prostaglandin A2), viprostol, 15-monohydroxyeicosatetraenoic, 15- monohydroxy-eicosatrienoic and 15-monohydroxyeicosapentaenoic acids, and leukotrienes B5, C5 and D5), compounds interfering with calcium flows, phenothiazines, diphenylbutylamines, verapamil, fuscoside, curcumin, chlorogenic acid, caffeic acid, 5,8,11,14-eicosatetrayenoic acid (ETYA), hydroxyphenylretinamide, I
  • chemotherapeutic agents include Erlotinib (TARCEVA ® , Genentech/OSI Pharm.), Bortezomib (VELCADE ® , Millennium Pharm.), Fulvestrant (FASLODEX ® , AstraZeneca), Sutent (SU11248, Pfizer), Letrozole (FEMARA ® , Novartis), Imatinib mesylate (GLEEVEC ® , Novartis), PTK787/ZK 222584 (Novartis), Oxaliplatin (Eloxatin ® , Sanofi), 5-FU (5-fluorouracil), Leucovorin, Rapamycin (Sirolimus, RAPAMUNE ® , Wyeth), Lapatinib (TYKERB ® , GSK572016, Glaxo Smith Kline), Lonafarnib (
  • chemotherapeutic agents Attorney Docket No. BRI-023WO include alkylating agents such as thiotepa and CYTOXAN ® (cyclosphosphamide); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (e.g., bullatacin and bullatacinone); a camptothecin (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (e.g., cryptophycin 1 and crypto
  • anti-cancer agents include aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5- oxo-L-norleucine, ADRIAMYCIN® (doxorubicin), morpholino-doxorubicin, cyanomorpholino- doxorubicin, 2-pyrrolino-doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tub
  • anti-cancer agents include anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamniprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid;
  • BRI-023WO polysaccharide complex JHS Natural Products, Eugene, Oreg.
  • razoxane rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL ® (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE ® (Cremophor-free), albumin-engineered nanoparticle formulations of
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • SERMs selective estrogen receptor modulators
  • tamoxifen including NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, 4- hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifine citrate)
  • aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE ® (megestrol acetate), AROMASIN ® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR ®
  • SERMs selective estrogen receptor modulators
  • anti-angiogenic agents include MMP-2 (matrix- metalloproteinase 2) inhibitors, MMP-9 (matrix-metalloproteinase 9) inhibitors, COX-II Attorney Docket No. BRI-023WO (cyclooxygenase II) inhibitors, and VEGF receptor tyrosine kinase inhibitors.
  • VEGF receptor tyrosine kinase inhibitors include 4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1- methylpiperidin-4-ylmethoxy)quinazoline (ZD6474), 4-(4-fluoro-2-methylindol-5-yloxy)-6- methoxy-7-(3-pyrrolidin-1-ylpropoxy)-quinazoline (AZD2171), vatalanib (PTK787) and SU11248 (sunitinib).
  • Additional exemplary molecules for conjugation include an amatoxin, chalicheamicin, DUBA, FAM, MMAD, PBD, and a toxoid.
  • an unnatural amino acid comprises a bioconjugation handle (also referred to herein as a bioconjugation site) to facilitate conjugation to another molecule (e.g., via use of a crosslinking agent).
  • a method disclosed herein can be used to site-specifically incorporate two different unnatural amino acids, each with a different bioconjugation handle, into a single protein (e.g., a single antibody).
  • the two bioconjugation handles can be chosen such that they each can be chemoselectively conjugated to two different labels using mutually orthogonal conjugation chemistries.
  • Such pairs of bioconjugation handles include, for example: azide and alkyne, azide and ketone/aldehyde, azide and cyclopropene, ketone/aldehyde and cyclopropene, 5-hydroxyindole and azide, 5-hydroxyindole and cyclopropene, and 5-hydroxyindole and ketone/aldehyde.
  • a payload e.g., a molecule used in accordance with the present disclosure can be conjugated through a variety of linking groups (linkers).
  • the linker may be a cleavable linker or a non-cleavable linker (e.g., a linker may comprises a cleavable or non-cleavable portion or region).
  • a linker may be a flexible linker or an inflexible linker.
  • the linker should be a length sufficiently long to allow the molecule and the antibody to be linked without steric hindrance from one another and sufficiently short to retain the intended activity of the antibody.
  • the linker preferably is sufficiently hydrophilic to avoid or minimize instability or insolubility of the antibody.
  • the linker should be sufficiently stable in vivo (e.g., it is not cleaved by serum, enzymes, etc.) to permit the antibody to be operative (e.g., selectively operative) in vivo.
  • the linker is a molecule which functions as a novel bifunctional fluorescence probe that allows self-elimination cleavage in the presence of proteases for payload release and fluorophore activation, such as 7-amino-3-hydroxyethyl-coumarin (7-AHC) based dipeptide linkers.
  • a linker used in accordance with the present disclosure may be from about 1 angstroms ( ⁇ ) to about 150 ⁇ in length, or from about 1 ⁇ to about 120 ⁇ in length, or from about 5 ⁇ to about 110 ⁇ in length, or from about 10 ⁇ to about 100 ⁇ in length.
  • a linker may be greater than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 27, 30 or greater angstroms in length and/or less than about 110, 100, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, or fewer ⁇ in length.
  • a linker may be about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, and 120 ⁇ in length.
  • the linker is any divalent or multivalent linker known to those of skill in the art.
  • the linker is capable of forming covalent bonds to the molecule and the unnatural amino acid.
  • Useful divalent linkers include alkylene, substituted alkylene, heteroalkylene, substituted heteroalkylene, arylene, substituted arylene, heteroarlyene and substituted heteroarylene linkers.
  • the linker is C1-10 alkylene or C1-10 heteroalkylene.
  • the linker may include a water soluble polymer.
  • the water soluble polymer may be any structural form including but not limited to linear, forked or branched.
  • the water soluble polymer is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG), but other water soluble polymers can also be employed.
  • PEG is used broadly to encompass any polyethylene glycol molecule, without regard to size or to modification at an end of the PEG.
  • a PEG used in accordance with the present disclosure terminates on one end with hydroxy or methoxy.
  • the PEG can terminate with a reactive group, thereby forming a bifunctional polymer.
  • Typical reactive groups can include those reactive groups that are commonly used to react with the functional groups found in the 20 common amino acids (including but not limited to, maleimide groups, activated carbonates (including but not limited to, p-nitrophenyl ester), activated esters (including but not limited to, N- hydroxysuccinimide, p-nitrophenyl ester) and aldehydes) as well as functional groups that are inert to the 20 common amino acids but that react specifically with complementary functional groups present in UAAs (including but not limited to, azide groups, and alkyne groups).
  • the functional groups found in the 20 common amino acids including but not limited to, maleimide groups, activated carbonates (including but not limited to, p-nitrophenyl ester), activated esters (including but not limited to, N- hydroxysuccinimide, p-nitrophenyl ester) and aldehydes) as well as functional groups that are inert to the 20 common amino acids but that react specifically with complementary functional groups present in UAA
  • Any molecular mass for a PEG can be used as practically desired, including but not limited to, from about 50 Daltons (Da) to 100,000 Da or more as desired (including but not limited to, sometimes 100 Da to 100,000 Da, 0.1-50 kDa, or 10-40 kDa).
  • Branched chain PEGs including but not limited to, PEG molecules with each chain having a MW ranging from 1-100 kDa (including but not limited to, 1-50 kDa or 5-20 kDa) can also be used.
  • a contemplated linker may Attorney Docket No.
  • BRI-023WO include any appropriate number of PEG units, e.g. ⁇ from 2 to 24 PEG units, e.g., PEG2, PEG4, PEG6, PEG8, PEG10, PEG12, or PEG24.
  • PEG2 e.g., PEG2, PEG4, PEG6, PEG8, PEG10, PEG12, or PEG24.
  • a wide range of PEG molecules are described in, including but not limited to, the Shearwater Polymers, Inc. catalog, Nektar Therapeutics catalog.
  • a linker comprises a spacer region or portion that comprises a PEG region.
  • a PEG region comprises PEG8.
  • at least one terminus of the PEG molecule is available for reaction with the unnatural amino acid.
  • PEG derivatives bearing alkyne and azide moieties for reaction with amino acid side chains can be used to attach PEG to unnatural amino acids as described herein.
  • the unnatural amino acid comprises an azide
  • the PEG will typically contain either an alkyne moiety to effect formation of the [3+2] cycloaddition product or an activated PEG species (i.e., ester, carbonate) containing a phosphine group to effect formation of the amide linkage.
  • the unnatural amino acid comprises an alkyne
  • the PEG will typically contain an azide moiety to effect formation of the [3+2] Huisgen cycloaddition product.
  • the PEG will typically contain a strained alkene.
  • the PEG will typically contain a tetrazine.
  • the PEG will typically comprise a potent nucleophile (including but not limited to, a hydrazide, hydrazine, hydroxylamine, or semicarbazide functionality) in order to effect formation of corresponding hydrazone, oxime, and semicarbazone linkages, respectively.
  • a reverse of the orientation of the reactive groups described above can be used, i.e., an azide moiety in the unnatural amino acid can be reacted with a PEG derivative containing an alkyne.
  • PEG derivative containing an alkyne a reverse of the orientation of the reactive groups described above.
  • Many other polymers are also suitable for use in the present disclosure.
  • polymer backbones that are water-soluble, with from 2 to about 300 termini, are particularly useful.
  • suitable polymers include, but are not limited to, other poly(alkylene glycols), such as poly(propylene glycol) (“PPG”), copolymers thereof (including but not limited to copolymers of ethylene glycol and propylene glycol), terpolymers thereof, mixtures thereof, and the like.
  • PEG and related polymers may include degradable linkages in the polymer backbone or in the linker group between the polymer backbone and one or more of the terminal functional groups of the polymer molecule.
  • ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on a Attorney Docket No. BRI-023WO biologically active agent generally hydrolyze under physiological conditions to release the agent.
  • hydrolytically degradable linkages include, but are not limited to, carbonate linkages; imine linkages resulted from reaction of an amine and an aldehyde; phosphate ester linkages formed by reacting an alcohol with a phosphate group; hydrazone linkages which are reaction product of a hydrazide and an aldehyde; acetal linkages that are the reaction product of an aldehyde and an alcohol; orthoester linkages that are the reaction product of a formate and an alcohol; peptide linkages formed by an amine group, including but not limited to, at an end of a polymer such as PEG, and a carboxyl group of a peptide; and oligonucleotide linkages formed by a phosphoramidite group, including but not limited to, at the end of a polymer, and a 5' hydroxyl group of an oligonucleotide.
  • Branched linkers may be used in antibodies of the disclosure.
  • a number of different cleavable linkers are known to those of skill in the art.
  • the mechanisms for release of an agent from these linker groups include, for example, irradiation of a photolabile bond and acid- catalyzed hydrolysis.
  • the length of the linker may be predetermined or selected depending upon a desired spatial relationship between the antibody and the molecule linked to it.
  • radiodiagnostic compounds, radiotherapeutic compounds, drugs, toxins, and other agents one skilled in the art will be able to determine a suitable method for attaching a given agent to an antibody.
  • Any hetero- or homo-bifunctional linker can be used to link the conjugates.
  • the linker may have a wide range of molecular weight or molecular length. Larger or smaller molecular weight linkers may be used to provide a desired spatial relationship or conformation between the antibody and the linked entity. Linkers having longer or shorter molecular length may also be used to provide a desired space or flexibility between the antibody and the linked entity. Similarly, a linker having a particular shape or conformation may be utilized to impart a particular shape or conformation to the antibody or the linked entity, either before or after the antibody reaches its target. The functional groups present on each end of the linker may be selected to modulate the release of an antibody or a payload under desired conditions.
  • Some examples of water-soluble bifunctional linkers have a dumbbell structure that includes: a) an azide, an alkyne, a hydrazine, a hydrazide, a hydroxylamine, a carbonyl, a tetrazine, or a strained alkene-containing moiety on at least a first end of a polymer backbone; and b) at least a second functional group on a second end of the polymer backbone.
  • the second functional group can be the same or different as the first functional group.
  • the second functional group in some examples, is not reactive with the first functional group.
  • water- soluble compounds that comprise at least one arm of a branched molecular structure.
  • the branched molecular structure can be dendritic.
  • Further illustrative linkers include, for example, malC, thioether, AcBut, valine- citrulline peptide, malC-valine-citrulline peptide, hydrazone, and disulfide.
  • Other illustrative linkers can include beta-glucuronide linkers.
  • coupling of antibody and molecule can be accomplished via a crosslinking agent. There are several intermolecular crosslinking agents which can be utilized, see for example, Means and Feeney, CHEMICAL MODIFICATION OF PROTEINS, Holden-Day, 1974, pp. 39-43.
  • N-succinimidyl3-(2-pyridyldithio) propionate SPDP
  • N, N’- (1,3-phenylene) bismaleimide both of which are highly specific for sulfhydryl groups and form irreversible linkages
  • N, N’-ethylene-bis-(iodoacetamide) or other such reagent having 6 to 11 carbon methylene bridges which are relatively specific for sulfhydryl groups
  • 1, 5-difluoro-2,4- dinitrobenzene which forms irreversible linkages with amino and tyrosine groups).
  • crosslinking agents useful for this purpose include: p,p’-difluoro-N,N’- dinitrodiphenylsulfone (which forms irreversible crosslinkages with amino and phenolic groups); dimethyl adipimidate (which is specific for amino groups); phenol-1,4- disulfonylchloride (which reacts principally with amino groups); hexamethylenediisocyanate or diisothiocyanate, or azophenyl-p-diisocyanate (which reacts principally with amino groups); glutaraldehyde (which reacts with several different side chains) and disdiazobenzidine (which reacts primarily with tyrosine and histidine); N-3- Maleimidopropanoic acid; N-6-Maleimidocaproic acid; N-11- Maleimidoundecanoic acid, 4- (N-maleimidomethyl)cyclohexane-1-carboxy-6-amidocaproic acid; 4-[(
  • the crosslinking agent may be homobifunctional, i.e., having two functional groups that undergo the same reaction.
  • An example of a homobifunctional crosslinking agent is bismaleimidohexane (“BMH”).
  • BMH contains two maleimide functional groups, which react specifically with sulfhydryl-containing compounds under mild conditions (pH 6.5-7.7). The two maleimide groups are connected by a hydrocarbon chain. Therefore, BMH is useful for irreversible crosslinking of polypeptides that contain cysteine residues.
  • homobifunctional crosslinking agents include: BSOCOES (Bis(2 [Succinimidooxycarbonyloxy]ethyl) sulfone; DPDPB (1,4-Di-(3’-[2pyridyldithio]-propionamido) butane; DSS (disuccinimidyl suberate); DST (disuccinimidyl tartrate); Sulfo DST (sulfodisuccinimidyl tartrate); DSP (dithiobis(succinimidyl propionate); DTSSP (3,3’- Dithiobis(sulfosuccinimidyl propionate); EGS (ethylene glycol bis(succinimidyl succinate)); BASED (Bis( ⁇ -[4-azidosalicylamido]-ethyl)disulfide iodinatable); homobifunctional NHS crosslinking reagents (e
  • Heterobifunctional crosslinking agents have two different functional groups, for example an amine-reactive group and a thiol-reactive group, that will crosslink two moieties having free amines and thiols, respectively.
  • the most common commercially available heterobifunctional crosslinking agents have an amine reactive N-hydroxysuccinimide ester as one functional group, and a sulfhydryl reactive group as the second functional group.
  • the most common sulfhydryl reactive groups are maleimides, pyridyl disulfides and active halogens.
  • One of the functional groups can be a photoactive aryl nitrene, which upon irradiation reacts with a variety of groups.
  • exemplary heterobifunctional crosslinking agents include succinimidyl 4-(N maleimidomethyl) cyclohexane-1-carboxylate (“SMCC”), Succinimidyl-4-(N maleimidomethyl)-cyclohexane-1- carboxy(6-amidocaproate) (“LC-SMCC”), N maleimidobenzoyl-N-hydroxysuccinimide ester (“MBS”), and succinimide 4-(p-maleimidophenyl) butyrate (“SMPB”), an extended chain analog of MBS.
  • SMCC succinimidyl 4-(N maleimidomethyl) cyclohexane-1-carboxylate
  • LC-SMCC Succinimidyl-4-(N maleimidomethyl)-cyclohexane-1- carboxy(6-amidoca
  • succinimidyl group of these crosslinking agents reacts with a primary amine forming an amide bond, and the thiol-reactive maleimide forms a covalent thioether bond with the thiol group (e.g., of a cysteine).
  • Additional exemplary crosslinking agents include: BS3 ([Bis(sulfosuccinimidyl)suberate], which is a homobifunctional N-hydroxysuccinimide ester that targets accessible primary amines; NHS/EDC (N-hydroxy-succinimide and N-ethyl- ‘(dimethylaminopropyl)carbodimide, which allows for the conjugation of primary amine groups with carboxyl groups); sulfoEMCS ([N-e-Maleimido-caproic acid]hydrazide, which includes heterobifunctional reactive groups (a maleimide and an NHS-ester) that are reactive toward sulfhydryl and amino groups; hydrazide, which is useful for useful for linking carboxyl groups on exposed carbohydrates to primary amines; SATA (N-succinimidyl-S-acetylthioacetate), which is reactive towards amines and adds protected sulfhydryl groups; monofluoride,
  • a crosslinking agent may be used in combination with an aminohexyl spacer or other short carbon spacer to connect the crosslinking agent to the main or linker (e.g., a crosslinking agent may comprise a DBCO and a linker containing carbons, a hexyl linker, or a heterohexyl linker).
  • a crosslinking agent may comprise a DBCO and a linker containing carbons, a hexyl linker, or a heterohexyl linker.
  • the length of these crosslinking agents can be varied by the use of polymeric regions between the two reactive groups, which typically take the form of chemical linkers such as polymeric ethylene glycol or simple carbon chains, but can also include sugars, amino acids or peptides, or oligonucleotides.
  • crosslinking agent may comprise a ⁇ 2 carbon chain arm, a 2-5 carbon chain arm, or a 3-6 carbon chain arm.
  • Crosslinking agents often have low solubility in water.
  • a hydrophilic moiety such as a sulfonate group, may be added to the crosslinking agent to improve its water solubility.
  • Sulfo- MBS and sulfo-SMCC are examples of crosslinking agents modified for water solubility.
  • Many crosslinking agents yield a conjugate that is essentially non-cleavable under cellular conditions.
  • crosslinking agents contain a covalent bond, such as a disulfide, that is cleavable under cellular conditions.
  • a disulfide such as a disulfide
  • DSP dithiobis(succinimidylpropionate)
  • SPDP N-succinimidyl 3-(2-pyridyldithio) propionate
  • DSP dithiobis(succinimidylpropionate)
  • SPDP N-succinimidyl 3-(2-pyridyldithio) propionate
  • SPDP N-succinimidyl 3-(2-pyridyldithio) propionate
  • the linker comprises a polypeptide linker that connects or fuses the molecule to the antibody.
  • the linker may comprise hydrophilic amino acid residues, such as Gln, Ser, Gly, Glu, Pro, His and Arg.
  • the linker is a peptide containing 1-25 amino acid residues, 1-20 amino acid residues, 2-15 amino acid residues, 3-10 amino acid residues, 3-7 amino acid residues, 4-25 amino acid residues, 4-20 amino acid residues, 4-15 amino acid residues, 4-10 amino acid residues, 5-25 amino acid residues, 5-20 amino acid residues, 5-15 amino acid residues, or 5-10 amino acid residues.
  • exemplary linkers include glycine and serine-rich linkers, e.g., (GlyGlyPro) n (SEQ ID NO: 63), or (GlyGlyGlyGlySer) n (SEQ ID NO: 64), where n is 1-5.
  • the linker comprises, consists, or consists essentially of GGGGS (SEQ ID NO: 65). In certain embodiments, the linker comprises, consists, or consists essentially of GGGGSGGGGS (SEQ ID NO: 66). Additional exemplary linker sequences are disclosed, e.g., in George et al. (2003) PROTEIN ENGINEERING, 15: 871–879, and U.S. Patent Nos.5,482,858 and 5,525,491.
  • an unnatural amino acid used in accordance with the present disclosure comprises a non-natural aromatic chemical moiety (e.g., a hydroxyl-indole group; an amino-indole group; an aminophenol group; or a hydroxyl-phenol group, e.g., the UAA is 5- hydroxytryptophan (5-HTP), or an analog thereof), and/or the linker comprises a diazonium group (e.g., the linker comprises 4-nitrobenzenediazonium (4NDz); 4-carboxybenzenediazonium (4NeDz) Attorney Docket No. BRI-023WO or 4-methoxybenzenediazonium (4MCDz).
  • a non-natural aromatic chemical moiety e.g., a hydroxyl-indole group; an amino-indole group; an aminophenol group; or a hydroxyl-phenol group, e.g., the UAA is 5- hydroxytryptophan (5-HTP), or an analog thereof
  • the linker comprises
  • the unnatural amino acid and linker may react under conditions suitable to form an azo-linkage via an azo-coupling reaction between the aromatic chemical moiety and the diazonium group. Further methods for conjugation of molecules to unnatural amino acids are described, for example, in U.S. Patent Application Publication No. 2018/0360984.
  • the antibody when a molecule is conjugated to an antibody, the antibody has an average drug-to-antibody ratio (DAR) of at least 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0, as measured by hydrophobic interaction chromatography (HIC) or liquid chromatography-mass spectrometry (LCMS).
  • DAR may refer to the ratio of any conjugated molecule to antibody (e.g., a detectable label as well as a drug).
  • the antibody has an average DAR that is within 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the number of unnatural amino acids in the antibody.
  • a payload is conjugated to an antibody or antigen binding fragment thereof using a linkage of A-U-U’-X1-L-X2-P, where A is an antibody or antigen binding fragment thereof, U is an unnatural amino acid bioconjugation handle (e.g., any suitable unnatural amino acid described herein), U’ is a crosslinking agent or crosslinker (e.g., any crosslinking agent or crosslinker described herein), X1 is a first spacer, L is a cleavable or non-cleavable linking spacer, X2 is a second spacer, and P is a payload (e.g., any payload described herein).
  • only one spacer (X1 or X2) is used to conjugate the payload to the antibody or antigen binding fragment.
  • linkage of an A-U portion to a U’-X1- L-X2-P portion are shown in FIGs.23A-23B.
  • a payload is conjugated to an antibody or antigen binding fragment thereof using a linkage of A-U-U’-X1-L-P.
  • a payload is conjugated to an antibody or antigen binding fragment thereof using a linkage of A-U-U’-L-X2-P.
  • a spacer comprises PEG2, PEG4, PEG6, PEG8, PEG10, PEG12, or PEG24.
  • a spacer comprises PEG2.
  • a spacer comprises PEG4.
  • a spacer comprises PEG6.
  • a spacer comprises PEG8.
  • a spacer comprises PEG10.
  • a spacer comprises PEG12.
  • a spacer comprises PEG24.
  • a spacer comprises a polysarcosine (PSAR). In some embodiments, a spacer comprises a monodisperse PSAR. In some Attorney Docket No. BRI-023WO embodiments, a spacer comprises a PAB. In some embodiments, a spacer comprises an aliphatic chain. [0184] In some embodiments, a linking spacer comprises a cleavable linking spacer portion. In some embodiments, a linking spacer comprises a non-cleavable spacer portion. In some embodiments, a linking spacer comprises a peptide region. In some embodiments, a linking spacer comprises a Val-Cit motif.
  • a linking spacer comprises a Val-Ala (VA) motif. In some embodiments, a linking spacer comprises a beta-glucuronide. In some embodiments, a linking spacer comprises a GGFG motif. [0185] In some embodiments, a linker comprises a structure of U’-X1-L-X2, wherein U’ comprises DBCO, X1 comprises a PEG, L comprises a VA motif, and X2 comprises a PAB. In some embodiments, a linker comprises a structure of U’-X1-L-X2, wherein U’ comprises DBCO, X1 comprises a PEG8, L comprises a VA motif, and X2 comprises a PAB.
  • a linker comprises a structure of U’-X1-L, wherein U’ comprises DBCO, X1 comprises a PEG, and L comprises a GGFG motif. In some embodiments, a linker comprises a structure of U’-X1-L, wherein U’ comprises DBCO, X1 comprises a PEG4, and L comprises a GGFG motif.
  • Other exemplary linkers that may be used in accordance with the present disclosure include those discussed in Zheng et al. (2021) ACTA PHARM SIN B, 11(12): 3889-3907.
  • the linkage is mediated by interchain cysteine-maleimide conjugation, for example, where linkage is between an A-InterchainCys portion and a Mal-Linker- Payload portion.
  • the linkage is mediated by THIOMAB conjugation, for example, where linkage is between an A-EngineeredCys portion and a Mal-Linker-Payload portion.
  • the antibody when a payload molecule (any payload described herein, e.g., those in FIGs.15-18) is conjugated to an antibody through an unnatural amino acid bioconjugation handle (U) to a U’-Linker-Payload portion, the antibody has an average-to-drug antibody ratio (DAR) of at least 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0, or 4-6 DAR in 0.1 DAR increments as measured by hydrophobic interaction chromatography (HIC) or LCMS. In some embodiments, the antibody has an average DAR of about 2.
  • DAR average-to-drug antibody ratio
  • DAR may refer to the ratio of any conjugated molecule to antibody (e.g., a detectable label as well as a drug).
  • the antibody has an average DAR that is within 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the number of unnatural amino acids with the corresponding bioconjugation handle in the antibody.
  • a conjugate e.g., any conjugate described herein
  • the divalent residue of the conjugating Attorney Docket No.
  • BRI-023WO group comprises a triazole ring or fused cyclic group comprising a triazole ring.
  • a conjugate is formed through a strain-promoted [3+2] alkyne-azide cycloaddition (SPAAC) reaction, or other tryptophan based conjugation chemistries (CRACR, eCLIC, etc.), see, e.g., FIGs.23A-23B.
  • an antibody provided herein comprises an unnatural amino acid, which facilitates conjugation to a molecule or payload, e.g., a detectable label or a drug (e.g., a small molecule drug).
  • the molecule is: AEB, AEVB, AFP, an amatoxin, an auristatin (e.g., auristatin E), a calicheamicin, CC-1065 or a CC-1065 analog, chalicheamicin, combretastatin, DM1, DM4, docetaxel, dolastatin-10, DUBA, a duocarmycin, echinomycin, FAM, maytansine, a maytansinoid, MMAD, MMAE, MMAF, a morpholino-doxorubicin (e.g., cyanomorpholino-doxorubicin), netropsin, an oligonucleotide (e.g., a DNA, RNA, or LNA oligonucleotide), paclitaxel, PBD, a peptide (e.g., a therapeutic peptide), rhizoxin, a small molecule (e
  • the molecule is conjugated to the unnatural amino acid by a linker, e.g., a cleavable linker, a non-cleavable linker, a peptide-based linker, or a PEG-based linker.
  • a linker e.g., a cleavable linker, a non-cleavable linker, a peptide-based linker, or a PEG-based linker.
  • an ADC of the present disclosure e.g., any ADC described herein, e.g., those described in FIG.5
  • Any amino acid sites in an antibody sequence e.g., those shown in TABLE 1 may be selected for incorporation of an unnatural amino acid and subsequent modification (e.g., conjugation to a payload described herein).
  • FIGs.19-22 Exemplary conjugated antibody structures are shown in FIGs.19-22.
  • Additional examples of therapeutic, diagnostic, and other proteins that can be modified to comprise one or more unnatural amino acids and/or branched linkers are described in U.S. Patent Application Publication Nos.2003/0082575 and 2005/0009049, and WO2022115625A1, and WO2022056318A1.
  • Non-limiting examples of payload and linker structures are described in FIGs.15-22 and 29A-29B.
  • FIG.19 is an example of the diversity of PBD payloads that can be site-selectively attached to an antibody (e.g., an anti-CD276 antibody) to form an ADC using the unnatural amino acid incorporation technology described herein.
  • One or more of the PBD cores shown in FIG.20 may be used, with the exact attachment site of the linker being determined through a variety of industry validated sites (i.e., imine, amine, A, B, or C ring, linker between dimers or other). Depending upon the circumstances, one or more of the PBD cores outlined in FIG.20 are attached as monomers. Any known stereochemical iteration of the structures shown in FIG.20 may be used Attorney Docket No. BRI-023WO in accordance with the present disclosure, see, e.g., Mantaj et al. (2016).
  • FIG.21 shows some exemplary PBD based ADC connectivity options for certain PBD dimers.
  • B rings are deactivated to prevent DNA crosslinking, limiting the PBD dimer to monoalkylation.
  • U and U’ correspond to the site-specific unnatural amino acid bioconjugation handle (U), and the corresponding crosslinking agent (U’).
  • FIG.22 describes exemplary pro-PBD strategies that, in some embodiments, are attached to the antibody via unnatural amino acid conjugation chemistry. Commonly used unnatural amino acid conjugation chemistries are outlined in FIGs.23A-23B. The exact structures in FIGs.23A-23B are not limiting to the unnatural amino acid structure described therein as long as the functional group remains similar. II.
  • the present disclosure provides an antibody conjugate that specifically binds to CD276 comprising a heavy chain and a light chain, wherein the heavy chain and the light chain are selected from Table 2.
  • the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 1.
  • the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 61.
  • the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 3.
  • the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 62.
  • the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 61
  • the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 62.
  • an antibody of the present disclosure comprises one or more unnatural amino acids at sites selected from Table 1. It is contemplated that antibody amino acid residue sites shown in Table 1 are useful for incorporating unnatural amino acids in any anti- CD276 antibody sequence, e.g., by aligning the sites in Table 1 with another anti-CD276 antibody sequence to identify each site (see, e.g., FIG 1 and 2).
  • an antibody conjugate of the present disclosure can contain one or more unnatural amino acids selected from any genetically encodable unnatural amino acid known in the art (see, e.g., Liu and Schultz, 2010; Dumas and Davis, 2015) including those described herein (see, e.g., FIG.3), particularly LCA or HTP.
  • BRI-023WO amino acid is LCA.
  • the antibody comprises one, two, three, four, or more than four unnatural amino acids.
  • the antibody containing an unnatural amino acid is made in a eukaryotic or prokaryotic expression host via stable integration or transient transfection.
  • a provided antibody comprises an Fc region with at least one of S242C (S239C by EU numbering), L234A, L235A, and P329G mutations.
  • a provided antibody comprises an Fc region with S242C (S239C by EU numbering), L234A, L235A, and P329G mutations.
  • engineered antibodies are made according to the general conjugation scheme where a bioconjugation ready antibody, A-U (where “A” is an anti-CD276 antibody or antigen binding fragment thereof and “U” is an unnatural amino acid bioconjugation handle) is conjugated to a Linker-Payload, U’-X1-L-X2-P (where “U’” is the corresponding crosslinking agent or crosslinker that enables the covalent linkage of the linker-payload, “X1” and “X2” are spacers (e.g., PEG (e.g., PEG2, PEG4, PEG8, etc.), aliphatic chains, polysarcosines (PSARs) (e.g., monodisperse polysarcosines), PAB, a cyclized carbon ring or substituent, etc.), “L” is a cleavable or non-cleavable linking spacer (e.g., Val-Cit, Val-Ala
  • the engineered antibodies described herein are not limited to this exact general conjugation scheme, as any combination of site (e.g., any combination of those provided in TABLE 1), linker structure (e.g., any linker described herein), and payload (e.g., any payload described herein) may be used.
  • linker structure e.g., any linker described herein
  • payload e.g., any payload described herein
  • spacers can be positioned differentially and branched, as shown in FIG.18.
  • only one spacer is used in the generic conjugation scheme, e.g., providing a Linker-Payload structure of U’-X1-L-P or U’-L- X2-P.
  • the payload moiety or molecule consists of any combination of the following: AEB, AEVB, AFP, an amatoxin, an auristatin (e.g., auristatin E), a calicheamicin, CC-1065 or a CC-1065 analog, chalicheamicin, combretastatin, DM1, DM4, docetaxel, dolastatin- 10, DUBA, a duocarmycin, echinomycin, FAM, maytansine, a maytansinoid, MMAD, MMAE, MMAF, a morpholino-doxorubicin (e.g., cyanomorpholino-doxorubicin), netropsin, an oligonucleotide (e.g., a DNA, RNA, or LNA oligonucleotide), paclitaxel, PBD, a peptide (e.g., a therapeutic peptide, e.g.,
  • the molecule is Attorney Docket No. BRI-023WO conjugated to the unnatural amino acid by a linker, e.g., a cleavable linker, a non-cleavable linker, a peptide-based linker, a PEG-based linker, a non-PEG based hydrophilic linker or any linker described herein, and can attach to a payload (e.g., any payload described herein) at any suitable attachment site (e.g., sites that do not perturb function of the linker (if cleavable) or the payload when released from the antibody or when the ADC enters the cell and exerts its therapeutic function).
  • a linker e.g., a cleavable linker, a non-cleavable linker, a peptide-based linker, a PEG-based linker, a non-PEG based hydrophilic linker or any linker described herein
  • a payload e.g
  • the present disclosure provides an ADC of structure A-U-U’- X1-L-X2-P where U can replace one or more residues on the heavy chain only, light chain only, or combinations of the two.
  • the present disclosure provides an ADC of structure A-U-U’-X1-L-X2-P where the linker contains a cleavable moiety (L), a PEG spacer (X1), and a PAB (X2) and the payload (P) comprises a PBD derivative.
  • an antibody of the present disclosure is conjugated at a site with a PBD derivative via methods common known in the art, e.g., as described in Lai et al.
  • an antibody of the present disclosure comprises one or more amino acid substitutions which are conservative amino acid substitutions to m276 antibody, DS antibody, and trastuzumab antibody sequences, or any antibody region (variable or constant) with 5-10, 10-15, 15-20, 20-25, 25-30, 35-40, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, or 95-100% sequence similarity.
  • a provided antibody or engineered antibody is a humanized or human antibody.
  • the antibody is glycosylated.
  • the antibody is not glycosylated.
  • the antibody has effector function.
  • the antibody does not have effector function.
  • an antibody of the present disclosure is linked site-specifically to at least one payload moiety, such that the drug-to-antibody ratio (DAR) is 1, but more typically the DAR is 2 and up to 8. In some embodiments, the DAR is 2.
  • DAR drug-to-antibody ratio
  • the present disclosure also provides methods of treating a cancer that expresses CD276 receptor.
  • the method comprises administering to the subject in need thereof an effective amount of an anti-CD276 antibody conjugate (e.g., any anti-CD276 antibody conjugate described herein).
  • the cancer can be selected from a group consisting of oncology indications, such as breast, prostate, ovarian, endometrial, sarcoma, melanoma, NSCLC, Sq-ESO, bladder cancer, HNSCC, HCC, Salivary Cancer, colon, pancreatic, RCC, gastric, thyroid, blood cancers such as Attorney Docket No. BRI-023WO leukemia or lymphoma, blastoma, glioma, or neuroblastoma, or any other malignancy due to their high expression in cancer cells and low expression in healthy tissue.
  • the present disclosure further provides methods of treating a subject who has an elevated level of CD276 expression on tumor vasculature.
  • the method comprises the step of administering an antibody conjugate of the present disclosure, wherein the level of CD276 expression is elevated relative to a suitable control (e.g., in a healthy tissue of the subject being treated or in another subject that is healthy).
  • a suitable control e.g., in a healthy tissue of the subject being treated or in another subject that is healthy.
  • the present disclosure also provides methods of treating a patient in need thereof with the ADC compositions described herein in combination with an immune checkpoint inhibitor. III.
  • tRNAS tRNAS
  • aminoacyl-tRNA synthetases and/or unnatural amino acids that may be used to incorporate an unnatural amino acid into an antibody of interest (e.g., an anti-CD276 antibody) using any appropriate translation system.
  • translation system refers to a system including components necessary to incorporate an amino acid into a growing polypeptide chain (protein).
  • Components of a translation system can include, e.g., ribosomes, tRNA's, synthetases, mRNA and the like.
  • Translation systems may be cellular or cell-free, and may be prokaryotic or eukaryotic.
  • translation systems may include, or be derived from, a non-eukaryotic cell, e.g., a bacterium (such as E. coli), a eukaryotic cell, e.g., a yeast cell, a mammalian cell, a plant cell, an algae cell, a fungus cell, or an insect cell.
  • a non-eukaryotic cell e.g., a bacterium (such as E. coli)
  • a eukaryotic cell e.g., a yeast cell, a mammalian cell, a plant cell, an algae cell, a fungus cell, or an insect cell.
  • Translation systems include host cells or cell lines, e.g., host cells or cell lines contemplated herein.
  • a polypeptide of interest with an unnatural amino acid in a host cell
  • Translation systems also include whole cell preparations such as permeabilized cells or cell cultures wherein a desired nucleic acid sequence can be transcribed to mRNA and the mRNA translated. Cell-free translation systems are commercially available and many different types and systems are well-known.
  • cell-free systems include, but are not limited to, prokaryotic lysates such as Escherichia coli lysates, and eukaryotic lysates such as wheat germ extracts, insect cell lysates, rabbit reticulocyte lysates, rabbit oocyte lysates and human cell lysates.
  • Reconstituted translation systems may also be used.
  • Reconstituted translation systems may include mixtures of purified translation factors as well as combinations of lysates or lysates supplemented with purified Attorney Docket No. BRI-023WO translation factors such as initiation factor-1 (IF-1), IF-2, IF-3 ( ⁇ or ⁇ ), elongation factor T (EF-Tu), or termination factors.
  • Cell-free systems may also be coupled transcription/translation systems wherein DNA is introduced to the system, transcribed into mRNA and the mRNA is translated.
  • the present disclosure provides, among other things, methods of expressing an antibody containing an unnatural amino acid at one or more specified positions in the antibody.
  • provided methods comprise incubating a translation system (e.g., culturing or growing a host cell or cell line, e.g., a host cell or cell line disclosed herein) under conditions that permit incorporation of the unnatural amino acid into the antibody being expressed in the cell.
  • the translation system may be contacted with (e.g.
  • the cell culture medium may be contacted with) one, or more, unnatural amino acids (e.g., leucyl analogs, tyrosyl analogs, pyrrolysyl analogs, tryptophanyl analogs, etc.) under conditions suitable for incorporation of the one, or more, unnatural amino acids into the antibody.
  • unnatural amino acids e.g., leucyl analogs, tyrosyl analogs, pyrrolysyl analogs, tryptophanyl analogs, etc.
  • the antibody can be expressed from a nucleic acid sequence comprising a premature stop codon.
  • the translation system may, for example, contain a leucyl- tRNA synthetase mutein (e.g., a leucyl-tRNA synthetase mutein disclosed herein) capable of charging a suppressor leucyl tRNA (e.g., a suppressor leucyl tRNA disclosed herein) with an unnatural amino acid (e.g., a leucyl analog) which is incorporated into the antibody at a position corresponding to the premature stop codon.
  • a leucyl- tRNA synthetase mutein e.g., a leucyl-tRNA synthetase mutein disclosed herein
  • an unnatural amino acid e.g., a leucyl analog
  • the leucyl suppressor tRNA comprises an anticodon sequence that hybridizes to the premature stop codon and permits the unnatural amino to be incorporated into the antibody at the position corresponding to the premature stop codon.
