Antibody-drug conjugates and methods of making thereof

Abstract

To provide novel biological materials, production methods and formulations for antibody-drug conjugates.SOLUTION: Provided herein is a method for producing partially re-oxidized antibody or antigen binding fragment thereof comprising the steps of: providing an antibody or antigen binding fragment thereof comprising one or more capped cysteine residues; reducing the cysteine residues to provide an antibody or antigen binding fragment thereof comprising one or more decapped cysteine residues; and storing the antibody or antigen binding fragment thereof comprising one or more decapped cysteine residues under oxidation conditions that enable recapping of one or more off-target cysteine residues thereby producing a partially re-oxidized antibody or antigen binding fragment thereof. Also provided is a method for producing an antibody-drug conjugate, the method comprising binding a linker-drug moiety to the partially re-oxidized antibody or antigen binding fragment thereof thereby producing an antibody-drug conjugate.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 176,046, filed April 16, 2021, and U.S. Provisional Patent Application No. 63 / 254,031, filed October 8, 2021, the contents of which are hereby incorporated by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on April 15, 2022, is named N2067-7179WO_SL.txt, and is 239,071 bytes in size. [Background technology]

[0003] Uveal melanoma (UM) is a rare malignancy with an incidence of 6–10 cases per million people and 2500–3000 new cases per year in the United States, accounting for 3–5% of all melanomas. Despite excellent local disease control rates (5-year OSR of 70%), up to 50% of patients will develop metastatic disease (liver 60%, lung 25%; median OS of 13 months, 2-year OSR of 8%). For patients who do develop metastatic disease, no proven standard of care is available. There is a significant medical need for specifically approved therapies and dedicated disease management strategies to improve outcomes for patients with metastatic UM. Summary of the Invention [Problem to be solved by the invention]

[0004] Oncogenic mutations in GNAQ or GNA11 are hallmark mutations of uveal melanoma. Reduction of GNAQ / 11 levels leads to inhibition of cell proliferation and apoptosis. This inhibitory effect can be achieved by targeted delivery of antibody-drug conjugates that inhibit GNAQ / 11 activity. Despite recent advances, there is still a need to develop new biological materials, manufacturing methods, and formulations for antibody-drug conjugates. [Means for solving the problem]

[0005] The present disclosure provides, at least in part, a genetically engineered microorganism (e.g., Chromobacterium vaccinii) that produces depsipeptide, e.g., compound (A1), in high titer and / or isolated yield. In some embodiments, the microorganism comprises a non-native promoter operably linked to the depsipeptide biosynthetic gene cluster (BGC). In some embodiments, the BGC of the natural product is disrupted, preventing isolation of the depsipeptide. Methods of producing depsipeptide, e.g., compound (A1), using the microorganisms described herein are also described. Depsipeptide, e.g., compound (A1), produced by the methods described herein can be used to produce antibody-drug conjugates (e.g., antibody-drug conjugates described herein), for example, according to the methods described herein.

[0006] The present disclosure also provides, at least in part, methods for producing a partially reoxidized antibody or antigen-binding fragment thereof. In some embodiments, the methods involve reducing one or more capped cysteine ​​residues in the antibody or antigen-binding fragment thereof to provide an antibody or antigen-binding fragment thereof comprising one or more decapped cysteine ​​residues. In some embodiments, the methods involve storing the antibody or antigen-binding fragment thereof comprising one or more decapped cysteine ​​residues under oxidizing conditions sufficient to produce a partially reoxidized antibody or antigen-binding fragment thereof. Also described herein are methods for making antibody-drug conjugates (e.g., antibody-drug conjugates described herein) using the methods for producing a partially reoxidized antibody or antigen-binding fragment thereof described herein. Antibody-drug conjugates produced by the methods described herein can be formulated into the formulations described herein.

[0007] The present disclosure also provides, at least in part, formulations of antibody-drug conjugates (e.g., antibody-drug conjugates described herein). In some embodiments, the formulations comprise an antibody-drug conjugate described herein, a buffer, a stabilizer, and a surfactant, and have a pH of about 4.5 to about 6.5. In some embodiments, the formulations are lyophilized. The formulations described herein can be used to treat or prevent disorders, such as cancer. In some embodiments, the cancer expresses PMEL17 and / or contains a mutation in the GNAQ or GNA11 gene. In some embodiments, the cancer is carcinoma (e.g., hepatocellular carcinoma), sarcoma, leukemia, lymphoma, eye cancer, ocular neoplasm, melanoma (e.g., uveal melanoma, non-uveal melanoma, malignant melanoma, ocular melanoma, subcutaneous melanoma, cutaneous melanoma, or mucosal melanoma), or metastatic cancer thereof.

[0008] Engineered microorganisms and methods for producing compounds In some aspects, the disclosure features a microorganism comprising a nucleic acid comprising a nucleotide sequence encoding a polypeptide involved in producing a compound having the structure of formula (A1), wherein the nucleotide sequence is operably linked to a non-native promoter.

[0009] In some embodiments, the microorganism is a genetically engineered form of a microorganism that naturally produces the compound. In some embodiments, the microorganism is a bacterium. In some embodiments, the microorganism is a Chromobacterium. In some embodiments, the microorganism is Chromobacterium vaccinii. In some embodiments, the microorganism is Chromobacterium vaccinii DSM 25150 (=ATCC BAA-2314, =NRRL B-50840, =MWU205). In some embodiments, the microorganism is an isolated microorganism. In some embodiments, the microorganism is a synthetic microorganism.

[0010] In some embodiments, the nucleic acid is located on the chromosome of the microorganism, for example, naturally located on the chromosome or stably integrated into the chromosome. In some embodiments, the nucleotide sequence encoding a polypeptide involved in the production of the compound is located in a biosynthetic gene cluster (BGC). In some embodiments, the BGC is compound (A1)-BGC. In some embodiments, compound (A1)-BGC has the genetic organization shown in Figure 1.

[0011] In some embodiments, the BGC comprises one or more (e.g., 2, 3, 4, 5, 6, 7, or all) of frsA, frsB, frsC, frsD, frsE, frsF, frsG, or frsH, or homologs thereof. In some embodiments, the BGC comprises frsA, frsB, frsC, frsD, frsE, frsF, frsG, and frsH, or homologs thereof.

[0012] In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound is located in frsA, frsB, frsC, frsD, frsE, frsF, frsG, or frsH, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound is located in a region comprising frsA or a homolog thereof. In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound is located in a region comprising frsA and frsB, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound is located in a region comprising frsA, frsB, and frsC, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound is located in a region comprising frsA, frsB, frsC, and frsD, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound is located in a region comprising frsA, frsB, frsC, frsD, and frsE, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide involved in the production of a compound is located in a region comprising frsA, frsB, frsC, frsD, frsE, and frsF, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide involved in the production of a compound is located in a region comprising frsA, frsB, frsC, frsD, frsE, frsF, and frsG, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide involved in the production of a compound is located in a region comprising frsA, frsB, frsC, frsD, frsE, frsF, frsG, and FrsH, or homologs thereof.

[0013] In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound comprises the coding sequence of frsA, frsB, frsC, frsD, frsE, frsF, frsG, or frsH, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound comprises the coding sequence of frsA or a homolog thereof. In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound comprises the coding sequence of frsA and frsB, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound comprises the coding sequence of frsA, frsB, and frsC, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound comprises the coding sequence of frsA, frsB, frsC, and frsD, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound comprises the coding sequence of frsA, frsB, frsC, frsD, and frsE, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound comprises the coding sequence of frsA, frsB, frsC, frsD, frsE, and frsF, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound comprises the coding sequence of frsA, frsB, frsC, frsD, frsE, frsF, and frsG, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound comprises the coding sequence of frsA, frsB, frsC, frsD, frsE, frsF, frsG, and FrsH, or homologs thereof.

[0014] In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 317, or a functional fragment thereof, or a nucleotide sequence that results from a homologous recombination event (e.g., for promoter exchange) using a nucleotide sequence that has at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto, or that differs therefrom by no more than about 100, about 90, about 80, about 70, about 60, about 50, about 40, about 30, about 20, about 15, about 10, about 5, or about 2 nucleotides.

[0015] In some embodiments, the nucleic acid comprises the nucleotide sequence associated with GenBank Accession Number: BankIt2437961 BSeq#1 MW732719, or a functional fragment thereof, or a nucleotide sequence having at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto.

[0016] In certain embodiments, the non-native promoters are from the same microorganism, while in other embodiments, the non-native promoters are from different microorganisms.

[0017] In some embodiments, the non-native promoter is a vioP promoter, a nptII promoter, a rbs promoter, a J23119 promoter, a pLpp promoter, a PS12burk promoter, an ErmE promoter, or a nucleotide sequence encoding the nucleotide sequence of interest. * In some embodiments, the non-native promoter comprises a vioP promoter, a nptII promoter, or a rbs promoter.

[0018] In some embodiments, the non-native promoter comprises the nucleotide sequence of any of SEQ ID NOs: 264-266, 268-271, or 316, or a functional fragment thereof, or a nucleotide sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto, or differing by no more than about 60, about 50, about 40, about 30, about 20, about 15, about 10, about 5, or about 2 nucleotides thereto. In some embodiments, the non-native promoter comprises or consists of the nucleotide sequence of any of SEQ ID NOs: 264-266, 268-271, or 316, or a functional fragment thereof.

[0019] In some embodiments, the non-native promoter comprises the vioP promoter. In some embodiments, the non-native promoter comprises the nucleotide sequence of SEQ ID NO:264, or a functional fragment thereof, or a nucleotide sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto, or differing by no more than about 60, about 50, about 40, about 30, about 20, about 15, about 10, about 5, or about 2 nucleotides thereto. In some embodiments, the non-native promoter comprises or consists of the nucleotide sequence of SEQ ID NO:264, or a functional fragment thereof.

[0020] In some embodiments, the non-native promoter comprises the nptII promoter. In some embodiments, the non-native promoter comprises the nucleotide sequence of SEQ ID NO: 265, or a functional fragment thereof, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or differing by no more than about 60, 50, 40, 30, 20, 15, 10, 5, or 2 nucleotides therefrom. In some embodiments, the non-native promoter comprises or consists of the nucleotide sequence of SEQ ID NO: 265, or a functional fragment thereof.

[0021] In some embodiments, the non-native promoter comprises the rbs promoter. In some embodiments, the non-native promoter comprises the nucleotide sequence of SEQ ID NO: 266, or a functional fragment thereof, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or differing by no more than 60, 50, 40, 30, 20, 15, 10, 5, or 2 nucleotides thereto. In some embodiments, the non-native promoter comprises or consists of the nucleotide sequence of SEQ ID NO: 266, or a functional fragment thereof.

[0022] In some embodiments, the non-native promoter comprises the J23119 promoter. In some embodiments, the non-native promoter comprises the nucleotide sequence of SEQ ID NO: 316, or a functional fragment thereof, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or differing by no more than 60, 50, 40, 30, 20, 15, 10, 5, or 2 nucleotides thereto. In some embodiments, the non-native promoter comprises or consists of the nucleotide sequence of SEQ ID NO: 316, or a functional fragment thereof.

[0023] In some embodiments, the non-native promoter comprises the pLpp promoter. In some embodiments, the non-native promoter comprises the nucleotide sequence of SEQ ID NO: 268, or a functional fragment thereof, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or differing by no more than 60, 50, 40, 30, 20, 15, 10, 5, or 2 nucleotides thereto. In some embodiments, the non-native promoter comprises or consists of the nucleotide sequence of SEQ ID NO: 268, or a functional fragment thereof.

[0024] In some embodiments, the non-native promoter comprises the Pem7 promoter. In some embodiments, the non-native promoter comprises the nucleotide sequence of SEQ ID NO: 269, or a functional fragment thereof, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or differing by no more than 60, 50, 40, 30, 20, 15, 10, 5, or 2 nucleotides thereto. In some embodiments, the non-native promoter comprises or consists of the nucleotide sequence of SEQ ID NO: 269, or a functional fragment thereof.

[0025] In some embodiments, the non-native promoter comprises the PS12burk promoter. In some embodiments, the non-native promoter comprises the nucleotide sequence of SEQ ID NO:270, or a functional fragment thereof, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or differing by no more than 60, 50, 40, 30, 20, 15, 10, 5, or 2 nucleotides thereto. In some embodiments, the non-native promoter comprises or consists of the nucleotide sequence of SEQ ID NO:270, or a functional fragment thereof.

[0026] In some embodiments, the non-native promoter is ErmE. * In some embodiments, the non-native promoter comprises the nucleotide sequence of SEQ ID NO: 271, or a functional fragment thereof, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or differing by no more than 60, 50, 40, 30, 20, 15, 10, 5, or 2 nucleotides thereto. In some embodiments, the non-native promoter comprises or consists of the nucleotide sequence of SEQ ID NO: 271, or a functional fragment thereof.

[0027] In some embodiments, the non-native promoter controls transcription of one or more (e.g., 2, 3, 4, 5, 6, 7, or all) of FrsA, FrsB, FrsC, FrsD, FrsE, FrsF, FrsG, or FrsH, or homologs thereof. In some embodiments, the non-native promoter controls transcription of FrsA, FrsB, FrsC, FrsD, FrsE, FrsF, FrsG, and FrsH, or homologs thereof. In some embodiments, the non-native promoter is inserted upstream of the coding region of FrsA, or a homolog thereof, for example, upstream of all of the coding regions of FrsA, FrsB, FrsC, FrsD, FrsE, FrsF, FrsG, and FrsH, or homologs thereof.

[0028] In some embodiments, the polypeptide involved in the production of the compound is a nonribosomal peptide synthetase (NRPS) or a functional fragment thereof. In some embodiments, the NRPS is FrsA, FrsD, FrsE, FrsF, or FrsG, or a homolog thereof.

[0029] In some embodiments, the polypeptide associated with the production of the compound is an MbtH-like protein or a functional fragment thereof, hi some embodiments, the MbtH-like protein is FrsB or a homolog thereof.

[0030] In some embodiments, the polypeptide associated with the production of the compound is malate dehydrogenase, hi some embodiments, the malate dehydrogenase is FrsC or a homolog thereof.

[0031] In some embodiments, the polypeptide associated with the production of the compound is a hydroxylase, hi some embodiments, the hydroxylase is FrsH or a homolog thereof.

[0032] In some embodiments, the nucleic acid comprises multiple nucleotide sequences encoding multiple polypeptides involved in the production of a compound. In some embodiments, the multiple polypeptides involved in the production of a compound comprise multiple NRPSs, or functional fragments thereof. In some embodiments, the multiple NRPSs comprise two or more (e.g., three, four, or all) of FrsA, FrsD, FrsE, FrsF, or FrsG, or homologs thereof. In some embodiments, the multiple polypeptides involved in the production of a compound comprise an NRPS, an MbtH-like protein, a malate dehydrogenase, and a hydroxylase, or functional fragments thereof.

[0033] In some embodiments, the plurality of polypeptides involved in the production of the compound comprises FrsA, FrsB, FrsC, FrsD, FrsE, FrsF, FrsG, and FrsH, or homologs thereof.

[0034] In some embodiments, the microorganism exhibits an increased titer of the compound. In some embodiments, when cultured under conditions permissive for production of the compound, the titer of the compound is increased by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold compared to a reference microorganism, e.g., an otherwise identical microorganism (e.g., wild-type) that does not include a non-native promoter in Compound (A1)-BGC. In some embodiments, the titer of the compound is increased by at least about 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, 500 mg / L, 600 mg / L, 700 mg / L, 800 mg / L, 900 mg / L, or 1000 mg / L compared to a reference microorganism, e.g., an otherwise identical microorganism (e.g., wild-type) that does not include a non-native promoter in Compound (A1)-BGC, when cultured under conditions permissive for production of the compound.

[0035] In some embodiments, the BGC of the natural product that prevents isolation of the compound is modified, e.g., disrupted. In some embodiments, the natural product includes one or more (e.g., two, three, or all) of violacein, Compound J, Compound F5, Compound F3, or Compound D. In some embodiments, at least a portion of the BGC of the natural product that prevents isolation of the compound is deleted. In some embodiments, the violacein-BGC is modified, e.g., disrupted. In some embodiments, the Compound J-BGC is modified, e.g., disrupted. In some embodiments, Compound J-BGC is associated with the production of Compound J, Compound F5, Compound F3, and Compound D. In some embodiments, both the violacein-BGC and Compound J-BGC are modified, e.g., disrupted. In some embodiments, production of the natural product is reduced, e.g., by at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.

[0036] In some embodiments, the microorganism exhibits an increased yield of isolated compound, ie, the yield of isolated compound is increased by about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more compared to a reference microorganism, e.g., an otherwise identical microorganism (e.g., wild-type) in which the BGC of the natural product has not been modified to prevent isolation of the compound.

[0037] In some embodiments, the BGC of the natural product is not altered, eg, not disrupted, to prevent isolation of the compound.

[0038] In another aspect, the disclosure features a microorganism that produces a compound having a structure of Formula (A1), wherein the BGC of the natural product has been modified to prevent isolation of the compound.

[0039] In some embodiments, the natural product includes one or more (e.g., two, three, or all) of violacein, Compound J, Compound F5, Compound F3, or Compound D. In some embodiments, at least a portion of the BGC of the natural product is deleted, preventing isolation of the compound. In some embodiments, the violacein-BGC is modified, e.g., disrupted. In some embodiments, the Compound J-BGC is modified, e.g., disrupted. In some embodiments, Compound J-BGC is associated with the production of Compound J, Compound F5, Compound F3, and Compound D. In some embodiments, both the violacein-BGC and Compound J-BGC are modified, e.g., disrupted. In some embodiments, production of the natural product is reduced, e.g., by at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%.

[0040] In some embodiments, the microorganism exhibits an increased yield of isolated compound, ie, the yield of isolated compound is increased by about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more compared to a reference microorganism, e.g., an otherwise identical microorganism (e.g., wild-type) in which the BGC of the natural product has not been modified to prevent isolation of the compound.

[0041] In another aspect, the disclosure features a method for producing a compound having a structure of formula (A1), the method including culturing (e.g., fermenting) a microbial organism described herein under conditions permissive for expression of the compound, thereby producing the compound.