  • the antibody is expressed from a nucleic acid sequence comprising a premature stop codon.
  • the translation system may, for example, contain a tryptophanyl-tRNA synthetase mutein (e.g., a tryptophanyl-tRNA synthetase mutein disclosed herein) capable of charging a suppressor tryptophanyl tRNA (e.g., a suppressor tryptophanyl tRNA disclosed herein) with an unnatural amino acid (e.g., a tryptophan analog) which is incorporated into the antibody at a position corresponding to the premature stop codon.
  • a tryptophanyl-tRNA synthetase mutein e.g., a tryptophanyl-tRNA synthetase mutein disclosed herein
  • an unnatural amino acid e.g., a tryptophan analog
  • the tryptophanyl suppressor tRNA comprises an anticodon sequence that hybridizes to the premature stop codon and permits the unnatural amino to be incorporated into the antibody at the position corresponding to the premature stop codon.
  • the antibody is expressed from a nucleic acid sequence comprising a premature stop codon.
  • the translation system e.g., host cell or cell line
  • the BRI-023WO disclosed herein capable of charging a suppressor tyrosyl tRNA (e.g., a suppressor tyrosyl tRNA disclosed herein) with an unnatural amino acid (e.g., a tyrosine analog) which is incorporated into the antibody at a position corresponding to the premature stop codon.
  • the tyrosyl suppressor tRNA comprises an anticodon sequence that hybridizes to the premature stop codon and permits the unnatural amino to be incorporated into the antibody at the position corresponding to the premature stop codon.
  • the antibody is expressed from a nucleic acid sequence comprising a premature stop codon.
  • the translation system may, for example, contain a pyrrolysl-tRNA synthetase mutein (e.g., a pyrrolysl-tRNA synthetase mutein disclosed herein) capable of charging a suppressor pyrrolysl tRNA (e.g., a suppressor pyrrolysl tRNA disclosed herein) with an unnatural amino acid (e.g., a pyrrolysine analog) which is incorporated into the antibody at a position corresponding to the premature stop codon.
  • a pyrrolysl-tRNA synthetase mutein e.g., a pyrrolysl-tRNA synthetase mutein disclosed herein
  • an unnatural amino acid e.g., a pyrrolysine analog
  • the pyrrolysl suppressor tRNA comprises an anticodon sequence that hybridizes to the premature stop codon and permits the unnatural amino to be incorporated into the antibody at the position corresponding to the premature stop codon.
  • a protein e.g., an antibody containing a unnatural amino acid
  • a eukaryotic cell e.g., a mammalian cell.
  • prokaryotic cells e.g., bacteria
  • eukaryotic cells e.g., mammalian cells
  • proteins produced in mammalian cells may undergo post- translational modifications, e.g., modifications that are dependent upon enzymes located in organelles, e.g., the endoplasmic reticulum or Golgi apparatus.
  • post-translational modifications include, without limitation, sulfation, amidation, palmitation, and glycosylation (e.g., N-linked glycosylation and O-linked glycosylation).
  • a protein e.g., an antibody containing a unnatural amino acid
  • the expression yield of an antibody comprising the unnatural amino acid is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the expression yield of a reference antibody.
  • the amount of antibody comprising the unnatural amino acid expressed by the host cell or cell line is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the Attorney Docket No. BRI-023WO amount of a reference antibody expressed by the same cell or a similar cell.
  • the reference antibody is an antibody that does not comprise the unnatural amino acid but is otherwise identical to the antibody comprising the unnatural amino acid.
  • the reference antibody may comprise a wild-type amino acid sequence, or comprise a wild-type amino acid residue at the position corresponding to the unnatural amino acid.
  • Antibody expression may be measured by any method known in the art, including for example, Western blot or ELISA.
  • a disclosed method further comprises purifying the antibody.
  • Specific expression and purification conditions will vary depending upon the expression system employed. Purification techniques known in the art include, e.g., those employing affinity tags such as glutathione-S-transferase (GST) or histidine tags.
  • an antibody may be purified by contacting the antibody with protein A and/or protein G.
  • a disclosed method further comprises conjugating a molecule or payload to a unnatural amino acid in the antibody.
  • the method comprises conjugating the molecule or payload to the unnatural amino acid within 5 minutes to 48 hours at room temperature (e.g., for less than 48 hours, less than 36 hours, less than 24 hours, less than 12 hours, less than 6 hours, less than 1 hour, less than 30 minutes, less than 15 minutes, or less than 10 minutes).
  • the invention provides a method of producing any of the following antibodies in preparation for bioconjugation to a therapeutic payload.
  • the method comprises culturing a cell with: (i) a nucleic acid comprising a nucleotide sequence encoding a tRNA comprising an anticodon that hybridizes to a codon selected from UAG, UGA, and UAA, and is capable of being charged with the unnatural amino acid (UAA); (ii) a nucleic acid comprising a nucleotide sequence encoding an aminoacyl-tRNA synthetase capable of charging the tRNA with the unnatural amino acid (UAA); and (iii) a nucleic acid comprising a nucleotide sequence encoding a heavy chain, a light chain, or a combination of a heavy chain and light chain of the antibody and comprising the codon selected from UAG, UGA, and UAA; under conditions that permit the tRNA, when expressed in the cell and charged with the unnatural amino acid (UAA), to Attorney Docket No.
  • the tRNA is an analog or derivative of a prokaryotic tryptophanyl-tRNA, e.g., an E. coli tryptophanyl-tRNA.
  • the aminoacyl- tRNA synthetase is an analog or derivative of a prokaryotic tryptophanyl-tRNA synthetase, e.g., an E. coli tryptophanyl-tRNA synthetase.
  • the codon is UAG, UGA, or UAA.
  • the UAA is a tryptophan analog, e.g., a non-naturally occurring tryptophan analog.
  • the UAA is 5-HTP or 5-AzW.
  • the tRNA is an analog or derivative of a prokaryotic leucyl- tRNA, e.g., an E. coli leucyl-tRNA.
  • the aminoacyl-tRNA synthetase is an analog or derivative of a prokaryotic leucyl-tRNA synthetase, e.g., an E. coli leucyl-tRNA synthetase.
  • the codon is UAG, UGA, or UAA.
  • the UAA is a leucine analog, e.g., a non-naturally occurring leucine analog.
  • the UAA is LCA or Cys-5-N3.
  • the tRNA is an analog or derivative of a prokaryotic tyrosyl- tRNA, e.g., an E. coli tyrosyl-tRNA.
  • the aminoacyl-tRNA synthetase is an analog or derivative of a prokaryotic tyrosyl-tRNA synthetase, e.g., an E.
  • the codon is UAG, UGA, or UAA.
  • the UAA is a tyrosine analog, e.g., a non-naturally occurring tyrosine analog.
  • the UAA is OmeY, AzF, pAMF, or OpropY (PCT/US2020/054859).
  • the tRNA is an analog or derivative of an archael pyrrolysyl- tRNA, e.g., an M. barkeri pyrrolysyl-tRNA.
  • the aminoacyl-tRNA synthetase is an analog or derivative of an archael pyrrolysyl-tRNA synthetase, e.g., an M. barkeri pyrrolysyl-tRNA synthetase.
  • the codon is UAG, UGA, or UAA.
  • the UAA is a pyrrolysine analog, e.g., a non-naturally occurring pyrrolysine analog.
  • the UAA is BocK, CpK, or AzK (PCT/US2020/054859).
  • the UAA is: (i) a tryptophan analog (e.g., 5- HTP and 5-AzW); (ii) a leucine analog (e.g., LCA and Cys-5-N3); (iii) a tyrosine analog (e.g., OmeY, AzF, and OpropY); or (iv) a pyrrolysine analog (e.g., BocK, CpK, and AzK).
  • a tryptophan analog e.g., 5- HTP and 5-AzW
  • a leucine analog e.g., LCA and Cys-5-N3
  • a tyrosine analog e.g., OmeY, AzF, and OpropY
  • a pyrrolysine analog e.g., BocK, CpK, and AzK
  • the UAA comprises a non-natural aromatic chemical moiety (e.g., a hydroxyl-indole group; an amino-indole group; an aminophenol group; or a hydroxyl- phenol group, e.g., the UAA is 5-hydroxytryptophan (5-HTP), or an analog thereof), and/or the Attorney Docket No. BRI-023WO linker comprises a diazonium group (e.g., the linker comprises 4-nitorbenzenediazonium (4NDz); 4-carboxybenzenediazonium (4NeDz) or 4-methoxybenzenediazonium (4MCDz).
  • a non-natural aromatic chemical moiety e.g., a hydroxyl-indole group; an amino-indole group; an aminophenol group; or a hydroxyl- phenol group, e.g., the UAA is 5-hydroxytryptophan (5-HTP), or an analog thereof
  • the UAA and linker may react under conditions suitable to form an azo-linkage via an azo-coupling reaction between the aromatic chemical moiety and the diazonium group. Further methods for conjugation of molecules to UAAs are described, for example, in U.S. Patent Application Publication No. 2018/0360984.
  • the amount of the antibody comprising the unnatural amino acid (UAA) expressed by the cell is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the amount of a reference antibody expressed by the same cell or a similar cell.
  • the reference antibody is an otherwise identical antibody that does not comprise the UAA, for example, the reference antibody comprises a wild-type amino acid residue at the position corresponding to the unnatural amino acid (UAA).
  • the cell is a human cell, e.g., a human embryonic kidney (HEK) or a Chinese hamster ovary (CHO) cell.
  • HEK human embryonic kidney
  • CHO Chinese hamster ovary
  • FIG.5 Exemplary ADCs provided by the present disclosure are described in FIG.5.
  • Conjugation ready antibodies were generated through unnatural amino acid incorporation to generate unconjugated 9-S1 to 11-S9 (FIG.5).
  • Sites outlined in column 3 of FIG.5 were mutated to contain either a TAG or TGA stop codon at one or multiple sites in order to introduce unnatural amino acids as previously described (LCA in this instance), using transient transfection screening methods. In some instances, sites were mutated to cysteine for thiomab based conjugation.
  • Antibodies that do not contain unnatural amino acid (FIG.5 ADC 6, 13, 14, 15, 16, 17) were expressed using industry standard transient transfection in EXPI293.
  • Antibodies were purified at small scale via drip-column Protein A, or at large scale by FPLC purification via HiTrap MabSelect PrismA column (Cat#09-928-070, Protocol). General transfection, UAA incorporation, and conjugation detail are included in the following patent (PCT/US2021/049953, PCT/US2021/045088). Proteins were purified prior to conjugation, as described herein. III.
  • Transfer RNAs [0230] During protein synthesis, e.g., synthesis of an antibody with an unnatural amino acid, a tRNA molecule delivers an amino acid to a ribosome for incorporation into a growing protein (polypeptide) chain.
  • tRNAs typically are about 70 to 100 nucleotides in length. Active tRNAs Attorney Docket No. BRI-023WO contain a 3' CCA sequence that may be transcribed into the tRNA during its synthesis or may be added later during post-transcriptional processing.
  • the amino acid that is attached to a given tRNA molecule is covalently attached to the 2' or 3' hydroxyl group of the 3'- terminal ribose to form an aminoacyl-tRNA (aa-tRNA). It is understood that an amino acid can spontaneously migrate from the 2'-hydroxyl group to the 3'-hydroxyl group and vice versa, but it is incorporated into a growing protein chain at the ribosome from the 3'-OH position.
  • a loop at the other end of the folded aa-tRNA molecule contains a sequence of three bases known as the anticodon.
  • this anticodon sequence hybridizes or base-pairs with a complementary three- base codon sequence in a ribosome-bound mRNA
  • the aa-tRNA binds to the ribosome and its amino acid is incorporated into the polypeptide chain being synthesized by the ribosome. Because all tRNAs that base-pair with a specific codon are aminoacylated with a single specific amino acid, the translation of the genetic code is affected by tRNAs.
  • Each of the 61 non-termination codons in an mRNA directs the binding of its cognate aa-tRNA and the addition of a single specific amino acid to the growing polypeptide chain being synthesized by the ribosome.
  • the term “cognate” refers to components that function together, e.g., a tRNA and an aminoacyl-tRNA synthetase.
  • Suppressor tRNAs are modified tRNAs that alter the reading of a mRNA in a given translation system. For example, a suppressor tRNA may read through a codon such as a stop codon, a four base codon, or a rare codon.
  • suppressor uses the word in suppressor to the fact, that under certain circumstance, the modified tRNA "suppresses" the typical phenotypic effect of the codon in the mRNA.
  • Suppressor tRNAs typically contain a mutation (modification) in either the anticodon, changing codon specificity, or at some position that alters the aminoacylation identity of the tRNA.
  • suppression activity refers to the ability of a tRNA, e.g., a suppressor tRNA, to read through a codon (e.g., a premature stop codon) that would not be read through by the endogenous translation machinery in a system of interest.
  • a tRNA (e.g., a suppressor tRNA) contains a modified anticodon region, such that the modified anticodon hybridizes with a different codon than the corresponding naturally occurring anticodon.
  • a tRNA comprises an anticodon that hybridizes to a codon selected from UAG (i.e., an “amber” termination codon), UGA (i.e., an “opal” termination codon), and UAA (i.e., an “ochre” termination codon).
  • a tRNA comprises an anticodon that hybridizes to a non-standard codon, e.g., a 4- or 5-nucleotide codon.
  • a non-standard codon e.g., a 4- or 5-nucleotide codon.
  • four base codons include AGGA, CUAG, UAGA, and CCCU.
  • five base codons include AGGAC, CCCCU, CCCUC, Attorney Docket No. BRI-023WO CUAGA, CUACU, and UAGGC.
  • tRNAs comprising an anticodon that hybridizes to a non-standard codon, e.g., a 4- or 5-nucleotide codon
  • a non-standard codon e.g., a 4- or 5-nucleotide codon
  • methods of using such tRNAs to incorporate unnatural amino acids into proteins are described, for example, in Moore et al. (2000) J. MOL. BIOL., 298: 195; Hohsaka et al. (1999) J. AM. CHEM. SOC., 121: 12194; Anderson et al. (2002) CHEMISTRY AND BIOLOGY, 9: 237-244; Magliery (2001) J. MOL. BIOL., 307: 755-769; and PCT Publication No. WO2005/007870.
  • tRNA includes variants having one or more mutations (e.g., nucleotide substitutions, deletions, or insertions) relative to a reference (e.g., a wild-type) tRNA sequence.
  • a tRNA may comprise, consist, or consist essentially of, a single mutation (e.g., a mutation contemplated herein), or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more than 15 mutations (e.g., mutations contemplated herein).
  • a tRNA may comprise, consist, or consist essentially 1-15, 1-10, 1-7, 1-6, 1-5, 1- 4, 1-3, 1-2, 2-15, 2-10, 2-7, 2-6, 2-5, 2-4, 2-3, 3-15, 3-10, 3-7, 3-6, 3-5, or 3-4 mutations (e.g., mutations contemplated herein).
  • a variant suppressor tRNA has increased activity to incorporate an unnatural amino acid (e.g., an unnatural amino acid contemplated herein) into a mammalian protein relative to a counterpart wild-type suppressor tRNA (in this context, a wild-type suppressor tRNA refers to a suppressor tRNA that corresponds to a wild-type tRNA molecule but for any modifications to the anti-codon region to impart suppression activity).
  • an unnatural amino acid e.g., an unnatural amino acid contemplated herein
  • a wild-type suppressor tRNA refers to a suppressor tRNA that corresponds to a wild-type tRNA molecule but for any modifications to the anti-codon region to impart suppression activity.
  • the activity of the variant suppressor tRNA may be increased relative to the wild type suppressor tRNA, for example, by about 2.5 to about 200 fold, about 2.5 to about 150 fold, about 2.5 to about 100 fold about 2.5 to about 80 fold, about 2.5 to about 60 fold, about 2.5 to about 40 fold, about 2.5 to about 20 fold, about 2.5 to about 10 fold, about 2.5 to about 5 fold, about 5 to about 200 fold, about 5 to about 150 fold, about 5 to about 100 fold, about 5 to about 80 fold, about 5 to about 60 fold, about 5 to about 40 fold, about 5 to about 20 fold, about 5 to about 10 fold, about 10 to about 200 fold, about 10 to about 150 fold, about 10 to about 100 fold, about 10 to about 80 fold, about 10 to about 60 fold, about 10 to about 40 fold, about 10 to about 20 fold, about 20 to about 200 fold, about 20 to about 150 fold, about 20 to about 100 fold, about 20 to about 80 fold, about 20 to about 60 fold, about 20 to about 40 fold, about 40 to about 200 fold, about 40 to about 150 fold, about 40 to
  • the tRNA may function in vitro or in vivo and can be provided to a translation system (e.g., an in vitro translation system or a cell) as a mature tRNA (e.g., an aminoacylated tRNA), or as a polynucleotide that encodes the tRNA.
  • a tRNA may be derived from a bacterial source, e.g., Escherichia coli, Thermus thermophilus, or Bacillus stearothermphilus.
  • a tRNA may also be derived from an archaeal source, e.g, from the Methanosarcinacaea or Desulfitobacterium families, any of the M. barkeri (Mb), M. alvus (Ma), M. mazei (Mm) or D. hafnisense (Dh) families, Methanobacterium thermoautotrophicum, Haloferax volcanii, Halobacterium species NRC-1, or Archaeoglobus fulgidus.
  • eukaryotic sources can also be used, for example, plants, algae, protists, fungi, yeasts, or animals (e.g., mammals, insects, arthropods, etc.).
  • a tRNA used in accordance with the present disclosure may comprise any nucleotide sequence as shown in Table 3.
  • the tRNA is derived from a prokaryotic leucyl-tRNA (e.g., an analog or derivative of a prokaryotic leucyl-tRNA).
  • the tRNA is derived from an E. coli leucyl tRNA and, for example, is preferentially charged with a leucine analog over the naturally-occurring leucine amino acid by an aminoacyl-tRNA synthetase derived from an E.
  • the tRNA may comprise, consist essentially of, or consist of the nucleotide sequence of any one of SEQ ID NOs: 22-49, or a nucleotide sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 22-49.
  • the tRNA is derived from a prokaryotic tryptophanyl-tRNA (e.g., an analog or derivative of a prokaryotic tryptophanyl-tRNA).
  • the tRNA is derived from an E. coli tryptophanyl tRNA and, for example, is preferentially charged with a tryptophan analog over the naturally-occurring tryptophan amino acid by an aminoacyl- tRNA synthetase derived from an E. coli tryptophanyl-tRNA synthetase, e.g., an aminoacyl-tRNA synthetase contemplated herein.
  • the tRNA may comprise, consist essentially of, or consist of the nucleotide sequence of any one of SEQ ID NOs: 55-60, or a nucleotide sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 55-60.
  • the tRNA is derived from a prokaryotic tyrosyl-tRNA (e.g., an analog or derivative of a prokaryotic tyrosyl-tRNA).
  • the tRNA is derived from an E.
  • the tRNA is derived from an archael pyrrolysyl-tRNA (e.g., an analog or derivative of a archael pyrrolysyl-tRNA).
  • the tRNA is derived from a M. barkeri pyrrolysyl tRNA and, for example, is preferentially charged with a pyrrolysine analog over the naturally-occurring pyrrolysine amino acid by an aminoacyl-tRNA synthetase derived from a M. barkeri pyrrolysyl-tRNA synthetase, e.g., an aminoacyl-tRNA synthetase contemplated herein.
  • a tRNA comprises, consists essentially of, or consists of a nucleotide sequence including one or more thymines (T)
  • a tRNA is also contemplated that comprises, consists essentially of, or consists of the same nucleotide sequence including a uracil (U) in place of one or more of the thymines (T), or a uracil (U) in place of all the thymines (T).
  • a tRNA comprises, consists essentially of, or consists of a nucleotide sequence including one or more uracils (U)
  • a tRNA is also contemplated that comprises, consists essentially of, or consists of a nucleotide sequence including a thymine (T) in place of the one or more of the uracils (U), or a thymine (T) in place of all the uracils (U).
  • T thymine
  • additional modifications to the bases can be present.
  • a tRNA may be aminoacylated (i.e., charged) with a desired unnatural amino acid by any method, including enzymatic or chemical methods.
  • Enzymatic molecules capable of charging a tRNA include aminoacyl-tRNA synthetases, e.g., aminoacyl-tRNA synthetases disclosed herein. Additional enzymatic molecules capable of charging tRNA include ribozymes, for example, as described in Illangakekare et al.
  • the enigeered antibodies are created using engineered aminoacyl-tRNA synthetases (or aaRSs) capable of charging a tRNA with an unnatural amino acid for incorporation into a protein of interest (e.g., an antibody, such as an anti-CD276 antibody).
  • aaRSs engineered aminoacyl-tRNA synthetases
  • aminoacyl-tRNA synthetase refers to any enzyme, or a functional fragment thereof, that charges, or is capable of charging, a tRNA with an amino acid (e.g., an unnatural amino acid) for incorporation into a protein.
  • the term “functional fragment” of an aminoacyl-tRNA synthetase refers to fragment of a full-length aminoacyl-tRNA synthetase that retains, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the enzymatic activity of the corresponding full- length tRNA synthetase (e.g., a naturally occurring tRNA synthetase). Aminoacyl-tRNA synthetase enzymatic activity may be assayed by any method known in the art.
  • the functional fragment comprises at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 consecutive amino acids present in a full-length tRNA synthetase (e.g., a naturally occurring aminoacyl-tRNA synthetase).
  • aminoacyl-tRNA synthetase includes variants (i.e., muteins) having one or more mutations (e.g., amino acid substitutions, deletions, or insertions) relative to a wild-type aminoacyl-tRNA synthetase sequence.
  • an aminoacyl-tRNA synthetase mutein may comprise, consist, or consist essentially of, a single mutation (e.g., a mutation contemplated herein), or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more than 15 mutations (e.g., mutations discussed herein).
  • an aminoacyl-tRNA synthetase mutein may comprise, consist, or consist essentially 1-15, 1-10, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-15, 2-10, 2-7, 2-6, 2-5, 2-4, 2-3, 3-15, 3-10, 3-7, 3-6, 3-5, or 4-10, 4-7, 4-6, 4-5, 5-10, 5-7, 5- 6, 6-10, 6-7, 7-10, 7-8, or 8-10 mutations (e.g., mutations contemplated herein).
  • An aminoacyl- tRNA synthetase mutein may comprise a conservative substitution relative to a wild-type sequence or a sequence disclosed herein.
  • the substrate specificity of the aminoacyl- tRNA synthetase mutein is altered relative to a corresponding (or template) wild-type aminoacyl- tRNA synthetase such that only a desired unnatural amino acid, but not any of the common 20 amino acids, is charged to the substrate tRNA.
  • an aminoacyl-tRNA synthetase may be derived from a bacterial source, e.g., Escherichia coli, Thermus thermophilus, or Bacillus stearothermphilus.
  • an aminoacyl-tRNA synthetase may be derived from an archaeal source, e.g, from the Methanosarcinacaea or Desulfitobacterium families, any of the M. barkeri (Mb), M. alvus (Ma), M. mazei (Mm) or D. hafnisense (Dh) families, Methanobacterium thermoautotrophicum, Haloferax volcanii, Halobacterium species NRC-1, or Archaeoglobus fulgidus.
  • an archaeal source e.g, from the Methanosarcinacaea or Desulfitobacterium families, any of the M. barkeri (Mb), M. alvus (Ma), M. mazei (Mm) or D. hafnisense (Dh) families, Methanobacterium thermoautotrophicum, Haloferax volcanii, Halobacterium species NRC-1, or Archaeoglobus fulgidus.
  • eukaryotic sources can be used, for example, plants, algae, protists, fungi, yeasts, or animals (e.g., mammals, insects, arthropods, etc.).
  • the terms “derivative” or “derived from” refer to a component that is isolated from or made using information from a specified molecule or organism.
  • analog refers to a component (e.g., a tRNA, tRNA synthetase, or unnatural amino acid) that is derived from or analogous with (in terms of structure and/or function) a reference component (e.g., a wild-type tRNA, a wild-type tRNA synthetase, or a natural amino acid).
  • a reference component e.g., a wild-type tRNA, a wild-type tRNA synthetase, or a natural amino acid.
  • derivatives or analogs have at least 40%, 50%, 60%, 70%, 80%, 90%, 100% or more of a given activity as a reference or originator component (e.g., wild type component).
  • aminoacyl-tRNA synthetase may aminoacylate a substrate tRNA in vitro or in vivo, and can be provided to a translation system (e.g., an in vitro translation system or a cell) as a polypeptide or protein, or as a polynucleotide that encodes the aminoacyl- tRNA synthetase.
  • a translation system e.g., an in vitro translation system or a cell
  • an aminoacyl-tRNA synthetase used in accordance with the present disclosure may comprise any amino acid sequence shown in TABLE 3, including functional fragments thereof.
  • the aminoacyl-tRNA synthetase is derived from a prokaryotic leucyl-tRNA synthetase (e.g., an analog or derivative of a prokaryotic leucyl-tRNA synthetase).
  • the aminoacyl-tRNA synthetase is derived from an E. coli leucyl-tRNA synthetase and, for example, the aminoacyl-tRNA synthetase preferentially aminoacylates an E. coli leucyl tRNA (or a variant thereof) with a leucine analog over the naturally-occurring leucine amino acid.
  • the aminoacyl-tRNA synthetase comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 7-21, or an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 7-21.
  • the aminoacyl-tRNA synthetase is derived from a prokaryotic tryptophanyl-tRNA synthetase (e.g., an analog or derivative of a prokaryotic tryptophanyl-tRNA Attorney Docket No. BRI-023WO synthetase).
  • the aminoacyl-tRNA synthetase is derived from an E. coli tryptophanyl-tRNA synthetase and, for example, the aminoacyl-tRNA synthetase preferentially aminoacylates an E. coli tryptophanyl tRNA (or a variant thereof) with a tryptophan analog over the naturally-occurring tryptophan amino acid.
  • the aminoacyl-tRNA synthetase comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 50-54, or an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 50-54.
  • the aminoacyl-tRNA synthetase is derived from a prokaryotic tyrosyl-tRNA synthetase (e.g., an analog or derivative of a prokaryotic tyrosyl-tRNA synthetase).
  • the aminoacyl-tRNA synthetase is derived from an E. coli tyrosyl-tRNA synthetase and, for example, the aminoacyl-tRNA synthetase preferentially aminoacylates an E. coli tyrosyl tRNA (or a variant thereof) with a tyrosine analog over the naturally-occurring tyrosine amino acid.
  • the aminoacyl-tRNA synthetase is derived form an archael pyrrolysyl-tRNA synthetase (e.g., an analog or derivative of an archael pyrrolysyl-tRNA synthetase).
  • the aminoacyl-tRNA synthetase is derived from an M. barkeri pyrrolysyl-tRNA synthetase and, for example, the aminoacyl-tRNA synthetase preferentially aminoacylates an M. barkeri pyrrolysyl tRNA (or a variant thereof) with a pyrrolysine analog over the naturally-occurring pyrrolysine amino acid.
  • Methods for producing proteins e.g., aminoacyl-tRNA synthetases, are known in the art. For example, DNA molecules encoding a protein of interest can be synthesized chemically or by recombinant DNA methodologies.
  • the resulting DNA molecules encoding the protein interest can be ligated to other appropriate nucleotide sequences, including, for example, expression control sequences, to produce conventional gene expression constructs (i.e., expression vectors) encoding the desired protein. Production of defined gene constructs is within routine skill in the art.
  • Nucleic acids encoding desired proteins e.g, aminoacyl-tRNA synthetases
  • expression vectors which can be introduced into host cells through conventional transfection or transformation techniques.
  • Exemplary host cells are E.
  • Transformed host cells can be grown under conditions that permit the host cells to express the desired protein.
  • coli it is first cloned into an expression vector by positioning the engineered gene downstream from a suitable bacterial promoter, e.g., Trp or Tac, and a prokaryotic signal sequence.
  • the expressed protein may be secreted.
  • the expressed protein may accumulate in refractile or inclusion bodies, which can be harvested after disruption of the cells by French press or sonication.
  • the refractile bodies then are solubilized, and the protein may be refolded and/or cleaved by methods known in the art.
  • the engineered gene is to be expressed in eukaryotic host cells, e.g., CHO cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, a secretion signal, a poly A sequence, and a stop codon.
  • the vector or gene construct may contain enhancers and introns.
  • the gene construct can be introduced into eukaryotic host cells using conventional techniques.
  • a protein of interest e.g, an aminoacyl-tRNA synthetase
  • a protein of interest can be produced by growing (culturing) a host cell transfected with an expression vector encoding such a protein under conditions that permit expression of the protein.
  • the protein can be harvested and purified or isolated using techniques known in the art, e.g., affinity tags such as glutathione-S-transferase (GST) or histidine tags.
  • affinity tags such as glutathione-S-transferase (GST) or histidine tags.
  • the present disclosure also encompasses nucleic acids encoding aminoacyl-tRNA synthetases disclosed herein (e.g., nucleic acids encoding any of the aminoacyl-tRNA synthetases described in TABLE 3).
  • nucleic acids encoding aminoacyl-tRNA synthetases disclosed herein e.g., nucleic acids encoding any of the aminoacyl-tRNA synthetases described in TABLE 3.
  • Exemplary tRNAs and aminoacyl-tRNA synthetases that may be used in accordance with the present disclosure are described in International (PCT) Publication Nos. WO2020257668, WO2022056318, and WO2022115625, the entire contents of each of which are hereby incorporated by reference. III.
  • tRNAs aminoacyl-tRNA synthetases, or any other molecules of interest may be expressed in a cell of interest by incorporating a gene encoding the molecule into an appropriate expression vector.
  • expression vector refers to a vector comprising Attorney Docket No. BRI-023WO a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • the tRNAs, aminoacyl-tRNA synthetases, or any other molecules of interest may be introduced to a cell of interest by incorporating a gene encoding the molecule into an appropriate transfer vector.
  • transfer vector refers to a vector comprising a recombinant polynucleotide which can be used to deliver the polynucleotide to the interior of a cell. It is understood that a vector may be both an expression vector and a transfer vector.
  • Vectors e.g., expression vectors or transfer vectors
  • Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular target nucleic acid.
  • the vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both (including but not limited to, shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems.
  • the vector comprises a regulatory sequence or promoter operably linked to the nucleotide sequence encoding the suppressor tRNA and/or the tRNA synthetase.
  • operably linked refers to a linkage of polynucleotide elements in a functional relationship.
  • a nucleic acid sequence is "operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a gene if it affects the transcription of the gene.
  • Operably linked nucleotide sequences are typically contiguous.
  • enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths
  • some polynucleotide elements may be operably linked but not directly flanked and may even function in trans from a different allele or chromosome.
  • Exemplary promoters which may be employed include, but are not limited to, the retroviral LTR, the SV40 promoter, the human cytomegalovirus (CMV) promoter, the U6 promoter, the EF1 ⁇ promoter, the CAG promoter, the H1 promoter, the UbiC promoter, the PGK promoter, the 7SK promoter, a pol II promoter, a pol III promoter, or any other promoter (e.g., Attorney Docket No. BRI-023WO cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and ⁇ -actin promoters).
  • CMV human cytomegalovirus
  • a vector comprises a nucleotide sequence encoding an aminoacyl-tRNA synthetase operably linked to a CMV or an EF1 ⁇ promoter and/or a nucleotide sequence encoding a suppressor tRNA operably linked to a U6 or an H1 promoter.
  • the vector is a viral vector.
  • virus is used herein to refer to an obligate intracellular parasite having no protein-synthesizing or energy-generating mechanism.
  • exemplary viral vectors include retroviral vectors (e.g., lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, herpesviruses vectors, epstein-barr virus (EBV) vectors, polyomavirus vectors (e.g., simian vacuolating virus 40 (SV40) vectors), poxvirus vectors, and pseudotype virus vectors.
  • retroviral vectors e.g., lentiviral vectors
  • adenoviral vectors e.g., adenoviral vectors, adeno-associated viral vectors, herpesviruses vectors, epstein-barr virus (EBV) vectors, polyomavirus vectors (e.g., simian vacuolating virus 40 (SV40) vectors), poxvirus vector
  • the viral vector is a DNA virus vector.
  • DNA viruses include parvoviruses (e.g., adeno-associated viruses), adenoviruses, asfarviruses, herpesviruses (e.g., herpes simplex virus 1 and 2 (HSV-1 and HSV-2), epstein-barr virus (EBV), cytomegalovirus (CMV)), papillomoviruses (e.g., HPV), polyomaviruses (e.g., simian vacuolating virus 40 (SV40)), and poxviruses (e.g., vaccinia virus, cowpox virus, smallpox virus, fowlpox virus, sheeppox virus, myxoma virus).
  • parvoviruses e.g., adeno-associated viruses
  • adenoviruses e.g., asfarviruses
  • herpesviruses e.g., her
  • the viral vector is a RNA virus vector.
  • RNA viruses include bunyaviruses (e.g., hantavirus), coronaviruses, flaviviruses (e.g., yellow fever virus, west nile virus, dengue virus), hepatitis viruses (e.g., hepatitis A virus, hepatitis C virus, hepatitis E virus), influenza viruses (e.g., influenza virus type A, influenza virus type B, influenza virus type C), measles virus, mumps virus, noroviruses (e.g., Norwalk virus), poliovirus, respiratory syncytial virus (RSV), retroviruses (e.g., human immunodeficiency virus-1 (HIV-1)) and toroviruses.
  • bunyaviruses e.g., hantavirus
  • coronaviruses e.g., flaviviruses (e.g., yellow fever virus, west nile virus,
  • Host cells or cell lines e.g., prokaryotic or eukaryotic host cells or cell lines
  • a tRNA, aminoacyl-tRNA synthetase, unnatural amino acid, nucleic acid, and/or vector disclosed herein can be used in the production of the of the proteins, e.g., antibodies, described herein.
  • the nucleic acid encoding the engineered tRNA and aminoacyl-tRNA synthetase can be expressed in an expression host cell either as an autonomously replicating vector within the Attorney Docket No.
  • BRI-023WO expression host cell e.g., a plasmid, or viral particle
  • a stable integrated element or series of stable integrated elements in the genome of the expression host cell e.g., a mammalian host cell.
  • Host cells are genetically engineered (including but not limited to, transformed, transduced or transfected), for example, using nucleic acids or vectors disclosed herein.
  • one or more vectors include coding regions for an orthogonal tRNA, an orthogonal aminoacyl-tRNA synthetase, and, optionally, a protein (e.g., an antibody) to be modified by the inclusion of one or more unnatural amino acids, which are operably linked to gene expression control elements that are functional in the desired host cell or cell line.
  • a protein e.g., an antibody
  • the genes encoding tRNA synthetase and tRNA and an optional selectable marker can be integrated in a transfer vector (e.g., a plasmid, which can be linearized prior to transfection), where for example, the genes encoding the tRNA synthetase can be under the control of a polymerase II promoter (e.g., CMV, EF1 ⁇ , UbiC, or PGK, e.g., CMV or EF1 ⁇ ) and the genes encoding the tRNA can be under the control of a polymerase III promoter (e.g., U6, 7SK, or H1, e.g., U6).
  • a polymerase II promoter e.g., CMV, EF1 ⁇ , UbiC, or PGK, e.g., CMV or EF1 ⁇
  • a polymerase III promoter e.g., U6, 7SK, or H1, e.g., U6
  • the vectors are transfected into cells and/or microorganisms by standard methods including electroporation or infection by viral vectors, and clones can be selected via expression of the selectable marker (for example, by antibiotic resistance).
  • exemplary prokaryotic host cells or cell lines include cells derived from a bacteria, e.g., E. coli, Thermus thermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa, and Pseudomonas putida.
  • Exemplary eukaryotic host cells or cell lines include cells derived from a plant (e.g., a complex plant such as a monocot or dicot), an algae, a protist, a fungus, a yeast (including Saccharomyces cerevisiae), or an animal (including a mammal, an insect, an arthropod, etc.).
  • a plant e.g., a complex plant such as a monocot or dicot
  • an algae e.g., a complex plant such as a monocot or dicot
  • a protist e.g., a fungus
  • yeast including Saccharomyces cerevisiae
  • animal including a mammal, an insect, an arthropod, etc.
  • Additional exemplary host cells or cell lines include HEK293, HEK293T, Expi293, CHO, CHOK1, Sf9, Sf21, HeLa, U20S, A549, HT1080, CAD, P19, NIH 3T3, L929, N2a, MCF-7, Y79, SO-RB50, HepG2, DUKX-X11, J558L, BHK, COS, Vero, NS0, or ESCs. It is understood that a host cell or cell line can include individual colonies, isolated populations (monoclonal), or a heterogeneous mixture of cells.
  • a contemplated cell or cell line includes, for example, one or multiple copies of an orthogonal tRNA/aminoacyl-tRNA synthetase pair, optionally stably maintained in the cell’s genome or another piece of DNA maintained by the cell.
  • the cell or cell line may contain one or more copies of (i) a tryptophanyl tRNA/aminoacyl-tRNA synthetase pair (wild type or engineered) stably maintained by the cell, and/or (ii) a leucyl tRNA/aminoacyl-tRNA synthetase pair (wild-type or engineered) stably maintained by the cell.
  • the cell line is a stable cell line and the cell line comprises a genome having stably integrated therein (i) a nucleic acid sequence encoding an aminoacyl-tRNA synthetase (e.g., a prokaryotic tryptophanyl-tRNA synthetase mutein capable of charging a tRNA with an unnatural amino acid or a prokaryotic leucyl-tRNA synthetase mutein capable of charging a tRNA with an unnatural amino acid, e.g., a tRNA synthetase mutein disclosed herein); and/or (ii) a nucleic acid sequence encoding a suppressor tRNA (e.g., prokaryotic suppressor tryptophanyl-tRNA capable of being charged with an unnatural amino acid or prokaryotic suppressor leucyl-tRNA capable of being charged with an unnatural amino acid
  • a suppressor tRNA e.g., prok
  • nucleic acid encoding a tRNA and/or an aminoacyl-tRNA synthetase into the genome of a cell of interest, or to stably maintain the nucleic acid in DNA replicated by the cell that is outside of the genome, are well known in the art.
  • the nucleic acid encoding the tRNA and/or an aminoacyl-tRNA synthetase can be provided to the cell in an expression vector, transfer vector, or DNA cassette, e.g., an expression vector, transfer vector, or DNA cassette disclosed herein.
  • the expression vector transfer vector, or DNA cassette encoding the tRNA and/or aminoacyl-tRNA synthetase can contain one or more copies of the tRNA and/or aminoacyl-tRNA synthetase optionally under the control of an inducible or constitutively active promoter.
  • the expression vector, transfer vector, or DNA cassette may, for example, contain other standard components (enhancers, terminators, etc.).
  • nucleic acid encoding the tRNA and the nucleic acid encoding the aminoacyl-tRNA synthetase may be on the same or different vector, may be present in the same or different ratios, and may be introduced into the cell, or stably integrated in the cellular genome, at the same time or sequentially.
  • One or multiple copies of a DNA cassette encoding the tRNA and/or aminoacyl-tRNA synthetase can be integrated into a host cell genome or stably maintained in the cell using a transposon system (e.g., PiggyBac), a viral vector (e.g., a lentiviral vector or other retroviral vector), CRISPR/Cas9 based recombination, electroporation and natural recombination, a BxB1 recombinase system, or using a replicating/maintained piece of DNA (such as one derived from Epstein-Barr virus).
  • a transposon system e.g., PiggyBac
  • a viral vector e.g., a lentiviral vector or other retroviral vector
  • CRISPR/Cas9 based recombination e.g., a lentiviral vector or other retroviral vector
  • electroporation and natural recombination
  • a selectable marker can be used.
  • exemplary selectable markers include zeocin, puromycin, neomycin, dihydrofolate reductase (DHFR), glutamine synthetase (GS), mCherry-EGFP fusion, or other fluorescent proteins.
  • DHFR dihydrofolate reductase
  • GS glutamine synthetase
  • mCherry-EGFP fusion or other fluorescent proteins.
  • BRI-023WO selectable marker protein (or a gene encoding a protein required for a detectable function, e.g., viability, in the presence of the selectable marker) may include a premature stop codon, such that the protein will only be expressed if the cell line is capable of incorporating a UAA at the site of the premature stop codon.
  • a premature stop codon such that the protein will only be expressed if the cell line is capable of incorporating a UAA at the site of the premature stop codon.
  • Host cells or cell lines which contain enhanced UAA incorporation efficiency, low background, and decreased toxicity can first be isolated via a selectable marker containing one or more stop codons. Subsequently, the host cells or cell lines can be subjected to a selection scheme to identify host cells or cell lines which contain the desired copies of tRNA/aminoacyl-tRNA synthetase pairs and express a gene of interest (either genomically integrated or not) containing one or more stop codons. Protein expression may be assayed using any method known in the art, including for example, Western blot using an antibody that binds the protein of interest or a C- terminal tag.
  • the host cells or cell lines be cultured in conventional nutrient media modified as appropriate for such activities as, for example, screening steps, activating promoters or selecting transformants. These cells can optionally be cultured into transgenic organisms.
  • Other useful references, e.g. for cell isolation and culture include Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley-Liss, New York; Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc.
  • the desired protein e.g., an antibody, (e.g., an anti-CD276 antibody) containing one or more unnatural amino acids
  • the resulting protein-payload conjugate e.g., antibody- conjugate (e.g., antibody drug conjugate) after standard purification
  • the pharmaceutical composition can be formulated for use in a variety of drug delivery systems.
  • One or more pharmaceutically acceptable excipients or carriers can also be included in the composition for proper formulation.
  • a pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition.
  • suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl- beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophiliric acid,
  • compositions containing a protein-payload conjugate can be presented in a dosage unit form and can be prepared by any suitable method.
  • a pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous, intradermal, inhalation, transdermal, topical, transmucosal, intrathecal and rectal administration.
  • routes of administration are intravenous, intradermal, inhalation, transdermal, topical, transmucosal, intrathecal and rectal administration.
  • Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl Attorney Docket No.
  • BRI-023WO parabens include antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the carrier should be stable under the conditions of manufacture and storage, and should be preserved against microorganisms.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol), and suitable mixtures thereof.
  • An intravenous drug delivery formulation of the present disclosure may be contained in a bag, a pen, or a syringe. In certain embodiments, the bag may be connected to a channel including a tube and/or a needle. [0293] Depending upon the circumstances, the formulation can be a liquid formulation.
  • An aqueous formulation can be prepared including the protein-payload conjugate, e.g., antibody- conjugate (e.g., antibody drug conjugate) in a pH-buffered solution.
  • the pH of the liquid formulation may be set by addition of a pharmaceutically acceptable acid and/or base.
  • the pharmaceutically acceptable acid may be hydrochloric acid.
  • the base may be sodium hydroxide.
  • a salt or buffer components may be added in an amount of 10 mM to 200 mM.
  • the salts and/or buffers are pharmaceutically acceptable and are derived from various known acids (inorganic and organic) with “base forming” metals or amines.
  • the buffer may be phosphate buffer.
  • the buffer may be glycinate, carbonate, citrate buffers, in which case, sodium, potassium or ammonium ions can serve as counterion.
  • Intravenous formulations can be diluted with 0.9% sodium chloride solution before administration.
  • the diluted drug product for injection is isotonic and suitable for administration by intravenous infusion.
  • the formulation can be a lyophilized formulation including the protein- payload conjugate, e.g., antibody-conjugate (e.g., antibody drug conjugate) and a lyoprotectant.
  • the lyoprotectant may be sugar, e.g., disaccharides.
  • the lyoprotectant may be sucrose or maltose.
  • the lyophilized formulation may also include one or more of a buffering agent, a surfactant, a bulking agent, and/or a preservative.
  • the amount of sucrose or maltose useful for stabilization of the lyophilized drug product may be in a weight ratio of at least 1:2 protein to sucrose or maltose.
  • the protein to sucrose or maltose weight ratio may be of from 1:2 to 1:5.
  • the pH of the solution containing the protein of the Attorney Docket No. BRI-023WO present disclosure may be adjusted between 6 to 8.
  • the pH range for the lyophilized drug product may be from 7 to 8.
  • a “bulking agent” may be added.
  • a “bulking agent” is a compound which adds mass to a lyophilized mixture and contributes to the physical structure of the lyophilized cake (e.g., facilitates the production of an essentially uniform lyophilized cake which maintains an open pore structure).
  • Illustrative bulking agents include mannitol, glycine, polyethylene glycol and sorbitol.
  • the lyophilized formulations of the present disclosure may contain such bulking agents.
  • the lyophilized drug product may be constituted with an aqueous carrier.
  • the aqueous carrier of interest herein is one which is pharmaceutically acceptable (e.g., safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation, after lyophilization.
  • Illustrative diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline solution, Ringer’s solution or dextrose solution.
  • the lyophilized protein product of the instant disclosure is constituted to about 4.5 mL water for injection and diluted with 0.9% saline solution (sodium chloride solution).
  • compositions preferably are sterile. Sterilization can be accomplished by any suitable method, e.g., filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.
  • the resulting aqueous solutions may be packaged for use as-is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration.
  • the pH of the preparations typically will be between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5.
  • the resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents.
  • composition in solid form can also be packaged in a container for a flexible quantity.
  • the resulting pharmaceutical composition is formulated as a liquid formulation in either a USP / Ph Eur type I 50R vial closed with a rubber stopper and sealed with an aluminum crimp seal closure.
  • the stopper may be made of elastomer complying with USP and Ph Eur.
  • the liquid formulation may be diluted with 0.9% saline solution prior to use.
  • a preservative may be optionally added to the formulations to reduce bacterial action. The addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation.
  • a pharmaceutical composition may contain a sustained- or controlled-delivery formulation.
  • sustained- or controlled-delivery means such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art.
  • Sustained-release preparations may include, e.g., porous polymeric microparticles or semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules.
  • Sustained release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, poly (2-hydroxyethyl- inethacrylate), ethylene vinyl acetate, or poly-D( ⁇ )-3-hydroxybutyric acid.
  • Sustained release compositions may also include liposomes that can be prepared by any of several methods known in the art. [0300]
  • the compositions described herein may be administered locally or systemically. Administration will generally be parenteral administration. In certain embodiments, the pharmaceutical composition is administered subcutaneously and in other embodiments the pharmaceutical composition is administered intravenously.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • a therapeutically effective amount of active component for example, an antibody
  • a therapeutically effective amount of active component is in the range of 0.1 mg/kg to 100 mg/kg, e.g., 1 mg/kg to 100 mg/kg, 1 mg/kg to 10 mg/kg. It is understood, however, that the amount administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health of the patient, the in vivo potency of the antibody, the pharmaceutical formulation, and the route of administration.
  • the initial dosage can be increased beyond the upper level in order to rapidly achieve the desired blood- level or tissue-level.
  • the initial dosage can be smaller than the optimum, and the daily dosage may be progressively increased during the course of treatment.
  • Human dosage can be optimized, e.g., in a conventional Phase I dose escalation study. Dosing frequency can vary, depending on factors such as route of administration, dosage amount, serum half-life of the antibody, fusion protein, and/or antibody conjugate, and the disease being treated. Exemplary dosing frequencies are once per day, once per week and once every two weeks. V. THERAPEUTIC USES [0302] Once produced, the proteins and pharmaceutical compositions described herein can used in in a variety of therapies and therapeutic methods.
  • the proteins and pharmaceutical compositions described herein can be used in a method of treating a subject with a disease or disorder (e.g., cancer).
  • the method comprises administering to the subject an effective amount of an antibody of the present disclosure (e.g., any Attorney Docket No. BRI-023WO antibody described herein, including engineered antibodies such as conjugated antibodies) or a pharmaceutical composition containing such antibody.
  • the antibody can be an anti-CD276 antibody (e.g., an anti-CD276 antibody described herein).
  • the antibody can be engineered antibody (e.g., an engineered anti-CD276 antibody) containing an unnatural amino acid incorporated at one or more amino acid residue sites (e.g., one or more sites selected from Table 1).
  • a method of treating a subject with a cancer that overexpresses CD276 comprises administering to the subject an effective amount of an anti-CD276 antibody of the present disclosure (e.g., an engineered anti-CD276 antibody comprising one or more conjugated payload molecules) or a pharmaceutical composition containing such an antibody.
  • an anti-CD276 antibody of the present disclosure e.g., an engineered anti-CD276 antibody comprising one or more conjugated payload molecules
  • a pharmaceutical composition containing such an antibody e.g., an engineered anti-CD276 antibody comprising one or more conjugated payload molecules
  • a cancer treatable by an antibody or an engineered antibody of the present disclosure is a cancer characterized by elevated CD276 expression compared to a suitable control (e.g., a healthy cell or tissue, for example, a non-cancerous cell or tissue).
  • a suitable control e.g., a healthy cell or tissue, for example, a non-cancerous cell or tissue.
  • the cancer can be breast cancer, prostate cancer, ovarian cancer, endometrial cancer, sarcoma, melanoma, non-small cell lung cancer (NSCLC), Sq-ESO, bladder cancer, head and neck squamous cell carcinoma (HNSCC), hepatocellular carcinoma (HCC), salivary gland cancer, colon cancer, pancreatic cancer, renal cell carcinoma (RCC), gastric cancer, thyroid cancer, blood cancer (such as leukemia or lymphoma), blastoma, glioma, or neuroblastoma.
  • a subject can be treated with a protein-conjugate described herein (e.g., an engineered anti-CD276 antibody comprising one or more conjugated payload molecules) in combination therapy, for example, a combination therapy that includes one or more checkpoint inhibitors.
  • a protein-conjugate described herein e.g., an engineered anti-CD276 antibody comprising one or more conjugated payload molecules
  • combination therapy for example, a combination therapy that includes one or more checkpoint inhibitors.
  • EXAMPLES [0308] The following Examples are merely illustrative and are not intended to limit the scope or content of the disclosure in any way.
  • Attorney Docket No. BRI-023WO Example 1 Methods of Making Anti-CD276 Antibody Drug Conjugates
  • This example describes use of methods and compositions for making antibody drug conjugates that have certain desirable characteristics that allow for enhanced therapeutic efficacy. This example further describes methods for screening antibody drug conjugates for said desirable characteristics.
  • This example describes variations of protocols, e.g., as outlined in PCT/US2021/049953 and PCT/US2021/045088, for modifying an antibody to form an antibody conjugate, including use of unnatural amino acids, sites of incorporation within the antibody, and payload conjugation.
  • the antibody used for these experiments was the m276 anti-CD276 antibody (see Table 2).
  • Ifinatamab (referred to as “DS” within) is used in experiments as an anti-CD276 antibody (sequence 85 (HC) and 77 (LC) derived from US patent 9,371,395 B2).
  • m276 antibody containing an unnatural amino acid at selected positions was performed transiently in HEK293 cells. All monoclonal antibody expression was performed using the Expi293 Expression System according to the manufacturer’s instructions. Briefly, before transfection, cells were split to a density of 2.7 ⁇ 10 6 to 3.0 ⁇ 10 6 cells/ml. An unnatural amino acid, LCA, was added to achieve a final concentration of 0.25 to 0.5 mM.
  • plasmid mix (equal parts suppressor plasmid, m276 heavy chain plasmid, and m276 light chain plasmid) was used for transfection into 1 L cell culture.
  • Plasmids were diluted in 50 ml Opti-MEM medium. A PEI stock solution was incubated at room temperature for 3 minutes, subsequently diluted to achieve a 6:1 PEI:DNA final ratio, and incubated for 15 minutes at room temperature. The Plasmid:PEI complex was then added dropwise to the culture. At the time of transfection, 0.25 to 1 mM LCA was added to the cells.
  • BRI-023WO expression levels were achieved with unnatural amino acid incorporation at any site, and in some cases yields were within 60% or greater of WT expression, indicating efficient production in the mammalian cell-based expression system for UAA incorporation (see, e.g., FIG.4).
  • Conjugated antibodies 9-S1 through 9-S9 and 11-S1 through 11-S9 (see, e.g., FIG.5), were made according to the general scheme, A-U-U’-X1-L-X2-P (where A is an antibody or antigen binding fragment thereof, U is an unnatural amino acid bioconjugation handle, U’ is a crosslinking agent or crosslinker, X1 is a first spacer, L is a linking spacer, X2 is a second spacer (X2 not present in 11-S1 through 11-S9), and P is a payload), where the payload was selected from the payloads described herein, e.g., those listed in FIG.5 (including PBD, also referred to as SG3199, and Dxd, also referred to as DX8951), and represented structurally in FIG.16 and FIG.
  • purified antibody (m276 or DS) was buffer exchanged into PBS with 2mM EDTA, pH 7.4. The antibody was diluted to a final concentration of 1 mg/mL in PBS with 2mM EDTA and warmed to 37°C in a heat block.
  • a stock solution of TCEP (10 mM) was freshly prepared in water and 2.5 molar equivalents (relative to the antibody concentration) was added. After 2 hrs the partially reduced antibody was removed from the heat block and cooled to room temperature.
  • Antibody drug conjugates (ADCs) 9-S1 through 9-S9 and 11-S1 through 11-S9 were assayed via HIC, SDS-PAGE, SEC, plasma stability, and cathepsin cleavage to select suitable ADC candidates for downstream production (see, e.g., FIGs.6-10).
  • ADCs Antibody drug conjugates 9-S1 through 9-S9 and 11-S1 through 11-S9 were assayed via HIC, SDS-PAGE, SEC, plasma stability, and cathepsin cleavage to select suitable ADC candidates for downstream production (see, e.g., FIGs.6-10).
  • HIC data FIG.6, top
  • Mobile phase A was 1.5 M ammonium sulfate, 25 mM sodium phosphate dibasic, pH 7 and a linear gradient of Attorney Docket No. BRI-023WO mobile phase B (25 mM sodium phosphate, pH 7) was introduced. Samples were separated at a flow rate of 0.8 mL/min and at a column temperature of 25 o C. The average DAR and distribution profile were determined by peak area percentage of each species. Exemplary analyses conducted by HIC are shown in FIG.6 and a summary of certain ADCs analyzed is shown in FIG.10.
  • RRT relative retention time
  • FIGs.7A-7B show exemplary in vitro characterization for two ADCs, including Cathepsin B kinetic assays, mouse plasma stability, and human plasma stability using standard protocols. Briefly, human plasma or mouse plasma was spiked with a final ADC concentration of 0.2 mg/mL and subsequently incubated at 37 °C in 100 ⁇ L aliquots for up to 72 hours; aliquots were immediately flash frozen and stored at -80 °C until further analysis. For analysis, plasma- incubated ADCs were thawed and subjected to affinity capture with Protein G magnetic beads. The eluted samples containing purified incubated ADC were then analyzed by HIC as described previously.
  • FIG.7C-7F show mass spectra profiles for the heavy chain and light chain fragments of 9-S3, while FIGs.7G-7J show the same for 9-S9.
  • FIG.7C shows the full LC/UV chromatogram after PNGase reduction LCMS.
  • FIG.7D shows the deconvoluted mass spectrum of 9-S37.230 min peak with expected modified heavy chain mass.
  • FIG.7E shows the ESI Mass spectrum of 9-S37.230 min peak.
  • FIG.7F shows the deconvoluted mass spectrum of 9-S35.570 min peak with the correct unmodified light chain mass.
  • FIG.7G shows the full LC/UV chromatogram after PNGase reduction LCMS.
  • FIG.7H shows the deconvoluted mass spectrum of 9-S96.840 min peak with expected modified heavy chain mass.
  • FIG.7I is the ESI mass spectrum of 9-S95.580 min peak.
  • FIG.7J shows the deconvoluted mass spectrum of 9-S95.580 min peak with expected unmodified light chain. Results, as shown in FIGs.7A-7J and summarized in FIG.
  • the present example shows that click chemistry mediated site-specific conjugation may produce ADCs with improved biophysical characteristics as compared to their thiomab-generated site counterparts. Further, in ADCs conjugated using certain methods hydrophobicity (HIC RRT) is improved, and target DAR is better achieved.
  • HIC RRT hydrophobicity
  • Interchain cysteine conjugation was also utilized to produce comparator ADCs. These data are summarized in FIG.10, with interchain cysteine conjugates denoted as “m276 (6)”, “DS- PBD (17)”, “m276 (13)”, “m276 (14)”, “DS-Dxd (15)” and “DS-Dxd (16)”.
  • site-specific conjugation by click chemistry produces ADCs with improved biophysical characteristics as compared to their thiomab- generated site counterparts.
  • hydrophobicity HEC RRT
  • target DAR is better achieved
  • aggregation % monomer by SEC
  • stability is more preserved.
  • Sites S1, S3, S5, S7, and S9 were chosen for further cytotoxicity assays after evaluation of target criteria.
  • FIG.8 outlines target product profiles of ADCs produced for in vivo studies. Attorney Docket No.
  • Cytotoxicity assays were performed in HCT-116 (CD276+ colon cancer), Daudi (CD276 negative lymphoma), A549 (CD276+ Lung cancer), SKBR (CD276+ breast cancer) and OVCAR3 (CD276+ ovarian cancer) using HCT-116 (ATCC CCL-247), OVCAR3 (ATCC HTB- 161), A549 (ATCC CCL-185), and SKBR3 (ATCC HTB-30).
  • HCT-116 ATCC CCL-247
  • OVCAR3 ATCC HTB- 161
  • A549 ATCC CCL-185
  • SKBR3 ATCC HTB-30
  • ADC derivatives demonstrated efficacy at multiple conjugation sites, with ADCs with site-specific PBD payloads demonstrating enhanced efficacy in CD276+ cancer cell lines compared to ADCs with Dxd payloads.
  • Off-target toxicity is equivalent for ADCs with site-specific PBD payloads as compared to ADCs with Dxd payload in DAUDI cells that lack CD276 expression (see FIG.11).
  • ADCs with PBD payloads demonstrated improved therapeutic index over ADCs with Dxd payloads conjugated at the same DAR of 2.
  • the efficacy of PBD conjugated ADCs were demonstrated across a range of cell lines representing various indications (see FIG.12).
  • the m276 antibody conjugates tested provide an increased therapeutic window over other anti-CD276 antibodies, by way of example, in comparison to Ifinatamab (“DS”), when the same DAR and payload are used.
  • m276 and Ifinatamab (“DS”) were conjugated with interchain cysteine compatible compound 8 (MA-PEG8-VA-SG3199) (FIG.13).
  • Example 2 Use of Anti-CD276 Antibody Conjugates in vivo
  • This example describes the assessment of antibody compositions generated in Example 1 for anti-tumor activity and toxicity in vivo.
  • Anti-Tumor Activity [0323] To investigate the anti-tumor activity of different provided ADC compositions, female 10 to 16-week old athymic NCr-nu/nu mice (Charles Rivers) that also carried the carboxylesterase 1C (Ces1c/ES1) mutation (B6(Cg)-Ces1ctm1.1Loc/J, The Jackson Laboratory, Stock No: 014096) were injected with HCT-116 cancer cells subcutaneously into the flank on day 0.
  • BRI-023WO At a high dose (0.5 mg/kg for PBD-based ADCs, or 10 mg/kg for Dxd-based ADC), tumor growth rate is delayed with all drug compositions, but was most pronounced with site- specific PBD-based ADC, 9-S3. Following administration at a low dose (0.1 mg/kg for PBD-based ADCs, or 3 mg/kg for Dxd-based ADC), the site-specific PBD-based ADC, 9-S3, is most effective at tumor regression. Taking aggregate results from both low and high dose groups, 9-S3 also displays the highest rate of tumor regression and complete responses (CR) (FIGs.24-25).
  • mice included in efficacy studies discussed herein were monitored for changes in body weight beginning after the first ADC administration and presented as the mean ⁇ SEM.
  • PBD-based site-specific conjugates 9-S3 and 9-S9 dosed at 0.5 mg/kg display maintenance of body weight similar to Vehicle control or 10 mg/kg of Ifinatamab deruxtecan (DS-Dxd, “16”), an ADC known to have low toxicity, indicating no toxicity is observed for PBD-based site-specific ADCs (9-S3 and 9-S9) as compared to PBD-based cysteine-conjugated ADC (6) that causes toxicity and a decrease in mouse body weight after administration.
  • DS-Dxd Ifinatamab deruxtecan
  • the results show that at a low dose (0.1 mg/kg) and high dose (0.5 mg/kg), tumor growth rate was delayed when administering m276 antibody interchain-cysteine PBD-based ADC (“6”) (FIG.27).
  • DS Ifinatamab
  • 17 core antibody

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Abstract

The invention relates generally to engineered anti-CD276 antibodies containing one or more unnatural amino acids (UAAs), their conjugation with a payload to generate an antibody-payload (e.g., drug) conjugate, drug substances containing such a conjugate, and methods of making and using such a conjugate.

Description

Attorney Docket No. BRI-023WO CD276 (B7-H3) ANTIBODY-DRUG CONJUGATES CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Patent Application No.63/418,730, filed October 24, 2022, which is hereby incorporated by reference in its entirety for all purposes. BACKGROUND [0002] The ability to produce homogenously modified proteins has been the subject of considerable research in the field, which has resulted in a variety of methods for creating proteins with differing characteristics with respect to site specificity, site selection, homogeneity, and whether these proteins are produced in vitro or in vivo. Efforts to specifically modify proteins expressed from eukaryotic cells are becoming increasingly popular, as eukaryotic cell expression has emerged as the standard for expression of therapeutically relevant proteins. However, challenges remain, in particular with respect to producing drug conjugated biologics, such as antibody-drug conjugates (ADCs). Indeed, obstacles in designing effective ADCs have historically included low blood residency time, low penetration capacity to tumor microenvironment, variable antibody-drug ratios, low payload potency, immunogenicity, off-target toxicity, drug resistance, and lack of stable drug linkage in blood circulation, among many other linker associate problems. [0003] Despite the efforts that have been made today, there remains a need for effective methods and compositions for producing effective ADCs. SUMMARY OF THE DISCLOSURE [0004] The disclosure described herein is based in part, upon the discovery of an anti-CD276 antibody containing one or more unnatural amino acids at one or more specific locations in an immunoglobulin heavy chain and/or immunoglobulin light chain of the anti-CD276 antibody that can be used to conjugate one or more payloads, e.g., one or more therapeutic molecules, to the antibody. A payload can be linked directly to the unnatural amino acid or indirectly to the unnatural amino acid by a linker. The resulting antibody conjugates (e.g., antibody-drug conjugate) can be used in methods for treating a disorder, e.g., cancer, associated with expression of elevated levels of CD276. [0005] In a first aspect, the disclosure provides, among other things, an engineered anti-CD276 antibody comprising: a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to one or more of: (i) A142 Attorney Docket No. BRI-023WO of SEQ ID NO: 61; (ii) T157 of SEQ ID NO: 61; (iii) T171 of SEQ ID NO: 61; (iv) A174 of SEQ ID NO: 61; (v) Y182 of SEQ ID NO: 61; (vi) T197 of SEQ ID NO: 61; (vii) D267 of SEQ ID NO: 61; or (viii) S241 of SEQ ID NO: 61. [0006] In certain embodiments of the first aspect, the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to one or more of: (i) A142 of SEQ ID NO: 61; (ii) T157 of SEQ ID NO: 61; (iii) T171 of SEQ ID NO: 61; (iv) A174 of SEQ ID NO: 61; (v) Y182 of SEQ ID NO: 61; (vi) T197 of SEQ ID NO: 61; (vii) D267 of SEQ ID NO: 61; or (viii) S241 of SEQ ID NO: 61. In one embodiment, the unnatural amino acid is incorporated at a position corresponding to T171 of SEQ ID NO: 61. [0007] In certain other embodiments of the first aspect, the antibody further comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 62. In certain embodiments, the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 62. In certain embodiments, the antibody further comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 62, with an unnatural amino acid incorporated at a position corresponding to Q162 of SEQ ID NO: 62. In certain embodiments, the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to Q162 of SEQ ID NO: 62. [0008] In a second aspect, the disclosure provides, an engineered anti-CD276 antibody comprising: a light chain sequence, wherein the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 62, with an unnatural amino acid incorporated at a position corresponding to Q162 of SEQ ID NO: 62. In certain embodiments, the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 62, with an unnatural amino acid incorporated at a position corresponding to Q162 of SEQ ID NO: 62. [0009] In certain embodiments of the second aspect, the antibody further comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at Attorney Docket No. BRI-023WO least 99%, or greater sequence identity to SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to one or more of: (i) A142 of SEQ ID NO: 61; (ii) T157 of SEQ ID NO: 61; (iii) T171 of SEQ ID NO: 61; (iv) A174 of SEQ ID NO: 61; (v) Y182 of SEQ ID NO: 61; (vi) T197 of SEQ ID NO: 61; (vii) D267 of SEQ ID NO: 61; or (viii) S241 of SEQ ID NO: 61. [0010] In certain embodiments, the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to one or more of: (i) A142 of SEQ ID NO: 61; (ii) T157 of SEQ ID NO: 61; (iii) T171 of SEQ ID NO: 61; (iv) A174 of SEQ ID NO: 61; (v) Y182 of SEQ ID NO: 61; (vi) T197 of SEQ ID NO: 61; (vii) D267 of SEQ ID NO: 61; (viii) S241 of SEQ ID NO: 61. In certain embodiments, the unnatural amino acid is incorporated at a position corresponding to T171 of SEQ ID NO: 61. [0011] In a third aspect, the disclosure provides, among other things, an engineered anti-CD276 antibody comprising: a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 137, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 138, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 139, and wherein the heavy chain and/or light chain comprises an unnatural amino acid incorporated at a position corresponding to one or more amino acid residue positions selected from TABLE 1. [0012] In a fourth aspect, the disclosure provides, among other things, an engineered anti- CD276 antibody comprising: a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 137, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 138, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 139, and wherein the heavy chain comprises an unnatural amino acid incorporated at a position corresponding to one or more of: (i) A142 of SEQ ID NO: 61; (ii) T157 of SEQ ID NO: 61; (iii) T171 of SEQ ID NO: 61; Attorney Docket No. BRI-023WO (iv) A174 of SEQ ID NO: 61; (v) Y182 of SEQ ID NO: 61; (vi) T197 of SEQ ID NO: 61; (vii) D267 of SEQ ID NO: 61; or (viii) S241 of SEQ ID NO: 61. [0013] In a fifth aspect, the disclosure provides, among other things, an engineered anti-CD276 antibody comprising: a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 137, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 138, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 139, and wherein the heavy chain comprises an unnatural amino acid incorporated at a position corresponding to T171 of SEQ ID NO: 61. [0014] In certain embodiments of aspects one through five, the antibody comprises at least one, at least two, at least three, at least four, at least five, or more unnatural amino acids. For example, the unnatural amino acid can be a tryptophan analog, a leucine analog, a tyrosine analog, or a pyrrolysine analog. In certain embodiments, the unnatural amino acid is a tryptophan analog (e.g., 5-HTP or 5-AzW). In certain embodiments, the unnatural amino acid is a leucine analog (e.g., LCA or Cys-5-N3, or LCA). In certain embodiments, the unnatural amino acid is a tyrosine analog (e.g., OmeY, AzF, or OpropY). In certain embodiments, the unnatural amino acid is a pyrrolysine analog (e.g., BocK, CpK, or AzK). [0015] In certain embodiments of aspects one through five, the unnatural amino acid is chemically modified. The chemical modification can comprise the unnatural amino acid conjugated to a molecule of interest, for example, a detectable label or a therapeutic molecule. In certain embodiments, the therapeutic molecule is: AEB, AEVB, AFP, an amatoxin, an auristatin (e.g., auristatin E), a calicheamicin, CC-1065 or a CC-1065 analog, chalicheamicin, combretastatin, docetaxel, dolastatin-10, DUBA, a duocarmycin, echinomycin, FAM, maytansine, a maytansinoid (e.g. DM1 or DM4), MMAD, MMAE, MMAF, a morpholino-doxorubicin (e.g., cyanomorpholino- doxorubicin), netropsin, an oligonucleotide (e.g., a DNA, RNA, or LNA oligonucleotide), paclitaxel, pyrrolobenzodiazepine dimer (PBD), a peptide (e.g., a therapeutic peptide), rhizoxin, a small molecule (e.g., a therapeutic small molecule), SN-38, topotecan, a topoisomerase inhibitor, or a toxoid. In certain embodiments, the therapeutic molecule is PBD. Attorney Docket No. BRI-023WO [0016] In certain embodiments of the foregoing aspects, the molecule is conjugated to the antibody by a crosslinking agent. Exemplary crosslinking agents can include bissuflosuccinimidyl suberate (BS3), N-hydroxy-succinimide/1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (NHS/EDC), N-ε-maleimidocaproyl-oxysulfosuccinimide ester (sulfoEMCS), N-ε- maleimidocaproic acid hydrazide (EMCH), hydrazide, N-succinimidyl-S-acetylthioacetate (SATA), monofluorocyclooctyne (MFCO), bicyclo[6.1.0]nonyne (BCN), N succinimidyl-S- acetylthiopropionate (SATP), a maleimido ester, a dibenzocyclooctyne (DBCO) ester, EDC, or any combination thereof. [0017] In certain embodiments of the foregoing aspects, the molecule is conjugated to the antibody by a linker, which can contain a cleavable linker region or a non-cleavable linker region, and optionally, one or more spacer regions. In certain embodiments, the cleavable or non-cleavable linker region is a peptide-linker region. In some embodiments, the peptide-linker region comprises a VA linker or a GGFG linker. In certain embodiments, the spacer region comprises a PEG-based region (e.g., a PEG8 region). [0018] In certain embodiments, the linker conjugates the molecule to the antibody via a cross- linking agent. Exemplary cross-linking agents include bissuflosuccinimidyl suberate (BS3), N- hydroxy-succinimide/1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (NHS/EDC), N-ε- maleimidocaproyl-oxysulfosuccinimide ester (sulfoEMCS), N-ε-maleimidocaproic acid hydrazide (EMCH), hydrazide, N-succinimidyl-S-acetylthioacetate (SATA), monofluorocyclooctyne (MFCO), bicyclo[6.1.0]nonyne (BCN), N succinimidyl-S-acetylthiopropionate (SATP), a maleimido ester, a dibenzocyclooctyne (DBCO) ester, EDC, or any combination thereof. [0019] In certain embodiments, the cross-linking agent or the linker conjugates the molecule to the antibody at the unnatural amino acid site. [0020] In certain embodiments of each of the foregoing aspects and embodiments, (a) the antibody has an average drug-to-antibody ratio (DAR) of at least 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0, as measured by hydrophobic interaction chromatography (HIC); (b) the antibody has an average DAR of about 2, as measured by HIC; (c) at least 40%, 50%, 60%, 70%, 80%, or 90% of the antibody remains following incubation in human plasma for 72 hours at 37 °C; (d) at least 40%, 50%, 60%, 70%, 80%, or 90% of the antibody remains following incubation with Cathepsin B for 240 minutes at 37 °C; (e) the antibody has a binding affinity for a target antigen of about 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.75 nM, 0.5 nM, 0.1 nM, 0.075 nM, or 0.05 nM or lower, as measured by enzyme-linked immunosorbent assay (ELISA); and/or (f) the antibody has a binding affinity for Attorney Docket No. BRI-023WO CD276, or a derivative or variant thereof, that is within 0.1 fold, 0.2 fold, 0.3 fold, 0.4 fold, 0.5 fold, 1.0 fold, 1.5 fold, 2.0 fold, 3.0 fold, 4.0 fold, 5.0 fold, 6.0 fold, 8.0 fold, or 10.0 fold of the binding affinity of a suitable reference antibody as measured by ELISA, wherein the suitable reference antibody is an otherwise identical antibody that does not comprise the unnatural amino acid. [0021] In certain embodiments of the foregoing aspects and embodiments, the antibody is derived from an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the antibody is derived from an IgG1 antibody. [0022] In a sixth aspect, the disclosure provides a method of making an antibody as described herein. The method comprising a step of culturing a cell comprising: (i) a first nucleotide sequence encoding a tRNA comprising an anticodon that hybridizes to a codon selected from UAG, UGA, and UAA, and is capable of being charged with an unnatural amino acid; (ii) a second nucleotide sequence encoding an aminoacyl-tRNA synthetase capable of charging the tRNA with the unnatural amino acid; and (iii) a third nucleotide sequence encoding the heavy chain sequence and/or a fourth nucleotide sequence encoding the light chain sequence, wherein the third nucleotide sequence and/or the fourth nucleotide sequence comprise a codon selected from UAG, UGA, and UAA; wherein the cell is cultured under conditions that permit the tRNA, when expressed in the cell and charged with the unnatural amino acid, to hybridize to the codon and direct incorporation of the unnatural amino acid into the antibody. [0023] In a seventh aspect, the disclosure provides a method of making an engineered anti-CD276 antibody. The method comprising a step of culturing a cell comprising: (i) a first nucleotide sequence encoding a tRNAs comprising an anticodon that hybridizes to a codon selected from UAG, UGA, and UAA, and is capable of being charged with an unnatural amino acid; (ii) a second nucleotide sequence encoding an aminoacyl-tRNA synthetase capable of charging the tRNA with the unnatural amino acid; and (iii) a third nucleotide sequence encoding a heavy chain sequence as set forth in SEQ ID NO: 61 and/or a fourth nucleotide sequence encoding a light chain sequence as set forth in SEQ ID NO: 62, wherein the third nucleotide sequence and/or the fourth nucleotide sequence comprise a codon selected from UAG, UGA, and UAA; wherein the cell is cultured under conditions that permit the tRNA, when expressed in the cell and charged with the unnatural amino acid, to hybridize to the codon and direct incorporation of the unnatural amino acid into the antibody. Attorney Docket No. BRI-023WO [0024] In certain embodiments of the seventh aspect, the third nucleotide sequence and/or the fourth nucleotide sequence comprise a codon selected from UAG, UGA, and UAA at a site that corresponds to any site shown in FIG.5 or Table 1. [0025] In each of the foregoing methods, (a) the amount of the antibody comprising the unnatural amino acid expressed by the cell is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the amount of a suitable reference antibody expressed by the same cell or a similar cell; and/or (b) following purification of the antibody, less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the antibody is aggregated, as measured by size exclusion chromatography (SEC). [0026] In certain embodiments of the foregoing methods, the tRNA is an analog or derivative of a prokaryotic tryptophanyl-tRNA (e.g., E. coli tryptophanyl-tRNA). In certain embodiments, the nucleotide sequence encoding the tRNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 56-60. [0027] In certain embodiments of the foregoing methods, the aminoacyl-tRNA synthetase is an analog or derivative of a prokaryotic tryptophanyl-tRNA synthetase (e.g., E. coli tryptophanyl- tRNA synthetase). In certain embodiments, the aminoacyl-tRNA synthetase comprises an amino acid sequence selected from any one of SEQ ID NOs: 51-54. [0028] In certain embodiments of the foregoing methods, the tRNA is an analog or derivative of a prokaryotic leucyl-tRNA (e.g., an E. coli leucyl-tRNA. In certain embodiments, the nucleotide sequence encoding the tRNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 22-49. [0029] In certain embodiments of the foregoing methods, the aminoacyl-tRNA synthetase is an analog or derivative of a prokaryotic leucyl-tRNA synthetase (e.g., an E. coli leucyl-tRNA synthetase). In certain embodiments, the aminoacyl-tRNA synthetase comprises an amino acid sequence selected from any one of SEQ ID NOs: 8-21. [0030] In certain embodiments of the foregoing methods, the tRNA is an analog or derivative of a prokaryotic tyrosyl-tRNA (e.g., an E. coli tyrosyl-tRNA). In some embodiments, the nucleotide sequence encoding the tRNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 140-144, 192, or 193. [0031] In certain embodiments of the foregoing methods, the aminoacyl-tRNA synthetase is an analog or derivative of the prokaryotic tyrosyl-tRNA synthetase (e.g., an E. coli tyrosyl-tRNA synthetase). In certain embodiments, the aminoacyl-tRNA synthetase comprises an amino acid sequence selected from any one of SEQ ID NOs: 145 or 147. Attorney Docket No. BRI-023WO [0032] In certain embodiments of the foregoing methods, the tRNA is an analog or derivative of an archael pyrrolysyl-tRNA (e.g., an M. barkeri pyrrolysyl-tRNA). In certain embodiments, the nucleotide sequence encoding the tRNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 148-178, 181, or 182. [0033] In certain embodiments of the foregoing methods, the aminoacyl-tRNA synthetase is an analog or derivative of the archael pyrrolysyl-tRNA synthetase (e.g., an M. barkeri pyrrolysyl- tRNA synthetase). In certain embodiments, the aminoacyl-tRNA synthetase comprises an amino acid sequence selected from any one of SEQ ID NO: 179. [0034] In certain embodiments of the foregoing methods, the reference antibody comprises a wild- type amino acid residue at the position corresponding to the unnatural amino acid. In certain embodiments, the cell is a mammalian cell (e.g., a human cell). In certain embodiments, the mammalian cell is a human embryonic kidney (HEK) cell or a Chinese hamster ovary (CHO) cell. [0035] In certain embodiments of the foregoing methods, the method further comprises a step of purifying the antibody. Alternatively or in addition, the method further comprises a step of chemically modifying the unnatural amino acid. The chemical modification can comprise conjugation to a molecule (e.g., a detectable label or a therapeutic molecule such as AEB, AEVB, AFP, an amatoxin, an auristatin (e.g., auristatin E), a calicheamicin, CC-1065 or a CC-1065 analog, chalicheamicin, combretastatin, docetaxel, dolastatin-10, DUBA, a duocarmycin, echinomycin, FAM, maytansine, a maytansinoid (e.g., DM1 or DM4), MMAD, MMAE, MMAF, a morpholino- doxorubicin (e.g., cyanomorpholino-doxorubicin), netropsin, an oligonucleotide (e.g., a DNA, RNA, or LNA oligonucleotide), paclitaxel, PBD, a peptide (e.g., a therapeutic peptide), rhizoxin, a small molecule (e.g., a therapeutic small molecule), SN-38, topotecan, a topoisomerase inhibitor, or a toxoid. In certain embodiments, the therapeutic molecule is PBD. [0036] In certain embodiments, the molecule is conjugated to the antibody by a crosslinking agent. Exemplary crosslinking agents include bissuflosuccinimidyl suberate (BS3), N-hydroxy- succinimide/1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (NHS/EDC), N-ε-maleimidocaproyl- oxysulfosuccinimide ester (sulfoEMCS), N-ε-maleimidocaproic acid hydrazide (EMCH), hydrazide, N-succinimidyl-S-acetylthioacetate (SATA), monofluorocyclooctyne (MFCO), bicyclo[6.1.0]nonyne (BCN), N succinimidyl-S-acetylthiopropionate (SATP), a maleimido ester, a dibenzocyclooctyne (DBCO) ester, EDC, or any combination thereof. [0037] In certain embodiments, the molecule is conjugated to the engineered antibody by a linker, which can comprise a cleavable or non-cleavable linker region, and optionally, one or more spacer regions. In certain embodiments, the cleavable or non-cleavable linker region is a peptide-linker Attorney Docket No. BRI-023WO region. In certain embodiments, the peptide-linker region comprises a VA linker or a GGFG linker. In certain embodiments, the spacer region comprises a PEG-based domain (e.g., a PEG8 region). [0038] In certain