[0042] In some embodiments, the microorganisms are cultured (e.g., fermented) at a scale of about 50 L to about 500 L, for example, about 50 L, about 100 L, about 150 L, about 200 L, about 250 L, about 300 L, about 350 L, about 400 L, about 450 L, or about 500 L. In some embodiments, the microorganisms are cultured (e.g., fermented) at a scale of about 1,000 L to about 20,000 L, for example, about 1,000 L to about 10,000 L or about 10,000 L to about 20,000 L, for example, about 1,000 L, about 2,000 L, about 5,000 L, about 7,500 L, about 10,000 L, about 15,000 L, or about 20,000 L.

[0043] In some embodiments, culturing (e.g., fermenting) produces at least about 200 mL of a compound, e.g., a titer of at least about 250 mg / mL, about 300 mg / L, about 400 mg / L, about 500 mg / L, about 600 mg / L, about 700 mg / L, about 800 mg / L, about 900 mg / L, about 1000 mg / L, about 1100 mg / L, about 1200 mg / L, about 1300 mg / L, about 1400 mg / L, about 1500 mg / L, about 1600 mg / L, about 1700 mg / L, about 1800 mg / L, about 1900 mg / L, or about 2000 mg / L of a compound.

[0044] In some embodiments, the method further includes isolating the compound to a purity of, e.g., at least about 90%, e.g., at least about 95%, about 96%, about 97%, about 98%, or about 99%, e.g., as determined by a method described herein.

[0045] In another aspect, the disclosure features a nucleic acid comprising a nucleotide sequence encoding a polypeptide involved in producing a compound having the structure of formula (A1), the nucleotide sequence being operably linked to a non-native promoter.

[0046] In some embodiments, the nucleic acid is located on the chromosome of the microorganism, for example, naturally located on the chromosome or stably integrated into the chromosome. In some embodiments, the nucleotide sequence encoding a polypeptide involved in the production of the compound is located in a biosynthetic gene cluster (BGC). In some embodiments, the BGC is compound (A1)-BGC. In some embodiments, compound (A1)-BGC has the genetic organization shown in Figure 1.

[0047] In some embodiments, the BGC comprises one or more (e.g., 2, 3, 4, 5, 6, 7, or all) of frsA, frsB, frsC, frsD, frsE, frsF, frsG, or frsH, or homologs thereof. In some embodiments, the BGC comprises frsA, frsB, frsC, frsD, frsE, frsF, frsG, and frsH, or homologs thereof.

[0048] In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound is located in frsA, frsB, frsC, frsD, frsE, frsF, frsG, frsH, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound is located in a region comprising frsA or a homolog thereof. In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound is located in a region comprising frsA and frsB, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound is located in a region comprising frsA, frsB, and frsC, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound is located in a region comprising frsA, frsB, frsC, and frsD, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound is located in a region comprising frsA, frsB, frsC, frsD, and frsE, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide involved in the production of a compound is located in a region comprising frsA, frsB, frsC, frsD, frsE, and frsF, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide involved in the production of a compound is located in a region comprising frsA, frsB, frsC, frsD, frsE, frsF, and frsG, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide involved in the production of a compound is located in a region comprising frsA, frsB, frsC, frsD, frsE, frsF, frsG, and FrsH, or homologs thereof.

[0049] In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound comprises the coding sequence of frsA, frsB, frsC, frsD, frsE, frsF, frsG, frsH, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound comprises the coding sequence of frsA or a homolog thereof. In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound comprises the coding sequence of frsA and frsB, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound comprises the coding sequence of frsA, frsB, and frsC, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound comprises the coding sequence of frsA, frsB, frsC, and frsD, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound comprises the coding sequence of frsA, frsB, frsC, frsD, and frsE, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound comprises the coding sequence of frsA, frsB, frsC, frsD, frsE, and frsF, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound comprises the coding sequence of frsA, frsB, frsC, frsD, frsE, frsF, and frsG, or homologs thereof. In some embodiments, the nucleotide sequence encoding a polypeptide associated with the production of a compound comprises the coding sequence of frsA, frsB, frsC, frsD, frsE, frsF, frsG, and FrsH, or homologs thereof.

[0050] In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 317, or a functional fragment thereof, or a nucleotide sequence having at least 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto, or differing by no more than about 100, about 90, about 80, about 70, about 60, about 50, about 40, about 30, about 20, about 15, about 10, about 5, or about 2 nucleotides thereto.

[0051] In some embodiments, the nucleic acid comprises the nucleotide sequence associated with GenBank Accession Number: BankIt2437961 BSeq#1 MW732719, or a functional fragment thereof, or a nucleotide sequence having at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto.

[0052] In certain embodiments, the non-native promoters are from the same microorganism, while in other embodiments, the non-native promoters are from different microorganisms.

[0053] In some embodiments, the non-native promoter is a vioP promoter, a nptII promoter, a rbs promoter, a J23119 promoter, a pLpp promoter, a PS12burk promoter, an ErmE promoter, or a nucleotide sequence encoding the nucleotide sequence of interest. * In some embodiments, the non-native promoter comprises a vioP promoter, a nptII promoter, or a rbs promoter.

[0054] In some embodiments, the non-native promoter comprises the nucleotide sequence of any of SEQ ID NOs: 264-266, 268-271, or 316, or a functional fragment thereof, or a nucleotide sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto, or differing by no more than about 60, about 50, about 40, about 30, about 20, about 15, about 10, about 5, or about 2 nucleotides thereto. In some embodiments, the non-native promoter comprises or consists of the nucleotide sequence of any of SEQ ID NOs: 264-266, 268-271, or 316, or a functional fragment thereof.

[0055] In some embodiments, the non-native promoter comprises the vioP promoter. In some embodiments, the non-native promoter comprises the nucleotide sequence of SEQ ID NO:264, or a functional fragment thereof, or a nucleotide sequence having at least 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto, or differing by no more than about 60, about 50, about 40, about 30, about 20, about 15, about 10, about 5, or about 2 nucleotides thereto. In some embodiments, the non-native promoter comprises or consists of the nucleotide sequence of SEQ ID NO:264, or a functional fragment thereof.

[0056] In some embodiments, the non-native promoter comprises the nptII promoter. In some embodiments, the non-native promoter comprises the nucleotide sequence of SEQ ID NO: 265, or a functional fragment thereof, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or differing by no more than about 60, 50, 40, 30, 20, 15, 10, 5, or 2 nucleotides therefrom. In some embodiments, the non-native promoter comprises or consists of the nucleotide sequence of SEQ ID NO: 265, or a functional fragment thereof.

[0057] In some embodiments, the non-native promoter comprises the rbs promoter. In some embodiments, the non-native promoter comprises the nucleotide sequence of SEQ ID NO: 266, or a functional fragment thereof, or a nucleotide sequence having at least 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto, or differing by no more than about 60, about 50, about 40, about 30, about 20, about 15, about 10, about 5, or about 2 nucleotides thereto. In some embodiments, the non-native promoter comprises or consists of the nucleotide sequence of SEQ ID NO: 266, or a functional fragment thereof.

[0058] In some embodiments, the non-native promoter comprises the J23119 promoter. In some embodiments, the non-native promoter comprises the nucleotide sequence of SEQ ID NO: 316, or a functional fragment thereof, or a nucleotide sequence having at least 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto, or differing by no more than about 60, about 50, about 40, about 30, about 20, about 15, about 10, about 5, or about 2 nucleotides thereto. In some embodiments, the non-native promoter comprises or consists of the nucleotide sequence of SEQ ID NO: 316, or a functional fragment thereof.

[0059] In some embodiments, the non-native promoter comprises the pLpp promoter. In some embodiments, the non-native promoter comprises the nucleotide sequence of SEQ ID NO:268, or a functional fragment thereof, or a nucleotide sequence having at least 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto, or differing by no more than about 60, about 50, about 40, about 30, about 20, about 15, about 10, about 5, or about 2 nucleotides thereto. In some embodiments, the non-native promoter comprises or consists of the nucleotide sequence of SEQ ID NO:268, or a functional fragment thereof.

[0060] In some embodiments, the non-native promoter comprises the Pem7 promoter. In some embodiments, the non-native promoter comprises the nucleotide sequence of SEQ ID NO:269, or a functional fragment thereof, or a nucleotide sequence having at least 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto, or differing by no more than about 60, about 50, about 40, about 30, about 20, about 15, about 10, about 5, or about 2 nucleotides thereto. In some embodiments, the non-native promoter comprises or consists of the nucleotide sequence of SEQ ID NO:269, or a functional fragment thereof.

[0061] In some embodiments, the non-native promoter comprises the PS12burk promoter. In some embodiments, the non-native promoter comprises the nucleotide sequence of SEQ ID NO:270, or a functional fragment thereof, or a nucleotide sequence having at least 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto, or differing by no more than about 60, about 50, about 40, about 30, about 20, about 15, about 10, about 5, or about 2 nucleotides thereto. In some embodiments, the non-native promoter comprises or consists of the nucleotide sequence of SEQ ID NO:270, or a functional fragment thereof.

[0062] In some embodiments, the non-native promoter is ErmE. * In some embodiments, the non-native promoter comprises a nucleotide sequence of SEQ ID NO: 271, or a functional fragment thereof, or a nucleotide sequence having at least 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto, or differing by no more than about 60, about 50, about 40, about 30, about 20, about 15, about 10, about 5, or about 2 nucleotides thereto. In some embodiments, the non-native promoter comprises or consists of the nucleotide sequence of SEQ ID NO: 271, or a functional fragment thereof.

[0063] In some embodiments, the non-native promoter controls transcription of one or more (e.g., 2, 3, 4, 5, 6, 7, or all) of FrsA, FrsB, FrsC, FrsD, FrsE, FrsF, FrsG, or FrsH, or homologs thereof. In some embodiments, the non-native promoter controls transcription of FrsA, FrsB, FrsC, FrsD, FrsE, FrsF, FrsG, and FrsH, or homologs thereof. In some embodiments, the non-native promoter is inserted upstream of the coding region of FrsA, or a homolog thereof, for example, upstream of all of the coding regions of FrsA, FrsB, FrsC, FrsD, FrsE, FrsF, FrsG, and FrsH, or homologs thereof.

[0064] In some embodiments, the polypeptide involved in the production of the compound is a nonribosomal peptide synthetase (NRPS) or a functional fragment thereof. In some embodiments, the NRPS is FrsA, FrsD, FrsE, FrsF, or FrsG, or a homolog thereof.

[0065] In some embodiments, the polypeptide associated with the production of the compound is an MbtH-like protein or a functional fragment thereof, hi some embodiments, the MbtH-like protein is FrsB or a homolog thereof.

[0066] In some embodiments, the polypeptide associated with the production of the compound is malate dehydrogenase, hi some embodiments, the malate dehydrogenase is FrsC or a homolog thereof.

[0067] In some embodiments, the polypeptide associated with the production of the compound is a hydroxylase, hi some embodiments, the hydroxylase is FrsH or a homolog thereof.

[0068] In some embodiments, the nucleic acid comprises multiple nucleotide sequences encoding multiple polypeptides involved in the production of a compound. In some embodiments, the multiple polypeptides involved in the production of a compound comprise multiple NRPSs, or functional fragments thereof. In some embodiments, the multiple NRPSs comprise two or more (e.g., three, four, or all) of FrsA, FrsD, FrsE, FrsF, or FrsG, or homologs thereof. In some embodiments, the multiple polypeptides involved in the production of a compound comprise an NRPS, an MbtH-like protein, a malate dehydrogenase, and a hydroxylase, or functional fragments thereof.

[0069] In some embodiments, the plurality of polypeptides involved in the production of the compound comprises FrsA, FrsB, FrsC, FrsD, FrsE, FrsF, FrsG, and FrsH, or homologs thereof.

[0070] In another aspect, the disclosure features a nucleic acid including a nucleotide sequence encoding a polypeptide related to the production of one or more of Compound J, Compound F5, Compound F3, or Compound D.

[0071] In some embodiments, the nucleic acid is an isolated nucleic acid. In some embodiments, the nucleic acid is a non-naturally occurring nucleic acid. In some embodiments, the nucleic acid is a synthetic nucleic acid. In some embodiments, the nucleic acid includes a mutation, e.g., a deletion.

[0072] In some embodiments, the nucleic acid comprises multiple nucleotide sequences encoding multiple polypeptides involved in the production of one or more of Compound J, Compound F5, Compound F3, or Compound D.

[0073] In some embodiments, the nucleotide sequence is from BGC. In some embodiments, the BGC is compound J-BGC. In some embodiments, compound J-BGC has the genetic organization shown in Figure 1.

[0074] In some embodiments, the nucleotide sequence comprises one or more (e.g., two, three, or all) of dis1, dis2, dis3, or dis4, or homologs thereof. In some embodiments, the BGC comprises dis1, dis2, dis3, and dis4, or homologs thereof.

[0075] In some embodiments, the nucleic acid comprises the nucleotide sequence associated with GenBank Accession Number: BankIt2437961 BSeq#1 MW732719, or a functional fragment thereof, or a nucleotide sequence having at least about 75%, e.g., at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto.

[0076] In another aspect, the disclosure features a vector including a nucleic acid described herein. In another aspect, the disclosure features a cell including a nucleic acid described herein or a vector described herein.

[0077] In another aspect, the disclosure features a method of engineering a cell, the method including modifying a nucleotide sequence encoding a polypeptide associated with the production of one or more of Compound J, Compound F5, Compound F3, or Compound D, thereby engineering the cell.

[0078] In some embodiments, the cell is a microorganism that naturally produces a compound having the structure of Formula (A1). In some embodiments, the microorganism is a bacterium, such as a Chromobacterium. In some embodiments, the microorganism is a Chromobacterium vaccinii, such as Chromobacterium vaccinii DSM 25150 (=ATCC BAA-2314, =NRRL B-50840, =MWU205).

[0079] In some embodiments, the nucleotide sequence is disrupted, eg, at least a portion of the nucleotide sequence is deleted.

[0080] In some embodiments, the nucleic acid comprises multiple nucleotide sequences encoding multiple polypeptides associated with the production of one or more of Compound J, Compound F5, Compound F3, or Compound D. In some embodiments, the production of one or more (e.g., two, three, or all) of Compound J, Compound F5, Compound F3, or Compound D is reduced, e.g., by at least about 50%, e.g., by at least about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.

[0081] In some embodiments, the nucleotide sequence is from BGC. In some embodiments, the BGC is Compound J-BGC. In some embodiments, Compound J-BGC has the genetic organization shown in FIG. 1. In some embodiments, the nucleotide sequence includes one or more (e.g., two, three, or all) of dis1, dis2, dis3, or dis4, or homologs thereof. In some embodiments, BGC includes dis1, dis2, dis3, and dis4, or homologs thereof. In some embodiments, the nucleic acid includes GenBank Accession Number: BankIt2437961 BSeq#1 MW732719, related to the nucleotide sequence, or a functional fragment thereof, or a nucleotide sequence having at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto.

[0082] In some embodiments, the modification increases the yield of isolated compound, ie, the yield of isolated compound is increased by about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more compared to a reference microorganism, e.g., an otherwise identical microorganism (e.g., wild-type) in which the natural product BGC has not been modified to prevent isolation of the compound.

[0083] Partial reoxidation and methods for preparing antibody-drug conjugates In another aspect, the disclosure features a method of producing a partially reoxidized antibody or antigen-binding fragment thereof, the method including: providing an antibody or antigen-binding fragment thereof that includes one or more decapped cysteine ​​residues; and storing the antibody or antigen-binding fragment thereof that includes the one or more decapped cysteine ​​residues under oxidizing conditions, thereby producing the partially reoxidized antibody or antigen-binding fragment thereof.

[0084] In some embodiments, the method further comprises providing an antibody or antigen-binding fragment thereof comprising one or more capped cysteine ​​residues. In some embodiments, the one or more capped cysteine ​​residues are located in the constant domain heavy chain of the antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises four capped cysteine ​​residues. In some embodiments, the antibody or antigen-binding fragment thereof comprises two capped cysteine ​​residues. In some embodiments, the antibody or antigen-binding fragment thereof comprises a capped cysteine ​​residue located in the CH1 region of the antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises a capped cysteine ​​residue located in each CH1 region of the antibody or antigen-binding fragment thereof. In some embodiments, the capped cysteine ​​residues are engineered surface-exposed cysteine ​​residues. In some embodiments, the antibody or antigen-binding fragment thereof comprises a capped cysteine ​​residue located at residue 152 (EU numbering) in the CH1 region of the antibody or antigen-binding fragment thereof. In some embodiments, the one or more capped cysteine ​​residues are capped with cysteine ​​or glutathione.

[0085] In some embodiments, the method further comprises reducing the one or more capped cysteine ​​residues to provide an antibody or antigen-binding fragment thereof comprising one or more decapped cysteine ​​residues. In some embodiments, the one or more decapped cysteine ​​residues are located in the constant domain heavy chain of the antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises four decapped cysteine ​​residues. In some embodiments, the antibody or antigen-binding fragment thereof comprises two decapped cysteine ​​residues. In some embodiments, the antibody or antigen-binding fragment thereof comprises a decapped cysteine ​​residue located in the CH1 region of the antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises a decapped cysteine ​​residue located in each CH1 region of the antibody or antigen-binding fragment thereof. In some embodiments, the decapped cysteine ​​residues are engineered surface-exposed cysteine ​​residues. In some embodiments, the antibody or antigen-binding fragment thereof comprises a decapped cysteine ​​residue located at residue 152 (EU numbering) of the CH1 region of the antibody or antigen-binding fragment thereof.

[0086] In some embodiments, the method comprises contacting an antibody or antigen-binding fragment thereof comprising one or more capped cysteine ​​residues with a reducing wash buffer to reduce the one or more capped cysteine ​​residues, thereby providing an antibody or antigen-binding fragment thereof comprising one or more decapped cysteine ​​residues.

[0087] In some embodiments, the reducing wash buffer contains about 5 mM to about 50 mM cysteine, for example, about 10 mM to about 40 mM, about 20 mM to about 30 mM, about 5 mM to about 15 mM, about 5 mM to about 25 mM, about 5 mM to about 35 mM, about 5 mM to about 45 mM, about 40 mM to about 50 mM, about 30 mM to about 50 mM, about In some embodiments, the reducing wash buffer contains about 5 mM to about 15 mM cysteine, for example, about 10 mM.