embodiments, the linker conjugates the molecule to the antibody via a cross- linking agent. In certain embodiments, the cross-linking agent comprises bissuflosuccinimidyl suberate (BS3), N-hydroxy-succinimide/1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (NHS/EDC), N-ε-maleimidocaproyl-oxysulfosuccinimide ester (sulfoEMCS), N-ε- maleimidocaproic acid hydrazide (EMCH), hydrazide, N-succinimidyl-S-acetylthioacetate (SATA), monofluorocyclooctyne (MFCO), bicyclo[6.1.0]nonyne (BCN), N succinimidyl-S- acetylthiopropionate (SATP), a maleimido ester, a dibenzocyclooctyne (DBCO) ester, EDC, or any combination thereof. In certain embodiments, the cross-linking agent or the linker conjugates the molecule to the antibody at the unnatural amino acid site. [0039] In an eight aspect, the disclosure provides an engineered anti-CD276 antibody comprising: a heavy chain sequence comprising an amino acid sequence as set forth in SEQ ID NO: 61, with an LCA incorporated at a position corresponding to T171 of SEQ ID NO: 61; a light chain sequence comprising an amino acid sequence as set forth in SEQ ID NO: 62; wherein a pyrrolobenzodiazepine dimer (PBD) is conjugated to the LCA via a linker and crosslinking agent, wherein the linker comprises a PEG8 spacer region and a VA linker region and the crosslinking agent is DBCO. Such an engineered anti-CD276 antibody can be produced by a method comprising a step of culturing a cell comprising: (i) a first nucleotide sequence encoding a tRNAs comprising an anticodon that hybridizes to a codon selected from UAG, UGA, and UAA, and is capable of being charged with an LCA; (ii) a second nucleotide sequence encoding an aminoacyl- tRNA synthetase capable of charging the tRNA with the LCA; and (iii) a third nucleotide sequence encoding the heavy chain sequence and a fourth nucleotide sequence encoding the light chain sequence, wherein the third nucleotide sequence comprises a codon selected from UAG, UGA, and UAA at a site corresponding to T171 of SEQ ID NO: 61; wherein the cell is cultured under conditions that permit the tRNA, when expressed in the cell and charged with the LCA, to hybridize to the codon and direct incorporation of the LCA. [0040] In a nineth aspect, the disclosure provides an engineered anti-CD276 antibody comprising: an immunoglobulin heavy chain sequence comprising an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to one or more sites selected from TABLE 1 or FIG.5; and an immunoglobulin light chain sequence comprising an amino acid sequence with at least 75%, at Attorney Docket No. BRI-023WO least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 62, with an unnatural amino acid incorporated at a position corresponding to one or more sites selected from TABLE 1 or FIG.5. In certain embodiments of the nineth aspect, the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to one or more sites selected from Table 1 or FIG.5; and the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 62, with an unnatural amino acid incorporated at a position corresponding to one or more sites selected from TABLE 1 or FIG.5. In certain embodiments, the at least one unnatural amino acid is: (a) a leucine analog, a tyrosine analog, a tryptophan analog, or a pyrrololysine analog; (b) 5-HTP or 5-AzW; (c) LCA or Cys-5-N43; (d) LCA; (e) OmeY, AzF, or OpropY; and/or (f) BocK, CpK, or AzK. [0041] In certain embodiments, the unnatural amino acid is chemically modified, wherein the chemical modification comprises conjugation of the unnatural amino acid to a molecule: (a) wherein the molecule is a detectable label; (b) wherein the molecule is a therapeutic molecule; (c) wherein the molecule is conjugated to the antibody by a crosslinking reagent; (d) wherein the molecule is conjugated to the antibody by a linker; (e) wherein the molecule is conjugated to the antibody by a linker forming a linker-payload structure of U’-X1-L-X2-P, where U’ is DBCO, X1 is PEG8, L is VA, X2 is PAB, and P is PBD, and DBCO crosslinks the linker-payload to the unnatural amino acid; and/or (f) wherein the molecule is conjugated to the antibody by a linker forming a linker-payload structure of U’-X1-L-P, where U’ is DBCO, X1 is PEG4, L is GGFG, and P is Dxd, and DBCO crosslinks the linker-payload to the unnatural amino acid. [0042] In a tenth aspect, the disclosure provides a pharmaceutical composition comprising an engineered antibody as described herein, and optionally comprising one or more pharmaceutically acceptable excipients or carriers. [0043] In an eleventh aspect, the disclosure provides a method of treating (a) a subject with a disease or a disorder, the method comprising administering to the subject an effective amount of an engineered antibody as described herein, a pharmaceutical composition as described herein, or an antibody made by a method described herein, (b) a subject with a cancer that overexpresses CD276, the method comprising administering to the subject an effective amount of an engineered antibody as described herein, a pharmaceutical composition as described herein, or an antibody made by a method as described herein, or (c) a subject with CD276 positive tumor vasculature, the method comprising administering to the subject an effective amount of an engineered antibody as described Attorney Docket No. BRI-023WO herein, a pharmaceutical composition as described herein, or an antibody made by a method as described herein. BRIEF DESCRIPTION OF THE DRAWINGS [0044] FIG.1 depicts a sequence alignment between an exemplary anti-CD276 antibody heavy chain amino acid sequence (SEQ ID NO: 61) that may be used in accordance with the present disclosure, and, for sequence comparison purposes, a Trastuzumab heavy chain amino acid sequence (SEQ ID NO: 183). [0045] FIG.2 depicts a sequence alignment between an exemplary anti-CD276 antibody light chain amino acid sequence (SEQ ID NO: 62) that may be used in accordance with the present disclosure, and, for sequence comparison purposes, a Trastuzumab light chain amino acid sequence (SEQ ID NO: 184). [0046] FIGs.3A-3E depict exemplary unnatural amino acids (UAAs) that may be used in accordance with the present disclosure. [0047] FIG.4 depict graphs showing % wild-type expression for certain UAA-containing antibodies generated in accordance with the present disclosure. [0048] FIG.5 depicts a list of exemplary ADCs generated in accordance with the present disclosure. [0049] FIG.6 depict exemplary QC profiles using biophysical assays for certain ADCs generated in accordance with the present disclosure. [0050] FIGs.7A-7J depict exemplary QC profiles using stability, cathepsin cleavage assays, and LCMS assays for certain ADCs generated in accordance with the present disclosure. [0051] FIG.8 depicts an analysis of drug-to-antibody ratio (DAR) in certain ADCs via LCMS. [0052] FIG.9 depicts an exemplary broad site-select screening panel for certain ADCs generated in accordance with the present disclosure. Panel assays included HIC, SDS-PAGE, SEC, DAR assessment, yield, human plasma stability, mouse plasma stability, and cathepsin cleavage. [0053] FIG.10 depicts an exemplary full site-panel of certain ADCs generated in accordance with the present disclosure. Panel assays included HIC, SDS-PAGE, SEC, DAR assessment, yield, human plasma stability, mouse plasma stability, and cathepsin cleavage. [0054] FIGs.11A-11B depict graphs showing cytotoxicity of certain ADCs in CD276+ cells and CD276- cells. Attorney Docket No. BRI-023WO [0055] FIG.12 depicts graphs showing cytotoxicity of certain ADCs in lung cancer cells, breast cancer cells, and ovarian cancer cells. [0056] FIG.13 depicts a graph showing cytotoxicity of certain ADCs in CD276+ cells and CD276- cells. [0057] FIG.14 depicts a schematic overview of genetic code expansion and unnatural amino acid (UAA) incorporation. [0058] FIG.15 depicts exemplary auristatin payloads that may be used in accordance with the present disclosure. [0059] FIG.16 depicts exemplary pyrrolobenzodiazepine (PBD) payloads that may be used in accordance with the present disclosure. [0060] FIG.17 depicts exemplary camptothecin payloads that may be used in accordance with the present disclosure. [0061] FIG.18 depicts exemplary branched payload adapters that may be used in accordance with the present disclosure. [0062] FIGs.19A-19C depict exemplary PBD structures that may be used in accordance with the present disclosure. Structures depicted are adapted from Angew. Chem. Int. Ed.2017, 56, 462-488. [0063] FIG.20 depicts exemplary PBD cores that may be used in accordance with the present disclosure. [0064] FIG.21 depicts an exemplary PBD based ADC connectivity that may be used in accordance with the present disclosure. [0065] FIGS.22A-B depict an exemplary pro-PBD based ADC connectivity that may be used in accordance with the present disclosure. [0066] FIGS.23A-23B depict an exemplary connectivity to ADCs via click chemistry that may be used in accordance with the present disclosure. [0067] FIG.24 depicts a table and graphs showing efficacy of certain ADCs in a HCT116 CDX in vivo mouse model. [0068] FIG.25 depicts graphs showing tumor regression in a HCT116 CDX in vivo mouse model. [0069] FIG.26 depicts a graph showing body weight of mice treated with certain ADCs at different time points. Attorney Docket No. BRI-023WO [0070] FIG.27 depicts a graph showing tumor volume reduction efficacy of certain interchain cysteine conjugate ADCs in a HCT116 CDX in vivo mouse model. [0071] FIG.28 depicts a graph showing differential cathepsin B cleavage rate as a function of site of conjugation in certain ADCs [0072] FIGs.29A-29B depict exemplary an connectivity between an exemplary antibody, e.g., an anti-CD276 antibody provided herein, containing an unnatural amino acid (A-U) modified with a linker-payload as described herein (e.g., U’-X1-L-X2-P). FIG.29A represents a schematic representation of an antibody with an exemplary payload attached, and FIG 29B represents an unnatural amino acid (with peptide bonds connecting the unnatural amino acid to the adjacent amino acids in remainder of the immunoglobulin chain, e.g., the immunoglobulin heavy chain, shown with wavy lines) with the payload attached. DETAILED DESCRIPTION [0073] The disclosure described herein is based in part, upon the discovery of an anti-CD276 antibody containing one or more unnatural amino acids at one or more specific locations in an immunoglobulin heavy chain and/or immunoglobulin light chain of the anti-CD276 antibody that can be used to conjugate one or more payloads, e.g., one or more therapeutic molecules, to the antibody. A payload can be linked directly to the unnatural amino acid or indirectly to the unnatural amino acid by a linker. The resulting antibody conjugates (e.g., antibody-drug conjugate) can be used in methods for treating a disorder, e.g., cancer, associated with expression of elevated levels of CD276. [0074] The approach described herein involves a method of modifying proteins via the site- specific incorporation of one or more unnatural amino acids (also referred to as UAAs) into a protein of interest in vivo. The ability to site-specifically incorporate unnatural amino acids (e.g., non-natural amino acids, non-canonical amino acids, nonstandard amino acids, etc.) into proteins in vivo is a powerful tool that can be used to augment protein function or introduce new chemical functionalities not found in nature, particularly for protein production or the production of novel therapeutics, biological reagents, or other protein subclasses. The core elements used to implement this technology include: an engineered tRNA/aminoacyl-tRNA synthetase (aaRS) pair and a unique codon directing the incorporation of the unnatural amino acid. An engineered tRNA/aaRS pair can derived from a tRNA/aaRS pair obtained from a different organism than that used as an expression host (to maintain orthogonality, and minimize cross-reactivity) that are imported into the desired expression host (e.g., a cell, such as a eukaryotic cell). Attorney Docket No. BRI-023WO [0075] The tRNA/aaRS pairs can be engineered to incorporate an unnatural amino acid to a site- specific locus in a protein of interest at the direction of the charged tRNA-unnatural amino acid complex towards a unique codon, such as stop codons (e.g., TAG, TGA, TAA, etc.), four-base frameshift codons, sense codons, or non-canonical codons. This technology has been established for a variety of tRNA/aaRS pairs for some of the 20 natural amino acids (e.g., E. coli TrpRS/tRNA, E. coli LeuRS/tRNA, E. coli TyrRS/tRNA, M. barkeri PylRS/tRNA, M. mazei PylRS/tRNA, etc.), facilitating expression of proteins containing site specific modifications such as the introduction of bioconjugation handles, small fluorescent/redox probes, photo-crosslinkers, post-translational modifications (PTMs), and photocaged amino acids. A variety of orthogonal tRNA/aaRS pairs have been produced for certain of the naturally occurring amino acids (see, e.g., U.S. Patent Publication US2017/0349891 A1, and Zheng et al. (2018) BIOCHEM.57:441-445). Many of these types of technologies can be applied to therapeutic proteins (e.g., antibody-drug conjugates (ADCs), bi-specific monoclonal antibodies (mAbs), nanobodies, chemokines, vaccines, coagulation factors, hormones, enzyme therapies, etc.). [0076] It has become apparent that antibody and protein conjugates have emerged as an effective method to improve therapeutic characteristics of biologics. The conjugation of payloads (e.g., drugs, oligos, half-life extenders, and other molecules) to biologics such as antibodies, cytokines, peptides, and other protein biologics have introduced a variety of novel functions and enhanced activity. The present disclosure is based, in part, upon the recognition that the site of conjugation can make drastic differences to molecular characteristics such as biophysical properties, PK/PD, efficacy, as well as the logistical challenges such as yield, conjugation efficiency, and modularity. In particular, the site-specific conjugation of cytotoxic payloads to targeting antibodies has been shown to increase the therapeutic window of these molecules. Furthermore, the site and location of conjugation can have a major impact on the overall characteristics of an ADC. For example, site and location of conjugation have been found to impact properties that prevent non-specific uptake (Mahalingaiah, PK. et al., (2019) PHARMACOL. THER.200:110-125), off-target and on-target adverse events, hydrophobicity, aggregation propensity (Lyon, RP. et al., (2015) NAT. BIOTECHNOL.33:733-735), and stability of the overall structural integrity of the antibody (Su, D. and Zhang, D., (2021) FRONT. PHARMCOL.23:12:687926; Ohri, R. et al., (2018) BIOCONJ. CHEM. 29:473-485). Site and location of conjugation can also tune the activity of the payload by affecting, or adjusting the cleavage rate (Tumey, NL. et al., (2017) AAPS J., 19:1123-1135). [0077] It has been found that CD276 is overexpressed in many solid tumors, but has minimal to no expression in healthy tissues. CD276 (also known as 4Ig-B7-H3, B7 homolog 3 costimulatory molecule, and B7-H3) is a transmembrane protein of the B7 family (e.g., PD-L1, etc.) which Attorney Docket No. BRI-023WO includes many immune checkpoint molecules. mRNA expression is broadly observed, but protein expression is low in healthy tissues and organs. The physiological function of CD276 is still under investigation but the receptor has implications towards stimulation of T-cells, as well as influencing tumor-infiltration, cancer progression, migration, and other tumorigenic functions. The present disclosure appreciates that not only is CD276 expressed on tumor cells, but it is also expressed heavily on the tumor vasculature and cancer initiating cells, of which targeted cell death could prevent future tumor relapse (Crispen, PL. et al., (2008) CLIN. CANCER RES.14:5150-5157). CD276 is an attractive target as a cancer therapy for which 50+ clinical trials have commenced, some progressing to Phase III. The broad expression profile of CD276 in malignant tissue has prompted investigation into therapeutic modalities across many different solid cancers, such as lung, esophageal, sarcomas, endometrial, prostate, breast, and colon cancers (Michelakos, T. et al., (2021) EXPERT OPIN. BIOL. THER.21:587-602; Du, H. et al., (2019) CANCER CELL.35:221-237; Seaman, S. et al., (2017) CANCER CELL.31:501-515). Notwithstanding the advances that have been made to date, ADCs provide an attractive strategy due to their high specificity and ability to selectively deliver highly potent payloads to the site of interest. Regardless of payload used, there is a balance between increasing and improving the efficacy of the ADC conjugate versus maintaining a high safety profile. The present disclosure appreciates that modular, tunable site- specific conjugation enabled by unnatural amino acid conjugation handles can provide best-in-class safety profiles while allowing for easy control over drug to antibody ratio (DAR), conjugation site location, and linker chemistry variation to enhance the desired properties of an ADC (e.g., any desired properties described herein). [0078] Accordingly, in certain aspects and embodiments, the present disclosure relates to an engineered anti-CD276 antibody comprising one or more unnatural amino acids (e.g., any unnatural amino acids described herein). The one or more UAAs can be incorporated into an engineered anti-CD276 antibody using one or more orthogonal tRNA/aminoacyl-tRNA synthetase pairs that are present in a desired expression system (e.g., in an in vitro system, in a host cell, etc.). The incorporated UAAs can subsequently be used to conjugate a payload (e.g., a cytotoxic payload, an immune modulatory payload, or any other payload described herein) to generate a therapeutic ADC for the treatment of disorders associated with elevated levels of CD276 expression, e.g., CD276 positive oncological malignancies. Accordingly, the resulting anti-CD276 antibody conjugate (e.g., an anti-CD276 antibody drug conjugate described herein) can be used to deliver one or more payload moieties to a target cell or tissue (e.g., a cancerous target cell, or cancerous tissue) expressing CD276. Attorney Docket No. BRI-023WO I. DEFINITIONS [0079] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. Generally, nomenclatures utilized in connection with, and techniques of, immunology, oncology, cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. [0080] As used herein, the terms “a,” and “an,” mean “one or more” and include the plural unless the context clearly indicates otherwise. Thus, for example, reference to “an antibody” includes a plurality of antibodies and reference to “an antibody” in some embodiments includes multiple antibodies, and so forth. [0081] As used herein, all numerical values or numerical ranges include whole integers within or encompassing such ranges and fractions of the values or the integers within or encompassing ranges unless the context clearly indicates otherwise. Thus, for example, reference to a range of 90-100%, includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth. In another example, reference to a range of 1-5,000 fold includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 fold, etc., as well as 1.1, 1.2, 1.3, 1.4, 1.5 fold, etc., 2.1, 2.2, 2.3, 2.4, 2.5 fold, etc., and so forth. [0082] Where the use of the term “about” is before a quantitative value, the present invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred. When values are expressed as approximations by use of the antecedent “about,” it is understood that the disclosure also contemplates embodiments that specify the particular values and ranges of values without the approximations. It is further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed in this disclosure, and that each value is also disclosed as “about” that particular value in addition to the value itself. Attorney Docket No. BRI-023WO [0083] The terms “polypeptide,” “protein,” and “peptide” refer to any chain of amino acid residues, regardless of its length or post-translational modification (e.g., glycosylation or phosphorylation). [0084] As used herein, the phrase “percent identity” and “% identity” refers to the extent to which two sequences e.g., two polypeptides or two nucleic acids have the same respective amino acid or nucleotide at the same positions in an alignment. As used herein, “percent identity” between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Similarly, “percent identity” between a nucleic acid sequence and a reference sequence is defined as the percentage of nucleotides in the nucleic acid sequence that are identical to the nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity (e.g., nucleic acid sequence identity or amino acid sequence identity) can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST (Basic Local Alignment Search Tool), BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. For a discussion of basic issues in searching sequence databases see Altschul et al., (1994) NATURE GENETICS 6:119-129, which is fully incorporated by reference herein. 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. [0085] The alignment algorithms above may take into account a scoring matrix to calculate an alignment score (see Chao et al. (2022) BIOMOLECULES, 12(4): 546). For example, for an amino acid sequence at least 85 amino acids in length, the scoring matrix recommended by the BLAST algorithm is BLOSUM-62. The BLOSUM-62 scoring matrix assigns positive, zero, or negative scores between each pair or standard amino acid residues (see Henikoff and Henikoff (1992) PROC. NATL. ACAD. SCI. USA, 89: 10915–19 at FIG.2). A positive score between two amino acid residues indicates that substitution of these amino acid residues for each other is conservative. As used herein, “similarity” between a subject amino acid sequence and a reference amino acid sequence refers to the percentage of amino acid residues in the subject amino acid sequence that are identical or have a conservative substitution according to the BLOSUM-62 scoring matrix, relative to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum sequence alignment score. Attorney Docket No. BRI-023WO [0086] As used herein, the terms “subject” and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably include humans. [0087] As used herein, the term “effective amount” refers to the amount of an active agent sufficient to effect beneficial or desired results (e.g., a desired prophylactic or therapeutic effect). An effective amount can be administered in one or more administration(s), application(s) or dosage(s) and is not intended to be limited to a particular formulation or administration route. [0088] As used herein, the term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vivo or ex vivo. [0089] The terms “treat,” “treating,” or “treatment,” and other grammatical equivalents as used in this disclosure, include alleviating, abating, ameliorating, or preventing a disease, condition or symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition, and are intended to include prophylaxis. The terms further include achieving a therapeutic benefit and/or a prophylactic benefit. The term “therapeutic benefit” refers to eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder. [0090] As used herein, the term “combination” in the context of therapies means that two or more different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, such that the effects of the treatments on the patient overlap at a point in time. In certain embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In certain embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second Attorney Docket No. BRI-023WO treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In certain embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered. [0091] Throughout the description, where compositions are described as having, including, containing, incorporating, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is intended that compositions and methods are inclusive or open-ended and do not exclude additional, unrecited components or steps. It is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited steps. [0092] Further, it should be understood that elements and/or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present invention, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions of the present invention and/or in methods of the present invention, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and invention(s). For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of the invention(s) described and depicted herein. [0093] It should be understood that the expression “at least one of” or “one or more of” includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and/or” in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context. [0094] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present invention remain operable. Moreover, two or more steps or actions may be conducted simultaneously. Attorney Docket No. BRI-023WO [0095] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present invention and does not pose a limitation on the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present invention. II. ANTIBODY CONJUGATE COMPOSITIONS [0096] The present disclosure provides, among other things, engineered antibodies (e.g., engineered anti-CD276 antibodies) that comprise at least one unnatural amino acid that functions as conjugation site (e.g., as a bioconjugation handle) for at least one payload molecule (e.g., a therapeutic payload, or any payload described herein), and methods of making and using the same. In various embodiments, the present disclosure provides engineered anti-CD276 antibodies, or anti- CD276 antibody drug conjugates (ADCs), that utilize unnatural amino acid conjugation sites to attach one or more payload molecules (e.g., any payload described herein, e.g., PBD or PBD dimers). The conjugated antibodies can have enhanced efficacy compared to similar antibodies that do not comprise a conjugated payload. The present disclosure further provides, among other things, methods of producing and methods of using the conjugated antibodies. [0097] Also provided are specific amino acid residue locations (e.g., sites or positions) in an antibody (e.g., an anti-CD276 antibody, such as an m276 antibody) are suitable for efficient incorporation of one, or more, unnatural amino acids into a given antibody sequence. These positions are amenable to modification with unnatural amino acids, which can then be conjugated to payload molecules (e.g., cytotoxic linker-payloads, or any other payload described herein). Also provided are methods that allow for efficient generation of antibody conjugates (e.g., ADCs, such as anti-CD276 ADCs) with desirable properties, including, increased expression yield, tunable and consistent drug-to-antibody ratio (DAR), lack of aggregation, increased stability, and increased binding affinity. Indeed, engineered antibodies of the present disclosure may provide, among other things, enhanced release kinetics for therapeutic use, increased cytotoxic windows between cancerous and healthy cells, improved toxicity profiles, lower uptake in healthy tissues, increased efficacy at lower dosages, increased therapeutic window (efficacy to toxicity dosing range), and increased maximum tolerated dose (MTD) while maintaining effective concentrations for therapeutic dosages. The advantages provided by the present disclosure may include reduced adverse events and serious adverse events commonly seen in current antibody and antibody conjugate regimens. Attorney Docket No. BRI-023WO [0098] In some embodiments, an antibody provided by the present disclosure (e.g., an engineered antibody, such as a conjugated antibody or ADC) comprises a heavy chain amino acid sequence as shown in TABLE 2. In some embodiments, an antibody provided by the present disclosure comprises a heavy chain amino acid sequence as shown in TABLE 2, with an unnatural amino acid incorporated at one or more positions in the heavy chain amino acid sequence. In some embodiments, an antibody provided by the present disclosure comprises a heavy chain amino acid sequence as shown in TABLE 2, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1. In some embodiments, an antibody provided by the present disclosure comprises a light chain amino acid sequence as shown in TABLE 2. In some embodiments, an antibody provided by the present disclosure comprises a light chain amino acid sequence as shown in TABLE 2, with an unnatural amino acid incorporated at one or more positions in the light chain amino acid sequence. In some embodiments, an antibody provided by the present disclosure comprises a light chain amino acid sequence as shown in TABLE 2, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1. [0099] In some embodiments, an antibody provided by the present disclosure comprises a heavy chain sequence as shown in TABLE 2, and a light chain sequence as shown in TABLE 2. In some embodiments, an antibody provided by the present disclosure comprises a heavy chain sequence as shown in TABLE 2, with an unnatural amino acid incorporated at one or more positions in the heavy chain, and a light chain sequence as shown in TABLE 2, with an unnatural amino acid incorporated at one or more positions in the light chain. In some embodiments, an antibody provided by the present disclosure comprises a heavy chain sequence as shown in TABLE 2, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1, and a light chain sequence as shown in TABLE 2, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1. [0100] In some embodiments, an antibody or engineered antibody provided by the present disclosure comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 1. In some embodiments, an antibody or engineered antibody comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 1. [0101] In some embodiments, an antibody or engineered antibody provided by the present disclosure comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at Attorney Docket No. BRI-023WO least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 1, with an unnatural amino acid incorporated at one or more amino acid positions of the heavy chain. In some embodiments, an antibody or engineered antibody comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 1, with an unnatural amino acid incorporated at one or more amino acid positions of the heavy chain. [0102] In some embodiments, an antibody or engineered antibody provided by the present disclosure comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 1, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1. In some embodiments, an antibody or engineered antibody comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 1, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1. [0103] In some embodiments, an antibody or engineered antibody provided by the present disclosure comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 61. In some embodiments, an antibody or engineered antibody comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 61. [0104] In some embodiments, an antibody or engineered antibody provided by the present disclosure comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 61, with an unnatural amino acid incorporated at one or more amino acid positions of the heavy chain. An exemplary payload can be attached to the unnatural amino acid, for example, as shown in FIGs. 29A-29B. In some embodiments, an antibody or engineered antibody comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 61, with an unnatural amino acid incorporated at one or more amino acid positions of the heavy chain. An exemplary payload can be attached to the unnatural amino acid, for example, as shown in FIGs.29A-29B. [0105] In some embodiments, an antibody or engineered antibody provided by the present disclosure comprises a heavy chain sequence, wherein the heavy chain sequence comprises an Attorney Docket No. BRI-023WO amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 61, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1. In some embodiments, an antibody or engineered antibody comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 61, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1. [0106] In some embodiments, an antibody or engineered antibody provided by the present disclosure comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to one or more of: (i) A142 of SEQ ID NO: 61; (ii) T157 of SEQ ID NO: 61; (iii) T171 of SEQ ID NO: 61; (iv) A174 of SEQ ID NO: 61; (v) Y182 of SEQ ID NO: 61; (vi) T197 of SEQ ID NO: 61; (vii) D267 of SEQ ID NO: 61; or (viii) S241 of SEQ ID NO: 61. An exemplary payload can be attached to the unnatural amino acid, for example, as shown in FIGs.29A-29B. In some embodiments, an antibody or engineered antibody comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to one or more of: (i) A142 of SEQ ID NO: 61; (ii) T157 of SEQ ID NO: 61; (iii) T171 of SEQ ID NO: 61; (iv) A174 of SEQ ID NO: 61; (v) Y182 of SEQ ID NO: 61; (vi) T197 of SEQ ID NO: 61; (vii) D267 of SEQ ID NO: 61; or (viii) S241 of SEQ ID NO: 61. An exemplary payload can be attached to the unnatural amino acid, for example, as shown in FIGs.29A-29B. [0107] In some embodiments, an antibody or engineered antibody provided by the present disclosure comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to T171 of SEQ ID NO: 61. An exemplary payload can be attached to T171, for example, as shown in FIG.29A and FIG.29B. In some embodiments, an antibody or engineered antibody comprises a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to T171 of SEQ ID NO: 61. An exemplary payload can be attached to T171, for example, as shown in FIG.29A and FIG. 29B. Attorney Docket No. BRI-023WO [0108] In some embodiments, an antibody or engineered antibody provided by the present disclosure comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 3. In some embodiments, an antibody or engineered antibody comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 3. [0109] In some embodiments, an antibody or engineered antibody provided by the present disclosure comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 3, with an unnatural amino acid incorporated at one or more amino acid positions of the light chain. In some embodiments, an antibody or engineered antibody comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 3, with an unnatural amino acid incorporated at one or more amino acid positions of the light chain. [0110] In some embodiments, an antibody or engineered antibody provided by the present disclosure comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 3, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1. In some embodiments, an antibody or engineered antibody comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 3, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1. [0111] In some embodiments, an antibody or engineered antibody provided by the present disclosure comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 62. In some embodiments, an antibody or engineered antibody comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 62. [0112] In some embodiments, an antibody or engineered antibody provided by the present disclosure comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 62, with an unnatural amino acid incorporated at one or more amino acid positions of the light chain. In some Attorney Docket No. BRI-023WO embodiments, an antibody or engineered antibody comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 62, with an unnatural amino acid incorporated at one or more amino acid positions of the light chain. [0113] In some embodiments, an antibody or engineered antibody provided by the present disclosure comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 62, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1. In some embodiments, an antibody or engineered antibody comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 62, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1. [0114] In some embodiments, an antibody or engineered antibody provided by the present disclosure comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 62, with an unnatural amino acid incorporated at a position corresponding to Q162 of SEQ ID NO: 62. In some embodiments, an antibody or engineered antibody comprises a light chain sequence, wherein the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 62, with an unnatural amino acid incorporated at a position corresponding to Q162 of SEQ ID NO: 62. [0115] In some embodiments, an antibody or engineered antibody provided by the present disclosure may comprises a combination of any heavy chain amino acid sequence and any light chain amino acid sequence described herein. [0116] In some embodiments, an antibody or engineered antibody provided by the present disclosure comprises a heavy chain sequence and a light chain sequence, wherein the heavy chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 1 or SEQ ID NO: 61, and the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 3 or SEQ ID NO: 62. In some embodiments, an antibody or engineered antibody comprises a heavy chain sequence and a light chain sequence, wherein the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 61, and the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 62. Attorney Docket No. BRI-023WO [0117] In some embodiments, an antibody or engineered antibody provided by the present disclosure comprises a heavy chain sequence and a light chain sequence, wherein the heavy chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 1 or SEQ ID NO: 61, with an unnatural amino acid incorporated at one or more positions in the heavy chain, and the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 3 or SEQ ID NO: 62, with an unnatural amino acid incorporated at one or more positions in the light chain. An exemplary payload can be attached to an unnatural amino acid, for example, as shown in FIGs.29A-29B. In some embodiments, an antibody or engineered antibody comprises a heavy chain sequence and a light chain sequence, wherein the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 61, with an unnatural amino acid incorporated at one or more positions in the heavy chain, and the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 62, with an unnatural amino acid incorporated at one or more positions in the light chain. An exemplary payload can be attached to an unnatural amino acid, for example, as shown in FIGs.29A-29B. [0118] In some embodiments, an antibody or engineered antibody provided by the present disclosure comprises a heavy chain sequence and a light chain sequence, wherein the heavy chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 1 or SEQ ID NO: 61, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1, and the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 3 or SEQ ID NO: 62, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1. In some embodiments, an antibody or engineered antibody comprises a heavy chain sequence and a light chain sequence, wherein the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 61, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1, and the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 62, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1. [0119] It is contemplated that an engineered anti-CD276 antibody may comprise or more unnatural amino acids at positions selected from TABLE 1. Attorney Docket No. BRI-023WO [0120] It will be understood by a skilled artisan that provided sites for incorporating an unnatural amino acid (e.g., positions identified in SEQ ID NOs 67-133 in TABLE 1, those identified in Table 2, etc.) can be identified in most known antibody sequences by aligning a provided sequence with an antibody to be engineered, see, e.g., FIGs.1 and 2. TABLE 1: Conjugation Sites Domain Trastuzumab m276 Site SEQ Trastuzumab SEQ m276 Context
Figure imgf000028_0001
Attorney Docket No. BRI-023WO
Figure imgf000029_0001
[0121] In some embodiments, an engineered antibody, as provided herein, comprises a modification at one or more unnatural amino acid residues. The modification can comprise a payload molecule or moiety (e.g., any payload molecule or moiety described herein) that is conjugated using any crosslinker agent and/or linker described herein to yield an antibody conjugate, or an antibody-drug conjugate (ADC). In some embodiments, an antibody provided herein comprises a heavy chain amino acid sequence as shown in TABLE 2. In some embodiments, an antibody provided herein comprises a heavy chain amino acid sequence as shown in TABLE 2, with an unnatural amino acid incorporated at one or more positions in the heavy chain amino acid sequence. In some embodiments, an antibody provided by the present disclosure comprises a heavy chain amino acid sequence as shown in TABLE 2, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1. In some embodiments, an antibody provided herein comprises a light chain amino acid sequence as shown in TABLE 2. In Attorney Docket No. BRI-023WO some embodiments, an antibody provided herein comprises a light chain amino acid sequence as shown in TABLE 2, with an unnatural amino acid incorporated at one or more positions in the light chain amino acid sequence. In some embodiments, an antibody provided by the present disclosure comprises a light chain amino acid sequence as shown in TABLE 2, with an unnatural amino acid incorporated at one or more positions selected from TABLE 1. [0122] In some embodiments, an antibody or engineered antibody (e.g., a conjugated antibody comprising one or more unnatural amino acids that are modified via conjugation to a payload molecule to form an ADC), exhibits enhanced stability compared to a similar antibody that does not comprise a modification such as conjugation to a payload molecule. For example, stability of an antibody can be assessed using any suitable method in the field. In some embodiments, antibody stability is assessed using a plasma stability assay. In some embodiments, antibody stability is assessed in vivo after administration in animals such as mice, rats or non-human primates. In some embodiments, a conjugated antibody exhibits enhanced payload release characteristics compared to a similar antibody where the payload is conjugated to different site. Release characteristics of an antibody can be assessed using any suitable method in the field. For example, payload release can be assessed using a Cathepsin B assay. [0123] In some embodiments, at least 40%, 50%, 60%, 70%, 80%, or 90% of an antibody provided herein (e.g., an ADC as provided herein) remains following incubation in human plasma for 72 hours at 37 °C. In some embodiments, at least 40%, 50%, 60%, 70%, 80%, or 90% of the conjugated payload of an antibody provided herein is released from the antibody following incubation with Cathepsin B for 240 minutes at 37 °C. In some embodiments, the antibody has a binding affinity for a target antigen of 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.75 nM, 0.5 nM, 0.1 nM, 0.075 nM, or 0.05 nM or lower, as measured by enzyme-linked immunosorbent assay (ELISA) or any other suitable method. In certain embodiments, the antibody has a binding affinity for a target antigen that is within 0.1 fold, 0.2 fold, 0.3 fold, 0.4 fold, 0.5 fold, 1.0 fold, 1.5 fold, 2.0 fold, 3.0 fold, 4.0 fold, 5.0 fold, 6.0 fold, 8.0 fold, or 10.0 fold of the binding affinity for the target antigen of a reference antibody, wherein the reference antibody is an otherwise identical antibody that does not comprise the unnatural amino acid, as measured by ELISA or any other suitable method. [0124] In some embodiments, an antibody provided herein has improved biophysical characteristics, such as enhanced or tuned half-life (increased or decreased) and correlated to a desired improved efficacy and toxicity profile. Furthermore it is contemplated that an antibody Attorney Docket No. BRI-023WO conjugate provided herein has improved efficacy compared to other antibodies using different conjugation strategies. [0125] Furthermore, the site-specifically conjugated drug substance can have reduced off target toxicity or non-specific uptake (see, e.g., FIG.26). In some embodiments, the antibodies provided herein have reduced platform toxicity of the payload with an increased maximum tolerated dose as compared to a similar antibody or therapeutic modality. The resulting antibody can exhibit a difference in activity, toxicity, and overall therapeutic window based on the site of conjugation and DAR that results in greater therapeutic potential. [0126] Depending upon the circumstances, an antibody provided herein, which may include an Antibody-UAA-Linker-PBD structure, has enhanced activity in cell lines where other payloads (e.g, camptothecins, auristatins, etc.) are less active or inactive, or visa versa. Ideally the antibody has optimal activity as an ADC. II. (a) Unnatural Amino Acids [0127] Many kinds of unnatural amino acids (also referred to as “UAAs”) may be used in accordance with the present disclosure. As used herein, an unnatural amino acid refers to any amino acid, modified amino acid, or amino acid analogue other than the following twenty genetically encoded alpha-amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine. See, e.g., Biochemistry by L. Stryer, 3rd ed. 1988, Freeman and Company, New York, for structures of the twenty natural amino acids. The term unnatural amino acid also includes amino acids that occur by modification (e.g., post- translational modifications) of a natural amino acid, but are not themselves naturally incorporated into a growing polypeptide chain by the translation complex. Any unnatural amino acid described herein (e.g., those depicted in FIGs.3A-3E), or any suitable unnatural amino acid may be used in accordance with the present disclosure. [0128] Because unnatural amino acids typically differ from natural amino acids only in the structure of the side chain, unnatural amino acids may, for example, form amide bonds with other amino acids in the same manner in which they are formed in naturally occurring proteins. However, unnatural amino acids may have side chain groups that distinguish them from the natural amino acids. For example, an unnatural amino acid side chain may comprise an alkyl-, aryl-, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkyl, ether, thiol, seleno-, sulfonyl-, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amine, and the like, or any combination thereof. Other non- Attorney Docket No. BRI-023WO naturally occurring amino acids include, but are not limited to, amino acids comprising a photoactivatable cross-linker, spin-labeled amino acids, fluorescent amino acids, metal binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids with novel functional groups, amino acids that covalently or noncovalently interact with other molecules, photocaged and/or photoisomerizable amino acids, amino acids comprising biotin or a biotin analogue, glycosylated amino acids such as a sugar substituted serine, other carbohydrate modified amino acids, keto-containing amino acids, amino acids comprising polyethylene glycol or polyether, heavy atom substituted amino acids, chemically cleavable and/or photocleavable amino acids, amino acids with an elongated side chains as compared to natural amino acids, including but not limited to, polyethers or long chain hydrocarbons, including but not limited to, greater than about 5 or greater than about 10 carbons, carbon-linked sugar-containing amino acids, redox-active amino acids, amino thioacid containing amino acids, and amino acids comprising one or more toxic moiety. [0129] In addition to unnatural amino acids that contain novel side chains, unnatural amino acids also optionally comprise modified backbone structures. [0130] Many unnatural amino acids are based on natural amino acids, such as tyrosine, glutamine, phenylalanine, and the like. Tyrosine analogs include para-substituted tyrosines, ortho- substituted tyrosines, and meta substituted tyrosines, wherein the substituted tyrosine comprises a keto group (including but not limited to, an acetyl group), a benzoyl group, an amino group, a hydrazine, an hydroxyamine, a thiol group, a carboxy group, an isopropyl group, a methyl group, a C6-C20 straight chain or branched hydrocarbon, a saturated or unsaturated hydrocarbon, an O- methyl group, a polyether group, a nitro group, or the like. In addition, multiply substituted aryl rings are also contemplated. Glutamine analogs include, but are not limited to, α-hydroxy derivatives, γ-substituted derivatives, cyclic derivatives, and amide substituted glutamine derivatives. Exemplary phenylalanine analogs include, but are not limited to, para-substituted phenylalanines, ortho-substituted phenylalanines, and meta-substituted phenylalanines, wherein the substituent comprises a hydroxy group, a methoxy group, a methyl group, an allyl group, an aldehyde, an azido, an iodo, a bromo, a keto group (including but not limited to, an acetyl group), or the like. Specific examples of unnatural amino acids include, but are not limited to, a p-acetyl-L- phenylalanine, a p-propargyl-phenylalanine, O-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine, a 3- methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-GlcNAcβ- serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L- phenylalanine, a p-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, a p-bromophenylalanine, a p- Attorney Docket No. BRI-023WO amino-L-phenylalanine, an isopropyl-L-phenylalanine, and a p-propargyloxy-phenylalanine, and the like. [0131] Examples of structures of a variety of unnatural amino acids are provided in U.S. Patent Application Publication Nos.2003/0082575 and 2003/0108885, PCT Publication No. WO 2002/085923, and Kiick et al. (2002) PROC. NATL. ACAD. SCI. USA 99:19-24. [0132] Any suitable unnatural amino acid can be used in the methods and compositions of the present disclosure for incorporation into a protein of interest (e.g., an antibody). [0133] In some embodiments, an unnatural amino acid used in accordance with the present disclosure may be a leucine analog (also referred to herein as a derivative), for example, a non- naturally occurring leucine analog. In some embodiments, methods and compositions of the present disclosure utilize a leucine analog depicted in FIG.3A, or a composition comprising the leucine analog. For example, Formula A in FIG.3A depicts an amino acid analog containing a side chain including a carbon containing chain n units (0-20 units) long. An O, S, CH2, or NH is present in at position X, and another carbon containing chain of n units (0-20 units) long can follow. A functional group Y is attached to the terminal carbon of second carbon containing chain (e.g., functional groups 1-12 as depicted in FIG.3A, where R represents a linkage to the terminal carbon atom the second carbon containing side chain). In one example, these functional groups can be used for bioconjugation of any amenable ligand to any protein of interest that is amenable to site-specific unnatural amino acid incorporation. Formula B in FIG.3A depicts a similar amino acid analog containing a side chain denoted as either Z-Y2 or Z-Y3 attached to the second carbon containing chain or the first carbon containing chain, respectively. Z represents a carbon chain comprising (CH2)n units, where n is any integer from 0-20. Y2 or Y3, independently, can be the same or different groups as those of Y1. [0134] In some embodiments, methods and compositions of the present disclosure utilize a leucine analog depicted in FIG.3B (e.g., LCA, LKET, or ACA), or a composition comprising the leucine analog depicted in FIG.3B. Additional exemplary leucine analogs include those selected from linear alkyl halides and linear aliphatic chains comprising a functional group, for example, an alkyne, azide, cyclopropene, alkene, ketone, aldehyde, diazirine, or tetrazine functional group, as well as structures 1-6 shown in FIG.3B. However, it is contemplated that the amino and carboxylate groups both attached to the first carbon of any amino acid shown in FIGs.3A-3B would constitute portions of peptide bonds when the leucine analog is incorporated into a protein or polypeptide chain. Attorney Docket No. BRI-023WO [0135] Alternatively or in addition, the leucine analogs set forth in FIG.3C, referred to as C5AzMe and LCA can be used in the practice of the invention. Methods for preparing leucine analogs, e.g., C5AzMe or LCA, are described in International (PCT) Publication No. WO2021026506. [0136] In certain embodiments, an unnatural amino acid used in accordance with the present disclosure is a tryptophan analog (also referred to herein as a derivative). In some embodiments, the tryptophan analog is a non-naturally occurring tryptophan analog. Exemplary tryptophan analogs include 5-azidotryptophan, 5-propargyloxytryptophan, 5-aminotryptophan, 5- methoxytryptophan, 5-O-allyltryptophan or 5-bromotryptophan. Additional exemplary tryptophan analogs are depicted in FIG.3A (right side). However, it is contemplated that the amino and carboxylate groups both attached to the first carbon of the tryptophan analogs in FIG.3B would constitute portions of peptide bonds when the tryptophan analog is incorporated into a protein or polypeptide chain. [0137] In some embodiments, the tryptophan analog set forth in FIG.3C, referred to as AzW, can be used in accordance with the present disclosure. Methods for preparing tryptophan analogs, e.g., AzW, are described in International (PCT) Publication No. WO2021026506. [0138] In some embodiments, an unnatural amino acid used in accordance with the present disclosure is a tyrosine analog (also referred to herein as a derivative). In some embodiments, the tyrosine analog is a non-naturally occurring tyrosine analog. Exemplary tyrosine analogs include o-methyltyrosine (OmeY), p-azidophenylalanine (AzF), o-propargyltyrosine (OpropY or PrY), and p-acetylphenylalanine (AcF). Exemplary tryptophan analogs are depicted in FIG.3D. In some embodiments, an unnatural amino acid used in accordance with the present disclosure is a pyrrolysine analog (also referred to herein as a derivative). In some embodiments, the pyrrolysine analog is a non-naturally occurring pyrrolysine analog. Exemplary pyrrolysine analogs include aminocaprylic acid (Cap), H-Lys(Boc)-OH (Boc-Lysine, BocK), azidolysine (AzK), H-propargyl- lysine (hPrK), and cyclopropenelysine (CpK). Exemplary pyrrolysine analogs are depicted in FIG. 3E. [0139] Any commercially available unnatural amino acid may be used in accordance with the present disclosure. Many unnatural amino acids are commercially available, e.g., from Sigma- Aldrich (St. Louis, Mo., USA), Novabiochem (Darmstadt, Germany), or Peptech (Burlington, Mass., USA). Those that are not commercially available can be synthesized for use in a method or composition of the present disclosure using standard methods known to those of ordinary skill in the art. For organic synthesis techniques, see, e.g., Organic Chemistry by Fessendon and Attorney Docket No. BRI-023WO Fessendon, (1982, Second Edition, Willard Grant Press, Boston Mass.); Advanced Organic Chemistry by March (Third Edition, 1985, Wiley and Sons, New York); and Advanced Organic Chemistry by Carey and Sundberg (Third Edition, Parts A and B, 1990, Plenum Press, New York). Additional exemplary publications describing the synthesis of unnatural amino acids appear in PCT Publication No. WO2002/085923, U.S. Patent Application Publication No.2004/0198637, Matsoukas et al. (1995) J. MED. CHEM., 38:4660-4669, King et al. (1949) J. CHEM. SOC., 3315- 3319, Friedman et al. (1959) J. AM. CHEM. SOC., 81: 3750-3752, Craig et al. (1988) J. ORG. CHEM., 53: 1167-1170, Azoulay et al. (1991) EUR. J. MED. CHEM., 26: 201-5, Koskinen et al. (1989) J. ORG. CHEM., 54: 1859-1866, Christie et al. (1985) J. ORG. CHEM., 50: 1239-1246, Barton et al. (1987) TETRAHEDRON, 43: 4297-4308, and Subasinghe et al. (1992) J. MED. CHEM., 35: 4602-7. II. (b) Conjugation Methods [0140] As discussed in more detail herein, unnatural amino acid in a polypeptide (e.g., an antibody) may be used to attach another molecule to the polypeptide. For example, in some embodiments, an antibody provided herein (e.g., anti-CD276 antibody) comprises an unnatural amino acid that is chemically modified, e.g., conjugated to a payload molecule (e.g., a therapeutic molecule, or any other payload). For example, a polypeptide, (e.g., an antibody) may comprise one or more unnatural amino acids (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more unnatural amino acids, each of which may be the same or different), and similarly, may be conjugated to one or more molecules (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more than ten payload molecules, each of which may be the same or different). In some embodiments, a polypeptide, (e.g., an antibody) may comprise one or more unnatural amino acids (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more unnatural amino acids, each of which may be the same or different) at positions selected from TABLE 1, and similarly, may be conjugated to one or more molecules (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more than ten payload molecules, each of which may be the same or different), e.g., selected from any molecules or payloads described herein. [0141] Exemplary molecules that can be conjugated to a polypeptide containing an unnatural amino acid include, for example, a label, a dye, a polymer, a water-soluble polymer, a stabilizing agent (e.g., a derivative of polyethylene glycol), a photoactivatable crosslinker, a radionuclide, a cytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound, a resin, a second protein or polypeptide or polypeptide analog (e.g., a therapeutic peptide or polypeptide), an antibody or antibody fragment (e.g., an anti-CD276 antibody or antibody fragment), a metal chelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide, a DNA (e.g., a DNA Attorney Docket No. BRI-023WO oligonucleotide), a RNA (e.g., a RNA oligonucleotide), a LNA (e.g., a LNA oligonucleotide), an antisense polynucleotide, a saccharide, a water-soluble dendrimer, a cyclodextrin, an inhibitory ribonucleic acid (e.g., a small interfering RNA (siRNA), a small nuclear RNA (snRNA), or a non- coding RNA), a biomaterial, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, a radioactive moiety, a novel functional group, a group that covalently or noncovalently interacts with other molecules, a photocaged moiety, an actinic radiation excitable moiety, a photoisomerizable moiety, biotin, a derivative of biotin, a biotin analogue, a moiety incorporating a heavy atom, a chemically cleavable group, a photocleavable group, an elongated side chain, a carbon-linked sugar, a redox-active agent, an amino thioacid, a toxic moiety, an isotopically labeled moiety, a biophysical probe or biochemical probe (e.g., a PET probe, a fluorescent probe or an EPR probe), a phosphorescent group, a chemiluminescent group, an electron dense group, a magnetic group, an intercalating group, a chromophore, an energy transfer agent, a biologically active agent, a detectable label (e.g., for analysis of uptake in viable cells versus non-viable cells), a small molecule (e.g., a therapeutic small molecule), an immunomodulatory molecule, a targeting agent, a lipid based structure (e.g., a lipid-based nanoparticle), a microsphere, or any combination of the above. [0142] Additional exemplary molecules or payloads for conjugation to a protein of interest (e.g., an antibody) in accordance with the present disclosure include any cytotoxic, cytostatic or immunomodulatory drug. Useful classes of cytotoxic or immunomodulatory agents include, for example, anti-tubulin agents, auristatins (e.g., vedotin or MMAE, mafodotin or MMAF, etc.), maytansinoids (e.g., Emtansine, soravtansine, etc.), calicheamicin (e.g., ozogamicin, etc.), anthramycin, SG2000, pyrrolobenzodiazapines (e.g., PBD, talirine (SGD-1910), tesirine (SG3249), etc.), indolobenzodiazapines (e.g., IGNs), pyrrolobenzodiazepine (PBD) and derivatives thereof (Lai et al. (2022) J. MED. CHEM., 65(19): 13041-13051; Mantaj et al. (2016) ANGEW CHEM. INT. ED., 26: 462-488; Hartley et al. (2020) EXPERT OPIN. BIOL. THERAPY, 21(7): 931-943), DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cis-platin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, calmodulin inhibitors, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, pore-forming compounds, purine antimetabolites, puromycins, radiation sensitizers, rapamycins, steroids, taxanes, topoisomerase inhibitors (e.g., camptothecin based topopisomerase inhibitors (e.g., deruxtecan, SN-38, etc.), duocarmycins, amanitins, vinca alkaloids, or the like. Attorney Docket No. BRI-023WO [0143] Individual cytotoxic or immunomodulatory agents that may be used in accordance with the present disclosure include, for example, an androgen, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan, buthionine sulfoximine, calicheamicin, calicheamicin derivatives, camptothecin, carboplatin, carmustine (BSNU), CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin (formerly actinomycin), daunorubicin, decarbazine, DM1, DM4, docetaxel, doxorubicin, etoposide, an estrogen, 5-fluordeoxyuridine, 5-fluorouracil, gemcitabine, gramicidin D, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU), maytansine, mechlorethamine, melphalan, 6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone, nitroimidazole, paclitaxel, palytoxin, plicamycin, procarbizine, a pyrrolobenzodiazepine (PBD) (including PBD dimers), rhizoxin, streptozotocin, tenoposide, 6- thioguanine, thioTEPA, topotecan, vinblastine, vincristine, vinorelbine, VP-16 and VM-26. [0144] In some embodiments, suitable cytotoxic agents for use in accordance with the present disclosure include, for example, DNA minor groove binders (e.g., enediynes and lexitropsins, a CBI compound), duocarmycins, taxanes (e.g., paclitaxel and docetaxel), puromycins, vinca alkaloids, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino- doxorubicin, echinomycin, combretastatin, netropsin, epothilone A and B, estramustine, cryptophycins, cemadotin, maytansinoids, discodermolide, eleutherobin, and mitoxantrone. [0145] In some embodiments, a molecule or payload used in accordance with the present disclosure is an anti-tubulin agent. Examples of anti-tubulin agents include taxanes (e.g., Taxol® (paclitaxel), Taxotere® (docetaxel)), T67 (Tularik) and vinca alkyloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine). Other antitubulin agents include, for example, baccatin derivatives, taxane analogs, epothilones (e.g., epothilone A and B), nocodazole, colchicine and colcimid, estramustine, cryptophycins, cemadotin, maytansinoids, combretastatins, discodermolide, and eleutherobin. [0146] In certain embodiments, the cytotoxic agent is a maytansinoid (e.g., maytansine or DM1). [0147] In some embodiments, a molecule or payload used in accordance with the present disclosure is an auristatin, such as auristatin E or a derivative thereof. For example, the auristatin E derivative can be an ester formed between auristatin E and a keto acid. For example, auristatin E can be reacted with paraacetyl benzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatin derivatives include AFP, MMAF, and MMAE. Attorney Docket No. BRI-023WO [0148] In some embodiments, a molecule or payload used in accordance with the present disclosure is an antimetabolite. The antimetabolite can be, for example, a purine antagonist (e.g., azothioprine or mycophenolate mofetil), a dihydrofolate reductase inhibitor (e.g., methotrexate), acyclovir, ganciclovir, zidovudine, vidarabine, ribavarin, azidothymidine, cytidine arabinoside, amantadine, dideoxyuridine, iododeoxyuridine, poscarnet, or trifluridine. [0149] In some embodiments, a molecule or payload used in accordance with the present disclosure is tacrolimus, cyclosporine, FU506 or rapamycin. In further examples, the molecule or payload is aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, bexarotene, bexarotene, calusterone, capecitabine, celecoxib, cladribine, Darbepoetin alfa, Denileukin diftitox, dexrazoxane, dromostanolone propionate, epirubicin, Epoetin alfa, estramustine, exemestane, Filgrastim, floxuridine, fludarabine, fulvestrant, gemcitabine, gemtuzumab ozogamicin (MYLOTARG), goserelin, idarubicin, ifosfamide, imatinib mesylate, Interferon alfa-2a, irinotecan, letrozole, leucovorin, levamisole, meclorethamine or nitrogen mustard, megestrol, mesna, methotrexate, methoxsalen, mitomycin C, mitotane, nandrolone phenpropionate, oprelvekin, oxaliplatin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pentostatin, pipobroman, plicamycin, porfimer sodium, procarbazine, quinacrine, rasburicase, Rituximab, Sargramostim, streptozocin, tamoxifen, temozolomide, teniposide, testolactone, thioguanine, toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine, or zoledronate. [0150] In some embodiments, a molecule or payload used in accordance with the present disclosure is an immunomodulatory agent. The immunomodulatory agent can be, for example, ganciclovir, etanercept, tacrolimus, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil or methotrexate. Alternatively, the immunomodulatory agent can be, for example, a glucocorticoid (e.g., cortisol or aldosterone) or a glucocorticoid analogue (e.g., prednisone or dexamethasone). Alternatively, the immunomodulatory agent can be, for example, a Toll-like receptor (TLR) agonist, e.g., a TLR7 or TLR8 agonist, e.g., imiquimod, 852A, hiltonol, resiquimod, 3M-052, CpG oligodeoxynucleotides (CpG ODN), 1V270, or SD-101. [0151] In some embodiments, the immunomodulatory agent is an anti-inflammatory agent, such as arylcarboxylic derivatives, pyrazole-containing derivatives, oxicam derivatives and nicotinic acid derivatives. Classes of anti-inflammatory agents include, for example, cyclooxygenase inhibitors, 5-lipoxygenase inhibitors, and leukotriene receptor antagonists. [0152] Suitable cyclooxygenase inhibitors may include meclofenamic acid, mefenamic acid, carprofen, diclofenac, diflunisal, fenbufen, fenoprofen, indomethacin, ketoprofen, nabumetone, Attorney Docket No. BRI-023WO sulindac, tenoxicam and tolmetin. Leukotriene receptor antagonists may include calcitriol, and ontazolast. [0153] Suitable lipoxygenase inhibitors include redox inhibitors (e.g., catechol butane derivatives, nordihydroguaiaretic acid (NDGA), masoprocol, phenidone, Ianopalen, indazolinones, naphazatrom, benzofuranol, alkylhydroxylamine), and non-redox inhibitors (e.g., hydroxythiazoles, methoxyalkylthiazoles, benzopyrans and derivatives thereof, methoxytetrahydropyran, boswellic acids and acetylated derivatives of boswellic acids, and quinolinemethoxyphenylacetic acids substituted with cycloalkyl radicals), and precursors of redox inhibitors. Other suitable lipoxygenase inhibitors include antioxidants (e.g., phenols, propyl gallate, flavonoids and/or naturally occurring substrates containing flavonoids, hydroxylated derivatives of the flavones, flavonol, dihydroquercetin, luteolin, galangin, orobol, derivatives of chalcone, 4,2',4'- trihydroxychalcone, ortho-aminophenols, N-hydroxyureas, benzofuranols, ebselen and species that increase the activity of the reducing selenoenzymes), iron chelating agents (e.g., hydroxamic acids and derivatives thereof, N-hydroxyureas, 2-benzyl-1-naphthol, catechols, hydroxylamines, carnosol trolox C, catechol, naphthol, sulfasalazine, zyleuton, 5-hydroxyanthranilic acid and 4-(omega- arylalkyl)phenylalkanoic acids), imidazole-containing compounds (e.g., ketoconazole and itraconazole), phenothiazines, and benzopyran derivatives. Yet other suitable lipoxygenase inhibitors include inhibitors of eicosanoids (e.g., octadecatetraenoic, eicosatetraenoic, docosapentaenoic, eicosahexaenoic and docosahexaenoic acids and esters thereof, PGE1 (prostaglandin E1), PGA2 (prostaglandin A2), viprostol, 15-monohydroxyeicosatetraenoic, 15- monohydroxy-eicosatrienoic and 15-monohydroxyeicosapentaenoic acids, and leukotrienes B5, C5 and D5), compounds interfering with calcium flows, phenothiazines, diphenylbutylamines, verapamil, fuscoside, curcumin, chlorogenic acid, caffeic acid, 5,8,11,14-eicosatetrayenoic acid (ETYA), hydroxyphenylretinamide, Ionapalen, esculin, diethylcarbamazine, phenantroline, baicalein, proxicromil, thioethers, diallyl sulfide and di-(1-propenyl) sulfide. [0154] Other useful molecules or payloads that may be used in accordance with present disclosure include chemical compounds used in the treatment of cancer. Examples of chemotherapeutic agents include Erlotinib (TARCEVA®, Genentech/OSI Pharm.), Bortezomib (VELCADE®, Millennium Pharm.), Fulvestrant (FASLODEX®, AstraZeneca), Sutent (SU11248, Pfizer), Letrozole (FEMARA®, Novartis), Imatinib mesylate (GLEEVEC®, Novartis), PTK787/ZK 222584 (Novartis), Oxaliplatin (Eloxatin®, Sanofi), 5-FU (5-fluorouracil), Leucovorin, Rapamycin (Sirolimus, RAPAMUNE®, Wyeth), Lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), Lonafarnib (SCH 66336), Sorafenib (BAY43-9006, Bayer Labs), and Gefitinib (IRESSA®, AstraZeneca), AG1478, AG1571 (SU 5271; Sugen). Further examples of chemotherapeutic agents Attorney Docket No. BRI-023WO include alkylating agents such as thiotepa and CYTOXAN® (cyclosphosphamide); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (e.g., bullatacin and bullatacinone); a camptothecin (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (e.g., cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ11 and calicheamicin omegall); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores). Further anti-cancer agents include aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5- oxo-L-norleucine, ADRIAMYCIN® (doxorubicin), morpholino-doxorubicin, cyanomorpholino- doxorubicin, 2-pyrrolino-doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin. Further anti-cancer agents include anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamniprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® Attorney Docket No. BRI-023WO polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE® (Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, I11.), and TAXOTERE® (doxetaxel; Rhone- Poulenc Rorer, Antony, France); chloranmbucil; GEMZAR® (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® (vinorelbine); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above. [0155] Other useful molecules or payloads that may be used in accordance with the present disclosure include: (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, 4- hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifine citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX® (anastrozole; AstraZeneca); (iii) antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-α, Ralf and H-Ras; (vii) ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors; (viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®, LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; a topoisomerase 1 inhibitor such as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such as bevacizumab (AVASTIN®, Genentech); and (x) pharmaceutically acceptable salts, acids and derivatives of any of the above. Other anti-angiogenic agents include MMP-2 (matrix- metalloproteinase 2) inhibitors, MMP-9 (matrix-metalloproteinase 9) inhibitors, COX-II Attorney Docket No. BRI-023WO (cyclooxygenase II) inhibitors, and VEGF receptor tyrosine kinase inhibitors. Examples of VEGF receptor tyrosine kinase inhibitors include 4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1- methylpiperidin-4-ylmethoxy)quinazoline (ZD6474), 4-(4-fluoro-2-methylindol-5-yloxy)-6- methoxy-7-(3-pyrrolidin-1-ylpropoxy)-quinazoline (AZD2171), vatalanib (PTK787) and SU11248 (sunitinib). [0156] Additional exemplary molecules for conjugation include an amatoxin, chalicheamicin, DUBA, FAM, MMAD, PBD, and a toxoid. [0157] In some embodiments, an unnatural amino acid comprises a bioconjugation handle (also referred to herein as a bioconjugation site) to facilitate conjugation to another molecule (e.g., via use of a crosslinking agent). In some embodiments, a method disclosed herein can be used to site-specifically incorporate two different unnatural amino acids, each with a different bioconjugation handle, into a single protein (e.g., a single antibody). In some embodiments, the two bioconjugation handles can be chosen such that they each can be chemoselectively conjugated to two different labels using mutually orthogonal conjugation chemistries. Such pairs of bioconjugation handles include, for example: azide and alkyne, azide and ketone/aldehyde, azide and cyclopropene, ketone/aldehyde and cyclopropene, 5-hydroxyindole and azide, 5-hydroxyindole and cyclopropene, and 5-hydroxyindole and ketone/aldehyde. [0158] A payload (e.g., a molecule) used in accordance with the present disclosure can be conjugated through a variety of linking groups (linkers). The linker may be a cleavable linker or a non-cleavable linker (e.g., a linker may comprises a cleavable or non-cleavable portion or region). Optionally or in addition, a linker may be a flexible linker or an inflexible linker. The linker should be a length sufficiently long to allow the molecule and the antibody to be linked without steric hindrance from one another and sufficiently short to retain the intended activity of the antibody. The linker preferably is sufficiently hydrophilic to avoid or minimize instability or insolubility of the antibody. The linker should be sufficiently stable in vivo (e.g., it is not cleaved by serum, enzymes, etc.) to permit the antibody to be operative (e.g., selectively operative) in vivo. [0159] In some embodiments, the linker is a molecule which functions as a novel bifunctional fluorescence probe that allows self-elimination cleavage in the presence of proteases for payload release and fluorophore activation, such as 7-amino-3-hydroxyethyl-coumarin (7-AHC) based dipeptide linkers. An exemplary scenario is shown in FIG.7, where Cathepsin B releases payload from ADC; however, the intact ADC remains stable upon incubation in serum (human or mouse origin). Attorney Docket No. BRI-023WO [0160] In some embodiments, a linker used in accordance with the present disclosure may be from about 1 angstroms (Å) to about 150 Å in length, or from about 1 Å to about 120 Å in length, or from about 5 Å to about 110 Å in length, or from about 10 Å to about 100 Å in length. In some embodiments, a linker may be greater than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 27, 30 or greater angstroms in length and/or less than about 110, 100, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, or fewer Å in length. In some embodiments, a linker may be about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, and 120 Å in length. [0161] In some embodiments, the linker is any divalent or multivalent linker known to those of skill in the art. Generally, the linker is capable of forming covalent bonds to the molecule and the unnatural amino acid. Useful divalent linkers include alkylene, substituted alkylene, heteroalkylene, substituted heteroalkylene, arylene, substituted arylene, heteroarlyene and substituted heteroarylene linkers. In some embodiments, the linker is C1-10 alkylene or C1-10 heteroalkylene. [0162] The linker may include a water soluble polymer. The water soluble polymer may be any structural form including but not limited to linear, forked or branched. Typically, the water soluble polymer is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG), but other water soluble polymers can also be employed. The term "PEG" is used broadly to encompass any polyethylene glycol molecule, without regard to size or to modification at an end of the PEG. [0163] In some embodiments, a PEG used in accordance with the present disclosure terminates on one end with hydroxy or methoxy. Alternatively, the PEG can terminate with a reactive group, thereby forming a bifunctional polymer. Typical reactive groups can include those reactive groups that are commonly used to react with the functional groups found in the 20 common amino acids (including but not limited to, maleimide groups, activated carbonates (including but not limited to, p-nitrophenyl ester), activated esters (including but not limited to, N- hydroxysuccinimide, p-nitrophenyl ester) and aldehydes) as well as functional groups that are inert to the 20 common amino acids but that react specifically with complementary functional groups present in UAAs (including but not limited to, azide groups, and alkyne groups). [0164] Any molecular mass for a PEG can be used as practically desired, including but not limited to, from about 50 Daltons (Da) to 100,000 Da or more as desired (including but not limited to, sometimes 100 Da to 100,000 Da, 0.1-50 kDa, or 10-40 kDa). Branched chain PEGs, including but not limited to, PEG molecules with each chain having a MW ranging from 1-100 kDa (including but not limited to, 1-50 kDa or 5-20 kDa) can also be used. A contemplated linker may Attorney Docket No. BRI-023WO include any appropriate number of PEG units, e.g.¸ from 2 to 24 PEG units, e.g., PEG2, PEG4, PEG6, PEG8, PEG10, PEG12, or PEG24. A wide range of PEG molecules are described in, including but not limited to, the Shearwater Polymers, Inc. catalog, Nektar Therapeutics catalog. In some embodiments, a linker comprises a spacer region or portion that comprises a PEG region. In some embodiments, a PEG region comprises PEG8. [0165] Generally, at least one terminus of the PEG molecule is available for reaction with the unnatural amino acid. For example, PEG derivatives bearing alkyne and azide moieties for reaction with amino acid side chains can be used to attach PEG to unnatural amino acids as described herein. If the unnatural amino acid comprises an azide, then the PEG will typically contain either an alkyne moiety to effect formation of the [3+2] cycloaddition product or an activated PEG species (i.e., ester, carbonate) containing a phosphine group to effect formation of the amide linkage. Alternatively, if the unnatural amino acid comprises an alkyne, then the PEG will typically contain an azide moiety to effect formation of the [3+2] Huisgen cycloaddition product. Similarly, if the unnatural amino acid comprises a tetrazine, then the PEG will typically contain a strained alkene. Alternatively, if the unnatural amino acid comprises a strained alkene, then the PEG will typically contain a tetrazine. If the unnatural amino acid comprises a carbonyl group, the PEG will typically comprise a potent nucleophile (including but not limited to, a hydrazide, hydrazine, hydroxylamine, or semicarbazide functionality) in order to effect formation of corresponding hydrazone, oxime, and semicarbazone linkages, respectively. In other alternatives, a reverse of the orientation of the reactive groups described above can be used, i.e., an azide moiety in the unnatural amino acid can be reacted with a PEG derivative containing an alkyne. [0166] Many other polymers are also suitable for use in the present disclosure. In some examples, polymer backbones that are water-soluble, with from 2 to about 300 termini, are particularly useful. Examples of suitable polymers include, but are not limited to, other poly(alkylene glycols), such as poly(propylene glycol) ("PPG"), copolymers thereof (including but not limited to copolymers of ethylene glycol and propylene glycol), terpolymers thereof, mixtures thereof, and the like. Although the molecular weight of each chain of the polymer backbone can vary, it is typically in the range of from about 800 Da to about 100,000 Da, often from about 6,000 Da to about 80,000 Da. [0167] As understood in the art, PEG and related polymers may include degradable linkages in the polymer backbone or in the linker group between the polymer backbone and one or more of the terminal functional groups of the polymer molecule. For example, ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on a Attorney Docket No. BRI-023WO biologically active agent generally hydrolyze under physiological conditions to release the agent. Other hydrolytically degradable linkages include, but are not limited to, carbonate linkages; imine linkages resulted from reaction of an amine and an aldehyde; phosphate ester linkages formed by reacting an alcohol with a phosphate group; hydrazone linkages which are reaction product of a hydrazide and an aldehyde; acetal linkages that are the reaction product of an aldehyde and an alcohol; orthoester linkages that are the reaction product of a formate and an alcohol; peptide linkages formed by an amine group, including but not limited to, at an end of a polymer such as PEG, and a carboxyl group of a peptide; and oligonucleotide linkages formed by a phosphoramidite group, including but not limited to, at the end of a polymer, and a 5' hydroxyl group of an oligonucleotide. Branched linkers may be used in antibodies of the disclosure. A number of different cleavable linkers are known to those of skill in the art. The mechanisms for release of an agent from these linker groups include, for example, irradiation of a photolabile bond and acid- catalyzed hydrolysis. The length of the linker may be predetermined or selected depending upon a desired spatial relationship between the antibody and the molecule linked to it. In view of the large number of methods that have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, drugs, toxins, and other agents to antibodies one skilled in the art will be able to determine a suitable method for attaching a given agent to an antibody. [0168] Any hetero- or homo-bifunctional linker can be used to link the conjugates. The linker may have a wide range of molecular weight or molecular length. Larger or smaller molecular weight linkers may be used to provide a desired spatial relationship or conformation between the antibody and the linked entity. Linkers having longer or shorter molecular length may also be used to provide a desired space or flexibility between the antibody and the linked entity. Similarly, a linker having a particular shape or conformation may be utilized to impart a particular shape or conformation to the antibody or the linked entity, either before or after the antibody reaches its target. The functional groups present on each end of the linker may be selected to modulate the release of an antibody or a payload under desired conditions. [0169] Some examples of water-soluble bifunctional linkers have a dumbbell structure that includes: a) an azide, an alkyne, a hydrazine, a hydrazide, a hydroxylamine, a carbonyl, a tetrazine, or a strained alkene-containing moiety on at least a first end of a polymer backbone; and b) at least a second functional group on a second end of the polymer backbone. The second functional group can be the same or different as the first functional group. The second functional group, in some examples, is not reactive with the first functional group. Provided, in some examples, are water- soluble compounds that comprise at least one arm of a branched molecular structure. For example, the branched molecular structure can be dendritic. Attorney Docket No. BRI-023WO [0170] Further illustrative linkers include, for example, malC, thioether, AcBut, valine- citrulline peptide, malC-valine-citrulline peptide, hydrazone, and disulfide. Other illustrative linkers can include beta-glucuronide linkers. [0171] In some embodiments, coupling of antibody and molecule can be accomplished via a crosslinking agent. There are several intermolecular crosslinking agents which can be utilized, see for example, Means and Feeney, CHEMICAL MODIFICATION OF PROTEINS, Holden-Day, 1974, pp. 39-43. Among these reagents are, for example, N-succinimidyl3-(2-pyridyldithio) propionate (SPDP) or N, N’- (1,3-phenylene) bismaleimide (both of which are highly specific for sulfhydryl groups and form irreversible linkages); N, N’-ethylene-bis-(iodoacetamide) or other such reagent having 6 to 11 carbon methylene bridges (which are relatively specific for sulfhydryl groups); and 1, 5-difluoro-2,4- dinitrobenzene (which forms irreversible linkages with amino and tyrosine groups). Other crosslinking agents useful for this purpose include: p,p’-difluoro-N,N’- dinitrodiphenylsulfone (which forms irreversible crosslinkages with amino and phenolic groups); dimethyl adipimidate (which is specific for amino groups); phenol-1,4- disulfonylchloride (which reacts principally with amino groups); hexamethylenediisocyanate or diisothiocyanate, or azophenyl-p-diisocyanate (which reacts principally with amino groups); glutaraldehyde (which reacts with several different side chains) and disdiazobenzidine (which reacts primarily with tyrosine and histidine); N-3- Maleimidopropanoic acid; N-6-Maleimidocaproic acid; N-11- Maleimidoundecanoic acid, 4- (N-maleimidomethyl)cyclohexane-1-carboxy-6-amidocaproic acid; 4-[(Nmaleimidoethyl)carboxamidoethyl(Peg)4 carboxamidomethyl]cyclohexanecarboxylic acid. [0172] The crosslinking agent may be homobifunctional, i.e., having two functional groups that undergo the same reaction. An example of a homobifunctional crosslinking agent is bismaleimidohexane (“BMH”). BMH contains two maleimide functional groups, which react specifically with sulfhydryl-containing compounds under mild conditions (pH 6.5-7.7). The two maleimide groups are connected by a hydrocarbon chain. Therefore, BMH is useful for irreversible crosslinking of polypeptides that contain cysteine residues. Additional commercially available homobifunctional crosslinking agents include: BSOCOES (Bis(2 [Succinimidooxycarbonyloxy]ethyl) sulfone; DPDPB (1,4-Di-(3’-[2pyridyldithio]-propionamido) butane; DSS (disuccinimidyl suberate); DST (disuccinimidyl tartrate); Sulfo DST (sulfodisuccinimidyl tartrate); DSP (dithiobis(succinimidyl propionate); DTSSP (3,3’- Dithiobis(sulfosuccinimidyl propionate); EGS (ethylene glycol bis(succinimidyl succinate)); BASED (Bis(β-[4-azidosalicylamido]-ethyl)disulfide iodinatable); homobifunctional NHS crosslinking reagents (e.g., Bis(NHS)PEO-5 (bis N-succinimidyl-[pentaethylene glycol] ester); and homobifuctional isothiocyanate derivatives of PEG or dextran polymers. Attorney Docket No. BRI-023WO [0173] Heterobifunctional crosslinking agents have two different functional groups, for example an amine-reactive group and a thiol-reactive group, that will crosslink two moieties having free amines and thiols, respectively. The most common commercially available heterobifunctional crosslinking agents have an amine reactive N-hydroxysuccinimide ester as one functional group, and a sulfhydryl