[0088] In some embodiments, the reducing wash buffer has a pH of about 6.0 to about 7.5, for example, about 6.5 to about 7.2 or about 6.8 to about 7.0. In some embodiments, the reducing wash buffer has a pH of about 6.8 to about 7.0, for example, about 6.9.

[0089] In some embodiments, contacting the antibody or antigen-binding fragment thereof comprising one or more capped cysteine ​​residues with a reducing wash buffer is performed on a column (eg, a cation exchange chromatography column).

[0090] In some embodiments, the method further comprises recovering the antibody or antigen-binding fragment thereof comprising one or more decapped cysteine ​​residues in the eluate after contact with the reducing wash buffer. In some embodiments, the eluate has a pH of about 5.5 to about 6.0, e.g., about 5.8. In some embodiments, the concentration of the antibody or antigen-binding fragment thereof comprising one or more decapped cysteine ​​residues in the eluate is about 5 g / L to 25 g / L, e.g., about 8 g / L to about 20 g / L, about 10 g / L to about 18 g / L, about 12 g / L to about 15 g / L, about 5 g / L to about 20 g / L, about 5 g / L to about 15 g / L, about 5 g / L to about 10 g / L, about 20 g / L to about 25 g / L, about 15 g / L to about 25 g / L, about 10 g / L to about 25 g / L, About 10g / L to about 20g / L, about 13g / L to about 14g / L, about 12g / L to about 15g / L, for example, about 5g / L, about 6g / L, about 7g / L, about 8g / L, about 9g / L, about 10g / L, about 11g / L, about 12g / L, about 13g / L, about 13.5g / L, about 14g / L, about 15g / L, about 16g / L, about 17g / L, about 18g / L, about 19g / L, about 20g / L, about 21g / L, about 22g / L, about 23g / L, about 24g / L, or about 25g / L.

[0091] In some embodiments, the antibody or antigen-binding fragment thereof comprising one or more decapped cysteine ​​residues is stored in the elution solution with agitation. In some embodiments, the antibody or antigen-binding fragment thereof comprising one or more decapped cysteine ​​residues is stored in the elution solution without agitation.

[0092] In some embodiments, an antibody or antigen-binding fragment thereof comprising one or more decapped cysteine ​​residues is incubated in a container filled to a maximum of about 50% to about 70% capacity for, for example, about 12 hours to about 96 hours, e.g., about 12 hours to about 84 hours, about 24 hours to about 84 hours, about 36 hours to about 72 hours, about 48 hours to about 60 hours, about 24 hours to about 72 hours, about 24 hours to about 60 hours, or about 24 hours to about 48 hours. The antibody or antigen-binding fragment thereof containing one or more decapped cysteine ​​residues is stored for about 24 hours to about 36 hours, about 72 hours to about 84 hours, about 60 hours to about 84 hours, about 48 hours to about 84 hours, about 36 hours to about 84 hours, about 12 hours to about 36 hours, about 24 hours to about 48 hours, about 36 hours to about 60 hours, about 48 hours to about 72 hours, or about 72 hours to about 96 hours, for example, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, or about 96 hours. In some embodiments, the antibody or antigen-binding fragment thereof containing one or more decapped cysteine ​​residues is stored in a container that is up to about 60% full. In some embodiments, the antibody or antigen-binding fragment thereof containing one or more decapped cysteine ​​residues is stored for about 48 hours to about 72 hours, for example, without stirring. In some embodiments, the antibody or antigen-binding fragment thereof comprising one or more decapped cysteine ​​residues is stored, eg, with agitation, for about 12 hours to about 36 hours, eg, about 24 hours.

[0093] In some embodiments, an antibody or antigen-binding fragment thereof comprising one or more decapped cysteine ​​residues is stored at room temperature, hi some embodiments, an antibody or antigen-binding fragment thereof comprising one or more decapped cysteine ​​residues is stored at about 2°C to about 8°C (e.g., about 4°C), for example, for at least 48 hours (e.g., at least about 48 hours to about 96 hours).

[0094] In some embodiments, antibodies or antigen-binding fragments thereof comprising one or more decapped cysteine ​​residues are stored under an air gap.

[0095] In some embodiments, the method further comprises purifying the partially reoxidized antibody or antigen-binding fragment thereof.

[0096] In some embodiments, the method further comprises conjugating a linker-drug moiety (e.g., a linker-drug moiety described herein) to the partially reoxidized antibody or antigen-binding fragment thereof to produce an antibody-drug conjugate (e.g., an antibody-drug conjugate described herein).

[0097] In some embodiments, the method comprises the step of: R 8 -L B -(D) n (B) (In the formula, D is a GNAQ inhibitor, a GNA11 inhibitor or an inhibitor of GNAQ and GNA11; R 8 is a reactive group; L B is a cleavable or non-cleavable linker, and n is 1, 2, 3 or 4 The method further comprises preforming the linker-drug moiety of

[0098] In some embodiments, D is a GNAQ inhibitor described herein. In some embodiments, D is a GNA11 inhibitor described herein. In some embodiments, D is an inhibitor of GNAQ and GNA11 described herein. In some embodiments, R is a reactive group described herein. In some embodiments, L B is a cleavable linker as described herein. In some embodiments, L B is a non-cleavable linker as described herein.

[0099] In some embodiments, the antibody or antigen-binding fragment thereof binds to PMEL17. In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof described herein.

[0100] In some embodiments, the method further comprises purifying the antibody drug conjugate.

[0101] In some embodiments, the method results in at least about 75%, e.g., at least about 80%, about 85%, about 90%, or about 95% on-site coupling, e.g., one linker-drug moiety ("HC1") per heavy chain. In some embodiments, the method results in less than about 25%, e.g., at least about 20%, about 15%, about 10%, or about 5% off-site coupling, e.g., one linker-drug moiety ("LC1") per light chain and / or two linker-drug moieties ("HC2") per heavy chain. In some embodiments, the method results in a purity of at least about 70%, e.g., at least about 75%, about 80%, about 85%, about 90%, or about 95%, as determined, e.g., by non-reducing CE-SDS.

[0102] In another aspect, the present disclosure provides a method of producing an anti-PMEL17 antibody drug conjugate, comprising: providing an antibody or antigen-binding fragment thereof comprising one or more capped cysteine ​​residues with a reducing wash buffer, thereby providing an antibody or antigen-binding fragment thereof comprising one or more decapped cysteine ​​residues; storing the antibody or fragment thereof containing one or more decapped cysteine ​​residues under oxidizing conditions, thereby producing a partially reoxidized antibody or antigen-binding fragment thereof; and attaching a linker-drug moiety to a partially reoxidized antibody or antigen-binding fragment thereof, thereby producing an anti-PMEL17 antibody drug conjugate, the antibody or antigen-binding fragment thereof binds to PMEL17; and A linker-drug moiety of formula (B): R 8 -L B -(D) n (B) (In the formula, D is a GNAQ inhibitor, a GNA11 inhibitor or an inhibitor of GNAQ and GNA11; R 8 is a reactive group; L B is a cleavable or non-cleavable linker, and n is 1, 2, 3, or 4.

[0103] Formulation of antibody-drug conjugates In another aspect, the disclosure features a formulation including an antibody drug conjugate, a buffer, a stabilizer, and a surfactant, the formulation having a pH of 4.5 to 6.5, wherein the antibody drug conjugate is a compound represented by formula (C): Ab-(L A -(D) n ) y (C) (In the formula, D is a GNAQ inhibitor, a GNA11 inhibitor or an inhibitor of GNAQ and GNA11; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; L A is a linker; n is 1, 2, 3 or 4, and and y is 1, 2, 3, or 4.

[0104] In certain embodiments, D is a GNAQ inhibitor described herein, eg, a GNA11 inhibitor described herein, or an inhibitor of GNAQ and GNA11 described herein.

[0105] In certain embodiments, the antibody-drug conjugate has a concentration of about 5 mg / mL to about 30 mg / mL, e.g., about 10 mg to about 30 mg, about 15 mg / mL to about 25 mg / mL, about 18 mg / mL to about 22 mg / mL, about 10 mg / mL to 25 mg / mL, about 10 mg / mL to about 20 mg / mL, about 10 mg / mL to about 15 mg / mL, about 25 mg to about 30 mg / mL, about 20 mg / mL to about 30 mg / mL, about 5 mg / mL to about 15 mg / mL, about 15 mg / mL to about 25 mg / mL, or about 1 It is present at a concentration of 8 mg / mL to about 22 mg / mL, for example, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, about 21 mg / mL, about 22 mg / mL, about 23 mg / mL, about 24 mg / mL, or about 25 mg / mL.

[0106] In certain embodiments, the antibody drug conjugate is present at a concentration of about 15 mg / mL to about 25 mg / mL, for example, about 20 mg / mL.

[0107] In some embodiments, the buffer contains histidine. In some embodiments, the buffer contains succinate. In some embodiments, the buffer contains about 5 mM to about 50 mM, for example, about 10 mM to about 40 mM, about 15 mM to about 35 mM, about 20 mM to about 30 mM, about 5 mM to about 40 mM, about 5 mM to about 30 mM, about 5 mM to about 20 mM, about 5 mM to about 15 mM, about 5 mM to about 10 mM, about 40 mM to about 50 mM, about 35 mM to about 50 mM, about 30 mM to about 50 mM, about 25 mM to about 50 mM, The buffer is present at a concentration of about 20 mM to about 50 mM, about 15 mM to about 50 mM, about 10 mM to about 50 mM, about 10 mM to about 20 mM, about 15 mM to about 25 mM, about 25 mM to about 35 mM, about 30 mM to about 40 mM, or about 35 mM to about 45 mM, for example, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM. In one embodiment, the buffer is present at a concentration of about 15 mM to about 25 mM, for example, about 20 mM.

[0108] In some embodiments, the stabilizer comprises sucrose. In some embodiments, the stabilizer comprises trehalose. In some embodiments, the stabilizer is present in a concentration of about 100 mM to about 500 mM, for example, about 150 mM to about 450 mM, about 200 mM to about 400 mM, about 250 mM to about 350 mM, about 100 mM to about 450 mM, about 100 mM to about 400 mM, about 100 mM to about 350 mM, about 100 mM to about 300 mM, about 100 mM to about 250 mM, about 100 mM to about 200 mM, about 100 mM to about 150 mM, about 450 mM to about 500 mM, about 400 mM to about 500 mM, or about 350 mM to about 500 mM. In one embodiment, the stabilizer is present at a concentration of about 200 mM to about 300 mM, for example, about 240 mM.

[0109] In certain embodiments, the surfactant comprises polysorbate 20. In certain embodiments, the surfactant comprises polysorbate 80. In certain embodiments, the surfactant is present at a concentration of about 0.01% to about 0.06%, e.g., about 0.02% to about 0.05%, about 0.03% to about 0.04%, about 0.01% to about 0.05%, about 0.01% to about 0.04%, about 0.01% to about 0.03%, about 0.01% to about 0.02%, about 0.05% to about 0.06%, about 0.04% to about 0.06%, about 0.03% to about 0.06%, about 0.02% to about 0.06%, about 0.02% to about 0.04%, about 0.03% to about 0.05%, e.g., about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, or about 0.06%. In certain embodiments, the surfactant is present at a concentration of about 0.01% to about 0.03%, for example, about 0.02%.

[0110] In certain embodiments, the formulation may comprise a 200 mg / kg dose of about 4.7 to about 5.3, about 5.0 to about 6.0, about 5.2 to about 5.8, about 5.4 to about 5.6, about 5.2 to about 6.0, about 5.4 to about 6.0, about 5.6 to about 6.0, about 5.8 to about 6.0, about 5 to about 5.8, about 5 to about 5.6.0, about 5 to about 5.4, about 5 to about 5.2, about 4.8 to about 5.2, about 5.1 to about 5.3, about 5.2 to about 5.4, about 5.3 to about 5.5, or about 5.5 The formulation has a pH of about 5.7, about 5.6 to about 5.8, about 5.7 to about 5.9, about 4.9 to about 5.5, about 5.5 to about 6.1, or about 5.7 to about 6.3, for example, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, or about 6.5. In one embodiment, the formulation has a pH of about 5.0 to about 5.6, for example, about 5.3.

[0111] In one embodiment, the formulation comprises about 15 mg / mL to about 25 mg / mL of antibody-drug conjugate, about 15 mM to about 25 mM of a buffer comprising histidine or succinate, about 200 mM to about 300 mM of a stabilizer comprising sucrose or trehalose, and about 0.01% to about 0.03% of a surfactant comprising polysorbate 20 or polysorbate 80, wherein the formulation has a pH of about 5.0 to about 5.6.

[0112] In one embodiment, the formulation comprises about 15 mg / mL to about 25 mg / mL of antibody-drug conjugate, about 15 mM to about 25 mM of a buffer comprising histidine, about 200 mM to about 300 mM of a stabilizer comprising sucrose, and about 0.01% to about 0.03% of a surfactant comprising polysorbate 20, wherein the formulation has a pH of about 5.0 to about 5.6.

[0113] In one embodiment, the formulation comprises about 20 mg / mL of antibody drug conjugate, about 20 mM of a buffer comprising histidine or succinate, about 240 mM of a stabilizer comprising sucrose or trehalose, and about 0.02% of a surfactant comprising polysorbate 20 or polysorbate 80, wherein the formulation has a pH of about 5.3.

[0114] In one embodiment, the formulation comprises about 20 mg / mL of antibody drug conjugate, about 20 mM of a buffer comprising histidine, about 240 mM of a stabilizer comprising sucrose, and about 0.02% of a surfactant comprising polysorbate 20, wherein the formulation has a pH of about 5.3.

[0115] In one embodiment, the formulation comprises about 15 mg / mL to about 25 mg / mL of antibody-drug conjugate, about 15 mM to about 25 mM of a buffer comprising histidine or succinate, about 200 mM to about 300 mM of a stabilizer comprising sucrose or trehalose, and about 0.01% to about 0.03% of a surfactant comprising polysorbate 20 or polysorbate 80, wherein the formulation has a pH of about 4.7 to about 5.3.

[0116] In one embodiment, the formulation comprises about 15 mg / mL to about 25 mg / mL of antibody-drug conjugate, about 15 mM to about 25 mM of a buffer comprising histidine, about 200 mM to about 300 mM of a stabilizer comprising sucrose, and about 0.01% to about 0.03% of a surfactant comprising polysorbate 20, wherein the formulation has a pH of about 4.7 to about 5.3.

[0117] In one embodiment, the formulation comprises about 20 mg / mL of antibody drug conjugate, about 20 mM of a buffer comprising histidine or succinate, about 240 mM of a stabilizer comprising sucrose or trehalose, and about 0.02% of a surfactant comprising polysorbate 20 or polysorbate 80, wherein the formulation has a pH of about 5.0.

[0118] In one embodiment, the formulation comprises about 20 mg / mL of antibody drug conjugate, about 20 mM of a buffer comprising histidine, about 240 mM of a stabilizer comprising sucrose, and about 0.02% of a surfactant comprising polysorbate 20, wherein the formulation has a pH of about 5.0.

[0119] In one embodiment, the formulation comprises about 15 mg / mL to about 25 mg / mL of antibody-drug conjugate, about 15 mM to about 25 mM of a buffer comprising histidine or succinate, about 200 mM to about 300 mM of a stabilizer comprising sucrose or trehalose, and about 0.01% to about 0.03% of a surfactant comprising polysorbate 20 or polysorbate 80, wherein the formulation has a pH of about 5.2 to about 5.8.

[0120] In one embodiment, the formulation comprises about 15 mg / mL to about 25 mg / mL of antibody-drug conjugate, about 15 mM to about 25 mM of a buffer comprising histidine, about 200 mM to about 300 mM of a stabilizer comprising sucrose, and about 0.01% to about 0.03% of a surfactant comprising polysorbate 20, wherein the formulation has a pH of about 5.2 to about 5.8.

[0121] In one embodiment, the formulation comprises about 20 mg / mL of antibody drug conjugate, about 20 mM of a buffer comprising histidine or succinate, about 240 mM of a stabilizer comprising sucrose or trehalose, and about 0.02% of a surfactant comprising polysorbate 20 or polysorbate 80, wherein the formulation has a pH of about 5.5.

[0122] In one embodiment, the formulation comprises about 20 mg / mL of antibody drug conjugate, about 20 mM of a buffer comprising histidine, about 240 mM of a stabilizer comprising sucrose, and about 0.02% of a surfactant comprising polysorbate 20, wherein the formulation has a pH of about 5.5.

[0123] In one embodiment, the formulation comprises about 15 mg / mL to about 25 mg / mL of antibody-drug conjugate, about 15 mM to about 25 mM of a buffer comprising histidine or succinate, about 200 mM to about 300 mM of a stabilizer comprising sucrose or trehalose, and about 0.03% to about 0.05% of a surfactant comprising polysorbate 20 or polysorbate 80, wherein the formulation has a pH of about 5.2 to about 5.8.

[0124] In one embodiment, the formulation comprises about 15 mg / mL to about 25 mg / mL of antibody-drug conjugate, about 15 mM to about 25 mM of a buffer comprising histidine, about 200 mM to about 300 mM of a stabilizer comprising sucrose, and about 0.03% to about 0.05% of a surfactant comprising polysorbate 20, wherein the formulation has a pH of about 5.2 to about 5.8.

[0125] In one embodiment, the formulation comprises about 20 mg / mL of antibody drug conjugate, about 20 mM of a buffer comprising histidine or succinate, about 240 mM of a stabilizer comprising sucrose or trehalose, and about 0.04% of a surfactant comprising polysorbate 20 or polysorbate 80, wherein the formulation has a pH of about 5.5.

[0126] In one embodiment, the formulation comprises about 20 mg / mL of antibody drug conjugate, about 20 mM of a buffer comprising histidine, about 240 mM of a stabilizer comprising sucrose, and about 0.04% of a surfactant comprising polysorbate 20, wherein the formulation has a pH of about 5.5.

[0127] In one embodiment, the formulation comprises about 5 mg / mL to about 15 mg / mL of antibody-drug conjugate, about 15 mM to about 25 mM of a buffer comprising histidine or succinate, about 200 mM to about 300 mM of a stabilizer comprising sucrose or trehalose, and about 0.03% to about 0.05% of a surfactant comprising polysorbate 20 or polysorbate 80, wherein the formulation has a pH of about 5.2 to about 5.8.