reactive group as the second functional group. The most common sulfhydryl reactive groups are maleimides, pyridyl disulfides and active halogens. One of the functional groups can be a photoactive aryl nitrene, which upon irradiation reacts with a variety of groups. Exemplary heterobifunctional crosslinking agents include succinimidyl 4-(N maleimidomethyl) cyclohexane-1-carboxylate (“SMCC”), Succinimidyl-4-(N maleimidomethyl)-cyclohexane-1- carboxy(6-amidocaproate) (“LC-SMCC”), N maleimidobenzoyl-N-hydroxysuccinimide ester (“MBS”), and succinimide 4-(p-maleimidophenyl) butyrate (“SMPB”), an extended chain analog of MBS. The succinimidyl group of these crosslinking agents reacts with a primary amine forming an amide bond, and the thiol-reactive maleimide forms a covalent thioether bond with the thiol group (e.g., of a cysteine). [0174] Additional exemplary crosslinking agents include: BS3 ([Bis(sulfosuccinimidyl)suberate], which is a homobifunctional N-hydroxysuccinimide ester that targets accessible primary amines; NHS/EDC (N-hydroxy-succinimide and N-ethyl- ‘(dimethylaminopropyl)carbodimide, which allows for the conjugation of primary amine groups with carboxyl groups); sulfoEMCS ([N-e-Maleimido-caproic acid]hydrazide, which includes heterobifunctional reactive groups (a maleimide and an NHS-ester) that are reactive toward sulfhydryl and amino groups; hydrazide, which is useful for useful for linking carboxyl groups on exposed carbohydrates to primary amines; SATA (N-succinimidyl-S-acetylthioacetate), which is reactive towards amines and adds protected sulfhydryl groups; monofluoro cyclooctyne (MFCO); bicyclo[6.1.0]nonyne (BCN); N succinimidyl-S-acetylthiopropionate (SATP); maleimido and dibenzocyclooctyne ester (a DBCO ester); and EDC (1-Ethyl-3-[3- dimethylaminopropyl]carbodiimide hydrochloride). In some embodiments, a crosslinking agent may be used in combination with an aminohexyl spacer or other short carbon spacer to connect the crosslinking agent to the main or linker (e.g., a crosslinking agent may comprise a DBCO and a linker containing carbons, a hexyl linker, or a heterohexyl linker). [0175] The length of these crosslinking agents can be varied by the use of polymeric regions between the two reactive groups, which typically take the form of chemical linkers such as polymeric ethylene glycol or simple carbon chains, but can also include sugars, amino acids or peptides, or oligonucleotides. Polymer chain lengths of from 5 to 50 nm are typical, but can be Attorney Docket No. BRI-023WO shorter or longer as needed. For example, the crosslinking agent may comprise a <2 carbon chain arm, a 2-5 carbon chain arm, or a 3-6 carbon chain arm. [0176] Crosslinking agents often have low solubility in water. A hydrophilic moiety, such as a sulfonate group, may be added to the crosslinking agent to improve its water solubility. Sulfo- MBS and sulfo-SMCC are examples of crosslinking agents modified for water solubility. [0177] Many crosslinking agents yield a conjugate that is essentially non-cleavable under cellular conditions. However, some crosslinking agents contain a covalent bond, such as a disulfide, that is cleavable under cellular conditions. For example, Traut’s reagent, dithiobis(succinimidylpropionate) (“DSP”), and N-succinimidyl 3-(2-pyridyldithio) propionate (“SPDP”) are well-known cleavable crosslinking agents. Direct disulfide linkage may also be useful. [0178] Numerous crosslinking agents, including the ones discussed above, are commercially available. Detailed instructions for their use are readily available from the commercial suppliers. A general reference on protein cross-linking and conjugate preparation is: Wong, CHEMISTRY OF PROTEIN CONJUGATION AND CROSS-LINKING, CRC Press (1991). [0179] In some embodiments, the linker comprises a polypeptide linker that connects or fuses the molecule to the antibody. When a polypeptide linker is employed, the linker may comprise hydrophilic amino acid residues, such as Gln, Ser, Gly, Glu, Pro, His and Arg. In certain embodiments, the linker is a peptide containing 1-25 amino acid residues, 1-20 amino acid residues, 2-15 amino acid residues, 3-10 amino acid residues, 3-7 amino acid residues, 4-25 amino acid residues, 4-20 amino acid residues, 4-15 amino acid residues, 4-10 amino acid residues, 5-25 amino acid residues, 5-20 amino acid residues, 5-15 amino acid residues, or 5-10 amino acid residues. Exemplary linkers include glycine and serine-rich linkers, e.g., (GlyGlyPro)n (SEQ ID NO: 63), or (GlyGlyGlyGlySer)n (SEQ ID NO: 64), where n is 1-5. In some embodiments, the linker comprises, consists, or consists essentially of GGGGS (SEQ ID NO: 65). In certain embodiments, the linker comprises, consists, or consists essentially of GGGGSGGGGS (SEQ ID NO: 66). Additional exemplary linker sequences are disclosed, e.g., in George et al. (2003) PROTEIN ENGINEERING, 15: 871–879, and U.S. Patent Nos.5,482,858 and 5,525,491. [0180] In some embodiments, an unnatural amino acid used in accordance with the present disclosure comprises a non-natural aromatic chemical moiety (e.g., a hydroxyl-indole group; an amino-indole group; an aminophenol group; or a hydroxyl-phenol group, e.g., the UAA is 5- hydroxytryptophan (5-HTP), or an analog thereof), and/or the linker comprises a diazonium group (e.g., the linker comprises 4-nitrobenzenediazonium (4NDz); 4-carboxybenzenediazonium (4NeDz) Attorney Docket No. BRI-023WO or 4-methoxybenzenediazonium (4MCDz). The unnatural amino acid and linker may react under conditions suitable to form an azo-linkage via an azo-coupling reaction between the aromatic chemical moiety and the diazonium group. Further methods for conjugation of molecules to unnatural amino acids are described, for example, in U.S. Patent Application Publication No. 2018/0360984. [0181] In some embodiments, when a molecule is conjugated to an antibody, the antibody has an average drug-to-antibody ratio (DAR) of at least 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0, as measured by hydrophobic interaction chromatography (HIC) or liquid chromatography-mass spectrometry (LCMS). In this context, it is understood that DAR may refer to the ratio of any conjugated molecule to antibody (e.g., a detectable label as well as a drug). In some embodiments, the antibody has an average DAR that is within 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the number of unnatural amino acids in the antibody. [0182] In some embodiments, a payload is conjugated to an antibody or antigen binding fragment thereof using a linkage of A-U-U’-X1-L-X2-P, where A is an antibody or antigen binding fragment thereof, U is an unnatural amino acid bioconjugation handle (e.g., any suitable unnatural amino acid described herein), U’ is a crosslinking agent or crosslinker (e.g., any crosslinking agent or crosslinker described herein), X1 is a first spacer, L is a cleavable or non-cleavable linking spacer, X2 is a second spacer, and P is a payload (e.g., any payload described herein). In some embodiments, only one spacer (X1 or X2) is used to conjugate the payload to the antibody or antigen binding fragment. Several non-limiting examples of linkage of an A-U portion to a U’-X1- L-X2-P portion are shown in FIGs.23A-23B. In some embodiment, a payload is conjugated to an antibody or antigen binding fragment thereof using a linkage of A-U-U’-X1-L-P. In some embodiment, a payload is conjugated to an antibody or antigen binding fragment thereof using a linkage of A-U-U’-L-X2-P. [0183] In some embodiments, a spacer (e.g., X1 and/or X2) used in accordance with the present disclosure comprises a PEG (e.g., any PEG described herein). In some embodiments, a spacer comprises PEG2, PEG4, PEG6, PEG8, PEG10, PEG12, or PEG24. In some embodiments, a spacer comprises PEG2. In some embodiments, a spacer comprises PEG4. In some embodiments, a spacer comprises PEG6. In some embodiments, a spacer comprises PEG8. In some embodiments, a spacer comprises PEG10. In some embodiments, a spacer comprises PEG12. In some embodiments, a spacer comprises PEG24. In some embodiments, a spacer comprises a polysarcosine (PSAR). In some embodiments, a spacer comprises a monodisperse PSAR. In some Attorney Docket No. BRI-023WO embodiments, a spacer comprises a PAB. In some embodiments, a spacer comprises an aliphatic chain. [0184] In some embodiments, a linking spacer comprises a cleavable linking spacer portion. In some embodiments, a linking spacer comprises a non-cleavable spacer portion. In some embodiments, a linking spacer comprises a peptide region. In some embodiments, a linking spacer comprises a Val-Cit motif. In some embodiments, a linking spacer comprises a Val-Ala (VA) motif. In some embodiments, a linking spacer comprises a beta-glucuronide. In some embodiments, a linking spacer comprises a GGFG motif. [0185] In some embodiments, a linker comprises a structure of U’-X1-L-X2, wherein U’ comprises DBCO, X1 comprises a PEG, L comprises a VA motif, and X2 comprises a PAB. In some embodiments, a linker comprises a structure of U’-X1-L-X2, wherein U’ comprises DBCO, X1 comprises a PEG8, L comprises a VA motif, and X2 comprises a PAB. In some embodiments, a linker comprises a structure of U’-X1-L, wherein U’ comprises DBCO, X1 comprises a PEG, and L comprises a GGFG motif. In some embodiments, a linker comprises a structure of U’-X1-L, wherein U’ comprises DBCO, X1 comprises a PEG4, and L comprises a GGFG motif. [0186] Other exemplary linkers that may be used in accordance with the present disclosure include those discussed in Zheng et al. (2021) ACTA PHARM SIN B, 11(12): 3889-3907. [0187] In some embodiments, the linkage is mediated by interchain cysteine-maleimide conjugation, for example, where linkage is between an A-InterchainCys portion and a Mal-Linker- Payload portion. In some embodiments, the linkage is mediated by THIOMAB conjugation, for example, where linkage is between an A-EngineeredCys portion and a Mal-Linker-Payload portion. In some embodiments, when a payload molecule (any payload described herein, e.g., those in FIGs.15-18) is conjugated to an antibody through an unnatural amino acid bioconjugation handle (U) to a U’-Linker-Payload portion, the antibody has an average-to-drug antibody ratio (DAR) of at least 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0, or 4-6 DAR in 0.1 DAR increments as measured by hydrophobic interaction chromatography (HIC) or LCMS. In some embodiments, the antibody has an average DAR of about 2. In this context, it is understood that DAR may refer to the ratio of any conjugated molecule to antibody (e.g., a detectable label as well as a drug). In some embodiments, the antibody has an average DAR that is within 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the number of unnatural amino acids with the corresponding bioconjugation handle in the antibody. [0188] In some embodiments, when a conjugate (e.g., any conjugate described herein) is formed through a [3+2] alkyne-azide cycloaddition reaction, the divalent residue of the conjugating Attorney Docket No. BRI-023WO group comprises a triazole ring or fused cyclic group comprising a triazole ring. In some embodiments, a conjugate is formed through a strain-promoted [3+2] alkyne-azide cycloaddition (SPAAC) reaction, or other tryptophan based conjugation chemistries (CRACR, eCLIC, etc.), see, e.g., FIGs.23A-23B. [0189] In some embodiments, an antibody provided herein comprises an unnatural amino acid, which facilitates conjugation to a molecule or payload, e.g., a detectable label or a drug (e.g., a small molecule drug). In some embodiments, the molecule is: AEB, AEVB, AFP, an amatoxin, an auristatin (e.g., auristatin E), a calicheamicin, CC-1065 or a CC-1065 analog, chalicheamicin, combretastatin, DM1, DM4, docetaxel, dolastatin-10, DUBA, a duocarmycin, echinomycin, FAM, maytansine, a maytansinoid, MMAD, MMAE, MMAF, a morpholino-doxorubicin (e.g., cyanomorpholino-doxorubicin), netropsin, an oligonucleotide (e.g., a DNA, RNA, or LNA oligonucleotide), paclitaxel, PBD, a peptide (e.g., a therapeutic peptide), rhizoxin, a small molecule (e.g., a therapeutic small molecule) SN-38, topotecan, a topoisomerase inhibitor, or a toxoid. In some embodiments, the molecule is conjugated to the unnatural amino acid by a linker, e.g., a cleavable linker, a non-cleavable linker, a peptide-based linker, or a PEG-based linker. In some embodiments, an ADC of the present disclosure (e.g., any ADC described herein, e.g., those described in FIG.5) may be generated using any payload or molecule described herein, e.g., those shown in FIGs.15-18. Any amino acid sites in an antibody sequence (e.g., those shown in TABLE 1) may be selected for incorporation of an unnatural amino acid and subsequent modification (e.g., conjugation to a payload described herein). Exemplary conjugated antibody structures are shown in FIGs.19-22. [0190] Additional examples of therapeutic, diagnostic, and other proteins that can be modified to comprise one or more unnatural amino acids and/or branched linkers are described in U.S. Patent Application Publication Nos.2003/0082575 and 2005/0009049, and WO2022115625A1, and WO2022056318A1. [0191] Non-limiting examples of payload and linker structures are described in FIGs.15-22 and 29A-29B. FIG.19 is an example of the diversity of PBD payloads that can be site-selectively attached to an antibody (e.g., an anti-CD276 antibody) to form an ADC using the unnatural amino acid incorporation technology described herein. One or more of the PBD cores shown in FIG.20 may be used, with the exact attachment site of the linker being determined through a variety of industry validated sites (i.e., imine, amine, A, B, or C ring, linker between dimers or other). Depending upon the circumstances, one or more of the PBD cores outlined in FIG.20 are attached as monomers. Any known stereochemical iteration of the structures shown in FIG.20 may be used Attorney Docket No. BRI-023WO in accordance with the present disclosure, see, e.g., Mantaj et al. (2016). FIG.21 shows some exemplary PBD based ADC connectivity options for certain PBD dimers. In some embodiments, B rings are deactivated to prevent DNA crosslinking, limiting the PBD dimer to monoalkylation. In these instances, U and U’ correspond to the site-specific unnatural amino acid bioconjugation handle (U), and the corresponding crosslinking agent (U’). FIG.22 describes exemplary pro-PBD strategies that, in some embodiments, are attached to the antibody via unnatural amino acid conjugation chemistry. Commonly used unnatural amino acid conjugation chemistries are outlined in FIGs.23A-23B. The exact structures in FIGs.23A-23B are not limiting to the unnatural amino acid structure described therein as long as the functional group remains similar. II. (c) Specific Antibody Conjugates [0192] In some embodiments, the present disclosure provides an antibody conjugate that specifically binds to CD276 comprising a heavy chain and a light chain, wherein the heavy chain and the light chain are selected from Table 2. In some embodiments, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 61. In some embodiments, the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 62. In some embodiments, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 61, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 62. [0193] In some embodiments, an antibody of the present disclosure comprises one or more unnatural amino acids at sites selected from Table 1. It is contemplated that antibody amino acid residue sites shown in Table 1 are useful for incorporating unnatural amino acids in any anti- CD276 antibody sequence, e.g., by aligning the sites in Table 1 with another anti-CD276 antibody sequence to identify each site (see, e.g., FIG 1 and 2). [0194] In some embodiments, antigen binding fragments of an antibody provided herein (e.g., an anti-CD276 antibody) are used in accordance with the present disclosure. In certain embodiments, an exemplary antigen binding fragment comprises an amino acid sequence in whole or in part of the sequences outlined in TABLE 2. [0195] In some embodiments, an antibody conjugate of the present disclosure can contain one or more unnatural amino acids selected from any genetically encodable unnatural amino acid known in the art (see, e.g., Liu and Schultz, 2010; Dumas and Davis, 2015) including those described herein (see, e.g., FIG.3), particularly LCA or HTP. In some embodiments, the unnatural Attorney Docket No. BRI-023WO amino acid is LCA. In some embodiments, the antibody comprises one, two, three, four, or more than four unnatural amino acids. In some embodiments, the antibody containing an unnatural amino acid is made in a eukaryotic or prokaryotic expression host via stable integration or transient transfection. [0196] In some embodiments, a provided antibody comprises an Fc region with at least one of S242C (S239C by EU numbering), L234A, L235A, and P329G mutations. In some embodiments, a provided antibody comprises an Fc region with S242C (S239C by EU numbering), L234A, L235A, and P329G mutations. [0197] In some embodiments, engineered antibodies are made according to the general conjugation scheme where a bioconjugation ready antibody, A-U (where “A” is an anti-CD276 antibody or antigen binding fragment thereof and “U” is an unnatural amino acid bioconjugation handle) is conjugated to a Linker-Payload, U’-X1-L-X2-P (where “U’” is the corresponding crosslinking agent or crosslinker that enables the covalent linkage of the linker-payload, “X1” and “X2” are spacers (e.g., PEG (e.g., PEG2, PEG4, PEG8, etc.), aliphatic chains, polysarcosines (PSARs) (e.g., monodisperse polysarcosines), PAB, a cyclized carbon ring or substituent, etc.), “L” is a cleavable or non-cleavable linking spacer (e.g., Val-Cit, Val-Ala, beta-glucuronide, etc.), and “P” is a payload (e.g., any payload that has cytotoxic activity, can stimulate an immune response in a target cell, or any payload described herein) to produce an antibody drug conjugate, A-U-U’-X1- L-X2-P. The engineered antibodies described herein (e.g., anti-CD276 ADCs) are not limited to this exact general conjugation scheme, as any combination of site (e.g., any combination of those provided in TABLE 1), linker structure (e.g., any linker described herein), and payload (e.g., any payload described herein) may be used. In some embodiments, spacers can be positioned differentially and branched, as shown in FIG.18. In some embodiments, only one spacer is used in the generic conjugation scheme, e.g., providing a Linker-Payload structure of U’-X1-L-P or U’-L- X2-P. [0198] In some embodiments, the payload moiety or molecule consists of any combination of the following: AEB, AEVB, AFP, an amatoxin, an auristatin (e.g., auristatin E), a calicheamicin, CC-1065 or a CC-1065 analog, chalicheamicin, combretastatin, DM1, DM4, docetaxel, dolastatin- 10, DUBA, a duocarmycin, echinomycin, FAM, maytansine, a maytansinoid, MMAD, MMAE, MMAF, a morpholino-doxorubicin (e.g., cyanomorpholino-doxorubicin), netropsin, an oligonucleotide (e.g., a DNA, RNA, or LNA oligonucleotide), paclitaxel, PBD, a peptide (e.g., a therapeutic peptide), rhizoxin, a small molecule (e.g., a therapeutic small molecule) SN-38, topotecan, a topoisomerase inhibitor, or a toxoid. In some embodiments, the molecule is Attorney Docket No. BRI-023WO conjugated to the unnatural amino acid by a linker, e.g., a cleavable linker, a non-cleavable linker, a peptide-based linker, a PEG-based linker, a non-PEG based hydrophilic linker or any linker described herein, and can attach to a payload (e.g., any payload described herein) at any suitable attachment site (e.g., sites that do not perturb function of the linker (if cleavable) or the payload when released from the antibody or when the ADC enters the cell and exerts its therapeutic function). [0199] In some embodiments, the present disclosure provides an ADC of structure A-U-U’- X1-L-X2-P where U can replace one or more residues on the heavy chain only, light chain only, or combinations of the two. In some embodiments, the present disclosure provides an ADC of structure A-U-U’-X1-L-X2-P where the linker contains a cleavable moiety (L), a PEG spacer (X1), and a PAB (X2) and the payload (P) comprises a PBD derivative. [0200] In some embodiments, an antibody of the present disclosure is conjugated at a site with a PBD derivative via methods common known in the art, e.g., as described in Lai et al. (2022) J. MED. CHEM.; Mantaj, et al. (2016) ANGEW CHEM. INT. ED.; Hartley, et al. (2020) EXPERT OPINION ON BIOLOGICAL THERAPY. [0201] In some embodiments, an antibody of the present disclosure comprises one or more amino acid substitutions which are conservative amino acid substitutions to m276 antibody, DS antibody, and trastuzumab antibody sequences, or any antibody region (variable or constant) with 5-10, 10-15, 15-20, 20-25, 25-30, 35-40, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, or 95-100% sequence similarity. [0202] In some embodiments, a provided antibody or engineered antibody is a humanized or human antibody. In some embodiments, the antibody is glycosylated. In some embodiments, the antibody is not glycosylated. In some embodiments, the antibody has effector function. In some embodiments, the antibody does not have effector function. [0203] In some embodiments, an antibody of the present disclosure is linked site-specifically to at least one payload moiety, such that the drug-to-antibody ratio (DAR) is 1, but more typically the DAR is 2 and up to 8. In some embodiments, the DAR is 2. [0204] The present disclosure also provides methods of treating a cancer that expresses CD276 receptor. The method comprises administering to the subject in need thereof an effective amount of an anti-CD276 antibody conjugate (e.g., any anti-CD276 antibody conjugate described herein). The cancer can be selected from a group consisting of oncology indications, such as breast, prostate, ovarian, endometrial, sarcoma, melanoma, NSCLC, Sq-ESO, bladder cancer, HNSCC, HCC, Salivary Cancer, colon, pancreatic, RCC, gastric, thyroid, blood cancers such as Attorney Docket No. BRI-023WO leukemia or lymphoma, blastoma, glioma, or neuroblastoma, or any other malignancy due to their high expression in cancer cells and low expression in healthy tissue. [0205] The present disclosure further provides methods of treating a subject who has an elevated level of CD276 expression on tumor vasculature. The method comprises the step of administering an antibody conjugate of the present disclosure, wherein the level of CD276 expression is elevated relative to a suitable control (e.g., in a healthy tissue of the subject being treated or in another subject that is healthy). The present disclosure also provides methods of treating a patient in need thereof with the ADC compositions described herein in combination with an immune checkpoint inhibitor. III. METHODS OF MAKING ANTIBODIES WITH UNNATURAL AMINO ACIDS [0206] Disclosed herein are tRNAS, aminoacyl-tRNA synthetases, and/or unnatural amino acids that may be used to incorporate an unnatural amino acid into an antibody of interest (e.g., an anti-CD276 antibody) using any appropriate translation system. [0207] The term “translation system” refers to a system including components necessary to incorporate an amino acid into a growing polypeptide chain (protein). Components of a translation system can include, e.g., ribosomes, tRNA's, synthetases, mRNA and the like. Translation systems may be cellular or cell-free, and may be prokaryotic or eukaryotic. For example, translation systems may include, or be derived from, a non-eukaryotic cell, e.g., a bacterium (such as E. coli), a eukaryotic cell, e.g., a yeast cell, a mammalian cell, a plant cell, an algae cell, a fungus cell, or an insect cell. [0208] Translation systems include host cells or cell lines, e.g., host cells or cell lines contemplated herein. To express a polypeptide of interest with an unnatural amino acid in a host cell, one may clone a polynucleotide encoding the polypeptide into an expression vector that contains, for example, a promoter to direct transcription, a transcription/translation terminator, and if for a nucleic acid encoding a protein, a ribosome binding site for translational initiation. [0209] Translation systems also include whole cell preparations such as permeabilized cells or cell cultures wherein a desired nucleic acid sequence can be transcribed to mRNA and the mRNA translated. Cell-free translation systems are commercially available and many different types and systems are well-known. Examples of cell-free systems include, but are not limited to, prokaryotic lysates such as Escherichia coli lysates, and eukaryotic lysates such as wheat germ extracts, insect cell lysates, rabbit reticulocyte lysates, rabbit oocyte lysates and human cell lysates. Reconstituted translation systems may also be used. Reconstituted translation systems may include mixtures of purified translation factors as well as combinations of lysates or lysates supplemented with purified Attorney Docket No. BRI-023WO translation factors such as initiation factor-1 (IF-1), IF-2, IF-3 (α or β), elongation factor T (EF-Tu), or termination factors. Cell-free systems may also be coupled transcription/translation systems wherein DNA is introduced to the system, transcribed into mRNA and the mRNA is translated. [0210] The present disclosure provides, among other things, methods of expressing an antibody containing an unnatural amino acid at one or more specified positions in the antibody. In some embodiments, provided methods comprise incubating a translation system (e.g., culturing or growing a host cell or cell line, e.g., a host cell or cell line disclosed herein) under conditions that permit incorporation of the unnatural amino acid into the antibody being expressed in the cell. The translation system may be contacted with (e.g. the cell culture medium may be contacted with) one, or more, unnatural amino acids (e.g., leucyl analogs, tyrosyl analogs, pyrrolysyl analogs, tryptophanyl analogs, etc.) under conditions suitable for incorporation of the one, or more, unnatural amino acids into the antibody. [0211] The antibody can be expressed from a nucleic acid sequence comprising a premature stop codon. The translation system (e.g., host cell or cell line) may, for example, contain a leucyl- tRNA synthetase mutein (e.g., a leucyl-tRNA synthetase mutein disclosed herein) capable of charging a suppressor leucyl tRNA (e.g., a suppressor leucyl tRNA disclosed herein) with an unnatural amino acid (e.g., a leucyl analog) which is incorporated into the antibody at a position corresponding to the premature stop codon. In some embodiments, the leucyl suppressor tRNA comprises an anticodon sequence that hybridizes to the premature stop codon and permits the unnatural amino to be incorporated into the antibody at the position corresponding to the premature stop codon. [0212] In some embodiments, the antibody is expressed from a nucleic acid sequence comprising a premature stop codon. The translation system (e.g., host cell or cell line) may, for example, contain a tryptophanyl-tRNA synthetase mutein (e.g., a tryptophanyl-tRNA synthetase mutein disclosed herein) capable of charging a suppressor tryptophanyl tRNA (e.g., a suppressor tryptophanyl tRNA disclosed herein) with an unnatural amino acid (e.g., a tryptophan analog) which is incorporated into the antibody at a position corresponding to the premature stop codon. In some embodiments, the tryptophanyl suppressor tRNA comprises an anticodon sequence that hybridizes to the premature stop codon and permits the unnatural amino to be incorporated into the antibody at the position corresponding to the premature stop codon. [0213] In some embodiments, the antibody is expressed from a nucleic acid sequence comprising a premature stop codon. The translation system (e.g., host cell or cell line) may, for example, contain a tyrosyl-tRNA synthetase mutein (e.g., a tyrosyl-tRNA synthetase mutein Attorney Docket No. BRI-023WO disclosed herein) capable of charging a suppressor tyrosyl tRNA (e.g., a suppressor tyrosyl tRNA disclosed herein) with an unnatural amino acid (e.g., a tyrosine analog) which is incorporated into the antibody at a position corresponding to the premature stop codon. In some embodiments, the tyrosyl suppressor tRNA comprises an anticodon sequence that hybridizes to the premature stop codon and permits the unnatural amino to be incorporated into the antibody at the position corresponding to the premature stop codon. [0214] In some embodiments, the antibody is expressed from a nucleic acid sequence comprising a premature stop codon. The translation system (e.g., host cell or cell line) may, for example, contain a pyrrolysl-tRNA synthetase mutein (e.g., a pyrrolysl-tRNA synthetase mutein disclosed herein) capable of charging a suppressor pyrrolysl tRNA (e.g., a suppressor pyrrolysl tRNA disclosed herein) with an unnatural amino acid (e.g., a pyrrolysine analog) which is incorporated into the antibody at a position corresponding to the premature stop codon. In some embodiments, the pyrrolysl suppressor tRNA comprises an anticodon sequence that hybridizes to the premature stop codon and permits the unnatural amino to be incorporated into the antibody at the position corresponding to the premature stop codon. [0215] In some embodiments, a protein (e.g., an antibody containing a unnatural amino acid) is expressed or produced in a eukaryotic cell (e.g., a mammalian cell). Features may distinguish proteins produced in prokaryotic cells (e.g., bacteria) from those produced in eukaryotic cells (e.g., mammalian cells). For example, proteins produced in mammalian cells may undergo post- translational modifications, e.g., modifications that are dependent upon enzymes located in organelles, e.g., the endoplasmic reticulum or Golgi apparatus. For example, disulfide bond formation in the endoplasmic reticulum may influence protein conformation and/or stabilization. Additional examples of such post-translational modifications include, without limitation, sulfation, amidation, palmitation, and glycosylation (e.g., N-linked glycosylation and O-linked glycosylation). Accordingly, in some embodiments, a protein (e.g., an antibody containing a unnatural amino acid) comprises one or more post-translational modifications selected from sulfation, amidation, palmitation, and glycosylation (e.g., N-linked glycosylation and O-linked glycosylation). [0216] In some embodiments, the expression yield of an antibody comprising the unnatural amino acid, for example, when expressed by a host cell or cell line, is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the expression yield of a reference antibody. For example, in some embodiments, the amount of antibody comprising the unnatural amino acid expressed by the host cell or cell line is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the Attorney Docket No. BRI-023WO amount of a reference antibody expressed by the same cell or a similar cell. In some embodiments, the reference antibody is an antibody that does not comprise the unnatural amino acid but is otherwise identical to the antibody comprising the unnatural amino acid. For example, the reference antibody may comprise a wild-type amino acid sequence, or comprise a wild-type amino acid residue at the position corresponding to the unnatural amino acid. Antibody expression may be measured by any method known in the art, including for example, Western blot or ELISA. Expression may be measured by measuring protein concentration (e.g., by ultraviolet (UV) absorption at 280 nm or Bradford assay) in a solution of defined volume and purity following purification of the antibody. [0217] In some embodiments, a disclosed method further comprises purifying the antibody. Specific expression and purification conditions will vary depending upon the expression system employed. Purification techniques known in the art include, e.g., those employing affinity tags such as glutathione-S-transferase (GST) or histidine tags. In some embodiments, an antibody may be purified by contacting the antibody with protein A and/or protein G. In some embodiments, following protein G purification (e.g., following only protein G purification) less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the antibody is aggregated, as measured by size exclusion chromatography (SEC). [0218] In certain embodiments, a disclosed method further comprises conjugating a molecule or payload to a unnatural amino acid in the antibody. In certain embodiments, the method comprises conjugating the molecule or payload to the unnatural amino acid within 5 minutes to 48 hours at room temperature (e.g., for less than 48 hours, less than 36 hours, less than 24 hours, less than 12 hours, less than 6 hours, less than 1 hour, less than 30 minutes, less than 15 minutes, or less than 10 minutes). [0219] In another aspect, the invention provides a method of producing any of the following antibodies in preparation for bioconjugation to a therapeutic payload. The method comprises culturing a cell with: (i) a nucleic acid comprising a nucleotide sequence encoding a tRNA comprising an anticodon that hybridizes to a codon selected from UAG, UGA, and UAA, and is capable of being charged with the unnatural amino acid (UAA); (ii) a nucleic acid comprising a nucleotide sequence encoding an aminoacyl-tRNA synthetase capable of charging the tRNA with the unnatural amino acid (UAA); and (iii) a nucleic acid comprising a nucleotide sequence encoding a heavy chain, a light chain, or a combination of a heavy chain and light chain of the antibody and comprising the codon selected from UAG, UGA, and UAA; under conditions that permit the tRNA, when expressed in the cell and charged with the unnatural amino acid (UAA), to Attorney Docket No. BRI-023WO hybridize to the codon and direct the incorporation of the unnatural amino acid (UAA) into the antibody. [0220] In certain embodiments, the tRNA is an analog or derivative of a prokaryotic tryptophanyl-tRNA, e.g., an E. coli tryptophanyl-tRNA. In certain embodiments, the aminoacyl- tRNA synthetase is an analog or derivative of a prokaryotic tryptophanyl-tRNA synthetase, e.g., an E. coli tryptophanyl-tRNA synthetase. In certain embodiments, the codon is UAG, UGA, or UAA. In certain embodiments, the UAA is a tryptophan analog, e.g., a non-naturally occurring tryptophan analog. In certain embodiments, the UAA is 5-HTP or 5-AzW. [0221] In certain embodiments, the tRNA is an analog or derivative of a prokaryotic leucyl- tRNA, e.g., an E. coli leucyl-tRNA. In certain embodiments, the aminoacyl-tRNA synthetase is an analog or derivative of a prokaryotic leucyl-tRNA synthetase, e.g., an E. coli leucyl-tRNA synthetase. In certain embodiments, the codon is UAG, UGA, or UAA. In certain embodiments, the UAA is a leucine analog, e.g., a non-naturally occurring leucine analog. In certain embodiments, the UAA is LCA or Cys-5-N3. [0222] In certain embodiments, the tRNA is an analog or derivative of a prokaryotic tyrosyl- tRNA, e.g., an E. coli tyrosyl-tRNA. In certain embodiments, the aminoacyl-tRNA synthetase is an analog or derivative of a prokaryotic tyrosyl-tRNA synthetase, e.g., an E. coli tyrosyl-tRNA synthetase. In certain embodiments, the codon is UAG, UGA, or UAA. In certain embodiments, the UAA is a tyrosine analog, e.g., a non-naturally occurring tyrosine analog. In certain embodiments, the UAA is OmeY, AzF, pAMF, or OpropY (PCT/US2020/054859). [0223] In certain embodiments, the tRNA is an analog or derivative of an archael pyrrolysyl- tRNA, e.g., an M. barkeri pyrrolysyl-tRNA. In certain embodiments, the aminoacyl-tRNA synthetase is an analog or derivative of an archael pyrrolysyl-tRNA synthetase, e.g., an M. barkeri pyrrolysyl-tRNA synthetase. In certain embodiments, the codon is UAG, UGA, or UAA. In certain embodiments, the UAA is a pyrrolysine analog, e.g., a non-naturally occurring pyrrolysine analog. In certain embodiments, the UAA is BocK, CpK, or AzK (PCT/US2020/054859). [0224] Summarizing in certain embodiments, the UAA is: (i) a tryptophan analog (e.g., 5- HTP and 5-AzW); (ii) a leucine analog (e.g., LCA and Cys-5-N3); (iii) a tyrosine analog (e.g., OmeY, AzF, and OpropY); or (iv) a pyrrolysine analog (e.g., BocK, CpK, and AzK). In any of the above tRNA/aaRS combinations, UAAs can be derived from FIG.3. [0225] In certain embodiments, the UAA comprises a non-natural aromatic chemical moiety (e.g., a hydroxyl-indole group; an amino-indole group; an aminophenol group; or a hydroxyl- phenol group, e.g., the UAA is 5-hydroxytryptophan (5-HTP), or an analog thereof), and/or the Attorney Docket No. BRI-023WO linker comprises a diazonium group (e.g., the linker comprises 4-nitorbenzenediazonium (4NDz); 4-carboxybenzenediazonium (4NeDz) or 4-methoxybenzenediazonium (4MCDz). The UAA and linker may react under conditions suitable to form an azo-linkage via an azo-coupling reaction between the aromatic chemical moiety and the diazonium group. Further methods for conjugation of molecules to UAAs are described, for example, in U.S. Patent Application Publication No. 2018/0360984. [0226] In certain embodiments, the amount of the antibody comprising the unnatural amino acid (UAA) expressed by the cell is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the amount of a reference antibody expressed by the same cell or a similar cell. In certain embodiments, the reference antibody is an otherwise identical antibody that does not comprise the UAA, for example, the reference antibody comprises a wild-type amino acid residue at the position corresponding to the unnatural amino acid (UAA). [0227] In certain embodiments, the cell is a human cell, e.g., a human embryonic kidney (HEK) or a Chinese hamster ovary (CHO) cell. [0228] In certain embodiments, following only affinity purification (e.g., following only protein A or G purification) less than 20-10%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the antibody is aggregated, as measured by size exclusion chromatography (SEC). [0229] Exemplary ADCs provided by the present disclosure are described in FIG.5. Conjugation ready antibodies were generated through unnatural amino acid incorporation to generate unconjugated 9-S1 to 11-S9 (FIG.5). Sites outlined in column 3 of FIG.5 were mutated to contain either a TAG or TGA stop codon at one or multiple sites in order to introduce unnatural amino acids as previously described (LCA in this instance), using transient transfection screening methods. In some instances, sites were mutated to cysteine for thiomab based conjugation. Antibodies that do not contain unnatural amino acid (FIG.5 ADC 6, 13, 14, 15, 16, 17) were expressed using industry standard transient transfection in EXPI293. Antibodies were purified at small scale via drip-column Protein A, or at large scale by FPLC purification via HiTrap MabSelect PrismA column (Cat#09-928-070, Protocol). General transfection, UAA incorporation, and conjugation detail are included in the following patent (PCT/US2021/049953, PCT/US2021/045088). Proteins were purified prior to conjugation, as described herein. III. (a) Transfer RNAs [0230] During protein synthesis, e.g., synthesis of an antibody with an unnatural amino acid, a tRNA molecule delivers an amino acid to a ribosome for incorporation into a growing protein (polypeptide) chain. tRNAs typically are about 70 to 100 nucleotides in length. Active tRNAs Attorney Docket No. BRI-023WO contain a 3' CCA sequence that may be transcribed into the tRNA during its synthesis or may be added later during post-transcriptional processing. During aminoacylation, the amino acid that is attached to a given tRNA molecule is covalently attached to the 2' or 3' hydroxyl group of the 3'- terminal ribose to form an aminoacyl-tRNA (aa-tRNA). It is understood that an amino acid can spontaneously migrate from the 2'-hydroxyl group to the 3'-hydroxyl group and vice versa, but it is incorporated into a growing protein chain at the ribosome from the 3'-OH position. A loop at the other end of the folded aa-tRNA molecule contains a sequence of three bases known as the anticodon. When this anticodon sequence hybridizes or base-pairs with a complementary three- base codon sequence in a ribosome-bound mRNA, the aa-tRNA binds to the ribosome and its amino acid is incorporated into the polypeptide chain being synthesized by the ribosome. Because all tRNAs that base-pair with a specific codon are aminoacylated with a single specific amino acid, the translation of the genetic code is affected by tRNAs. Each of the 61 non-termination codons in an mRNA directs the binding of its cognate aa-tRNA and the addition of a single specific amino acid to the growing polypeptide chain being synthesized by the ribosome. The term “cognate” refers to components that function together, e.g., a tRNA and an aminoacyl-tRNA synthetase. [0231] Suppressor tRNAs are modified tRNAs that alter the reading of a mRNA in a given translation system. For example, a suppressor tRNA may read through a codon such as a stop codon, a four base codon, or a rare codon. The use of the word in suppressor is based on the fact, that under certain circumstance, the modified tRNA "suppresses" the typical phenotypic effect of the codon in the mRNA. Suppressor tRNAs typically contain a mutation (modification) in either the anticodon, changing codon specificity, or at some position that alters the aminoacylation identity of the tRNA. The term “suppression activity” refers to the ability of a tRNA, e.g., a suppressor tRNA, to read through a codon (e.g., a premature stop codon) that would not be read through by the endogenous translation machinery in a system of interest. [0232] In some embodiments, a tRNA (e.g., a suppressor tRNA) contains a modified anticodon region, such that the modified anticodon hybridizes with a different codon than the corresponding naturally occurring anticodon. [0233] In some embodiments, a tRNA comprises an anticodon that hybridizes to a codon selected from UAG (i.e., an “amber” termination codon), UGA (i.e., an “opal” termination codon), and UAA (i.e., an “ochre” termination codon). [0234] In some embodiments, a tRNA comprises an anticodon that hybridizes to a non- standard codon, e.g., a 4- or 5-nucleotide codon. Examples of four base codons include AGGA, CUAG, UAGA, and CCCU. Examples of five base codons include AGGAC, CCCCU, CCCUC, Attorney Docket No. BRI-023WO CUAGA, CUACU, and UAGGC. tRNAs comprising an anticodon that hybridizes to a non- standard codon, e.g., a 4- or 5-nucleotide codon, and methods of using such tRNAs to incorporate unnatural amino acids into proteins are described, for example, in Moore et al. (2000) J. MOL. BIOL., 298: 195; Hohsaka et al. (1999) J. AM. CHEM. SOC., 121: 12194; Anderson et al. (2002) CHEMISTRY AND BIOLOGY, 9: 237-244; Magliery (2001) J. MOL. BIOL., 307: 755-769; and PCT Publication No. WO2005/007870. [0235] As used herein, the term “tRNA” includes variants having one or more mutations (e.g., nucleotide substitutions, deletions, or insertions) relative to a reference (e.g., a wild-type) tRNA sequence. In certain embodiments, a tRNA may comprise, consist, or consist essentially of, a single mutation (e.g., a mutation contemplated herein), or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more than 15 mutations (e.g., mutations contemplated herein). It is contemplated that a tRNA may comprise, consist, or consist essentially 1-15, 1-10, 1-7, 1-6, 1-5, 1- 4, 1-3, 1-2, 2-15, 2-10, 2-7, 2-6, 2-5, 2-4, 2-3, 3-15, 3-10, 3-7, 3-6, 3-5, or 3-4 mutations (e.g., mutations contemplated herein). [0236] In some embodiments, a variant suppressor tRNA has increased activity to incorporate an unnatural amino acid (e.g., an unnatural amino acid contemplated herein) into a mammalian protein relative to a counterpart wild-type suppressor tRNA (in this context, a wild-type suppressor tRNA refers to a suppressor tRNA that corresponds to a wild-type tRNA molecule but for any modifications to the anti-codon region to impart suppression activity). The activity of the variant suppressor tRNA may be increased relative to the wild type suppressor tRNA, for example, by about 2.5 to about 200 fold, about 2.5 to about 150 fold, about 2.5 to about 100 fold about 2.5 to about 80 fold, about 2.5 to about 60 fold, about 2.5 to about 40 fold, about 2.5 to about 20 fold, about 2.5 to about 10 fold, about 2.5 to about 5 fold, about 5 to about 200 fold, about 5 to about 150 fold, about 5 to about 100 fold, about 5 to about 80 fold, about 5 to about 60 fold, about 5 to about 40 fold, about 5 to about 20 fold, about 5 to about 10 fold, about 10 to about 200 fold, about 10 to about 150 fold, about 10 to about 100 fold, about 10 to about 80 fold, about 10 to about 60 fold, about 10 to about 40 fold, about 10 to about 20 fold, about 20 to about 200 fold, about 20 to about 150 fold, about 20 to about 100 fold, about 20 to about 80 fold, about 20 to about 60 fold, about 20 to about 40 fold, about 40 to about 200 fold, about 40 to about 150 fold, about 40 to about 100 fold, about 40 to about 80 fold, about 40 to about 60 fold, about 60 to about 200 fold, about 60 to about 150 fold, about 60 to about 100 fold, about 60 to about 80 fold, about 80 to about 200 fold, about 80 to about 150 fold, about 80 to about 100 fold, about 100 to about 200 fold, about 100 to about 150 fold, or about 150 to about 200 fold. Attorney Docket No. BRI-023WO [0237] It is contemplated that the tRNA may function in vitro or in vivo and can be provided to a translation system (e.g., an in vitro translation system or a cell) as a mature tRNA (e.g., an aminoacylated tRNA), or as a polynucleotide that encodes the tRNA. [0238] A tRNA may be derived from a bacterial source, e.g., Escherichia coli, Thermus thermophilus, or Bacillus stearothermphilus. A tRNA may also be derived from an archaeal source, e.g, from the Methanosarcinacaea or Desulfitobacterium families, any of the M. barkeri (Mb), M. alvus (Ma), M. mazei (Mm) or D. hafnisense (Dh) families, Methanobacterium thermoautotrophicum, Haloferax volcanii, Halobacterium species NRC-1, or Archaeoglobus fulgidus. In other embodiments, eukaryotic sources can also be used, for example, plants, algae, protists, fungi, yeasts, or animals (e.g., mammals, insects, arthropods, etc.). [0239] In some embodiments, a tRNA used in accordance with the present disclosure may comprise any nucleotide sequence as shown in Table 3. [0240] In some embodiments, the tRNA is derived from a prokaryotic leucyl-tRNA (e.g., an analog or derivative of a prokaryotic leucyl-tRNA). In some embodiments, the tRNA is derived from an E. coli leucyl tRNA and, for example, is preferentially charged with a leucine analog over the naturally-occurring leucine amino acid by an aminoacyl-tRNA synthetase derived from an E. coli leucyl-tRNA synthetase, e.g., an aminoacyl-tRNA synthetase contemplated herein. [0241] For example, the tRNA may comprise, consist essentially of, or consist of the nucleotide sequence of any one of SEQ ID NOs: 22-49, or a nucleotide sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 22-49. [0242] In some embodiments, the tRNA is derived from a prokaryotic tryptophanyl-tRNA (e.g., an analog or derivative of a prokaryotic tryptophanyl-tRNA). In some embodiments, the tRNA is derived from an E. coli tryptophanyl tRNA and, for example, is preferentially charged with a tryptophan analog over the naturally-occurring tryptophan amino acid by an aminoacyl- tRNA synthetase derived from an E. coli tryptophanyl-tRNA synthetase, e.g., an aminoacyl-tRNA synthetase contemplated herein. [0243] For example, the tRNA may comprise, consist essentially of, or consist of the nucleotide sequence of any one of SEQ ID NOs: 55-60, or a nucleotide sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 55-60. [0244] In some embodiments, the tRNA is derived from a prokaryotic tyrosyl-tRNA (e.g., an analog or derivative of a prokaryotic tyrosyl-tRNA). In certain embodiments, the tRNA is derived from an E. coli tyrosyl tRNA and, for example, is preferentially charged with a tyrosine analog Attorney Docket No. BRI-023WO over the naturally-occurring tyrosine amino acid by an aminoacyl-tRNA synthetase derived from an E. coli tyrosyl-tRNA synthetase, e.g., an aminoacyl-tRNA synthetase described herein. [0245] In some embodiments, the tRNA is derived from an archael pyrrolysyl-tRNA (e.g., an analog or derivative of a archael pyrrolysyl-tRNA). In certain embodiments, the tRNA is derived from a M. barkeri pyrrolysyl tRNA and, for example, is preferentially charged with a pyrrolysine analog over the naturally-occurring pyrrolysine amino acid by an aminoacyl-tRNA synthetase derived from a M. barkeri pyrrolysyl-tRNA synthetase, e.g., an aminoacyl-tRNA synthetase contemplated herein. [0246] It is understood that, throughout the description, in each instance where a tRNA comprises, consists essentially of, or consists of a nucleotide sequence including one or more thymines (T), a tRNA is also contemplated that comprises, consists essentially of, or consists of the same nucleotide sequence including a uracil (U) in place of one or more of the thymines (T), or a uracil (U) in place of all the thymines (T). Similarly, in each instance where a tRNA comprises, consists essentially of, or consists of a nucleotide sequence including one or more uracils (U), a tRNA is also contemplated that comprises, consists essentially of, or consists of a nucleotide sequence including a thymine (T) in place of the one or more of the uracils (U), or a thymine (T) in place of all the uracils (U). In addition, additional modifications to the bases can be present. [0247] Methods for producing recombinant tRNA are described in U.S. Patent Application Publication Nos.2003/0108885 and 2005/0009049, Forster et al. (2003) PROC. NATL. ACAD. SCI. USA 100(11):6353-6357, and Feng et al. (2003), PROC. NATL. ACAD. SCI. USA 100(10): 5676- 5681. [0248] A tRNA may be aminoacylated (i.e., charged) with a desired unnatural amino acid by any method, including enzymatic or chemical methods. [0249] Enzymatic molecules capable of charging a tRNA include aminoacyl-tRNA synthetases, e.g., aminoacyl-tRNA synthetases disclosed herein. Additional enzymatic molecules capable of charging tRNA include ribozymes, for example, as described in Illangakekare et al. (1995) SCIENCE, 267: 643-647, Lohse et al. (1996) NATURE, 381: 442-444, Murakami et al. (2003) CHEMISTRY AND BIOLOGY, 10: 1077-1084, U.S. Patent Application Publication No.2003/0228593, Chemical aminoacylation methods include those described in Hecht (1992) ACC. CHEM. RES., 25: 545, Heckler et al. (1988) BIOCHEM., 1988, 27: 7254, Hecht et al. (1978) J. BIOL. CHEM., 253: 4517, Cornish et al. (1995) ANGEW. CHEM. INT. ED. ENGL., 34: 621, Robertson et al. (1991) J. AM. CHEM. SOC., 113: 2722, Noren et al. (1989) SCIENCE, 244: 182, Bain et al. (1989) J. AM. CHEM. SOC., 111: 8013, Bain et al. (1992) NATURE, 356: 537, Gallivan et al. (1997) CHEM. BIOL., 4: 740, Attorney Docket No. BRI-023WO Turcatti et al. (1996) J. BIOL. CHEM., 271: 19991, Nowak et al. (1995) SCIENCE, 268: 439, Saks et al. (1996) J. BIOL. CHEM., 271: 23169, and Hohsaka et al. (1999) J. AM. CHEM. SOC., 121: 34. III. (b) Aminoacyl-tRNA Synthetases [0250] The enigeered antibodies are created using engineered aminoacyl-tRNA synthetases (or aaRSs) capable of charging a tRNA with an unnatural amino acid for incorporation into a protein of interest (e.g., an antibody, such as an anti-CD276 antibody). As used herein, the term “aminoacyl-tRNA synthetase” refers to any enzyme, or a functional fragment thereof, that charges, or is capable of charging, a tRNA with an amino acid (e.g., an unnatural amino acid) for incorporation into a protein. As used herein, the term “functional fragment” of an aminoacyl-tRNA synthetase refers to fragment of a full-length aminoacyl-tRNA synthetase that retains, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the enzymatic activity of the corresponding full- length tRNA synthetase (e.g., a naturally occurring tRNA synthetase). Aminoacyl-tRNA synthetase enzymatic activity may be assayed by any method known in the art. For example, in vitro aminoacylation assays are described in Hoben et al. (1985) METHODS ENZYMOL., 113: 55-59 and in U.S. Patent Application Publication No.2003/0228593 and cell-based aminoacylation assays are described in U.S. Patent Application Publication Nos.2003/0082575 and 2005/0009049. In some embodiments, the functional fragment comprises at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 consecutive amino acids present in a full-length tRNA synthetase (e.g., a naturally occurring aminoacyl-tRNA synthetase). [0251] The term aminoacyl-tRNA synthetase includes variants (i.e., muteins) having one or more mutations (e.g., amino acid substitutions, deletions, or insertions) relative to a wild-type aminoacyl-tRNA synthetase sequence. In some embodiments, an aminoacyl-tRNA synthetase mutein may comprise, consist, or consist essentially of, a single mutation (e.g., a mutation contemplated herein), or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more than 15 mutations (e.g., mutations discussed herein). It is contemplated that an aminoacyl-tRNA synthetase mutein may comprise, consist, or consist essentially 1-15, 1-10, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-15, 2-10, 2-7, 2-6, 2-5, 2-4, 2-3, 3-15, 3-10, 3-7, 3-6, 3-5, or 4-10, 4-7, 4-6, 4-5, 5-10, 5-7, 5- 6, 6-10, 6-7, 7-10, 7-8, or 8-10 mutations (e.g., mutations contemplated herein). An aminoacyl- tRNA synthetase mutein may comprise a conservative substitution relative to a wild-type sequence or a sequence disclosed herein. In some embodiments, the substrate specificity of the aminoacyl- tRNA synthetase mutein is altered relative to a corresponding (or template) wild-type aminoacyl- tRNA synthetase such that only a desired unnatural amino acid, but not any of the common 20 amino acids, is charged to the substrate tRNA. Attorney Docket No. BRI-023WO [0252] In some embodiments, an aminoacyl-tRNA synthetase may be derived from a bacterial source, e.g., Escherichia coli, Thermus thermophilus, or Bacillus stearothermphilus. In some embodiments, an aminoacyl-tRNA synthetase may be derived from an archaeal source, e.g, from the Methanosarcinacaea or Desulfitobacterium families, any of the M. barkeri (Mb), M. alvus (Ma), M. mazei (Mm) or D. hafnisense (Dh) families, Methanobacterium thermoautotrophicum, Haloferax volcanii, Halobacterium species NRC-1, or Archaeoglobus fulgidus. In some embodiments, eukaryotic sources can be used, for example, plants, algae, protists, fungi, yeasts, or animals (e.g., mammals, insects, arthropods, etc.). As used herein, the terms “derivative” or “derived from” refer to a component that is isolated from or made using information from a specified molecule or organism. As used herein, the term “analog” refers to a component (e.g., a tRNA, tRNA synthetase, or unnatural amino acid) that is derived from or analogous with (in terms of structure and/or function) a reference component (e.g., a wild-type tRNA, a wild-type tRNA synthetase, or a natural amino acid). In some embodiments, derivatives or analogs have at least 40%, 50%, 60%, 70%, 80%, 90%, 100% or more of a given activity as a reference or originator component (e.g., wild type component). [0253] It is contemplated that the aminoacyl-tRNA synthetase may aminoacylate a substrate tRNA in vitro or in vivo, and can be provided to a translation system (e.g., an in vitro translation system or a cell) as a polypeptide or protein, or as a polynucleotide that encodes the aminoacyl- tRNA synthetase. [0254] In some embodiments, an aminoacyl-tRNA synthetase used in accordance with the present disclosure may comprise any amino acid sequence shown in TABLE 3, including functional fragments thereof. [0255] In some embodiments, the aminoacyl-tRNA synthetase is derived from a prokaryotic leucyl-tRNA synthetase (e.g., an analog or derivative of a prokaryotic leucyl-tRNA synthetase). In some embodiments, the aminoacyl-tRNA synthetase is derived from an E. coli leucyl-tRNA synthetase and, for example, the aminoacyl-tRNA synthetase preferentially aminoacylates an E. coli leucyl tRNA (or a variant thereof) with a leucine analog over the naturally-occurring leucine amino acid. [0256] In some embodiments, the aminoacyl-tRNA synthetase comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 7-21, or an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 7-21. [0257] In some embodiments, the aminoacyl-tRNA synthetase is derived from a prokaryotic tryptophanyl-tRNA synthetase (e.g., an analog or derivative of a prokaryotic tryptophanyl-tRNA Attorney Docket No. BRI-023WO synthetase). In some embodiments, the aminoacyl-tRNA synthetase is derived from an E. coli tryptophanyl-tRNA synthetase and, for example, the aminoacyl-tRNA synthetase preferentially aminoacylates an E. coli tryptophanyl tRNA (or a variant thereof) with a tryptophan analog over the naturally-occurring tryptophan amino acid. [0258] In some embodiments, the aminoacyl-tRNA synthetase comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 50-54, or an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 50-54. [0259] In some embodiments, the aminoacyl-tRNA synthetase is derived from a prokaryotic tyrosyl-tRNA synthetase (e.g., an analog or derivative of a prokaryotic tyrosyl-tRNA synthetase). In some embodiments, the aminoacyl-tRNA synthetase is derived from an E. coli tyrosyl-tRNA synthetase and, for example, the aminoacyl-tRNA synthetase preferentially aminoacylates an E. coli tyrosyl tRNA (or a variant thereof) with a tyrosine analog over the naturally-occurring tyrosine amino acid. [0260] In some embodiments, the aminoacyl-tRNA synthetase is derived form an archael pyrrolysyl-tRNA synthetase (e.g., an analog or derivative of an archael pyrrolysyl-tRNA synthetase). In some embodiments, the aminoacyl-tRNA synthetase is derived from an M. barkeri pyrrolysyl-tRNA synthetase and, for example, the aminoacyl-tRNA synthetase preferentially aminoacylates an M. barkeri pyrrolysyl tRNA (or a variant thereof) with a pyrrolysine analog over the naturally-occurring pyrrolysine amino acid. [0261] Methods for producing proteins, e.g., aminoacyl-tRNA synthetases, are known in the art. For example, DNA molecules encoding a protein of interest can be synthesized chemically or by recombinant DNA methodologies. The resulting DNA molecules encoding the protein interest can be ligated to other appropriate nucleotide sequences, including, for example, expression control sequences, to produce conventional gene expression constructs (i.e., expression vectors) encoding the desired protein. Production of defined gene constructs is within routine skill in the art. [0262] Nucleic acids encoding desired proteins (e.g, aminoacyl-tRNA synthetases) can be incorporated (ligated) into expression vectors, which can be introduced into host cells through conventional transfection or transformation techniques. Exemplary host cells are E. coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells. Transformed host cells can be grown under conditions that permit the host cells to express the desired protein. Attorney Docket No. BRI-023WO [0263] Specific expression and purification conditions will vary depending upon the expression system employed. For example, if a gene is to be expressed in E. coli, it is first cloned into an expression vector by positioning the engineered gene downstream from a suitable bacterial promoter, e.g., Trp or Tac, and a prokaryotic signal sequence. The expressed protein may be secreted. The expressed protein may accumulate in refractile or inclusion bodies, which can be harvested after disruption of the cells by French press or sonication. The refractile bodies then are solubilized, and the protein may be refolded and/or cleaved by methods known in the art. [0264] If the engineered gene is to be expressed in eukaryotic host cells, e.g., CHO cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, a secretion signal, a poly A sequence, and a stop codon. Optionally, the vector or gene construct may contain enhancers and introns. The gene construct can be introduced into eukaryotic host cells using conventional techniques. [0265] A protein of interest (e.g, an aminoacyl-tRNA synthetase) can be produced by growing (culturing) a host cell transfected with an expression vector encoding such a protein under conditions that permit expression of the protein. Following expression, the protein can be harvested and purified or isolated using techniques known in the art, e.g., affinity tags such as glutathione-S-transferase (GST) or histidine tags. [0266] Additional methods for producing aminoacyl-tRNA synthetases, and for altering the substrate specificity of the synthetase can be found in U.S. Patent Application Publication Nos. 2003/0108885 and 2005/0009049, Hamano-Takaku et al. (2000) JOURNAL OF BIOL. CHEM., 275(51): 40324-40328, Kiga et al. (2002) PROC. NATL. ACAD. SCI. USA, 99(15): 9715-9723, and Francklyn et al. (2002) RNA, 8: 1363-1372. [0267] The present disclosure also encompasses nucleic acids encoding aminoacyl-tRNA synthetases disclosed herein (e.g., nucleic acids encoding any of the aminoacyl-tRNA synthetases described in TABLE 3). [0268] Exemplary tRNAs and aminoacyl-tRNA synthetases that may be used in accordance with the present disclosure are described in International (PCT) Publication Nos. WO2020257668, WO2022056318, and WO2022115625, the entire contents of each of which are hereby incorporated by reference. III. (c) Vectors [0269] It is contemplated that tRNAs, aminoacyl-tRNA synthetases, or any other molecules of interest may be expressed in a cell of interest by incorporating a gene encoding the molecule into an appropriate expression vector. As used herein, "expression vector" refers to a vector comprising Attorney Docket No. BRI-023WO a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. [0270] The tRNAs, aminoacyl-tRNA synthetases, or any other molecules of interest may be introduced to a cell of interest by incorporating a gene encoding the molecule into an appropriate transfer vector. The term "transfer vector" refers to a vector comprising a recombinant polynucleotide which can be used to deliver the polynucleotide to the interior of a cell. It is understood that a vector may be both an expression vector and a transfer vector. [0271] Vectors (e.g., expression vectors or transfer vectors) include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), retrotransposons (e.g. piggyback, sleeping beauty), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno- associated viruses) that incorporate the recombinant polynucleotide of interest. [0272] Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular target nucleic acid. The vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both (including but not limited to, shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems. [0273] In some embodiments, the vector comprises a regulatory sequence or promoter operably linked to the nucleotide sequence encoding the suppressor tRNA and/or the tRNA synthetase. The term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a gene if it affects the transcription of the gene. Operably linked nucleotide sequences are typically contiguous. However, as enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not directly flanked and may even function in trans from a different allele or chromosome. [0274] Exemplary promoters which may be employed include, but are not limited to, the retroviral LTR, the SV40 promoter, the human cytomegalovirus (CMV) promoter, the U6 promoter, the EF1α promoter, the CAG promoter, the H1 promoter, the UbiC promoter, the PGK promoter, the 7SK promoter, a pol II promoter, a pol III promoter, or any other promoter (e.g., Attorney Docket No. BRI-023WO cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and β-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, TK promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein. In certain embodiments, a vector comprises a nucleotide sequence encoding an aminoacyl-tRNA synthetase operably linked to a CMV or an EF1α promoter and/or a nucleotide sequence encoding a suppressor tRNA operably linked to a U6 or an H1 promoter. [0275] In some embodiments, the vector is a viral vector. The term "virus" is used herein to refer to an obligate intracellular parasite having no protein-synthesizing or energy-generating mechanism. Exemplary viral vectors include retroviral vectors (e.g., lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, herpesviruses vectors, epstein-barr virus (EBV) vectors, polyomavirus vectors (e.g., simian vacuolating virus 40 (SV40) vectors), poxvirus vectors, and pseudotype virus vectors. [0276] The virus may be a RNA virus (having a genome that is composed of RNA) or a DNA virus (having a genome composed of DNA). In certain embodiments, the viral vector is a DNA virus vector. Exemplary DNA viruses include parvoviruses (e.g., adeno-associated viruses), adenoviruses, asfarviruses, herpesviruses (e.g., herpes simplex virus 1 and 2 (HSV-1 and HSV-2), epstein-barr virus (EBV), cytomegalovirus (CMV)), papillomoviruses (e.g., HPV), polyomaviruses (e.g., simian vacuolating virus 40 (SV40)), and poxviruses (e.g., vaccinia virus, cowpox virus, smallpox virus, fowlpox virus, sheeppox virus, myxoma virus). In certain embodiments, the viral vector is a RNA virus vector. Exemplary RNA viruses include bunyaviruses (e.g., hantavirus), coronaviruses, flaviviruses (e.g., yellow fever virus, west nile virus, dengue virus), hepatitis viruses (e.g., hepatitis A virus, hepatitis C virus, hepatitis E virus), influenza viruses (e.g., influenza virus type A, influenza virus type B, influenza virus type C), measles virus, mumps virus, noroviruses (e.g., Norwalk virus), poliovirus, respiratory syncytial virus (RSV), retroviruses (e.g., human immunodeficiency virus-1 (HIV-1)) and toroviruses. III. (d) Host Cells and Cell Lines [0277] Host cells or cell lines (e.g., prokaryotic or eukaryotic host cells or cell lines) that include a tRNA, aminoacyl-tRNA synthetase, unnatural amino acid, nucleic acid, and/or vector disclosed herein can be used in the production of the of the proteins, e.g., antibodies, described herein. The nucleic acid encoding the engineered tRNA and aminoacyl-tRNA synthetase can be expressed in an expression host cell either as an autonomously replicating vector within the Attorney Docket No. BRI-023WO expression host cell (e.g., a plasmid, or viral particle) or via a stable integrated element or series of stable integrated elements in the genome of the expression host cell, e.g., a mammalian host cell. [0278] Host cells are genetically engineered (including but not limited to, transformed, transduced or transfected), for example, using nucleic acids or vectors disclosed herein. For example, in some embodiments, one or more vectors include coding regions for an orthogonal tRNA, an orthogonal aminoacyl-tRNA synthetase, and, optionally, a protein (e.g., an antibody) to be modified by the inclusion of one or more unnatural amino acids, which are operably linked to gene expression control elements that are functional in the desired host cell or cell line. For example, the genes encoding tRNA synthetase and tRNA and an optional selectable marker (e.g., an antibiotic resistance gene, e.g., a puromycin resistance cassette) can be integrated in a transfer vector (e.g., a plasmid, which can be linearized prior to transfection), where for example, the genes encoding the tRNA synthetase can be under the control of a polymerase II promoter (e.g., CMV, EF1α, UbiC, or PGK, e.g., CMV or EF1α) and the genes encoding the tRNA can be under the control of a polymerase III promoter (e.g., U6, 7SK, or H1, e.g., U6). The vectors are transfected into cells and/or microorganisms by standard methods including electroporation or infection by viral vectors, and clones can be selected via expression of the selectable marker (for example, by antibiotic resistance). [0279] Exemplary prokaryotic host cells or cell lines include cells derived from a bacteria, e.g., E. coli, Thermus thermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa, and Pseudomonas putida. Exemplary eukaryotic host cells or cell lines include cells derived from a plant (e.g., a complex plant such as a monocot or dicot), an algae, a protist, a fungus, a yeast (including Saccharomyces cerevisiae), or an animal (including a mammal, an insect, an arthropod, etc.). Additional exemplary host cells or cell lines include HEK293, HEK293T, Expi293, CHO, CHOK1, Sf9, Sf21, HeLa, U20S, A549, HT1080, CAD, P19, NIH 3T3, L929, N2a, MCF-7, Y79, SO-RB50, HepG2, DUKX-X11, J558L, BHK, COS, Vero, NS0, or ESCs. It is understood that a host cell or cell line can include individual colonies, isolated populations (monoclonal), or a heterogeneous mixture of cells. [0280] A contemplated cell or cell line includes, for example, one or multiple copies of an orthogonal tRNA/aminoacyl-tRNA synthetase pair, optionally stably maintained in the cell’s genome or another piece of DNA maintained by the cell. For example, the cell or cell line may contain one or more copies of (i) a tryptophanyl tRNA/aminoacyl-tRNA synthetase pair (wild type or engineered) stably maintained by the cell, and/or (ii) a leucyl tRNA/aminoacyl-tRNA synthetase pair (wild-type or engineered) stably maintained by the cell. Attorney Docket No. BRI-023WO [0281] For example, in certain embodiments, the cell line is a stable cell line and the cell line comprises a genome having stably integrated therein (i) a nucleic acid sequence encoding an aminoacyl-tRNA synthetase (e.g., a prokaryotic tryptophanyl-tRNA synthetase mutein capable of charging a tRNA with an unnatural amino acid or a prokaryotic leucyl-tRNA synthetase mutein capable of charging a tRNA with an unnatural amino acid, e.g., a tRNA synthetase mutein disclosed herein); and/or (ii) a nucleic acid sequence encoding a suppressor tRNA (e.g., prokaryotic suppressor tryptophanyl-tRNA capable of being charged with an unnatural amino acid or prokaryotic suppressor leucyl-tRNA capable of being charged with an unnatural amino acid). [0282] Methods to introduce a nucleic acid encoding a tRNA and/or an aminoacyl-tRNA synthetase into the genome of a cell of interest, or to stably maintain the nucleic acid in DNA replicated by the cell that is outside of the genome, are well known in the art. [0283] The nucleic acid encoding the tRNA and/or an aminoacyl-tRNA synthetase can be provided to the cell in an expression vector, transfer vector, or DNA cassette, e.g., an expression vector, transfer vector, or DNA cassette disclosed herein. The expression vector transfer vector, or DNA cassette encoding the tRNA and/or aminoacyl-tRNA synthetase can contain one or more copies of the tRNA and/or aminoacyl-tRNA synthetase optionally under the control of an inducible or constitutively active promoter. The expression vector, transfer vector, or DNA cassette may, for example, contain other standard components (enhancers, terminators, etc.). It is contemplated that the nucleic acid encoding the tRNA and the nucleic acid encoding the aminoacyl-tRNA synthetase may be on the same or different vector, may be present in the same or different ratios, and may be introduced into the cell, or stably integrated in the cellular genome, at the same time or sequentially. [0284] One or multiple copies of a DNA cassette encoding the tRNA and/or aminoacyl-tRNA synthetase can be integrated into a host cell genome or stably maintained in the cell using a transposon system (e.g., PiggyBac), a viral vector (e.g., a lentiviral vector or other retroviral vector), CRISPR/Cas9 based recombination, electroporation and natural recombination, a BxB1 recombinase system, or using a replicating/maintained piece of DNA (such as one derived from Epstein-Barr virus). [0285] In order to select for cell lines which stably maintain the nucleic acid encoding the tRNA and/or aminoacyl-tRNA synthetase and/or are efficient at incorporating UAAs into a protein (e.g., an antibody)of interest, a selectable marker can be used. Exemplary selectable markers include zeocin, puromycin, neomycin, dihydrofolate reductase (DHFR), glutamine synthetase (GS), mCherry-EGFP fusion, or other fluorescent proteins. In certain embodiments, a gene encoding a Attorney Docket No. BRI-023WO selectable marker protein (or a gene encoding a protein required for a detectable function, e.g., viability, in the presence of the selectable marker) may include a premature stop codon, such that the protein will only be expressed if the cell line is capable of incorporating a UAA at the site of the premature stop codon. [0286] In order to create a host cell or cell line including two or more tRNA/aminoacyl-tRNA synthetase pairs, one can use multiple identical or distinct UAA directing codons in order to identify host cells or cell lines which have incorporated multiple copies of the two or more tRNA/aminoacyl-tRNA synthetase pairs through iterative rounds of genomic integration and selection. Host cells or cell lines which contain enhanced UAA incorporation efficiency, low background, and decreased toxicity can first be isolated via a selectable marker containing one or more stop codons. Subsequently, the host cells or cell lines can be subjected to a selection scheme to identify host cells or cell lines which contain the desired copies of tRNA/aminoacyl-tRNA synthetase pairs and express a gene of interest (either genomically integrated or not) containing one or more stop codons. Protein expression may be assayed using any method known in the art, including for example, Western blot using an antibody that binds the protein of interest or a C- terminal tag. [0287] The host cells or cell lines be cultured in conventional nutrient media modified as appropriate for such activities as, for example, screening steps, activating promoters or selecting transformants. These cells can optionally be cultured into transgenic organisms. Other useful references, e.g. for cell isolation and culture (e.g., for subsequent nucleic acid isolation) include Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley-Liss, New York; Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg N.Y.) and Atlas and Parks (eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla. IV. PHARMACEUTICAL COMPOSITIONS [0288] Once the desired protein, e.g., an antibody, (e.g., an anti-CD276 antibody) containing one or more unnatural amino acids has been produced and subsequently conjugated to a payload of interest, either directly or through a linker, the resulting protein-payload conjugate, e.g., antibody- conjugate (e.g., antibody drug conjugate) after standard purification can be formulated into a pharmaceutical composition of use. Attorney Docket No. BRI-023WO [0289] The pharmaceutical composition can be formulated for use in a variety of drug delivery systems. One or more pharmaceutically acceptable excipients or carriers can also be included in the composition for proper formulation. Suitable formulations for use in the present disclosure are found in Adeboye Adejare, Remington: The Science and Practice of Pharmacy (23rd ed.2020). [0290] In certain embodiments, a pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl- beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants (see, Adeboye Adejare, Remington: The Science and Practice of Pharmacy (23rd ed.2020)). [0291] Pharmaceutical compositions containing a protein-payload conjugate, e.g., antibody- conjugate (e.g., antibody drug conjugate) can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous, intradermal, inhalation, transdermal, topical, transmucosal, intrathecal and rectal administration. Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl Attorney Docket No. BRI-023WO parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. [0292] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage, and should be preserved against microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol), and suitable mixtures thereof. An intravenous drug delivery formulation of the present disclosure may be contained in a bag, a pen, or a syringe. In certain embodiments, the bag may be connected to a channel including a tube and/or a needle. [0293] Depending upon the circumstances, the formulation can be a liquid formulation. An aqueous formulation can be prepared including the protein-payload conjugate, e.g., antibody- conjugate (e.g., antibody drug conjugate) in a pH-buffered solution. The pH of the liquid formulation may be set by addition of a pharmaceutically acceptable acid and/or base. In certain embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In certain embodiments, the base may be sodium hydroxide. In certain embodiments, a salt or buffer components may be added in an amount of 10 mM to 200 mM. The salts and/or buffers are pharmaceutically acceptable and are derived from various known acids (inorganic and organic) with “base forming” metals or amines. In certain embodiments, the buffer may be phosphate buffer. In certain embodiments, the buffer may be glycinate, carbonate, citrate buffers, in which case, sodium, potassium or ammonium ions can serve as counterion. Intravenous formulations can be diluted with 0.9% sodium chloride solution before administration. In certain embodiments, the diluted drug product for injection is isotonic and suitable for administration by intravenous infusion. [0294] Alternatively, the formulation can be a lyophilized formulation including the protein- payload conjugate, e.g., antibody-conjugate (e.g., antibody drug conjugate) and a lyoprotectant. The lyoprotectant may be sugar, e.g., disaccharides. In certain embodiments, the lyoprotectant may be sucrose or maltose. The lyophilized formulation may also include one or more of a buffering agent, a surfactant, a bulking agent, and/or a preservative. The amount of sucrose or maltose useful for stabilization of the lyophilized drug product may be in a weight ratio of at least 1:2 protein to sucrose or maltose. In certain embodiments, the protein to sucrose or maltose weight ratio may be of from 1:2 to 1:5. Before lyophilization, the pH of the solution containing the protein of the Attorney Docket No. BRI-023WO present disclosure may be adjusted between 6 to 8. In certain embodiments, the pH range for the lyophilized drug product may be from 7 to 8. In certain embodiments, a “bulking agent” may be added. A “bulking agent” is a compound which adds mass to a lyophilized mixture and contributes to the physical structure of the lyophilized cake (e.g., facilitates the production of an essentially uniform lyophilized cake which maintains an open pore structure). Illustrative bulking agents include mannitol, glycine, polyethylene glycol and sorbitol. The lyophilized formulations of the present disclosure may contain such bulking agents. [0295] In certain embodiments, the lyophilized drug product may be constituted with an aqueous carrier. The aqueous carrier of interest herein is one which is pharmaceutically acceptable (e.g., safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation, after lyophilization. Illustrative diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline solution, Ringer’s solution or dextrose solution. In certain embodiments, the lyophilized protein product of the instant disclosure is constituted to about 4.5 mL water for injection and diluted with 0.9% saline solution (sodium chloride solution). [0296] Pharmaceutical formulations preferably are sterile. Sterilization can be accomplished by any suitable method, e.g., filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution. The resulting aqueous solutions may be packaged for use as-is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents. The composition in solid form can also be packaged in a container for a flexible quantity. [0297] Depending upon the circumstances, the resulting pharmaceutical composition is formulated as a liquid formulation in either a USP / Ph Eur type I 50R vial closed with a rubber stopper and sealed with an aluminum crimp seal closure. The stopper may be made of elastomer complying with USP and Ph Eur. In certain embodiments, the liquid formulation may be diluted with 0.9% saline solution prior to use. [0298] A preservative may be optionally added to the formulations to reduce bacterial action. The addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation. Attorney Docket No. BRI-023WO [0299] In certain embodiments, a pharmaceutical composition may contain a sustained- or controlled-delivery formulation. Techniques for formulating sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. Sustained-release preparations may include, e.g., porous polymeric microparticles or semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, poly (2-hydroxyethyl- inethacrylate), ethylene vinyl acetate, or poly-D(−)-3-hydroxybutyric acid. Sustained release compositions may also include liposomes that can be prepared by any of several methods known in the art. [0300] The compositions described herein may be administered locally or systemically. Administration will generally be parenteral administration. In certain embodiments, the pharmaceutical composition is administered subcutaneously and in other embodiments the pharmaceutical composition is administered intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. [0301] Generally, a therapeutically effective amount of active component, for example, an antibody, is in the range of 0.1 mg/kg to 100 mg/kg, e.g., 1 mg/kg to 100 mg/kg, 1 mg/kg to 10 mg/kg. It is understood, however, that the amount administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health of the patient, the in vivo potency of the antibody, the pharmaceutical formulation, and the route of administration. The initial dosage can be increased beyond the upper level in order to rapidly achieve the desired blood- level or tissue-level. Alternatively, the initial dosage can be smaller than the optimum, and the daily dosage may be progressively increased during the course of treatment. Human dosage can be optimized, e.g., in a conventional Phase I dose escalation study. Dosing frequency can vary, depending on factors such as route of administration, dosage amount, serum half-life of the antibody, fusion protein, and/or antibody conjugate, and the disease being treated. Exemplary dosing frequencies are once per day, once per week and once every two weeks. V. THERAPEUTIC USES [0302] Once produced, the proteins and pharmaceutical compositions described herein can used in in a variety of therapies and therapeutic methods. [0303] For example, the proteins and pharmaceutical compositions described herein can be used in a method of treating a subject with a disease or disorder (e.g., cancer). The method comprises administering to the subject an effective amount of an antibody of the present disclosure (e.g., any Attorney Docket No. BRI-023WO antibody described herein, including engineered antibodies such as conjugated antibodies) or a pharmaceutical composition containing such antibody. The antibody can be an anti-CD276 antibody (e.g., an anti-CD276 antibody described herein). The antibody can be engineered antibody (e.g., an engineered anti-CD276 antibody) containing an unnatural amino acid incorporated at one or more amino acid residue sites (e.g., one or more sites selected from Table 1). [0304] Also provided is a method of treating a subject with a cancer that overexpresses CD276. The method comprises administering to the subject an effective amount of an anti-CD276 antibody of the present disclosure (e.g., an engineered anti-CD276 antibody comprising one or more conjugated payload molecules) or a pharmaceutical composition containing such an antibody. [0305] Also provided is a method of treating a subject with CD276 positive tumor vasculature. The method comprises administering to the subject an effective amount of an anti- CD276 antibody of the present disclosure (e.g., an engineered anti-CD276 antibody comprising one or more conjugated payload molecules) or a pharmaceutical composition containing such an antibody. [0306] In some embodiments, a cancer treatable by an antibody or an engineered antibody of the present disclosure is a cancer characterized by elevated CD276 expression compared to a suitable control (e.g., a healthy cell or tissue, for example, a non-cancerous cell or tissue). Depending upon the circumstances, the cancer can be breast cancer, prostate cancer, ovarian cancer, endometrial cancer, sarcoma, melanoma, non-small cell lung cancer (NSCLC), Sq-ESO, bladder cancer, head and neck squamous cell carcinoma (HNSCC), hepatocellular carcinoma (HCC), salivary gland cancer, colon cancer, pancreatic cancer, renal cell carcinoma (RCC), gastric cancer, thyroid cancer, blood cancer (such as leukemia or lymphoma), blastoma, glioma, or neuroblastoma. [0307] It is contemplated a subject can be treated with a protein-conjugate described herein (e.g., an engineered anti-CD276 antibody comprising one or more conjugated payload molecules) in combination therapy, for example, a combination therapy that includes one or more checkpoint inhibitors. EXAMPLES [0308] The following Examples are merely illustrative and are not intended to limit the scope or content of the disclosure in any way. Attorney Docket No. BRI-023WO Example 1: Methods of Making Anti-CD276 Antibody Drug Conjugates [0309] This example describes use of methods and compositions for making antibody drug conjugates that have certain desirable characteristics that allow for enhanced therapeutic efficacy. This example further describes methods for screening antibody drug conjugates for said desirable characteristics. This example describes variations of protocols, e.g., as outlined in PCT/US2021/049953 and PCT/US2021/045088, for modifying an antibody to form an antibody conjugate, including use of unnatural amino acids, sites of incorporation within the antibody, and payload conjugation. The antibody used for these experiments was the m276 anti-CD276 antibody (see Table 2). In some instances, Ifinatamab (referred to as “DS” within) is used in experiments as an anti-CD276 antibody (sequence 85 (HC) and 77 (LC) derived from US patent 9,371,395 B2). Expression Testing of Anti-CD276 Antibodies With Unnatural Amino Acids Incorporated [0310] Expression of m276 antibody containing an unnatural amino acid at selected positions (see, e.g., FIG.5 and Table 1) was performed transiently in HEK293 cells. All monoclonal antibody expression was performed using the Expi293 Expression System according to the manufacturer’s instructions. Briefly, before transfection, cells