[0128] In one embodiment, the formulation comprises about 5 mg / mL to about 15 mg / mL of antibody-drug conjugate, about 15 mM to about 25 mM of a buffer comprising histidine, about 200 mM to about 300 mM of a stabilizer comprising sucrose, and about 0.03% to about 0.05% of a surfactant comprising polysorbate 20, wherein the formulation has a pH of about 5.2 to about 5.8.

[0129] In one embodiment, the formulation comprises about 10 mg / mL of antibody drug conjugate, about 20 mM of a buffer comprising histidine or succinate, about 240 mM of a stabilizer comprising sucrose or trehalose, and about 0.02% of a surfactant comprising polysorbate 20 or polysorbate 80, wherein the formulation has a pH of about 5.5.

[0130] In one embodiment, the formulation comprises about 10 mg / mL of antibody drug conjugate, about 20 mM of a buffer comprising histidine, about 240 mM of a stabilizer comprising sucrose, and about 0.02% of a surfactant comprising polysorbate 20, wherein the formulation has a pH of about 5.5.

[0131] In one embodiment, the formulation comprises about 15 mg / mL to about 25 mg / mL of antibody-drug conjugate, about 15 mM to about 25 mM of a buffer comprising histidine or succinate, about 200 mM to about 300 mM of a stabilizer comprising sucrose or trehalose, and about 0.01% to about 0.03% of a surfactant comprising polysorbate 20 or polysorbate 80, wherein the formulation has a pH of about 5.7 to about 6.3.

[0132] In one embodiment, the formulation comprises about 15 mg / mL to about 25 mg / mL of antibody-drug conjugate, about 15 mM to about 25 mM of a buffer comprising histidine, about 200 mM to about 300 mM of a stabilizer comprising sucrose, and about 0.01% to about 0.03% of a surfactant comprising polysorbate 20, wherein the formulation has a pH of about 5.7 to about 6.3.

[0133] In one embodiment, the formulation comprises about 20 mg / mL of antibody drug conjugate, about 20 mM of a buffer comprising histidine or succinate, about 240 mM of a stabilizer comprising sucrose or trehalose, and about 0.02% of a surfactant comprising polysorbate 20 or polysorbate 80, wherein the formulation has a pH of about 6.0.

[0134] In one embodiment, the formulation comprises about 20 mg / mL of antibody drug conjugate, about 20 mM of a buffer comprising histidine, about 240 mM of a stabilizer comprising sucrose, and about 0.02% of a surfactant comprising polysorbate 20, wherein the formulation has a pH of about 6.0.

[0135] In one embodiment, the formulation comprises about 15 mg / mL to about 25 mg / mL of antibody-drug conjugate, about 15 mM to about 25 mM of a buffer comprising histidine or succinate, about 200 mM to about 300 mM of a stabilizer comprising sucrose or trehalose, and about 0.01% to about 0.03% of a surfactant or polysorbate comprising polysorbate 20, wherein the formulation has a pH of about 4.9 to about 5.5.

[0136] In one embodiment, the formulation comprises about 15 mg / mL to about 25 mg / mL of antibody-drug conjugate, about 15 mM to about 25 mM of a buffer comprising histidine, about 200 mM to about 300 mM of a stabilizer comprising sucrose, and about 0.01% to about 0.03% of a surfactant comprising polysorbate 20, wherein the formulation has a pH of about 4.9 to about 5.5.

[0137] In one embodiment, the formulation comprises about 20 mg / mL of antibody drug conjugate, about 20 mM of a buffer comprising histidine or succinate, about 240 mM of a stabilizer comprising sucrose or trehalose, and about 0.02% of a surfactant comprising polysorbate 20 or polysorbate 80, wherein the formulation has a pH of about 5.2.

[0138] In one embodiment, the formulation comprises about 20 mg / mL of antibody drug conjugate, about 20 mM of a buffer comprising histidine, about 240 mM of a stabilizer comprising sucrose, and about 0.02% of a surfactant comprising polysorbate 20, wherein the formulation has a pH of about 5.2.

[0139] In one embodiment, the formulation comprises about 15 mg / mL to about 25 mg / mL of antibody-drug conjugate, about 15 mM to about 25 mM of a buffer comprising histidine or succinate, about 200 mM to about 300 mM of a stabilizer comprising sucrose or trehalose, and about 0.01% to about 0.03% of a surfactant comprising polysorbate 20 or polysorbate 80, wherein the formulation has a pH of about 5.5 to about 6.1.

[0140] In one embodiment, the formulation comprises about 15 mg / mL to about 25 mg / mL of antibody-drug conjugate, about 15 mM to about 25 mM of a buffer comprising histidine, about 200 mM to about 300 mM of a stabilizer comprising sucrose, and about 0.01% to about 0.03% of a surfactant comprising polysorbate 20, wherein the formulation has a pH of about 5.5 to about 6.1.

[0141] In one embodiment, the formulation comprises about 20 mg / mL of antibody drug conjugate, about 20 mM of a buffer comprising histidine or succinate, about 240 mM of a stabilizer comprising sucrose or trehalose, and about 0.02% of a surfactant comprising polysorbate 20 or polysorbate 80, wherein the formulation has a pH of about 5.8.

[0142] In one embodiment, the formulation comprises about 20 mg / mL of antibody drug conjugate, about 20 mM of a buffer comprising histidine, about 240 mM of a stabilizer comprising sucrose, and about 0.02% of a surfactant comprising polysorbate 20, wherein the formulation has a pH of about 5.8.

[0143] In another aspect, the disclosure features a lyophilized formulation, the lyophilized formulation being lyophilized from a formulation described herein.

[0144] In one embodiment, about 5 mL to about 5.5 mL of the formulation is lyophilized.

[0145] In certain embodiments, the D of the lyophilized formulation is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times more stable than an otherwise identical non-lyophilized formulation, as determined, e.g., by CZE, after about 0, about 4, or about 8 weeks of storage at about 5° C., 25° C., or 40° C. In certain embodiments, the fraction of D having an open ring structure in the lyophilized formulation is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times less stable than an otherwise identical non-lyophilized formulation, as determined, e.g., by CZE, after about 0, about 4, or about 8 weeks of storage at about 5° C., 25° C., or 40° C.

[0146] In one embodiment, the lyophilized formulation contains about 80 mg to about 120 mg (eg, about 107 mg) of antibody-drug conjugate.

[0147] In certain embodiments, the monomer level of the lyophilized formulation is at least about 95%, e.g., at least about 96%, 97%, 98%, or 99%, as determined, e.g., by SEC, after about 0, about 4, or about 8 weeks of storage at about 5°C, 25°C, or 40°C.

[0148] In certain embodiments, the fragment level of the lyophilized formulation is less than about 3%, e.g., less than about 2.5%, 2%, 1.5%, 1%, or 0.5%, as determined, e.g., by SEC, after about 0, about 4, or about 8 weeks of storage at about 5°C, 25°C, or 40°C.

[0149] In certain embodiments, the aggregate level of the lyophilized formulation is less than about 3%, e.g., less than about 2.5%, 2%, 1.5%, 1%, or 0.5%, as determined, e.g., by SEC, after about 0, about 4, or about 8 weeks of storage at about 5°C, 25°C, or 40°C.

[0150] In certain embodiments, the degradation level of the lyophilized formulation is less than about 2%, e.g., less than about 1.5%, 1%, or 0.5%, as determined, e.g., by SEC, after about 4 weeks or about 8 weeks of storage at about 5°C, 25°C, or 40°C.

[0151] In certain embodiments, the level of particles of 10 μm or larger in the lyophilized formulation is less than about 300 particles / mL, e.g., less than about 280 particles / mL, less than about 260 particles / mL, less than about 240 particles / mL, less than about 220 particles / mL, less than about 200 particles / mL, less than about 180 particles / mL, less than about 160 particles / mL, less than about 140 particles / mL, 120 particles / mL, less than about 100 particles / mL, 80 particles / mL, 60 particles / mL, 40 particles / mL, 20 particles / mL, or 10 particles / mL, as determined, for example, by light obscuration, after about 0, about 4, or about 8 weeks of storage at about 5°C, 25°C, or 40°C.

[0152] In certain embodiments, the level of particles larger than 10 μm in the lyophilized formulation does not increase by more than about 3-fold, e.g., does not increase by more than about 2.5, 2, 1.5, 1, or 0.5-fold, after about 4 or about 8 weeks of storage at about 5° C., 25° C., or 40° C., as determined, for example, by light obscuration.

[0153] In certain embodiments, the level of particles larger than 25 μm in the lyophilized formulation does not increase by more than about 3-fold, e.g., does not increase by more than about 2.5, 2, 1.5, 1, or 0.5-fold, after about 4 or about 8 weeks of storage at about 5° C., 25° C., or 40° C., as determined, e.g., by light obscuration.

[0154] In certain embodiments, the level of particles greater than 25 μm in the lyophilized formulation is less than about 20 particles / mL, e.g., less than about 15 particles / mL, 10 particles / mL, 5 particles / mL, or 2 particles / mL, as determined, e.g., by light obscuration, after about 0, about 4, or about 8 weeks of storage at about 5° C., 25° C., or 40° C.

[0155] In certain embodiments, the lyophilized formulation has an impurity level of less than about 15%, e.g., less than about 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, as determined, for example, by capillary electrophoresis, after about 0, about 4, or about 8 weeks of storage at about 5°C, 25°C, or 40°C.

[0156] In certain embodiments, the impurity levels of the lyophilized formulation do not increase by more than 20%, e.g., do not increase by more than about 15%, 10%, 5%, 2%, or 1%, after about 4 weeks or about 8 weeks of storage at about 5°C, 25°C, or 40°C, as determined, for example, by capillary electrophoresis.

[0157] In certain embodiments, the neutral variant level of the lyophilized formulation is greater than about 50%, e.g., greater than about 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, as determined, for example, by capillary zone electrophoresis (CZE), after about 0, about 4, or about 8 weeks of storage at about 5°C, 25°C, or 40°C.

[0158] In certain embodiments, the neutral variant level of the lyophilized formulation does not decrease by more than about 20%, e.g., does not decrease by more than about 15%, 10%, 5%, or 2%, after about 4 weeks or about 8 weeks of storage at about 5°C, 25°C, or 40°C, e.g., as determined by CZE.

[0159] In certain embodiments, the acidic variant level of the lyophilized formulation is less than about 30%, e.g., less than about 25%, 20%, 15%, 5%, or 2%, as determined, e.g., by CZE, after about 0, about 4, or about 8 weeks of storage at about 5°C, 25°C, or 40°C.

[0160] In certain embodiments, the acidic variant level of the lyophilized formulation does not increase by more than about 50%, e.g., does not increase by more than about 40%, 30%, 20%, 10%, or 5%, after about 4 weeks or about 8 weeks of storage at about 5°C, 25°C, or 40°C, e.g., as determined by CZE.

[0161] In certain embodiments, the lyophilized formulation has a basic variant level of less than about 30%, e.g., less than about 25%, 20%, 15%, 5%, or 2%, as determined, e.g., by CZE, after about 0, about 4, or about 8 weeks of storage at about 5°C, 25°C, or 40°C.

[0162] In certain embodiments, the basic variant level of the lyophilized formulation does not increase by more than about 50%, e.g., does not increase by more than about 40%, 30%, 20%, 10%, or 5%, after about 4 weeks or about 8 weeks of storage at about 5°C, 25°C, or 40°C, e.g., as determined by CZE.

[0163] In certain embodiments, the potency of the lyophilized formulation does not decrease by more than about 25%, e.g., not by more than about 20%, 15%, 10%, 5%, or 2%, after about 8 weeks of storage at about 5°C, 25°C, or 40°C, e.g., as determined by a biological activity assay.

[0164] In certain embodiments, the osmolality of the lyophilized formulation is about 200 mOsm / L to 400 mOsm / L, e.g., 200 mOsm / L to 300 mOsm / L, 250 mOsm / L to 350 mOsm / L, 270 mOsm / L to 330 mOsm / L, 290 mOsm / L to 31 ... / L~350mOsm / L, 290mOsm / L~350mOsm / L, 310mOsm / L~350mOsm / L, 330mOsm / L~350mOs m / L, 250mOsm / L~330mOsm / L, 250mOsm / L~310mOsm / L, 250mOsm / L~290mOsm / L, 250mO sm / L~270mOsm / L, 260mOsm / L~280mOsm / L, 270mOsm / L~290mOsm / L, 280mOsm / L~300m Osm / L, 300mOsm / L~320mOsm / L, 310mOsm / L~330mOsm / L, or 320mOsm / L~340mOsm / L, e.g. For example, 200 mOsm / L, 250 mOsm / L, 260 mOsm / L, 270 mOsm / L, 280 mOsm / L, 290 mOsm / L, 300 mOsm / L, 310 mOsm / L, 320 mOsm / L, 330 mOsm / L, 340 mOsm / L, 350 mOsm / L, or 400 mOsm / L.

[0165] In another aspect, the disclosure features a liquid formulation, the liquid formulation being reconstituted from a lyophilized formulation described herein.

[0166] In another aspect, the disclosure features a container containing a lyophilized formulation described herein.

[0167] In some embodiments, the container is a vial, for example, a 25R glass vial. In some embodiments, the container further comprises a stopper and a cap (e.g., a flip-off aluminum cap).

[0168] Treatment method In another aspect, the disclosure features a method of treating cancer, the method including administering to a subject in need thereof an antibody drug conjugate (ADC) described herein, or a formulation described herein, at a dose of 1 mg / kg to 16 mg / kg, thereby treating the cancer.

[0169] In some embodiments, the ADC is administered at a dose of 2 mg / kg to 15 mg / kg. In some embodiments, the ADC is administered at a dose of 1 mg / kg, 2 mg / kg, 4 mg / kg, 8 mg / kg, 12 mg / kg, or 16 mg / kg. In some embodiments, the ADC is administered once weekly, once every two weeks, or once every four weeks. In some embodiments, the ADC is administered once every two weeks. In some embodiments, the ADC is administered intravenously.

[0170] In certain embodiments, the subject has been treated with tebentafusp prior to administration of the ADC. In certain embodiments, the subject has not been treated with tebentafusp prior to administration of the ADC.

[0171] In some embodiments, the cancer expresses PMEL17 and contains a mutation in the GNAQ or GNA11 gene, or the melanoma expresses PMEL17 and contains a mutation in GNAQ, GNA11, or both. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is malignant melanoma, uveal melanoma, ocular melanoma, eye cancer, ocular neoplasm, cutaneous melanoma, or mucosal melanoma.

[0172] In one embodiment, the ADC has the following structure: [ka] (In the formula, Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein, and y is 1, 2, 3, or 4. In certain embodiments, y is 2.

[0173] In one embodiment, Ab is (a) a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, a heavy chain CDR3 of SEQ ID NO: 3, a light chain CDR1 of SEQ ID NO: 14, a light chain CDR2 of SEQ ID NO: 15, and a light chain CDR3 of SEQ ID NO: 16; (b) a heavy chain CDR1 of SEQ ID NO: 4, a heavy chain CDR2 of SEQ ID NO: 2, a heavy chain CDR3 of SEQ ID NO: 3, a light chain CDR1 of SEQ ID NO: 14, a light chain CDR2 of SEQ ID NO: 15, and a light chain CDR3 of SEQ ID NO: 16; (c) a heavy chain CDR1 of SEQ ID NO: 5, a heavy chain CDR2 of SEQ ID NO: 6, a heavy chain CDR3 of SEQ ID NO: 3, a light chain CDR1 of SEQ ID NO: 17, a light chain CDR2 of SEQ ID NO: 18, and a light chain CDR3 of SEQ ID NO: 19; or (d) a heavy chain CDR1 of SEQ ID NO: 7, a heavy chain CDR2 of SEQ ID NO: 8, a heavy chain CDR3 of SEQ ID NO: 9, a light chain CDR1 of SEQ ID NO: 20, a light chain CDR2 of SEQ ID NO: 18, and a light chain CDR3 of SEQ ID NO: 16 Includes.

[0174] In one embodiment, the Ab comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 25. In one embodiment, the Ab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 283 and a light chain comprising the amino acid sequence of SEQ ID NO: 27.

[0175] In certain embodiments, the Ab comprises a heavy chain amino acid sequence that includes an N-glycosylation site. In certain embodiments, the N-glycosylation site is located at Asn306.

[0176] In some embodiments, the method further comprises determining the expression of one or more biomarkers, in some embodiments, the one or more biomarkers are selected from premelanosome protein 17 (PMEL17), phosphorylated ERK (pERK), cluster of differentiation 8 (CD8), programmed death-ligand 1 (PD-L1), dual specificity phosphatase 6 (DUSP6), Ras guanyl-releasing protein 3 (RASGRP3), or any combination thereof.

[0177] In some embodiments, the expression of one or more biomarkers is determined in a sample from the subject. In some embodiments, the sample is obtained from the subject before, during, and / or after administration of the ADC. In some embodiments, the sample is a tumor sample. For example, the tumor sample may be an archival tissue block or may be obtained by needle biopsy. In some embodiments, the mRNA expression of one or more biomarkers is determined. In some embodiments, the protein expression of one or more biomarkers is determined. In some embodiments, the method further comprises detecting one or more cancer-associated genes by next-generation DNA sequencing, by performing whole transcriptome analysis, by determining the expression of one or more immune- and cancer-associated genes, or a combination thereof.

[0178] In some embodiments, the method further comprises assessing cell-free DNA (cfDNA) in a sample from the subject. In some embodiments, the sample is obtained from the subject before, during, and / or after administration of the ADC. In some embodiments, the sample is a blood sample. In some embodiments, assessing the cell-free DNA determines one or more of driver mutation status, predicted pharmacodynamics, tumor mutation burden (TMB), or resistance markers.

[0179] In certain aspects, the disclosure features a method of evaluating a treatment (e.g., a treatment described herein) for a disease (e.g., a disease described herein) in a subject (e.g., a subject described herein), the method including determining expression of one or more biomarkers according to a method described herein and / or evaluating cell-free DNA (cfDNA) in a sample (e.g., a sample described herein) from the subject.

[0180] In certain aspects, the disclosure features a method of assessing the progression of a disease (e.g., a disease described herein) in a subject (e.g., a subject described herein), the method including determining expression of one or more biomarkers according to a method described herein and / or assessing cell-free DNA (cfDNA) in a sample from the subject (e.g., a sample described herein).

[0181] In certain aspects, the disclosure features a method of selecting a treatment (e.g., a treatment described herein) for a disease (e.g., a disease described herein) in a subject (e.g., a subject described herein), the method comprising determining expression of one or more biomarkers according to a method described herein and / or assessing cell-free DNA (cfDNA) in a sample (e.g., a sample described herein) from the subject.

[0182] In an aspect, the disclosure features a method of selecting a subject (e.g., a subject described herein) for a treatment (e.g., a treatment described herein) for a disease (e.g., a disease described herein), the method comprising determining expression of one or more biomarkers according to a method described herein and / or assessing cell-free DNA (cfDNA) in a sample (e.g., a sample described herein) from the subject.

[0183] Other embodiments Aspects and embodiments described herein may include any of the following embodiments.

[0184] In some embodiments, the antibody or antigen-binding fragment thereof that binds PMEL17 is (a) a heavy chain variable region comprising a heavy chain CDR1 (complementarity determining region 1) of SEQ ID NO: 1, 4, 5 or 7, a heavy chain CDR2 (complementarity determining region 2) of SEQ ID NO: 2, 6 or 8, and a heavy chain CDR3 (complementarity determining region 3) of SEQ ID NO: 3 or 9; and a light chain variable region comprising a light chain CDR1 (complementarity determining region 1) of SEQ ID NO: 14, 17 or 20, a light chain CDR2 (complementarity determining region 2) of SEQ ID NO: 15 or 18, and a light chain CDR3 (complementarity determining region 3) of SEQ ID NO: 16 or 19; (b) a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 33, 36, 37, or 39, a heavy chain CDR2 of SEQ ID NO: 34, 38, or 40, a heavy chain CDR3 of SEQ ID NO: 35 or 41; a light chain CDR1 of SEQ ID NO: 46, 49, or 52; a light chain CDR2 of SEQ ID NO: 47 or 50; and a light chain CDR3 of SEQ ID NO: 48 or 51; (c) a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 5, 7, 57, or 60, a heavy chain CDR2 of SEQ ID NO: 58, 61, or 62, a heavy chain CDR3 of SEQ ID NO: 59 or 63; a light chain CDR1 of SEQ ID NO: 68, 71, or 74; a light chain CDR2 of SEQ ID NO: 69 or 72; and a light chain CDR3 of SEQ ID NO: 70 or 73; (d) a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 79, 82, 83, or 85, a heavy chain CDR2 of SEQ ID NO: 80, 84, or 86, a heavy chain CDR3 of SEQ ID NO: 81 or 87; a light chain CDR1 of SEQ ID NO: 92, 95, or 98; a light chain CDR2 of SEQ ID NO: 93 or 96; and a light chain CDR3 of SEQ ID NO: 94 or 97; (e) a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 103, 106, 107, or 109, a heavy chain CDR2 of SEQ ID NO: 104, 108, or 110, a heavy chain CDR3 of SEQ ID NO: 105 or 111; a light chain CDR1 of SEQ ID NO: 49, 52, or 116; a light chain CDR2 of SEQ ID NO: 47 or 50; and a light chain CDR3 of SEQ ID NO: 117 or 118; (f) a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 123, 126, 127, or 129, a heavy chain CDR2 of SEQ ID NO: 124, 128, or 130, a heavy chain CDR3 of SEQ ID NO: 125 or 131; a light chain CDR1 of SEQ ID NO: 136, 139, or 142; a light chain CDR2 of SEQ ID NO: 137 or 140; and a light chain CDR3 of SEQ ID NO: 138 or 141; (g) a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 123, 126, 127, or 129, a heavy chain CDR2 of SEQ ID NO: 124, 128, or 130, a heavy chain CDR3 of SEQ ID NO: 147 or 148; a light chain CDR1 of SEQ ID NO: 153, 156, or 158; a light chain CDR2 of SEQ ID NO: 50 or 154; and a light chain CDR3 of SEQ ID NO: 155 or 157; (h) a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 103, 106, 107, or 109, a heavy chain CDR2 of SEQ ID NO: 104, 108, or 110, a heavy chain CDR3 of SEQ ID NO: 163 or 164; a light chain CDR1 of SEQ ID NO: 49, 52, or 116; a light chain CDR2 of SEQ ID NO: 47 or 50; and a light chain CDR3 of SEQ ID NO: 169 or 170; (i) a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 175, 178, 179, or 181, a heavy chain CDR2 of SEQ ID NO: 176, 180, or 182, a heavy chain CDR3 of SEQ ID NO: 177 or 183; a light chain CDR1 of SEQ ID NO: 49, 52, or 116; a light chain CDR2 of SEQ ID NO: 47 or 50; and a light chain CDR3 of SEQ ID NO: 188 or 189; (j) a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 103, 106, 107, or 109, a heavy chain CDR2 of SEQ ID NO: 104, 108, or 110, a heavy chain CDR3 of SEQ ID NO: 194 or 195; a light chain CDR1 of SEQ ID NO: 49, 52, or 116; a light chain CDR2 or 50 of SEQ ID NO: 47; and a light chain CDR3 of SEQ ID NO: 200 or 201; (k) a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 206, 209, 210, or 212, a heavy chain CDR2 of SEQ ID NO: 207, 211, or 213, a heavy chain CDR3 of SEQ ID NO: 208 or 214; a light chain CDR1 of SEQ ID NO: 153, 156, or 158; a light chain CDR2 of SEQ ID NO: 50 or 154; and a light chain CDR3 of SEQ ID NO: 219 or 220; (l) a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 206, 209, 210, or 212, a heavy chain CDR2 of SEQ ID NO: 207, 211, or 213, a heavy chain CDR3 of SEQ ID NO: 225 or 226; a light chain CDR1 of SEQ ID NO: 136, 139, or 142; a light chain CDR2 of SEQ ID NO: 137 or 140; and a light chain CDR3 of SEQ ID NO: 231 or 232; (m) a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 206, 209, 210, or 212, an HCDR2 of SEQ ID NO: 207, 211, or 213, and an HCDR3 of SEQ ID NO: 237 or 238; and a light chain variable region comprising an LCDR1 of SEQ ID NO: 243, 245, or 247, an LCDR2 of SEQ ID NO: 47 or 50, and an LCDR3 of SEQ ID NO: 244 or 246; (n) a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 206, 209, 210, or 212, an HCDR2 of SEQ ID NO: 207, 211, or 213, and an HCDR3 of SEQ ID NO: 252 or 253; and a light chain variable region comprising an LCDR1 of SEQ ID NO: 153, 156, or 158, an LCDR2 of SEQ ID NO: 50 or 154, and an LCDR3 of SEQ ID NO: 258 or 259; (o) a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, a heavy chain CDR3 of SEQ ID NO: 3, a light chain CDR1 of SEQ ID NO: 14, a light chain CDR2 of SEQ ID NO: 15, and a light chain CDR3 of SEQ ID NO: 16; (p) a heavy chain CDR1 of SEQ ID NO: 4, a heavy chain CDR2 of SEQ ID NO: 2, a heavy chain CDR3 of SEQ ID NO: 3, a light chain CDR1 of SEQ ID NO: 14, a light chain CDR2 of SEQ ID NO: 15, and a light chain CDR3 of SEQ ID NO: 16; (q) a heavy chain CDR1 of SEQ ID NO: 5, a heavy chain CDR2 of SEQ ID NO: 6, a heavy chain CDR3 of SEQ ID NO: 3, a light chain CDR1 of SEQ ID NO: 17, a light chain CDR2 of SEQ ID NO: 18, and a light chain CDR3 of SEQ ID NO: 19; (r) a heavy chain CDR1 of SEQ ID NO: 7, a heavy chain CDR2 of SEQ ID NO: 8, a heavy chain CDR3 of SEQ ID NO: 9, a light chain CDR1 of SEQ ID NO: 20, a light chain CDR2 of SEQ ID NO: 18, and a light chain CDR3 of SEQ ID NO: 16; (s) a heavy chain CDR1 of SEQ ID NO: 33, a heavy chain CDR2 of SEQ ID NO: 34, a heavy chain CDR3 of SEQ ID NO: 35, a light chain CDR1 of SEQ ID NO: 46, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 48; (t) a heavy chain CDR1 of SEQ ID NO: 36, a heavy chain CDR2 of SEQ ID NO: 34, a heavy chain CDR3 of SEQ ID NO: 35, a light chain CDR1 of SEQ ID NO: 46, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 48; (u) a heavy chain CDR1 of SEQ ID NO: 37, a heavy chain CDR2 of SEQ ID NO: 38, a heavy chain CDR3 of SEQ ID NO: 35, a light chain CDR1 of SEQ ID NO: 49, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 51; (v) a heavy chain CDR1 of SEQ ID NO: 39, a heavy chain CDR2 of SEQ ID NO: 40, a heavy chain CDR3 of SEQ ID NO: 41, a light chain CDR1 of SEQ ID NO: 52, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 48; (w) a heavy chain CDR1 of SEQ ID NO: 57, a heavy chain CDR2 of SEQ ID NO: 58, a heavy chain CDR3 of SEQ ID NO: 59, a light chain CDR1 of SEQ ID NO: 68, a light chain CDR2 of SEQ ID NO: 69, and a light chain CDR3 of SEQ ID NO: 70; (x) a heavy chain CDR1 of SEQ ID NO: 60, a heavy chain CDR2 of SEQ ID NO: 58, a heavy chain CDR3 of SEQ ID NO: 59, a light chain CDR1 of SEQ ID NO: 68, a light chain CDR2 of SEQ ID NO: 69, and a light chain CDR3 of SEQ ID NO: 70; (y) a heavy chain CDR1 of SEQ ID NO: 5, a heavy chain CDR2 of SEQ ID NO: 61, a heavy chain CDR3 of SEQ ID NO: 59, a light chain CDR1 of SEQ ID NO: 71, a light chain CDR2 of SEQ ID NO: 72, and a light chain CDR3 of SEQ ID NO: 73; (z) a heavy chain CDR1 of SEQ ID NO: 7, a heavy chain CDR2 of SEQ ID NO: 62, a heavy chain CDR3 of SEQ ID NO: 63, a light chain CDR1 of SEQ ID NO: 74, a light chain CDR2 of SEQ ID NO: 72, and a light chain CDR3 of SEQ ID NO: 70; (aa) a heavy chain CDR1 of SEQ ID NO: 79, a heavy chain CDR2 of SEQ ID NO: 80, a heavy chain CDR3 of SEQ ID NO: 81, a light chain CDR1 of SEQ ID NO: 92, a light chain CDR2 of SEQ ID NO: 93, and a light chain CDR3 of SEQ ID NO: 94; (bb) a heavy chain CDR1 of SEQ ID NO: 82, a heavy chain CDR2 of SEQ ID NO: 80, a heavy chain CDR3 of SEQ ID NO: 81, a light chain CDR1 of SEQ ID NO: 92, a light chain CDR2 of SEQ ID NO: 93, and a light chain CDR3 of SEQ ID NO: 94; (cc) a heavy chain CDR1 of SEQ ID NO: 83, a heavy chain CDR2 of SEQ ID NO: 84, a heavy chain CDR3 of SEQ ID NO: 81, a light chain CDR1 of SEQ ID NO: 95, a light chain CDR2 of SEQ ID NO: 96, and a light chain CDR3 of SEQ ID NO: 97; (dd) a heavy chain CDR1 of SEQ ID NO: 85, a heavy chain CDR2 of SEQ ID NO: 86, a heavy chain CDR3 of SEQ ID NO: 87, a light chain CDR1 of SEQ ID NO: 98, a light chain CDR2 of SEQ ID NO: 96, and a light chain CDR3 of SEQ ID NO: 94; (ee) heavy chain CDR1 of SEQ ID NO: 103, heavy chain CDR2 of SEQ ID NO: 104, heavy chain CDR3 of SEQ ID NO: 105, light chain CDR1 of SEQ ID NO: 116; light chain CDR2 of SEQ ID NO: 47; and light chain CDR3 of SEQ ID NO: 117; (ff) a heavy chain CDR1 of SEQ ID NO: 106, a heavy chain CDR2 of SEQ ID NO: 104, a heavy chain CDR3 of SEQ ID NO: 105, a light chain CDR1 of SEQ ID NO: 116, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 117; (gg) a heavy chain CDR1 of SEQ ID NO: 107, a heavy chain CDR2 of SEQ ID NO: 108, a heavy chain CDR3 of SEQ ID NO: 105, a light chain CDR1 of SEQ ID NO: 49, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 118; (hh) a heavy chain CDR1 of SEQ ID NO: 109, a heavy chain CDR2 of SEQ ID NO: 110, a heavy chain CDR3 of SEQ ID NO: 111, a light chain CDR1 of SEQ ID NO: 52, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 117; (ii) a heavy chain CDR1 of SEQ ID NO: 123, a heavy chain CDR2 of SEQ ID NO: 124, a heavy chain CDR3 of SEQ ID NO: 125, a light chain CDR1 of SEQ ID NO: 136, a light chain CDR2 of SEQ ID NO: 137, and a light chain CDR3 of SEQ ID NO: 138; (jj) a heavy chain CDR1 of SEQ ID NO: 126, a heavy chain CDR2 of SEQ ID NO: 124, a heavy chain CDR3 of SEQ ID NO: 125, a light chain CDR1 of SEQ ID NO: 136, a light chain CDR2 of SEQ ID NO: 137, and a light chain CDR3 of SEQ ID NO: 138; (kk) a heavy chain CDR1 of SEQ ID NO: 127, a heavy chain CDR2 of SEQ ID NO: 128, a heavy chain CDR3 of SEQ ID NO: 125, a light chain CDR1 of SEQ ID NO: 139, a light chain CDR2 of SEQ ID NO: 140, and a light chain CDR3 of SEQ ID NO: 141; (ll) a heavy chain CDR1 of SEQ ID NO: 129, a heavy chain CDR2 of SEQ ID NO: 130, a heavy chain CDR3 of SEQ ID NO: 131, a light chain CDR1 of SEQ ID NO: 142, a light chain CDR2 of SEQ ID NO: 140, and a light chain CDR3 of SEQ ID NO: 138; (mm) heavy chain CDR1 of SEQ ID NO: 123, heavy chain CDR2 of SEQ ID NO: 124, heavy chain CDR3 of SEQ ID NO: 147, light chain CDR1 of SEQ ID NO: 153, light chain CDR2 of SEQ ID NO: 154, and light chain CDR3 of SEQ ID NO: 155; (nn) a heavy chain CDR1 of SEQ ID NO: 126, a heavy chain CDR2 of SEQ ID NO: 124, a heavy chain CDR3 of SEQ ID NO: 147, a light chain CDR1 of SEQ ID NO: 153, a light chain CDR2 of SEQ ID NO: 154, and a light chain CDR3 of SEQ ID NO: 155; (oo) a heavy chain CDR1 of SEQ ID NO: 127, a heavy chain CDR2 of SEQ ID NO: 128, a heavy chain CDR3 of SEQ ID NO: 147, a light chain CDR1 of SEQ ID NO: 156, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 157; (pp) a heavy chain CDR1 of SEQ ID NO: 129, a heavy chain CDR2 of SEQ ID NO: 130, a heavy chain CDR3 of SEQ ID NO: 148, a light chain CDR1 of SEQ ID NO: 158, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 155; (qq) a heavy chain CDR1 of SEQ ID NO: 103, a heavy chain CDR2 of SEQ ID NO: 104, a heavy chain CDR3 of SEQ ID NO: 163, a light chain CDR1 of SEQ ID NO: 116, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 169; (rr) a heavy chain CDR1 of SEQ ID NO: 106, a heavy chain CDR2 of SEQ ID NO: 104, a heavy chain CDR3 of SEQ ID NO: 163, a light chain CDR1 of SEQ ID NO: 116, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 169; (ss) a heavy chain CDR1 of SEQ ID NO: 107, a heavy chain CDR2 of SEQ ID NO: 108, a heavy chain CDR3 of SEQ ID NO: 163, a light chain CDR1 of SEQ ID NO: 49, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 170; (tt) heavy chain CDR1 of SEQ ID NO: 109, heavy chain CDR2 of SEQ ID NO: 110, heavy chain CDR3 of SEQ ID NO: 164, light chain CDR1 of SEQ ID NO: 52, light chain CDR2 of SEQ ID NO: 50, and light chain CDR3 of SEQ ID NO: 169; (uu) a heavy chain CDR1 of SEQ ID NO: 175, a heavy chain CDR2 of SEQ ID NO: 176, a heavy chain CDR3 of SEQ ID NO: 177, a light chain CDR1 of SEQ ID NO: 116, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 188; (vv) a heavy chain CDR1 of SEQ ID NO: 178, a heavy chain CDR2 of SEQ ID NO: 176, a heavy chain CDR3 of SEQ ID NO: 177, a light chain CDR1 of SEQ ID NO: 116, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 188; (ww) a heavy chain CDR1 of SEQ ID NO: 179, a heavy chain CDR2 of SEQ ID NO: 180, a heavy chain CDR3 of SEQ ID NO: 177, a light chain CDR1 of SEQ ID NO: 49, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 189; (xx) a heavy chain CDR1 of SEQ ID NO: 181, a heavy chain CDR2 of SEQ ID NO: 182; a heavy chain CDR3 of SEQ ID NO: 183, a light chain CDR1 of SEQ ID NO: 52, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 188; (yy) a heavy chain CDR1 of SEQ ID NO: 103, a heavy chain CDR2 of SEQ ID NO: 104, a heavy chain CDR3 of SEQ ID NO: 194, a light chain CDR1 of SEQ ID NO: 116, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 200; (zz) a heavy chain CDR1 of SEQ ID NO: 106, a heavy chain CDR2 of SEQ ID NO: 104, a heavy chain CDR3 of SEQ ID NO: 194, a light chain CDR1 of SEQ ID NO: 116, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 200; (aaa) a heavy chain CDR1 of SEQ ID NO: 107, a heavy chain CDR2 of SEQ ID NO: 108, a heavy chain CDR3 of SEQ ID NO: 194, a light chain CDR1 of SEQ ID NO: 49, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 201; (bbb) a heavy chain CDR1 of SEQ ID NO: 109, a heavy chain CDR2 of SEQ ID NO: 110, a heavy chain CDR3 of SEQ ID NO: 195, a light chain CDR1 of SEQ ID NO: 52, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 200; (ccc) a heavy chain CDR1 of SEQ ID NO: 206, a heavy chain CDR2 of SEQ ID NO: 207, a heavy chain CDR3 of SEQ ID NO: 208, a light chain CDR1 of SEQ ID NO: 153, a light chain CDR2 of SEQ ID NO: 154, and a light chain CDR3 of SEQ ID NO: 219; (ddd) a heavy chain CDR1 of SEQ ID NO: 209, a heavy chain CDR2 of SEQ ID NO: 207, a heavy chain CDR3 of SEQ ID NO: 208, a light chain CDR1 of SEQ ID NO: 153, a light chain CDR2 of SEQ ID NO: 154, and a light chain CDR3 of SEQ ID NO: 219; (eee) a heavy chain CDR1 of SEQ ID NO: 210, a heavy chain CDR2 of SEQ ID NO: 211, a heavy chain CDR3 of SEQ ID NO: 208, a light chain CDR1 of SEQ ID NO: 156, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 220; (fff) a heavy chain CDR1 of SEQ ID NO: 212, a heavy chain CDR2 of SEQ ID NO: 213, a heavy chain CDR3 of SEQ ID NO: 214, a light chain CDR1 of SEQ ID NO: 158, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 219; (ggg) heavy chain CDR1 of SEQ ID NO: 206, heavy chain CDR2 of SEQ ID NO: 207, heavy chain CDR3 of SEQ ID NO: 225, light chain CDR1 of SEQ ID NO: 136, light chain CDR2 of SEQ ID NO: 137, and light chain CDR3 of SEQ ID NO: 231; (hhh) a heavy chain CDR1 of SEQ ID NO: 209, a heavy chain CDR2 of SEQ ID NO: 207, a heavy chain CDR3 of SEQ ID NO: 225, a light chain CDR1 of SEQ ID NO: 136, a light chain CDR2 of SEQ ID NO: 137, and a light chain CDR3 of SEQ ID NO: 231; (iii) a heavy chain CDR1 of SEQ ID NO: 210, a heavy chain CDR2 of SEQ ID NO: 211, a heavy chain CDR3 of SEQ ID NO: 225, a light chain CDR1 of SEQ ID NO: 139, a light chain CDR2 of SEQ ID NO: 140, and a light chain CDR3 of SEQ ID NO: 232; (jjj) heavy chain CDR1 of SEQ ID NO: 212, heavy chain CDR2 of SEQ ID NO: 213, heavy chain CDR3 of SEQ ID NO: 226, light chain CDR1 of SEQ ID NO: 142; light chain CDR2 of SEQ ID NO: 140; and light chain CDR3 of SEQ ID NO: 231; (kkk) a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 206, an HCDR2 of SEQ ID NO: 207, and an HCDR3 of SEQ ID NO: 237, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 243, an LCDR2 of SEQ ID NO: 47, and an LCDR3 of SEQ ID NO: 244; (lll) a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 209, an HCDR2 of SEQ ID NO: 207, and an HCDR3 of SEQ ID NO: 237, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 243, an LCDR2 of SEQ ID NO: 47, and an LCDR3 of SEQ ID NO: 244; (mmm) a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 210, an HCDR2 of SEQ ID NO: 211, and an HCDR3 of SEQ ID NO: 237, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 245, an LCDR2 of SEQ ID NO: 50, and an LCDR3 of SEQ ID NO: 246; (nnn) a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 212, an HCDR2 of SEQ ID NO: 213, and an HCDR3 of SEQ ID NO: 238; and a light chain variable region comprising an LCDR1 of SEQ ID NO: 247, an LCDR2 of SEQ ID NO: 50, and an LCDR3 of SEQ ID NO: 244; (ooo) a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 206, an HCDR2 of SEQ ID NO: 207, and an HCDR3 of SEQ ID NO: 252, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 153, an LCDR2 of SEQ ID NO: 154, and an LCDR3 of SEQ ID NO: 258; (ppp) a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 209, an HCDR2 of SEQ ID NO: 207, and an HCDR3 of SEQ ID NO: 252, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 153, an LCDR2 of SEQ ID NO: 154, and an LCDR3 of SEQ ID NO: 258; (qqq) a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 210, an HCDR2 of SEQ ID NO: 211, and an HCDR3 of SEQ ID NO: 252, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 156, an LCDR2 of SEQ ID NO: 50, and an LCDR3 of SEQ ID NO: 259; or (rrr) a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 212, an HCDR2 of SEQ ID NO: 213, and an HCDR3 of SEQ ID NO: 253; and a light chain variable region comprising an LCDR1 of SEQ ID NO: 158, an LCDR2 of SEQ ID NO: 50, and an LCDR3 of SEQ ID NO: 258. Includes.