were split to a density of 2.7 × 106 to 3.0 × 106 cells/ml. An unnatural amino acid, LCA, was added to achieve a final concentration of 0.25 to 0.5 mM. A total of 1 mg of plasmid mix (equal parts suppressor plasmid, m276 heavy chain plasmid, and m276 light chain plasmid) was used for transfection into 1 L cell culture. Heavy chain or light chain plasmids were mutated to contain a TAG (LCA-directed) stop codon for the incorporation of the unnatural amino acid at sites S1-S9 on the heavy or light chain plasmid (By trastuzumab numbering, S1 = A143, S2 = T158, S3 = T172, S4 = TA175, S5 = Y183, S6 = T198, S7 = D268, S8 = LC-Q160, S9 = S242, or unmodified “WT” m276. By m276 numbering, S1 = A142, S2 = T157, S3 = T171, S4 = TA174, S5 = Y182, S6 = T197, S7 = D267, S8 = LC-Q162, S9 = S242). [0311] Plasmids were diluted in 50 ml Opti-MEM medium. A PEI stock solution was incubated at room temperature for 3 minutes, subsequently diluted to achieve a 6:1 PEI:DNA final ratio, and incubated for 15 minutes at room temperature. The Plasmid:PEI complex was then added dropwise to the culture. At the time of transfection, 0.25 to 1 mM LCA was added to the cells. Cells were incubated on an orbital shaking platform at 37 °C with 8% CO2 at a speed of 80 to 125 rpm for 5 to 8 days. Protein was purified using a PrismA column (Cytiva: 17549801) for use in characterization assays described herein. Protein titers were measured via Bradford- and nanodrop-based protein quantification methods. Reported titers were normalized to wild type (WT) protein expression (absence of TAG stop codon or unnatural amino acid). Suitable Attorney Docket No. BRI-023WO expression levels were achieved with unnatural amino acid incorporation at any site, and in some cases yields were within 60% or greater of WT expression, indicating efficient production in the mammalian cell-based expression system for UAA incorporation (see, e.g., FIG.4). Generating Anti-CD276 Antibody Drug Conjugates [0312] Conjugated antibodies, 9-S1 through 9-S9 and 11-S1 through 11-S9 (see, e.g., FIG.5), were made according to the general scheme, A-U-U’-X1-L-X2-P (where A is an antibody or antigen binding fragment thereof, U is an unnatural amino acid bioconjugation handle, U’ is a crosslinking agent or crosslinker, X1 is a first spacer, L is a linking spacer, X2 is a second spacer (X2 not present in 11-S1 through 11-S9), and P is a payload), where the payload was selected from the payloads described herein, e.g., those listed in FIG.5 (including PBD, also referred to as SG3199, and Dxd, also referred to as DX8951), and represented structurally in FIG.16 and FIG. 17 and conjugation was carried out using standard click chemistry conjugation methods, interchain cysteine conjugation methods, or thiomab conjugation methods to obtain the desired drug-to- antibody (DAR) specified in FIG.5. [0313] To generate ADCs from unconjugated LCA-containing antibodies, 9-S1 through 9-S9 and 11-S1 through 11-S9 were individually diluted to a final concentration of 1 mg/ml to 5 mg/ml in PBS.10 mM to 50 mM of payload stock solutions were made in buffer or organic solvent (DMSO, DMF, etc.) as appropriate. Payload was added at a molar excess of 2 to 75 fold and incubated at room temperature for 1 hour to 16 hours. For cysteine conjugates, purified antibody (m276 or DS) was buffer exchanged into PBS with 2mM EDTA, pH 7.4. The antibody was diluted to a final concentration of 1 mg/mL in PBS with 2mM EDTA and warmed to 37°C in a heat block. A stock solution of TCEP (10 mM) was freshly prepared in water and 2.5 molar equivalents (relative to the antibody concentration) was added. After 2 hrs the partially reduced antibody was removed from the heat block and cooled to room temperature. A stock solution of the maleimide payload 8 (FIG.16) or 10 (FIG.17) at 20 mM in DMSO was freshly prepared and 5-10 molar equivalents were added to the antibody. After 2 hrs the reaction mixture was buffer exchanged into PBS to remove excess payload and stored at 4°C. Screening Of Anti-CD276 Antibody Drug Conjugates [0314] Antibody drug conjugates (ADCs) 9-S1 through 9-S9 and 11-S1 through 11-S9 were assayed via HIC, SDS-PAGE, SEC, plasma stability, and cathepsin cleavage to select suitable ADC candidates for downstream production (see, e.g., FIGs.6-10). [0315] HIC data (FIG.6, top) was obtained using a MabPac Butyl column. Mobile phase A was 1.5 M ammonium sulfate, 25 mM sodium phosphate dibasic, pH 7 and a linear gradient of Attorney Docket No. BRI-023WO mobile phase B (25 mM sodium phosphate, pH 7) was introduced. Samples were separated at a flow rate of 0.8 mL/min and at a column temperature of 25 oC. The average DAR and distribution profile were determined by peak area percentage of each species. Exemplary analyses conducted by HIC are shown in FIG.6 and a summary of certain ADCs analyzed is shown in FIG.10. In general, a lower relative retention time (RRT) is desired, as calculated by shift in HIC elution time (in minutes) between the unconjugated (unmodified) and post-conjugated (labeled compound) antibody. A lower RRT indicates a more hydrophilic final ADC drug substance is produced and multiple site-specific conjugates produced final drug substances with minimal hydrophobic shift (i.e., <2 min RRT, in some instances <1 min RRT). SEC data (FIG.6, bottom) was obtained to determine protein purity and assess aggregation. Samples were diluted in mobile phase buffer (20 mM sodium phosphate, pH 6.8) at a concentration of 2 µM and injected onto a SEC Yarra-3000 column at 0.4 mL/min for 20 minutes. The percent monomer species, as compared to total species including all aggregate and fragment species, was reported as the percent of the total area for all protein-related peaks. Exemplary analyses conducted by SEC are shown in FIG.6 and a summary of certain ADCs analyzed is shown in FIG.10. Most site-specific conjugates resulted in little to no protein aggregation (e.g., less than or equal to 1% aggregation). The exemplary results in FIG.6, and summarized in FIG.10, indicate site-specific conjugation produces ADCs with improved biophysical characteristics. [0316] FIGs.7A-7B show exemplary in vitro characterization for two ADCs, including Cathepsin B kinetic assays, mouse plasma stability, and human plasma stability using standard protocols. Briefly, human plasma or mouse plasma was spiked with a final ADC concentration of 0.2 mg/mL and subsequently incubated at 37 °C in 100 µL aliquots for up to 72 hours; aliquots were immediately flash frozen and stored at -80 °C until further analysis. For analysis, plasma- incubated ADCs were thawed and subjected to affinity capture with Protein G magnetic beads. The eluted samples containing purified incubated ADC were then analyzed by HIC as described previously. Stability was measured by calculating the percentage of ADC remaining post plasma incubation. All site-specific conjugates maintain robust stability in plasma with the majority of ADCs ≥90%, and in most cases ≥95%, intact after incubation. Cathepsin cleavage kinetics was performed by incubating activated Cathepsin B with ADCs in reaction buffer (25 mM sodium acetate and 1 mM EDTA at pH 5.0) at 37°C for up to 240 minutes. Samples were quenched by adding E-64 protease inhibitor to a final concentration of 10 μM and stored at -80°C. Samples were thawed individually and analyzed by HIC analysis as described above. The percentage of cleavage is reported for each ADC; for example, 100% cleavage signifies that an ADC payload was Attorney Docket No. BRI-023WO completely liberated at the endpoint incubation period by cathepsin. A summary of certain ADCs analyzed is included in FIG.10. [0317] FIGs.7C-7F show mass spectra profiles for the heavy chain and light chain fragments of 9-S3, while FIGs.7G-7J show the same for 9-S9. FIG.7C shows the full LC/UV chromatogram after PNGase reduction LCMS. FIG.7D shows the deconvoluted mass spectrum of 9-S37.230 min peak with expected modified heavy chain mass. FIG.7E shows the ESI Mass spectrum of 9-S37.230 min peak. FIG.7F shows the deconvoluted mass spectrum of 9-S35.570 min peak with the correct unmodified light chain mass. FIG.7G shows the full LC/UV chromatogram after PNGase reduction LCMS. FIG.7H shows the deconvoluted mass spectrum of 9-S96.840 min peak with expected modified heavy chain mass. FIG.7I is the ESI mass spectrum of 9-S95.580 min peak. FIG.7J shows the deconvoluted mass spectrum of 9-S95.580 min peak with expected unmodified light chain. Results, as shown in FIGs.7A-7J and summarized in FIG. 10, demonstrate successful conjugation of a stable and homogenous product, with the DAR corroborated by HIC, and payload is efficiently released upon appropriate enzymatic activation. [0318] Thiomab conjugates were generated and assessed at Site 6 (S6) and Site 9 (S9) to compare thiomab site-specific conjugation to site-specific conjugation by click chemistry methods (e.g., by azide click chemistry, see, e.g., FIG.23A, top). These data are summarized in FIG.10, with thiomab ADCs denoted as “Site 6” and “Site 9”. The present example shows that click chemistry mediated site-specific conjugation may produce ADCs with improved biophysical characteristics as compared to their thiomab-generated site counterparts. Further, in ADCs conjugated using certain methods hydrophobicity (HIC RRT) is improved, and target DAR is better achieved. [0319] Interchain cysteine conjugation was also utilized to produce comparator ADCs. These data are summarized in FIG.10, with interchain cysteine conjugates denoted as “m276 (6)”, “DS- PBD (17)”, “m276 (13)”, “m276 (14)”, “DS-Dxd (15)” and “DS-Dxd (16)”. In some examples, site-specific conjugation by click chemistry (e.g., by azide click chemistry, see, e.g., FIG.23A, top) produces ADCs with improved biophysical characteristics as compared to their thiomab- generated site counterparts. In certain ADCs, hydrophobicity (HIC RRT) is improved, target DAR is better achieved, aggregation (% monomer by SEC) is reduced, and stability (mouse or human plasma stability) is more preserved. Sites S1, S3, S5, S7, and S9 were chosen for further cytotoxicity assays after evaluation of target criteria. For clarity, FIG.8 outlines target product profiles of ADCs produced for in vivo studies. Attorney Docket No. BRI-023WO Cell Cytotoxicity Assays [0320] Cytotoxicity assays were performed in HCT-116 (CD276+ colon cancer), Daudi (CD276 negative lymphoma), A549 (CD276+ Lung cancer), SKBR (CD276+ breast cancer) and OVCAR3 (CD276+ ovarian cancer) using HCT-116 (ATCC CCL-247), OVCAR3 (ATCC HTB- 161), A549 (ATCC CCL-185), and SKBR3 (ATCC HTB-30). The antibody-drug conjugates, alongside free payloads or naked antibody as controls, were tested via 8-point serial dilutions on the selected cell lines. After 96 hrs incubation in the presence of ADC or control, cell viability was measured using a CellTiter-Glo® assay (Fisher Catalog: PRG9242). As shown in FIG.11, ADC derivatives demonstrated efficacy at multiple conjugation sites, with ADCs with site-specific PBD payloads demonstrating enhanced efficacy in CD276+ cancer cell lines compared to ADCs with Dxd payloads. Off-target toxicity is equivalent for ADCs with site-specific PBD payloads as compared to ADCs with Dxd payload in DAUDI cells that lack CD276 expression (see FIG.11). Generated ADCs with PBD payloads demonstrated improved therapeutic index over ADCs with Dxd payloads conjugated at the same DAR of 2. The efficacy of PBD conjugated ADCs were demonstrated across a range of cell lines representing various indications (see FIG.12). [0321] The m276 antibody conjugates tested provide an increased therapeutic window over other anti-CD276 antibodies, by way of example, in comparison to Ifinatamab (“DS”), when the same DAR and payload are used. In this example, m276 and Ifinatamab (“DS”) were conjugated with interchain cysteine compatible compound 8 (MA-PEG8-VA-SG3199) (FIG.13). Example 2: Use of Anti-CD276 Antibody Conjugates in vivo [0322] This example describes the assessment of antibody compositions generated in Example 1 for anti-tumor activity and toxicity in vivo. Anti-Tumor Activity [0323] To investigate the anti-tumor activity of different provided ADC compositions, female 10 to 16-week old athymic NCr-nu/nu mice (Charles Rivers) that also carried the carboxylesterase 1C (Ces1c/ES1) mutation (B6(Cg)-Ces1ctm1.1Loc/J, The Jackson Laboratory, Stock No: 014096) were injected with HCT-116 cancer cells subcutaneously into the flank on day 0. Tumors were measured with a digital caliper, and tumor volumes were calculated using the formula LxW2x0.5 and presented as the median. Tumors less than 50 mm3 at the study endpoint were considered complete responses (CR). Mice were sorted into groups (N=8 mice per group) containing the same average size tumors (100-150 mm3) prior to initiation of therapy. Mice were treated with anti- CD276 ADCs or Vehicle via i.p. injection qw x 2 at the dose indicated in FIG.24. Attorney Docket No. BRI-023WO [0324] At a high dose (0.5 mg/kg for PBD-based ADCs, or 10 mg/kg for Dxd-based ADC), tumor growth rate is delayed with all drug compositions, but was most pronounced with site- specific PBD-based ADC, 9-S3. Following administration at a low dose (0.1 mg/kg for PBD-based ADCs, or 3 mg/kg for Dxd-based ADC), the site-specific PBD-based ADC, 9-S3, is most effective at tumor regression. Taking aggregate results from both low and high dose groups, 9-S3 also displays the highest rate of tumor regression and complete responses (CR) (FIGs.24-25). These results indicate that site-specific conjugation, particularly exemplified by Site 3, for PBD leads to improved in vivo anti-tumor activity. [0325] This example further demonstrates that the m276 antibody conjugates tested provide improved anti-tumor activity in a mouse model of colon cancer whereby a particular site of conjugation (S3) outperforms another site of conjugation (S9) providing an advantage for minimum efficacious dose with optimal conjugation site selection (FIGs.24-25). Furthermore, 9-S3 displays improved efficacy over interchain-cysteine conjugated ADCs (6) or other ADCs with alternative payloads (16). Toxicity [0326] To investigate the toxicity of different ADC compositions, mice included in efficacy studies discussed herein (FIG.24 and 25) were monitored for changes in body weight beginning after the first ADC administration and presented as the mean ± SEM. The results show that at a 0.5 mg/kg dose, mice administered an interchain cysteine-conjugated PBD-based ADC (m276-PBD, “6”) display a consistent decrease in body weight over the course of 8 weeks following treatment (FIG.26). Conversely, PBD-based site-specific conjugates 9-S3 and 9-S9 dosed at 0.5 mg/kg display maintenance of body weight similar to Vehicle control or 10 mg/kg of Ifinatamab deruxtecan (DS-Dxd, “16”), an ADC known to have low toxicity, indicating no toxicity is observed for PBD-based site-specific ADCs (9-S3 and 9-S9) as compared to PBD-based cysteine-conjugated ADC (6) that causes toxicity and a decrease in mouse body weight after administration. [0327] These results indicate that site specific conjugation of PBD payloads, as exemplified by Site 3 and Site 9, provide an improved toxicity profile as compared to other conjugation methodologies. Anti-tumor Activity Comparison [0328] To investigate the impact of antibody choice on tumor growth for ADCs, female 10 to 16-week old athymic NCr-nu/nu mice (Charles Rivers) that also carried the carboxylesterase 1C (Ces1c/ES1) mutation (B6(Cg)-Ces1ctm1.1Loc/J, The Jackson Laboratory, Stock No: 014096) were injected with HCT-116 cancer cells subcutaneously into the flank on day 0. Tumors were Attorney Docket No. BRI-023WO measured with a digital caliper, and tumor volumes were calculated using the formula LxW2x0.5 and presented as the mean ± SEM. Mice were sorted into groups (N=8 mice per group) containing the same average size tumors (100-150 mm3) prior to initiation of therapy. Mice were treated with anti-CD276 ADCs or Vehicle via i.p. injection qw x 2 at the dose indicated in FIG.27. [0329] The results show that at a low dose (0.1 mg/kg) and high dose (0.5 mg/kg), tumor growth rate was delayed when administering m276 antibody interchain-cysteine PBD-based ADC (“6”) (FIG.27). When an identical linker-payload composition utilizing Ifinatamab (“DS”) as the core antibody (“17”) was administered, tumor growth rate was only delayed at the high dose (0.5 mg/kg). These results indicate m276 is a particularly effective antibody scaffold when used as a PBD-based ADC. INCORPORATION BY REFERENCE [0330] The entire disclosure of each of the patent and scientific documents referred to herein is incorporated by reference for all purposes. EQUIVALENTS [0331] The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The embodiments described herein are therefore to be considered in all respects illustrative rather than limiting on the disclosure described herein. Scope of the disclosure is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.
Attorney Docket No. BRI-023WO SEQUENCE APPENDIX TABLE 2: CD276 binding proteins
Figure imgf000086_0001
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TABLE 3: tRNA/aaRS sequences
Figure imgf000089_0002
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Figure imgf000103_0001

Claims

Attorney Docket No. BRI-023WO CLAIMS What is claimed is: 1. An engineered anti-CD276 antibody comprising: a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to one or more of: (i) A142 of SEQ ID NO: 61; (ii) T157 of SEQ ID NO: 61; (iii) T171 of SEQ ID NO: 61; (iv) A174 of SEQ ID NO: 61; (v) Y182 of SEQ ID NO: 61; (vi) T197 of SEQ ID NO: 61; (vii) D267 of SEQ ID NO: 61; or (viii) S241 of SEQ ID NO: 61. 2 The antibody of claim 1, wherein the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to one or more of: (i) A142 of SEQ ID NO: 61; (ii) T157 of SEQ ID NO: 61; (iii) T171 of SEQ ID NO: 61; (iv) A174 of SEQ ID NO: 61; (v) Y182 of SEQ ID NO: 61; (vi) T197 of SEQ ID NO: 61; (vii) D267 of SEQ ID NO: 61; or (viii) S241 of SEQ ID NO: 61. 3 The antibody of claim 1 or claim 2, wherein the unnatural amino acid is incorporated at a position corresponding to T171 of SEQ ID NO: 61. 4 The antibody of any one of claims 1-3, further comprising a light chain sequence, wherein the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, Attorney Docket No. BRI-023WO at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 62. 5. The antibody of claim 4, wherein the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 62. 6 The antibody of any one of claims 1-3, further comprising a light chain sequence, wherein the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 62, with an unnatural amino acid incorporated at a position corresponding to Q162 of SEQ ID NO: 62. 7 The antibody of claim 6, wherein the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to Q162 of SEQ ID NO: 62. 8 An engineered anti-CD276 antibody comprising: a light chain sequence, wherein the light chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 62, with an unnatural amino acid incorporated at a position corresponding to Q162 of SEQ ID NO: 62. 9 The antibody of claim 8, wherein the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 62, with an unnatural amino acid incorporated at a position corresponding to Q162 of SEQ ID NO: 62. 10 The antibody of claim 8 or claim 9, further comprising a heavy chain sequence, wherein the heavy chain sequence comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to one or more of: (i) A142 of SEQ ID NO: 61; (ii) T157 of SEQ ID NO: 61; Attorney Docket No. BRI-023WO (iii) T171 of SEQ ID NO: 61; (iv) A174 of SEQ ID NO: 61; (v) Y182 of SEQ ID NO: 61; (vi) T197 of SEQ ID NO: 61; (vii) D267 of SEQ ID NO: 61; or (viii) S241 of SEQ ID NO: 61. 11. The antibody of claim 10, wherein the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to one or more of: (i) A142 of SEQ ID NO: 61; (ii) T157 of SEQ ID NO: 61; (iii) T171 of SEQ ID NO: 61; (iv) A174 of SEQ ID NO: 61; (v) Y182 of SEQ ID NO: 61; (vi) T197 of SEQ ID NO: 61; (vii) D267 of SEQ ID NO: 61; (viii) S241 of SEQ ID NO: 61. 12. The antibody of claim 10 or claim 11, wherein the unnatural amino acid is incorporated at a position corresponding to T171 of SEQ ID NO: 61. 13. An engineered anti-CD276 antibody comprising: an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 137, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 138, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 139, and wherein the heavy chain and/or the light chain comprises an unnatural amino acid incorporated at a position corresponding to one or more amino acid residue positions set forth in TABLE 1. Attorney Docket No. BRI-023WO 14. An engineered anti-CD276 antibody comprising: a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 137, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 138, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 139, and wherein the heavy chain comprises an unnatural amino acid incorporated at a position corresponding to one or more of: (i) A142 of SEQ ID NO: 61; (ii) T157 of SEQ ID NO: 61; (iii) T171 of SEQ ID NO: 61; (iv) A174 of SEQ ID NO: 61; (v) Y182 of SEQ ID NO: 61; (vi) T197 of SEQ ID NO: 61; (vii) D267 of SEQ ID NO: 61; or (viii) S241 of SEQ ID NO: 61. 15. An engineered anti-CD276 antibody comprising: a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 134, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 136, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 137, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 138, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 139, and wherein the heavy chain comprises an unnatural amino acid incorporated at a position corresponding to T171 of SEQ ID NO: 61. 16. The antibody of any one of claims 1-15, wherein the antibody comprises at least one, at least two, at least three, at least four, at least five, or more unnatural amino acids. 17. The antibody of any one of claims 1-16, wherein the unnatural amino acid is a tryptophan analog, a leucine analog, a tyrosine analog, or a pyrrolysine analog. 18. The antibody of any one of claims 1-17, wherein the unnatural amino acid is a tryptophan Attorney Docket No. BRI-023WO analog. 19. The antibody of claim 18, wherein the tryptophan analog is 5-HTP or 5-AzW. 20. The antibody of any one of claims 1-17, wherein the unnatural amino acid is a leucine analog. 21. The antibody of claim 20, wherein the leucine analog is LCA or Cys-5-N3. 22. The antibody of claim 20, wherein the leucine analog is LCA. 23. The antibody of any one of claims 1-17, wherein the unnatural amino acid is a tyrosine analog. 24. The antibody of claim 23, wherein the tyrosine analog is OmeY, AzF, or OpropY. 25. The antibody of any one of claims 1-17, wherein the unnatural amino acid is a pyrrolysine analog. 26. The antibody of claim 25, wherein the pyrrolysine analog is BocK, CpK, or AzK. 27. The antibody of any one of claims 1-26, wherein the unnatural amino acid is chemically modified. 28. The antibody of claim 27, wherein the chemical modification comprises conjugation of the unnatural amino acid to a molecule. 29. The antibody of claim 28, wherein the molecule is a detectable label. 30. The antibody of claim 28, wherein the molecule is a therapeutic molecule. 31. The antibody of claim 28, wherein the therapeutic molecule is: AEB, AEVB, AFP, an amatoxin, an auristatin (e.g., auristatin E), a calicheamicin, CC-1065 or a CC-1065 analog, chalicheamicin, combretastatin, docetaxel, dolastatin-10, DUBA, a duocarmycin, Attorney Docket No. BRI-023WO echinomycin, FAM, maytansine, a maytansinoid (e.g., DM1 or DM4), MMAD, MMAE, MMAF, a morpholino-doxorubicin (e.g., cyanomorpholino-doxorubicin), netropsin, an oligonucleotide (e.g., a DNA, RNA, or LNA oligonucleotide), paclitaxel, pyrrolobenzodiazepine dimer (PBD), a peptide (e.g., a therapeutic peptide), rhizoxin, a small molecule (e.g., a therapeutic small molecule), SN-38, topotecan, a topoisomerase inhibitor, or a toxoid. 32. The antibody of claim 31, wherein the molecule is a therapeutic molecule is PBD. 33. The antibody of any one of claims 28-32, wherein the molecule is conjugated to the antibody by a crosslinking agent. 34. The antibody of claim 33, wherein the crosslinking agent comprises bissuflosuccinimidyl suberate (BS3), N-hydroxy-succinimide/1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (NHS/EDC), N-ε-maleimidocaproyl-oxysulfosuccinimide ester (sulfoEMCS), N-ε- maleimidocaproic acid hydrazide (EMCH), hydrazide, N-succinimidyl-S-acetylthioacetate (SATA), monofluorocyclooctyne (MFCO), bicyclo[6.1.0]nonyne (BCN), N succinimidyl-S- acetylthiopropionate (SATP), a maleimido ester, a dibenzocyclooctyne (DBCO) ester, EDC, or any combination thereof. 35. The antibody of any one of claims 28-34, wherein the molecule is conjugated to the antibody by a linker. 36. The antibody of claim 35, wherein the linker comprises a cleavable or non-cleavable linker region, and optionally, one or more spacer regions. 37. The antibody of claim 36, wherein the cleavable or non-cleavable linker region is a peptide- linker region. 38. The antibody of claim 37, wherein the peptide-linker region comprises a VA linker or a GGFG linker. 39. The antibody of any one of claims 36-38, wherein the spacer region comprises a PEG-based region. Attorney Docket No. BRI-023WO 40. The antibody of claim 39, wherein the PEG-based region comprises a PEG8 region. 41. The antibody of any one of claims 35-40, wherein linker conjugates the molecule to the antibody via a cross-linking agent. 42. The antibody of claim 41, wherein the cross-linking agent comprises bissuflosuccinimidyl suberate (BS3), N-hydroxy-succinimide/1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (NHS/EDC), N-ε-maleimidocaproyl-oxysulfosuccinimide ester (sulfoEMCS), N-ε- maleimidocaproic acid hydrazide (EMCH), hydrazide, N-succinimidyl-S-acetylthioacetate (SATA), monofluorocyclooctyne (MFCO), bicyclo[6.1.0]nonyne (BCN), N succinimidyl-S- acetylthiopropionate (SATP), a maleimido ester, a dibenzocyclooctyne (DBCO) ester, EDC, or any combination thereof. 43. The antibody of any one of claims 33-42, wherein the cross-linking agent or the linker conjugates the molecule to the antibody at the unnatural amino acid site. 44. The antibody of any one of claims 1-43, wherein: (a) the antibody has an average drug-to-antibody ratio (DAR) of at least 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0, as measured by hydrophobic interaction chromatography (HIC) or LCMS; (b) the antibody has an average DAR of about 2, as measured by HIC or LCMS; (c) at least 40%, 50%, 60%, 70%, 80%, or 90% of the antibody remains following incubation in human plasma for 72 hours at 37 °C; (d) at least 40%, 50%, 60%, 70%, 80%, or 90% of the antibody remains following incubation with Cathepsin B for 240 minutes at 37 °C; (e) the antibody has a binding affinity for a target antigen of about 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.75 nM, 0.5 nM, 0.1 nM, 0.075 nM, or 0.05 nM or lower, as measured by enzyme-linked immunosorbent assay (ELISA); and/or (f) the antibody has a binding affinity for CD276, or a derivative or variant thereof, that is within 0.1 fold, 0.2 fold, 0.3 fold, 0.4 fold, 0.5 fold, 1.0 fold, 1.5 fold, 2.0 fold, 3.0 fold, 4.0 fold, 5.0 fold, 6.0 fold, 8.0 fold, or 10.0 fold of the binding affinity of a suitable reference antibody as measured by ELISA, wherein the suitable reference antibody is an Attorney Docket No. BRI-023WO otherwise identical antibody that does not comprise the unnatural amino acid. 45. The antibody of any one of claims 1-44, wherein the antibody is derived from an IgG1, IgG2, IgG3, or IgG4 antibody. 46. The antibody of claim 45, wherein the antibody is derived from an IgG1 antibody. 47. A method of making an antibody of any one of claims 1-46, the method comprising a step of culturing a cell comprising: (i) a first nucleotide sequence encoding a tRNA comprising an anticodon that hybridizes to a codon selected from UAG, UGA, and UAA, and is capable of being charged with an unnatural amino acid; (ii) a second nucleotide sequence encoding an aminoacyl-tRNA synthetase capable of charging the tRNA with the unnatural amino acid; and (iii) a third nucleotide sequence encoding the heavy chain sequence and/or a fourth nucleotide sequence encoding the light chain sequence, wherein the third nucleotide sequence and/or the fourth nucleotide sequence comprise a codon selected from UAG, UGA, and UAA; wherein the cell is cultured under conditions that permit the tRNA, when expressed in the cell and charged with the unnatural amino acid, to hybridize to the codon and direct incorporation of the unnatural amino acid into the antibody. 48. A method of making an engineered anti-CD276 antibody, the method comprising a step of culturing a cell comprising: (i) a first nucleotide sequence encoding a tRNAs comprising an anticodon that hybridizes to a codon selected from UAG, UGA, and UAA, and is capable of being charged with an unnatural amino acid; (ii) a second nucleotide sequence encoding an aminoacyl-tRNA synthetase capable of charging the tRNA with the unnatural amino acid; and (iii) a third nucleotide sequence encoding a heavy chain sequence as set forth in SEQ ID NO: 61 and/or a fourth nucleotide sequence encoding a light chain sequence as set forth in SEQ ID NO: 62, wherein the third nucleotide sequence and/or the fourth nucleotide sequence comprise a codon selected from UAG, UGA, and UAA; wherein the cell is cultured under conditions that permit the tRNA, when expressed Attorney Docket No. BRI-023WO in the cell and charged with the unnatural amino acid, to hybridize to the codon and direct incorporation of the unnatural amino acid into the antibody. 49. The method of claim 48, wherein the third nucleotide sequence and/or the fourth nucleotide sequence comprise a codon selected from UAG, UGA, and UAA at a site that corresponds to any site shown in FIG.5 or Table 1. 50. The method of any one of claims 47-49, wherein: (a) the amount of the antibody comprising the unnatural amino acid expressed by the cell is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the amount of a suitable reference antibody expressed by the same cell or a similar cell; and/or (b) following purification of the antibody, less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the antibody is aggregated, as measured by size exclusion chromatography (SEC). 51. The method of any one of claims 47-50, wherein the tRNA is an analog or derivative of a prokaryotic tryptophanyl-tRNA. 52. The method of claim 51, wherein the prokaryotic tryptophanyl-tRNA is an E. coli tryptophanyl-tRNA. 53. The method of any one of claims 47-50, wherein the nucleotide sequence encoding the tRNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 56-60. 54. The method of any one of claims 47-50, wherein the aminoacyl-tRNA synthetase is an analog or derivative of a prokaryotic tryptophanyl-tRNA synthetase. 55. The method of claim 54, wherein the prokaryotic tryptophanyl-tRNA synthetase is an E. coli tryptophanyl-tRNA synthetase. 56. The method of any one of claims 47-50, wherein the aminoacyl-tRNA synthetase comprises an amino acid sequence selected from any one of SEQ ID NOs: 51-54. 57. The method of any one of claims 47-50, wherein the tRNA is an analog or derivative of a Attorney Docket No. BRI-023WO prokaryotic leucyl-tRNA. 58. The method of claim 57, wherein the prokaryotic leucyl-tRNA is an E. coli leucyl-tRNA. 59. The method of any one of claims 47-50, wherein the nucleotide sequence encoding the tRNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 22-49. 60. The method of any one of claims 47-50, wherein the aminoacyl-tRNA synthetase is an analog or derivative of a prokaryotic leucyl-tRNA synthetase. 61. The method of claim 60, wherein the prokaryotic leucyl-tRNA synthetase is an E. coli leucyl-tRNA synthetase. 62. The method of any one of claims 47-50, wherein the aminoacyl-tRNA synthetase comprises an amino acid sequence selected from any one of SEQ ID NOs: 8-21. 63. The method of any one of claims 47-50, wherein the tRNA is an analog or derivative of a prokaryotic tyrosyl-tRNA. 64. The method of claim 63, wherein the prokaryotic tyrosyl-tRNA is an E. coli tyrosyl-tRNA. 65. The method of any one of claims 47-50, wherein the nucleotide sequence encoding the tRNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 140-144, 192, or 193. 66. The method of any one of claims 47-50, wherein the aminoacyl-tRNA synthetase is an analog or derivative of the prokaryotic tyrosyl-tRNA synthetase. 67. The method of claim 66, wherein the prokaryotic tyrosyl-tRNA synthetase is an E. coli tyrosyl-tRNA synthetase. 68. The method of any one of claims 47-50, wherein the aminoacyl-tRNA synthetase comprises an amino acid sequence selected from any one of SEQ ID NOs: 145 or 147. Attorney Docket No. BRI-023WO 69. The method of any one of claims 47-50, wherein the tRNA is an analog or derivative of an archael pyrrolysyl-tRNA. 70. The method of claim 69, wherein the archael pyrrolysyl-tRNA is an M. barkeri pyrrolysyl- tRNA. 71. The method of any one of claims 47-50, wherein the nucleotide sequence encoding the tRNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 148-178, 181, or 182. 72. The method of any one of claims 47-50, wherein the aminoacyl-tRNA synthetase is an analog or derivative of the archael pyrrolysyl-tRNA synthetase. 73. The method of claim 72, wherein the archael pyrrolysyl-tRNA synthetase is an M. barkeri pyrrolysyl-tRNA synthetase. 74. The method of any one of claims 47-50, wherein the aminoacyl-tRNA synthetase comprises an amino acid sequence selected from any one of SEQ ID NO: 179. 75. The method of any one of claims 50-74, wherein the reference antibody comprises a wild- type amino acid residue at the position corresponding to the unnatural amino acid. 76. The method of any one of claim 47-75, wherein the cell is a mammalian cell. 77. The method of any one of claims 47-75, wherein the cell is a human cell. 78. The method of any one of claims 47-75, wherein the cell is a human embryonic kidney (HEK) cell or a Chinese hamster ovary (CHO) cell. 79. The method of any one of claims 47-75, further comprising a step of purifying the antibody. 80. The method of any one of claims 47-75, further comprising a step of chemically modifying the unnatural amino acid. Attorney Docket No. BRI-023WO 81. The method of claim 80, wherein the chemical modification comprises conjugation to a molecule. 82. The method of claim 81, wherein the molecule is a detectable label. 83. The method of claim 81, wherein the molecule is a therapeutic molecule. 84. The method of claim 83, wherein the therapeutic molecule is: AEB, AEVB, AFP, an amatoxin, an auristatin (e.g., auristatin E), a calicheamicin, CC-1065 or a CC-1065 analog, chalicheamicin, combretastatin, docetaxel, dolastatin-10, DUBA, a duocarmycin, echinomycin, FAM, maytansine, a maytansinoid (e.g., DM1 or DM4), MMAD, MMAE, MMAF, a morpholino-doxorubicin (e.g., cyanomorpholino-doxorubicin), netropsin, an oligonucleotide (e.g., a DNA, RNA, or LNA oligonucleotide), paclitaxel, PBD, a peptide (e.g., a therapeutic peptide), rhizoxin, a small molecule (e.g., a therapeutic small molecule) SN-38, topotecan, a topoisomerase inhibitor, or a toxoid. 85. The method of claim 84, wherein the therapeutic molecule is PBD. 86. The method of any one of claims 81-85, wherein the molecule is conjugated to the antibody by a crosslinking agent. 87. The method of claim 86, wherein the crosslinking agent comprises bissuflosuccinimidyl suberate (BS3), N-hydroxy-succinimide/1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (NHS/EDC), N-ε-maleimidocaproyl-oxysulfosuccinimide ester (sulfoEMCS), N-ε- maleimidocaproic acid hydrazide (EMCH), hydrazide, N-succinimidyl-S-acetylthioacetate (SATA), monofluorocyclooctyne (MFCO), bicyclo[6.1.0]nonyne (BCN), N succinimidyl-S- acetylthiopropionate (SATP), a maleimido ester, a dibenzocyclooctyne (DBCO) ester, EDC, or any combination thereof. 88. The method of any one of claims 81-87, wherein the molecule is conjugated to the engineered antibody by a linker. 89. The method of claim 88, wherein the linker comprises a cleavable or non-cleavable linker region, and optionally, one or more spacer regions. Attorney Docket No. BRI-023WO 90. The method of claim 89, wherein the cleavable or non-cleavable linker region is a peptide- linker region. 91. The method of claim 90, wherein the peptide-linker region comprises a VA linker or a GGFG linker. 92. The method of any one of claims 89-91, wherein the spacer region comprises a PEG-based domain. 93. The method of claim 92, wherein the PEG-based domain comprises a PEG8 region. 94. The method of any one of claims 88-93, wherein linker conjugates the molecule to the antibody via a cross-linking agent. 95. The method of claim 94, wherein the cross-linking agent comprises bissuflosuccinimidyl suberate (BS3), N-hydroxy-succinimide/1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (NHS/EDC), N-ε-maleimidocaproyl-oxysulfosuccinimide ester (sulfoEMCS), N-ε- maleimidocaproic acid hydrazide (EMCH), hydrazide, N-succinimidyl-S-acetylthioacetate (SATA), monofluorocyclooctyne (MFCO), bicyclo[6.1.0]nonyne (BCN), N succinimidyl-S- acetylthiopropionate (SATP), a maleimido ester, a dibenzocyclooctyne (DBCO) ester, EDC, or any combination thereof. 96. The method of any one of claims 86-95, wherein the cross-linking agent or the linker conjugates the molecule to the antibody at the unnatural amino acid site. 97. An engineered anti-CD276 antibody comprising: a heavy chain sequence comprising an amino acid sequence as set forth in SEQ ID NO: 61, with an LCA incorporated at a position corresponding to T171 of SEQ ID NO: 61; a light chain sequence comprising an amino acid sequence as set forth in SEQ ID NO: 62; wherein a pyrrolobenzodiazepine dimer (PBD) is conjugated to the LCA using a linker and crosslinking agent, wherein the linker comprises a PEG8 spacer region and a VA linker region and the crosslinking agent is DBCO. Attorney Docket No. BRI-023WO 98. A method of making the engineered anti-CD276 antibody of claim 97, the method comprising a step of culturing a cell comprising: (i) a first nucleotide sequence encoding a tRNAs comprising an anticodon that hybridizes to a codon selected from UAG, UGA, and UAA, and is capable of being charged with an LCA; (ii) a second nucleotide sequence encoding an aminoacyl-tRNA synthetase capable of charging the tRNA with the LCA; and (iii) a third nucleotide sequence encoding the heavy chain sequence and a fourth nucleotide sequence encoding the light chain sequence, wherein the third nucleotide sequence comprises a codon selected from UAG, UGA, and UAA at a site corresponding to T171 of SEQ ID NO: 61; wherein the cell is cultured under conditions that permit the tRNA, when expressed in the cell and charged with the LCA, to hybridize to the codon and direct incorporation of the LCA. 99. An engineered anti-CD276 antibody comprising: an immunoglobulin heavy chain sequence comprising an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to one or more sites selected from TABLE or FIG.5; and/or an immunoglobulin light chain sequence comprising an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 62, with an unnatural amino acid incorporated at a position corresponding to one or more sites selected from TABLE or FIG.5. 100. The antibody of claim 99, wherein: the heavy chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 61, with an unnatural amino acid incorporated at a position corresponding to one or more sites selected from TABLE 1 or FIG.5; and/or the light chain sequence comprises an amino acid sequence as set forth in SEQ ID NO: 62, with an unnatural amino acid incorporated at a position corresponding to one or Attorney Docket No. BRI-023WO more sites selected from TABLE 1 or FIG.5. 101. The antibody of claim 99 or claim 100, wherein at least one unnatural amino acid is: (a) a leucine analog, a tyrosine analog, a tryptophan analog, or a pyrrololysine analog; (b) 5-HTP or 5-AzW; (c) LCA or Cys-5-N43; (d) LCA; (e) OmeY, AzF, or OpropY; or (d) BocK, CpK, or AzK. 102. The antibody of claim 101, wherein the unnatural amino acid is chemically modified, wherein the chemical modification comprises conjugation of the unnatural amino acid to a molecule: (a) wherein the molecule is a detectable label; (b) wherein the molecule is a therapeutic molecule; (c) wherein the molecule is conjugated to the antibody by a crosslinking reagent; (d) wherein the molecule is conjugated to the antibody by a linker; (e) wherein the molecule is conjugated to the antibody by a linker forming a linker-payload structure of U’-X1-L-X2-P, where U’ is DBCO, X1 is PEG8, L is VA, X2 is PAB, and P is PBD, and DBCO crosslinks the linker-payload to the unnatural amino acid; (f) wherein the molecule is conjugated to the antibody by a linker forming a linker-payload structure of U’- X1-L-P, where U’ is DBCO, X1 is PEG4, L is GGFG, and P is Dxd, and DBCO crosslinks the linker-payload to the unnatural amino acid. 103. A pharmaceutical composition comprising an engineered antibody of any one of claims 1- 47, 97, or 99-102. 104. The pharmaceutical composition of claim 103, further comprising one or more pharmaceutically acceptable excipients or carriers. 105. A method of treating a subject with a disease or a disorder, the method comprising administering to the subject an effective amount of an engineered antibody of any one of claims 1-46, 97, or 99-102, a pharmaceutical composition of claim 103 or claim 104, or an antibody made by a method of any one of claims 47-96, or 98. 106. A method of treating a subject with a cancer that overexpresses CD276, the method comprising administering to the subject an effective amount of an engineered antibody of any one of claims 1-46, 97, or 99-102, a pharmaceutical composition of claim 103 or claim 104, or an antibody made by a method of any one of claims 47-96, or 98. Attorney Docket No. BRI-023WO 107. A method of treating a subject with CD276 positive tumor vasculature, the method comprising administering to the subject an effective amount of an engineered antibody of any one of claims 1-46, 97, or 99-102, a pharmaceutical composition of claim 103 or claim 104, or an antibody made by a method of any one of claims 47-96, or 98.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190127471A1 (en) * 2016-04-15 2019-05-02 Macrogenics, Inc. Novel b7-h3 binding molecules, antibody drug conjugates thereof and methods of use thereof
WO2021118968A1 (en) * 2019-12-12 2021-06-17 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Antibody-drug conjugates specific for cd276 and uses thereof
WO2022056318A1 (en) * 2020-09-10 2022-03-17 Brickbio, Inc. Antibodies containing unnatural amino acids and methods of making and using the same

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190127471A1 (en) * 2016-04-15 2019-05-02 Macrogenics, Inc. Novel b7-h3 binding molecules, antibody drug conjugates thereof and methods of use thereof
WO2021118968A1 (en) * 2019-12-12 2021-06-17 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Antibody-drug conjugates specific for cd276 and uses thereof
WO2022056318A1 (en) * 2020-09-10 2022-03-17 Brickbio, Inc. Antibodies containing unnatural amino acids and methods of making and using the same

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