[0185] In some embodiments, the antibody or antigen-binding fragment thereof that binds PMEL17 is (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 21; (b) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 25; (c) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 29; (d) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 42, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 53; (e) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 64, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 75; (f) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 88, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 99; (g) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 112, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 119; (h) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 132, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 143; (i) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 149, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 159; (j) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 165, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 171; (k) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 184, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 190; (l) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 196, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 202; (m) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 215, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 221; (n) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 227, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 233; (o) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 239, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 248; or (p) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 254, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 260 Includes.

[0186] In some embodiments, the antibody or antigen-binding fragment thereof that binds PMEL17 is (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 12 and a light chain comprising the amino acid sequence of SEQ ID NO: 23; (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 12 and a light chain comprising the amino acid sequence of SEQ ID NO: 27; (c) a heavy chain comprising the amino acid sequence of SEQ ID NO: 12 and a light chain comprising the amino acid sequence of SEQ ID NO: 31; (d) a heavy chain comprising the amino acid sequence of SEQ ID NO: 44, and a light chain comprising the amino acid sequence of SEQ ID NO: 55; (e) a heavy chain comprising the amino acid sequence of SEQ ID NO: 66, and a light chain comprising the amino acid sequence of SEQ ID NO: 77; (f) a heavy chain comprising the amino acid sequence of SEQ ID NO: 90, and a light chain comprising the amino acid sequence of SEQ ID NO: 101; (g) a heavy chain comprising the amino acid sequence of SEQ ID NO: 114, and a light chain comprising the amino acid sequence of SEQ ID NO: 121; (h) a heavy chain comprising the amino acid sequence of SEQ ID NO: 134, and a light chain comprising the amino acid sequence of SEQ ID NO: 145; (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 151, and a light chain comprising the amino acid sequence of SEQ ID NO: 161; (j) a heavy chain comprising the amino acid sequence of SEQ ID NO: 167, and a light chain comprising the amino acid sequence of SEQ ID NO: 173; (k) a heavy chain comprising the amino acid sequence of SEQ ID NO: 186, and a light chain comprising the amino acid sequence of SEQ ID NO: 192; (l) a heavy chain comprising the amino acid sequence of SEQ ID NO: 198, and a light chain comprising the amino acid sequence of SEQ ID NO: 204; (m) a heavy chain comprising the amino acid sequence of SEQ ID NO: 217, and a light chain comprising the amino acid sequence of SEQ ID NO: 223; (n) a heavy chain comprising the amino acid sequence of SEQ ID NO: 229, and a light chain comprising the amino acid sequence of SEQ ID NO: 235; (o) a heavy chain comprising the amino acid sequence of SEQ ID NO: 241, and a light chain comprising the amino acid sequence of SEQ ID NO: 250; (p) a heavy chain comprising the amino acid sequence of SEQ ID NO: 256, and a light chain comprising the amino acid sequence of SEQ ID NO: 262; (q) a heavy chain comprising the amino acid sequence of SEQ ID NO: 283, and a light chain comprising the amino acid sequence of SEQ ID NO: 27; (r) a heavy chain comprising the amino acid sequence of SEQ ID NO: 283, and a light chain comprising the amino acid sequence of SEQ ID NO: 31; (s) a heavy chain comprising the amino acid sequence of SEQ ID NO: 315, and a light chain comprising the amino acid sequence of SEQ ID NO: 101. Includes.

[0187] In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more cysteine ​​substitutions. In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more cysteine ​​substitutions selected from E152C, S375C, or both E152C and S375C in the heavy chain of the antibody or antigen-binding fragment thereof, where positions are numbered according to the EU system. In some embodiments, the antibody or antigen-binding fragment thereof comprises an E152C substitution in the heavy chain of the antibody or antigen-binding fragment thereof, where positions are numbered according to the EU system.

[0188] In some embodiments, the antibody molecule is a monoclonal antibody. In some embodiments, the antibody molecule is an isolated antibody. In some embodiments, the antibody molecule is a synthetic antibody. In some embodiments, the antibody molecule is an engineered antibody.

[0189] In some embodiments, the antibody or antigen-binding fragment is encoded by a nucleic acid described herein. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 13, 24, 28, 32, 45, 56, 67, 78, 91, 102, 115, 122, 135, 146, 152, 162, 168, 174, 187, 193, 199, 205, 218, 224, 230, 236, 242, 251, 257, 263, 319, or 320. In some embodiments, the nucleic acid is present in a vector, e.g., a vector described herein. In some embodiments, the nucleic acid or vector is present in a host cell, e.g., a host cell described herein.

[0190] In some embodiments, the antibody or antigen-binding fragment is produced by a method comprising culturing host cells and recovering the antibody or antigen-binding fragment from the cell culture. In some embodiments, recovering the antibody from the cell culture comprises removing the cells and filtering the culture; purifying the culture by affinity chromatography; inactivating any viruses by adjusting the pH to 3.4-3.6, then readjusting the pH to 5.8-6.2 and filtering the culture; purifying the culture by cation exchange chromatography and performing on-column reduction of the culture; performing anion exchange chromatography on the culture; removing viruses by nanofiltration; filtering the culture containing the antibody; and obtaining the purified antibody.

[0191] In some embodiments, the antibody drug conjugate is an antibody drug conjugate made by the methods described herein.

[0192] In some embodiments, the antibody drug conjugate has formula (C): Ab-(L A -(D) n ) y (C) (In the formula, D is a GNAQ inhibitor, a GNA11 inhibitor or an inhibitor of GNAQ and GNA11; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; L A is a linker; n is 1, 2, 3 or 4, and y is 1, 2, 3 or 4 Includes.

[0193] In some embodiments, n is 1. In some embodiments, y is about 1 to about 4. In some embodiments, the linker is a cleavable linker or a non-cleavable linker. In some embodiments, the linker comprises a ValCit peptide linker.

[0194] In some embodiments, the drug moiety is an inhibitor of GNAQ and GNA11.

[0195] In some embodiments, D is [ka] is.

[0196] In some embodiments, the antibody drug conjugate has the following structure: [ka] It has.

[0197] In some embodiments, the antibody-drug conjugate has the following formula (C-2): [ka] (In the formula, R 0 is methyl or ethyl; R 1 is methyl or isopropyl; R 2 is methyl or ethyl; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; X1 is a divalent linking group; X2 is a self-sacrificing spacer; L1 is a bivalent peptide linker; L2 is a bond or linker, and y is 1, 2, 3 or 4 It has.

[0198] In some embodiments, the antibody drug conjugate is present in a pharmaceutical composition comprising the antibody drug conjugate and a pharmaceutically acceptable carrier. In some embodiments, the antibody drug conjugate is present in a formulation described herein.

[0199] In some embodiments, the antibody drug conjugate or formulation is used as a pharmaceutical.

[0200] In some embodiments, the antibody drug conjugate or formulation is used in a method of treating or preventing cancer in a patient in need thereof, comprising administering the antibody drug conjugate or formulation to the patient, wherein the cancer expresses PMEL17 and contains a mutation in the GNAQ or GNA11 gene, or the cancer expresses PMEL17 and contains a mutation in GNAQ, GNA11, or both. In some embodiments, the cancer is a carcinoma (e.g., hepatocellular carcinoma), sarcoma, leukemia, lymphoma, eye cancer, ocular neoplasm, melanoma (e.g., uveal melanoma, non-uveal melanoma, malignant melanoma, ocular melanoma, subcutaneous melanoma, cutaneous melanoma, or mucosal melanoma), or a metastatic cancer thereof.

[0201] In some embodiments, the antibody drug conjugate or formulation is administered to a patient in combination with one or more additional therapeutic compounds.

[0202] In some embodiments, the one or more additional therapeutic compounds are selected from a standard of care chemotherapeutic agent, an MDM2 inhibitor, an MRC2 inhibitor, a PKC inhibitor, a MAPK inhibitor, a costimulatory molecule, or a checkpoint inhibitor.

[0203] In some embodiments, the costimulatory molecule is selected from OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, STING, or an agonist of CD83 ligand.

[0204] In some embodiments, the checkpoint inhibitor is selected from an inhibitor of PD-1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and / or TGFR beta. [Brief explanation of the drawings]

[0205] [Figure 1] 1 shows the gene organization and transposable elements containing the BGC of compound (A1) (frsA to H) and compound J (dis1 to 4) in C. vaccinii (76,974 bp). [Figure 2] A scheme of the insertion strategy used for promoter exchange is shown. [Figure 3] The production of compound J in the disruption mutant compared to the wild type is shown. [Figure 4] The MS / MS spectrum of compound J is shown. [Figure 5] 1 shows the MS / MS spectrum of compound F5. [Figure 6] 1 shows the MS / MS spectrum of compound F3. [Figure 7] 1 shows the MS / MS spectrum of compound D. [Figure 8] 1 shows a schematic diagram of the biosynthesis of Compound J. The diagram discloses "Ser Val His Val Leu Val" as per SEQ ID NO: 324. [Figure 9-1] 1 shows an exemplary on-site and off-side combination. [Figure 9-2]1 shows an exemplary on-site and off-side combination. [Figure 10] Figure 10A: The composition of Compound (A1)-BGC (white: NRPS genes, black: genes encoding modifying enzymes) and the expression of the exchanged promoters. Figure 10B: A bar graph of Compound (A1) potency from different C. vaccinii promoter exchange mutants, including fold change compared to the wild type. [Figure 11] Figure 11A: HPLC-MS extracted ion chromatogram (EIC) 1002.535-1002.545 Da of culture extracts of C. vaccinii wild-type and C. vaccinii vioP mutant. Figure 11B: HPLC-MS EIC 1020.49-1020.50 Da of culture extracts of L-Met-fed C. vaccinii vioP mutant, revealing three novel analogs of compound (A1). [Figure 12] Figure 12A: HPLC-MS extracted ion chromatogram (EIC) of compound (A1) (bottom) at m / z 1002.52-1002.57 after 8 hours of incubation at 5 μg / mL in human plasma and 37° C. and the same sample further spiked with 100 ng of compound 5 (final concentration 6 μg / mL). Figure 12B: Absolute quantification and fractionation of compounds (A1) and 5 after 24 hours of incubation of compound (A1) at 5 μg / mL in human plasma and 37° C. [Figure 13A-1] The effects of compounds A1 and 2-8 on the proliferation of Gq or G11 mutant uveal melanoma (UM) cell lines and Gq or G11 wild-type cutaneous melanoma cell lines are shown. Growth of UM cell lines driven by constitutively active Gq or G11 mutants was inhibited by compound A1 with a sub-nM GI50 for the 92.1 and MP41 cell lines, whereas growth of the cutaneous melanoma cell lines A375, harboring a homozygous BRAF V600E mutation, and SK-MEL-30, harboring a heterozygous NRAS Q61K mutation, was unaffected by compound A1. [Figure 13A-2]The effects of compounds A1 and 2-8 on the proliferation of Gq or G11 mutant uveal melanoma (UM) cell lines and Gq or G11 wild-type cutaneous melanoma cell lines are shown. Growth of UM cell lines driven by constitutively active Gq or G11 mutants was inhibited by compound A1 with a sub-nM GI50 for the 92.1 and MP41 cell lines, whereas growth of the cutaneous melanoma cell lines A375, harboring a homozygous BRAF V600E mutation, and SK-MEL-30, harboring a heterozygous NRAS Q61K mutation, was unaffected by compound A1. [Figure 13B-1] The effects of compounds 9-15 on the proliferation of Gq or G11 mutant UM cell lines and Gq or G11 wild-type cutaneous melanoma cell lines are shown. Growth of UM cell lines driven by constitutively active Gq or G11 mutants was inhibited by compound (A1) with a sub-nM GI50 for the 92.1 and MP41 cell lines, whereas growth of the cutaneous melanoma cell lines A375, harboring a homozygous BRAF V600E mutation, and SK-MEL-30, harboring a heterozygous NRAS Q61K mutation, was unaffected by compound (A1). [Figure 13B-2] The effects of compounds 9-15 on the proliferation of Gq or G11 mutant UM cell lines and Gq or G11 wild-type cutaneous melanoma cell lines are shown. Growth of UM cell lines driven by constitutively active Gq or G11 mutants was inhibited by compound (A1) with a sub-nM GI50 for the 92.1 and MP41 cell lines, whereas growth of the cutaneous melanoma cell lines A375, harboring a homozygous BRAF V600E mutation, and SK-MEL-30, harboring a heterozygous NRAS Q61K mutation, was unaffected by compound (A1). [Figure 14] SEC data for exemplary ADC formulations is shown. [Figure 15] 1 shows light shielding data for exemplary ADC formulations. [Figure 16-1] 1 shows capillary electrophoresis analysis of exemplary ADC formulations. [Figure 16-2] 1 shows capillary electrophoresis analysis of exemplary ADC formulations. [Figure 16-3] 1 shows capillary electrophoresis analysis of exemplary ADC formulations. [Figure 17-1] 1 shows capillary zone electrophoresis (CZE) analysis of exemplary ADC formulations. [Figure 17-2] 1 shows capillary zone electrophoresis (CZE) analysis of exemplary ADC formulations. [Figure 17-3] 1 shows capillary zone electrophoresis (CZE) analysis of exemplary ADC formulations. [Figure 18-1] 1 shows an analysis of an exemplary lyophilized ADC formulation. [Figure 18-2] 1 shows an analysis of an exemplary lyophilized ADC formulation. [Figure 18-3] 1 shows an analysis of an exemplary lyophilized ADC formulation. [Figure 19] Payload ring opening data using pH and (CZE) are shown. [Figure 20] 1 shows potency decay data for exemplary ADC formulations after a 2-month storage period. [Figure 21] 1 shows the mean exposure profiles of total antibody (tmAb), active ADC (tADCa), and inactive ADC (tADCi) following a single iv dose of 3 mg / kg of Compound (E) in cynomolgus monkeys. DETAILED DESCRIPTION OF THE INVENTION

[0206] The present disclosure is based, at least in part, on the discovery that genetic engineering of certain microorganisms (e.g., Chromobacterium) can increase production of certain depsipeptides (e.g., compound A1). While compound A1 can be isolated from A. crenata leaves, the extremely low abundance and complex substrate may prevent compound A1 from being available in sufficient quantities to enable drug development efforts and commercial production. The genetically engineered microorganisms (e.g., Chromobacterium), nucleic acids, and methods described herein provide exploitable manufacturing routes to produce compound A1 with increased potency and / or increased isolated yield, such that sufficient quantities of the drug substance precursor are available.

[0207] The present disclosure is also based, at least in part, on the discovery that by targeting higher over-reduction and designing a robust partial re-oxidation step, efficient decapping of engineered cysteine ​​residues and a desired drug-to-antibody ratio can be achieved without compromising antibody purity. Over-reduction and ring-opening of intrachain disulfide bridges can lead to undesired off-site coupling. In some embodiments, inherent LC-HC instability can make over-reduction more likely. Without wishing to be bound by theory, in some embodiments, efficient partial re-oxidation of opened intrachain disulfide bridges is believed to be important for minimizing off-site binding to achieve high in vivo efficacy and reduce linker payload release. Factors that can be evaluated with respect to the partial re-oxidation step include, but are not limited to, one or more of the following: differences in vessel geometry, the presence or absence of a defined headspace (e.g., 60%), the presence or absence of mixing (e.g., 100 rpm), or room temperature versus cold room. In some embodiments, the inclusion of the partial reoxidation step described herein in a GMP manufacturing process can increase the purity of the final drug substance to about 95%, for example, as determined by non-reducing CE SDS. In certain embodiments, a drug-to-antibody ratio of about 2 is achieved. In other embodiments, a drug-to-antibody ratio of about 4 is achieved.

[0208] definition Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings:

[0209] As used herein, the singular forms "a" or "an" include plural references unless otherwise indicated.

[0210] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly dictates otherwise.

[0211] "About" and "approximately" are generally intended to mean an acceptable degree of error for the quantity measured, given the nature or precision of the measurement. Exemplary degrees of error are within 20 percent (%), typically within 10%, and more typically within 5% of a given value or range of values.

[0212] The term "biosynthetic gene cluster" or "BGC" refers synonymously to a group of two or more locally clustered genes that together encode a biosynthetic pathway for the production of a metabolite. In some embodiments, the metabolite is a secondary or specialized metabolite that is not directly involved in the normal growth, development, or reproduction of the organism that produces it.

[0213] The term "alkyl" refers to a monovalent saturated hydrocarbon chain having the specified number of carbon atoms. For example, C1-C6 alkyl refers to an alkyl group having from 1 to 6 carbon atoms. Alkyl groups can be straight or branched. Representative branched alkyl groups have one, two, or three branches. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl, sec-butyl, and t-butyl), pentyl (n-pentyl, isopentyl, and neopentyl), and hexyl.

[0214] As used herein, "cleavable" refers to a linking group or linker moiety that connects two moieties by a covalent bond but degrades under physiologically relevant conditions to sever the covalent bond between the moieties; typically, a cleavable linking group is cleaved more rapidly in the intracellular environment than outside the cell in vivo, causing release of the payload to occur preferentially inside the targeted cell. Cleavage can be enzymatic or non-enzymatic, but generally releases the payload from the antibody without degrading the antibody. Cleavage can leave some portion of the linking group or linker moiety attached to the payload, or it can release the payload without any residue of the linking group.

[0215] As used herein, "non-cleavable" refers to a linking group or linker moiety that is not particularly susceptible to degradation under physiological conditions, e.g., it is at least as stable as the antibody or antigen-binding fragment portion of the conjugate. Such linking groups are sometimes referred to as "stable," meaning that they are sufficiently resistant to degradation to retain the payload linked to the antibody or antigen-binding fragment until the antibody or antigen-binding fragment itself is at least partially degraded, i.e., degradation of the antibody or antigen-binding fragment precedes cleavage of the linking group in vivo. Degradation of the antibody portion of an ADC having a stable or non-cleavable linking group may leave some or all of the linking group, e.g., one or more amino acid groups, from the antibody attached to the payload or drug moiety that is delivered in vivo.

[0216] The term "antibody" refers to a polypeptide of the immunoglobulin family that can non-covalently, reversibly, and specifically bind to a corresponding antigen. For example, naturally occurring IgG antibodies are tetramers containing at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). VH and VL each consist of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0217] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies (e.g., anti-Id antibodies to an antibody of the present disclosure). Antibodies can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0218] The terms "complementarity-determining domain" or "complementarity-determining region" ("CDR") interchangeably refer to the hypervariable regions of VL and VH. CDRs are the target protein-binding sites of antibody chains, which confers specificity for such target proteins. Three CDRs (numbered sequentially from the N-terminus, CDR1 to CDR3) are present in each human VL or VH, constituting approximately 15 to 20% of the variable domain. CDRs are structurally complementary to the epitope of the target protein and are therefore directly responsible for binding specificity. The remaining regions of VL or VH, the so-called framework regions, show less variation in amino acid sequence (Kuby, Immunology, 4th ed., Chapter 4. W.H. Freeman & Co., New York, 2000).

[0219] The locations of CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat, Chothia, the International ImMunoGeneTics database (IMGT) (on the World Wide Web at www.imgt.org / ), and AbM (see, e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997)). The definition of an antigen-binding site is also described in Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); and Lefranc, MP, Nucleic Acids Res., 29:207-209 (2001); MacCallum et al., J. Mol. Biol., 262:732-745 (1996); and Martin et al., Proc. Natl. Acad Sci. USA, 86:9268-9272 (1989); Martin et al., Methods Enzymol., 203:121-153 (1991); and Rees et al., In Sternberg MJE (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996).

[0220] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it is understood that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light (CL) and heavy (CH1, CH2, or CH3) chains confer important biological properties, such as secretion, transplacental motility, Fc receptor binding, complement binding, etc. By convention, the numbering of constant region domains increases distally from the antigen-binding site or amino-terminus of the antibody. The N-terminus is the variable region, and the C-terminus is the constant region; the CH3 and CL domains actually comprise the carboxy-terminal domains of the heavy and light chains, respectively.

[0221] The term "antigen-binding fragment" as used herein refers to one or more portions of an antibody that retain the ability to specifically interact with an epitope of an antigen (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of binding fragments include, but are not limited to, single-chain Fv (scFv), camelid antibody, disulfide-bridged Fv (sdFv), Fab fragment, F(ab') fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragment consisting of the VH and CH1 domains; Fv fragment consisting of the VL and VH domains of a single arm of an antibody; dAb fragment consisting of the VH domain (Ward et al., Nature 341:544-546, 1989); and isolated complementarity-determining regions (CDRs) or other epitope-binding fragments of an antibody.

[0222] Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods with a synthetic linker, allowing the two domains to be produced as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain Fv ("scFv"); see, e.g., Bird et al., Science 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. 85:5879-5883, 1988). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment." These antigen-binding fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0223] Antigen-binding fragments may also be incorporated into single domain antibodies, maxibodies, minibodies, single domain antibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments may be grafted into polypeptide-based scaffolds, such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies).

[0224] The antigen-binding fragment may be incorporated into a single-chain molecule comprising a pair of tandem Fv segments (VH-CH1-VH-CH1), which, together with complementary light chain polypeptides, form a pair of antigen-binding regions (Zapata et al., Protein Eng. 8:1057-1062, 1995; and U.S. Pat. No. 5,641,870).

[0225] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to polypeptides, including antibodies and antigen-binding fragments, having substantially the same amino acid sequence or derived from the same genetic source. The terms also include preparations of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0226] As used herein, the term "human antibody" includes antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region also is derived from a human sequence, e.g., a human germline sequence, or a mutated version of a human germline sequence, or an antibody-containing consensus framework sequence derived from human framework sequence analysis, e.g., as described in Knappik et al., J. Mol. Biol. 296:57-86, 2000. Also included are antibodies derived from human sequences in which one or more CDRs have been mutated for affinity maturation or manufacturing / payload-binding purposes. See Kilpatrick et al., "Rapid development of affinity matured monoclonal antibodies using RIMMS," Hybridoma. 1997 Aug;16(4):381-9.

[0227] The human antibodies of this disclosure may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo, or conservative substitutions that enhance stability or manufacturing).

[0228] As used herein, the term "recognize" refers to an antibody or antigen-binding fragment thereof finding and interacting with (e.g., binding to) its epitope, whether linear or conformational. The term "epitope" refers to a site on an antigen to which an antibody or antigen-binding fragment of the present disclosure specifically binds. Epitopes can be formed both from contiguous amino acids or from noncontiguous amino acids arranged by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of epitopes include techniques in the art, such as x-ray crystallography and two-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)). A "paratope" is the portion of an antibody that recognizes an epitope of an antigen.

[0229] The term "affinity" refers to the strength of the interaction between an antibody and an antigen at a single antigenic site. Within each antigenic site, the variable regions of the antibody "arms" interact with the antigen at multiple sites through weak non-covalent forces; the more interactions, the stronger the affinity.

[0230] The term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities. However, an isolated antibody that specifically binds to an antigen may have cross-reactivity to other antigens. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0231] The term "corresponding human germline sequence" refers to a nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that shares the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence, as determined in comparison with all other known variable region amino acid sequences encoded by human germline immunoglobulin variable region sequences. Corresponding human germline sequence may also refer to the human variable region amino acid sequence or subsequence that shares the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence, as determined in comparison with all other variable region amino acid sequences evaluated. The corresponding human germline sequence may be framework regions only, complementarity determining regions only, framework and complementarity determining regions, variable segments (as defined above), or other combinations of sequences or subsequences that comprise variable regions. Sequence identity can be determined using methods described herein, such as aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art. The corresponding human germline nucleic acid or amino acid sequence can have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid or amino acid sequence of the reference variable region. Corresponding human germline sequences can be determined, for example, through the publicly available International ImMunoGeneTics database (IMGT) (on the World Wide Web at www.imgt.org / ) and V-base (on the World Wide Web at vbase.mrc-cpe.cam.ac.uk).

[0232] The phrases "specifically bind" or "selectively bind," when used in the context of describing the interaction of an antigen (e.g., a protein) with an antibody, antibody fragment, or antibody-derived binding agent, refer to a binding reaction that is determinative of the presence of the antigen in a heterogeneous population of proteins and other biologics, e.g., in a biological sample, such as a blood, serum, plasma, or tissue sample. Thus, under specified, specific immunoassay conditions, an antibody or binding agent with a specific binding specificity will bind to the specific antigen at least twice as much as background and will not significantly bind to other antigens present in the sample. In one embodiment, under specified, specific immunoassay conditions, an antibody or binding agent with a specific binding specificity will bind to the specific antigen at least 10 times as much as background and will not significantly bind to other antigens present in the sample. Specific binding to an antibody or binding agent under such conditions may require that the antibody or agent be selected for its specificity for a particular protein. If desired or appropriate, this selection can be achieved by subtracting out antibodies from other species (e.g., mouse or rat) or other subtypes that cross-react with the molecule. Additionally, in some embodiments, antibodies or antibody fragments are selected that cross-react with a particular molecule of interest.

[0233] A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, *Using Antibodies*, *A Laboratory Manual* (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, a specific or selective binding reaction will produce a signal at least two times the background signal, more typically at least 10-100 times the background signal.

[0234] The term "equilibrium dissociation constant (Kd, M)" refers to the dissociation rate constant (kd, time-1) divided by the association rate constant (ka, time-1, M-1). The equilibrium dissociation constant can be measured using any method known in the art. Antibodies of the present disclosure typically have an equilibrium dissociation constant of about 10 -7 or 10 -8 Less than M, e.g., about 10 -9 M or 10 -10 less than about 10 M, in some embodiments -11 M, 10 -12 M or 10 -13 It will be less than M.

[0235] The term "bioavailability" refers to the systemic availability (i.e., blood / plasma levels) of a given amount of drug administered to a patient. Bioavailability is an absolute value that indicates measurement of both the time (rate) and total amount (extent) of drug that reaches the systemic circulation from an administered dosage form.

[0236] As used herein, the phrase "consisting essentially of" refers to the genus or species of active agent included in a method or composition and any excipients that are inert for the intended use of the method or composition. In some embodiments, the phrase "consisting essentially of" explicitly excludes the inclusion of one or more additional active agents other than the antibody drug conjugates of the present disclosure. In some embodiments, the phrase "consisting essentially of" explicitly excludes the inclusion of one or more additional active agents other than the antibody drug conjugates of the present disclosure and a second co-administered agent.

[0237] The term "amino acid" refers to naturally occurring, synthetic, and unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. An amino acid analog refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, or methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. An amino acid mimetic refers to a compound that has a structure different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid.

[0238] The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to nucleic acids that encode the same or essentially the same amino acid sequence, or, if the nucleic acid does not encode an amino acid sequence, to essentially the same sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variation. Additionally, all nucleic acid sequences herein that encode a polypeptide describe all possible silent variations of the nucleic acid. Those of skill in the art will recognize that each codon in a nucleic acid (except AUG, which is normally the only codon for methionine, and TGG, which is normally the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0239] With respect to polypeptide sequences, "conservatively modified variants" include individual substitutions, deletions, or additions to a polypeptide sequence that substitute an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the present disclosure. The following eight groups contain amino acids that are conservative substitutions for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, e.g., Creighton, Proteins (1984)). In some embodiments, the term "conservative sequence modifications" is used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence.

[0240] As used herein, the term "optimized" refers to a nucleotide sequence that has been altered to encode an amino acid sequence using codons preferred in a production cell or organism, usually a eukaryotic cell, such as a yeast cell, a Pichia cell, a fungal cell, a Trichoderma cell, a Chinese hamster ovary cell (CHO), or a human cell. An optimized nucleotide sequence has been engineered to retain, entirely, or as closely as possible, the amino acid sequence originally encoded by the starting nucleotide sequence, also known as the "parent" sequence.

[0241] The terms "percent identical" or "percent identity," in the context of two or more nucleic acid or polypeptide sequences, refer to the degree to which two or more sequences or subsequences are the same. Two sequences are "identical" if the amino acid or nucleotide sequence is the same over the region being compared. Two sequences are "substantially identical" if a specified percentage of amino acid residues or nucleotides are the same (i.e., 60% identity, or in some cases 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, if not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using one of the sequence comparison algorithms described below or by manual alignment and visual inspection. In some cases, identity exists over a region that is at least about 30 nucleotides (or 10 amino acids) in length, more preferably over a region that is 100-500 nucleotides or 1000 nucleotides or more (or 20, 50, 200 amino acids or more) in length.

[0242] For sequence comparison, typically, one sequence serves as a reference sequence to which test sequences are compared. Using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

[0243] As used herein, a "comparison window" includes reference to any one segment of the number of contiguous positions selected from the group consisting of 20 to 600, usually about 50 to about 200, and more usually about 100 to about 150, within which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of sequence alignment for comparison are well known in the art. Optimal sequence alignment for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482c (1970), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443, by the similarity search method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Brent et al., Current Protocols in Molecular Biology, 2003).

[0244] Two examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST algorithm and the BLAST 2.0 algorithm, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively. Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in a database sequence, match or meet some positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. Word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction is halted when: the cumulative alignment score falls by an amount X from its maximum achieved value; the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program defaults to a word length of 3 and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915) uses an alignment (B) of 50, an expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

[0245] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0246] The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4:11-17 (1988), which is incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48:444-453 (1970), which is incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a BLOSUM62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0247] Another indication that two nucleic acid sequences or polypeptides are substantially identical, other than the percentage of sequence identity noted above, is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, when the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.

[0248] The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, including synthetic, naturally occurring, and non-naturally occurring nucleic acids, which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs).

[0249] Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly set forth. Specifically, as detailed below, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al., (1985) J. Biol. Chem. 260:2605-2608; and Rossolini et al., (1994) Mol. Cell. Probes 8:91-98).

[0250] The term "operably linked" in the context of nucleic acids refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence with a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. Typically, a promoter transcriptional regulatory sequence that is operably linked to a transcribed sequence is physically contiguous to the transcribed sequence, i.e., cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically adjacent to or positioned closely adjacent to the coding sequence whose transcription they enhance.

[0251] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise specified, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

[0252] As used herein, the term "antibody-drug conjugate" or "immunoconjugate" refers to the attachment of an antibody or antigen-binding fragment thereof to another agent, such as a chemotherapeutic agent, a toxin, an immunotherapeutic agent, an imaging probe, etc. Attachment can be via a covalent bond or a non-covalent interaction, such as electrostatic forces. Various linkers known in the art can be used to form antibody-drug conjugates. Furthermore, antibody-drug conjugates can be provided in the form of a fusion protein that can be expressed from a polynucleotide encoding the immunoconjugate. As used herein, "fusion protein" refers to a protein created through the joining of two or more genes or gene fragments that originally encoded separate proteins (e.g., peptides and polypeptides). Translation of the fusion gene results in a single protein possessing functional properties derived from each of the original proteins.

[0253] The term "subject" includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. Except where indicated, the terms "patient" or "subject" are used interchangeably herein.

[0254] The term "cytotoxin" or "cytotoxic agent" as used herein refers to any agent that is detrimental to the growth and proliferation of cells and can act to reduce, inhibit, or destroy cells or malignancies.

[0255] As used herein, the term "anti-cancer agent" refers to any agent that can be used to treat or prevent a cell proliferative disorder, e.g., cancer, including, but not limited to, cytotoxic agents, chemotherapeutic agents, radiation therapy and radiotherapeutic agents, targeted anti-cancer agents, and immunotherapeutic agents.

[0256] The term "drug moiety" or "payload," as used herein, refers to a chemical moiety attached to an antibody or antigen-binding fragment of the present disclosure and can include any therapeutic or diagnostic agent, such as an anti-cancer, anti-inflammatory, anti-infective (e.g., anti-fungal, anti-bacterial, anti-parasitic, anti-viral), or anesthetic. For example, the drug moiety can be an anti-cancer agent, such as a cytotoxin. In certain embodiments, the drug moiety is a target inhibitor compound. Furthermore, the payload can be a biophysical probe, a fluorescent label, a spin label, an infrared probe, an affinity probe, a chelator, a spectroscopic probe, a radioactive probe, a lipid molecule, polyethylene glycol, a polymer, a spin label, DNA, RNA, a protein, a peptide, a surface, an antibody, an antibody fragment, a nanoparticle, a quantum dot, a liposome, a PLGA particle, a sugar, or a polysaccharide.

[0257] In some embodiments, the drug moiety or payload is a GNAQ inhibitor, a GNA11 inhibitor, or an inhibitor of GNAQ and GNA11 (GNAQ / GNA11 inhibitor). In some embodiments, the GNAQ / 11 inhibitor is a molecule that inhibits GNAQ / 11-mediated production of IP3 and / or exhibits a dose-response antiproliferative effect in cells dependent on GNAQ / 11 signaling (i.e., GNAQ / 11-mutant uveal melanoma cells). In some embodiments, the GNAQ / 11 inhibitor is a compound that stabilizes GNAQ / 11 in an inactive GDP-bound state, preventing GDP release, or binds to an active GTP-bound state, preventing GNAQ / 11 interaction with downstream effectors. In some embodiments, the GNAQ / 11 inhibitor functions by inhibiting mutant GNAQ and / or GNA11, e.g., those containing the Q209L / P mutation. Methods for attaching such drug moieties to linkers compatible with targeting moieties are listed in this disclosure along with methods known in the art, see, e.g., Singh et al., (2009) Therapeutic Antibodies: Methods and Protocols, vol. 525, 445-457.

[0258] GNAQ (guanine nucleotide-binding protein G(q) subunit alpha, also known as CMC1, G-ALPHA-q, GAQ, SWS, and G protein subunit alpha q) and GNA11 (guanine nucleotide-binding protein subunit alpha-11, also known as FBH, FBH2, FHH2, GNA-11, HHC2, HYPOC2, and G protein subunit alpha 11) are closely related GTPases that constitute the α subunits of heterotrimeric G proteins that act downstream of G protein-coupled receptors (GPCRs). The α subunit acts as a switch for G protein activation by exchanging guanosine diphosphate (GDP) for guanosine triphosphate (GTP), leading to the activation of distinct downstream effectors. Activation is terminated by intrinsic GTPase activity. This is because GTP is hydrolyzed to GDP (Van Raamsdonk et al., 2010, N Engl J Med.;363(23):2191-9). Classical activation of the Gq protein cascade occurs via phospholipase C-β (PLC-β), which hydrolyzes the phospholipid phosphatidylinositol 4,5-bisphosphate to release two potent second messengers: D-myo-inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). Intracellular Ca 2+ After a transient increase in DAG, IP3 is rapidly converted to IP2, IP1, and myo-inositol. Meanwhile, DAG activates protein kinase C (PKC), leading to a cascade of phosphorylation of RAF, MEK, and ERK, which translocate to the nucleus and regulate cell proliferation and survival (Krantz et al., 2017, Clin Ophthalmol.;11:279-289).

[0259] Exemplary amino acid and nucleotide sequences of human GNAQ are published in GenBank under accession numbers NP_002063 and NM_002072, respectively. Exemplary amino acid and nucleotide sequences of human GNAQ are also listed in WO 2020 / 128612 as SEQ ID NOs: 268 and 269, respectively.

[0260] Exemplary amino acid and nucleotide sequences of human GNA11 are published in GenBank under accession numbers NP_002058 and NM_002067, respectively. Exemplary amino acid and nucleotide sequences of human GNA11 are also listed in WO 2020 / 128612 as SEQ ID NOs: 270 and 271, respectively.

[0261] "Tumor" refers to neoplastic cell growth and proliferation, whether malignant or benign, including all pre-cancerous and cancerous cells and tissues.

[0262] The term "anti-tumor activity" refers to a reduction in the rate of tumor cell proliferation, survival, or metastatic activity. For example, anti-tumor activity can be demonstrated by a decrease in the rate of growth of abnormal cells or a stable or reduced tumor size during treatment, or a longer survival period resulting from treatment compared to a control without treatment. Such activity can be assessed using recognized in vitro or in vivo tumor models, including, but not limited to, xenograft models, allograft models, MMTV models, and other known models known in the art for investigating anti-tumor activity.

[0263] The term "malignant tumor" refers to a non-benign tumor or cancer. As used herein, the term "cancer" includes malignant tumors characterized by deregulated or uncontrolled cell growth. Exemplary cancers include, but are not limited to, carcinoma (e.g., hepatocellular carcinoma), sarcoma, leukemia, lymphoma, eye cancer, ocular neoplasm, and melanoma (e.g., uveal melanoma, non-uveal melanoma, malignant melanoma, ocular melanoma, subcutaneous melanoma, cutaneous melanoma, or mucosal melanoma).

[0264] The term "cancer" includes primary malignant tumors (e.g., those whose cells have not migrated to sites in a subject's body other than the site of the original tumor) and secondary malignant tumors (e.g., those resulting from metastasis, which is the migration of tumor cells to secondary sites different from the site of the original tumor).

[0265] The term "PMEL17" (also known as pre-melanosome protein (PMEL), D12S53E, ME20, ME20-M, ME20M, P1, P100, gp100, SI, SIL, and silver locus protein homolog (SILV)) refers to a type I single-pass transmembrane protein produced by melanocytes and involved in melanin synthesis. Exemplary amino acid and nucleic acid sequences of human PMEL17 have been published in GenBank under the following accession numbers: NP_008859, NP_001307050, NP_001307051, NP_001186982, NP_001186983 (amino acid sequences), and NM_006928, NM_001200053, NM_001200054, NM_001320121, NM_001320122 (nucleotide sequences). Exemplary amino acid and nucleotide sequences of human PMEL17 are also set forth in WO 2020 / 128612 as SEQ ID NOs: 272-276 (amino acid sequences) and SEQ ID NOs: 277-281 (nucleotide sequences). As used herein, the term "PMEL17" is used collectively to refer to any naturally occurring isoform of the PMEL17 protein, or variants thereof.

[0266] The term "variant" refers to a polypeptide that has substantially the same amino acid sequence as a reference polypeptide, or is encoded by substantially the same nucleotide sequence, and may have one or more activities of the reference polypeptide. For example, a variant may have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the reference polypeptide, while retaining one or more activities of the reference polypeptide.

[0267] As used herein, the terms "treat," "treating," or "treatment" of any disease or disorder refer, in one embodiment, to ameliorating the disease or disorder (i.e., slowing, inhibiting, or alleviating the progression of the disease or at least one of its clinical symptoms). In another embodiment, "treat," "treating," or "treatment" refers to alleviating or improving at least one physical parameter, including those that may not be discernible by the patient. In yet another embodiment, "treat," "treating," or "treatment" refers to modulating the disease or disorder physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of a physical parameter), or both.

[0268] As used herein, the terms "prevent," "preventing," and "prevention" of any disease or disorder refer to prophylactic treatment of a disease or disorder or delaying the progression of a disease or disorder.

[0269] The terms "therapeutically acceptable amount" or "therapeutically effective dose" interchangeably refer to an amount sufficient to produce a desired result (i.e., reduction in tumor size, inhibition of tumor growth, prevention of metastasis, inhibition or prevention of viral, bacterial, fungal, or parasitic infection). In some embodiments, a therapeutically acceptable amount does not induce or cause undesirable side effects. In some embodiments, a therapeutically acceptable amount induces or causes side effects, but only side effects that are tolerable by a healthcare provider in light of the patient's condition. A therapeutically acceptable amount may be determined by administering an initial low dose and gradually increasing the dose until the desired effect is achieved. A "prophylactically effective dose" and a "therapeutically effective dose" of a molecule of the present disclosure may prevent the onset of or reduce the severity of disease symptoms, for example, cancer-related disease symptoms, respectively.

[0270] The term "co-administration" refers to the simultaneous presence of two active agents in the blood of an individual. The active agents may be delivered simultaneously or sequentially.

[0271] The terms "inhibition," "inhibitor," "binding antagonist," or "antagonist" include a decrease in some parameter, e.g., activity, of a given molecule. For example, at least 5%, 10%, 20%, 30%, 40% or more inhibition of the activity of a given molecule is included within the term. Thus, inhibition need not be 100%.

[0272] The terms "activation," "activator," or "agonist" include an increase in some parameter, e.g., activity, of a given molecule. For example, an increase in the activity of a given molecule by at least 5%, 10%, 25%, 50%, 75%, or more is included in the term.

[0273] Chromobacterium Chromobacterium is a genus of Gram-negative bacilli. Currently, 11 species are known within this genus: C. alkanivorans, C. amazonense, C. aquaticum, C. haemolyticum, C. phragmitis, C. piscinae, C. pseudoviolaceum, C. rhizoryzae, C. subtsugae, C. vaccinii, and C. violaceum.

[0274] In some embodiments, the Chromobacterium is C. vaccinii. C. vaccinii was isolated from cranberry plant soil (Soby et al. (2013) Int J Syst Evol Microbiol.;63(5):1840-6) and deposited in both the DSMZ and ATCC strain collections (Chromobacterium vaccinii DSM 25150 = ATCC BAA-2314, = NRRL B-50840, = MWU205). The GenBank accession number for the 16S rRNA gene sequence of this strain is JN120869. The draft genome of C. vaccinii DSM 25150 was published by Voeing et al. (2015) Genome Announc 3, e00477-15 (GenBank accession number: NZ_JZJL00000000).

[0275] C. vaccinii grows readily on nutrient agar, producing distinctive smooth, low-convex colonies with a dark purple metallic luster due to the production of violacein after 2 days of incubation at 28°C. C. vaccinii does not appear to cause any disease in healthy adult humans and poses minimal risk to laboratory personnel and the environment.

[0276] Microorganisms described herein include, for example, mutant forms of C. vaccinii that naturally produce depsipeptides, such as compound (A1). In some embodiments, the C. vaccinii mutant comprises a mutation that increases the titer of compound (A1). In some embodiments, the C. vaccinii mutant comprises a mutation that increases the isolated yield of compound (A1). In some embodiments, the C. vaccinii mutant comprises a mutation that increases the titer of compound (A1) and a mutation that increases the isolated yield of compound (A1). In some embodiments, the C. vaccinii mutant comprises a promoter insertion mutation (e.g., a promoter insertion mutation described herein). In some embodiments, the C. vaccinii mutant comprises a disruption mutation (e.g., a disruption mutation described herein). In some embodiments, the C. vaccinii mutant comprises multiple disruption mutations (e.g., multiple disruption mutations described herein). In some embodiments, the C. vaccinii mutant comprises a promoter insertion mutation (e.g., a promoter insertion mutation described herein) and a disruption mutation (e.g., a disruption mutation described herein). In some embodiments, the C. vaccinii mutant comprises a promoter insertion mutation (e.g., a promoter insertion mutation described herein) and multiple disruption mutations (e.g., multiple disruption mutations described herein).

[0277] Promoter insertion mutants In some embodiments, the C. vaccinii mutant exhibits increased titer of Compound (A1) compared to a reference C. vaccinii, e.g., wild-type C. vaccinii, when cultured under the same or similar conditions. For example, the native promoter upstream of Compound (A1)-BGC can be modified, e.g., replaced, with a non-native promoter, e.g., a stronger non-native promoter. Without wishing to be bound by theory, Compound (A1)-BGC is believed to contain a single operon, the transcription of which is believed to be driven by a single promoter.

[0278] A non-native promoter may be any promoter sequence that is not operably linked to compound (A1)-BGC in nature and that, when so operably linked, increases the transcriptional activity of compound (A1)-BGC.

[0279] In some embodiments, the non-native promoter is from C. vaccinii, e.g., a promoter controlling expression of a BGC other than compound (A1)-BGC, e.g., the vioP promoter. In other embodiments, the non-native promoter is not from C. vaccinii. In some embodiments, the non-native promoter is from E. coli.

[0280] In some embodiments, the non-native promoter is a constitutive promoter. In some embodiments, the non-native promoter is an inducible promoter. In some embodiments, the promoter, for example, the vioP promoter, is induced by the production of homoserine lactones. In some embodiments, the non-native promoter is a violacein promoter. In some embodiments, the non-native promoter is a ribosomal promoter. In some embodiments, the non-native promoter is located in a transposon.

[0281] In certain embodiments, the non-native promoter is a naturally occurring promoter. In other embodiments, the non-native promoter is a synthetic promoter. In other embodiments, the non-native promoter is a consensus promoter, e.g., one that comprises the consensus sequence of a group of related naturally occurring promoters.

[0282] In some embodiments, the non-native promoter is the vioP promoter. In some embodiments, the non-native promoter is the nptII promoter. In some embodiments, the non-native promoter is the rbs promoter.

[0283] Other non-native promoters include, but are not limited to, the J23119 promoter (e.g., as described in parts.igem.org), the pLpp promoter (e.g., as described in parts.igem.org), the PS12burk promoter (e.g., as described in Choi et al. (2008) App Environ Microbiol 74(4):1064-75), the Erme ... pLpp promoter (e.g., as described in parts.ig * promoter (e.g., as described in Bibb et al. (1994) Mol Microbiol 14(3):533-45), or the Pem7 promoter (e.g., as described in Zobel et al. (2015) ACS Synthetic Biology 4:1341-51).

[0284] In some embodiments, the non-native promoter is a promoter listed in Table 1. In some embodiments, the heterologous promoter comprises a nucleotide sequence listed in Table 1, or a functional fragment thereof. In some embodiments, the heterologous promoter comprises the nucleotide sequence of SEQ ID NO: 264, or a sequence having at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto, or a functional fragment thereof. In some embodiments, the heterologous promoter comprises the nucleotide sequence of SEQ ID NO: 265, or a sequence having at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto, or a functional fragment thereof. In some embodiments, the heterologous promoter comprises the nucleotide sequence of SEQ ID NO: 266, or a sequence having at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto, or a functional fragment thereof. In some embodiments, the heterologous promoter comprises the nucleotide sequence of SEQ ID NO: 316, or a sequence having at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto, or a functional fragment thereof. In some embodiments, the heterologous promoter comprises the nucleotide sequence of SEQ ID NO: 268, or a sequence having at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto, or a functional fragment thereof. In some embodiments, the heterologous promoter comprises the nucleotide sequence of SEQ ID NO: 269, or a sequence having at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto, or a functional fragment thereof.In some embodiments, the heterologous promoter comprises the nucleotide sequence of SEQ ID NO: 270, or a sequence having at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto, or a functional fragment thereof. In some embodiments, the heterologous promoter comprises the nucleotide sequence of SEQ ID NO: 271, or a sequence having at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto, or a functional fragment thereof.

[0285] [Table 1]

[0286] For example, the strain Chromobacterium vaccinii vioP-frsA is a mutant generated from the strain Chromobacterium vaccinii DSM 25150 by inserting the promoter vioP in front of the compound (A1)-BGC to create a translational fusion. The promoter vioP was extracted from the native violacein BGC of this strain. The nucleotide sequence of the promoter vioP is shown in Table 1. The generation of this mutant is described in Example 1.

[0287] The violacein promoter from C. vaccinii and that from C. violaceum share essentially the same function and approximately 82% sequence identity. The vioP promoter from C. violaceum is described, for example, in Morohoshi et al. (2010) Bioscie. Biotechnol. Biochem.; 74(10), 2116-2119 and Zhang et al. (2011) Appl Microbiol Biotechnol; 90:1963-1971.

[0288] As another example, the strain Chromobacterium vaccinii nptII-frsA is a mutant generated from the strain Chromobacterium vaccinii DSM 25150 by inserting the promoter nptII in front of compound (A1)-BGC to create a translational fusion. The promoter nptII is a constitutive promoter. The nucleotide sequence of the promoter nptII is shown in Table 1. The generation of this mutant is described in Example 1.

[0289] As yet another example, the strain Chromobacterium vaccinii rbs-frsA is a mutant generated from the strain Chromobacterium vaccinii DSM 25150 by inserting the promoter rbs in front of compound (A1)-BGC to create a translational fusion. The ribosomal promoter rbs is a constitutive promoter. The nucleotide sequence of the promoter rbs is shown in Table 1. The generation of this mutant is described in Example 1.

[0290] As yet another example, the promoter vioP, nptII, or rbs may be S12 burk, Erme * , or pLpp. Promoter J23119 is a synthetic promoter. The nucleotide sequence of promoter J23119 is shown in Table 1. Promoter P S12 burk is a ribosomal promoter from Burkholderia thailandensis. S12 The nucleotide sequence of burk is shown in Table 1. S12 Burk is also described in Choi et al. (2008) App Environ Microbiol 74(4):1064-75).S12 Burk may also be known as S12burk. Promoter ErmE * is the 23S rRNA methylase promoter from Saccharopolyspora erythraea. Promoter ErmE * The nucleotide sequence of the promoter ErmE is shown in Table 1. * is also described and described in Bibb et al. (1994) Mol Microbiol 14(3):533-45. Promoter pLpp is a synthetic promoter. The nucleotide sequence of promoter pLpp is shown in Table 1. Promoter pLpp is also described in parts.igem.org. pLpp may also be known as Plpp.

[0291] In some embodiments, the potency of Compound (A1) in the C. vaccinii promoter insertion mutant is increased by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold compared to the potency of a reference C. vaccinii, e.g., C. vaccinii not having the promoter insertion mutation in Compound (A1)-BGC or wild-type C. vaccinii, when cultured under identical conditions. In some embodiments, the potency of Compound (A1) in the C. vaccinii promoter insertion mutant is increased by at least about 3-fold, e.g., at least about 3.5-fold, compared to the potency of Compound (A1) in wild-type C. vaccinii.

[0292] In some embodiments, the potency of Compound (A1) of the C. vaccinii promoter insertion mutant is increased by at least about 100 mg / L, about 200 mg / L, about 300 mg / L, about 400 mg / L, about 500 mg / L, about 600 mg / L, about 700 mg / L, about 800 mg / L, about 900 mg / L, or about 1000 mg / L compared to the potency of a reference C. vaccinii, e.g., a C. vaccinii that does not have a promoter insertion mutation in Compound (A1)-BGC or a wild-type C. vaccinii, when cultured under identical conditions. In some embodiments, the potency of Compound (A1) in a C. vaccinii promoter insertion mutant is increased, for example, from about 300 mg / L or less to at least about 600 mg / L or at least about 800 mg / L, compared to the potency of Compound (A1) in wild-type C. vaccinii. In some embodiments, the potency of Compound (A1) in a C. vaccinii promoter insertion mutant is increased, for example, to at least about 200 mg / L, about 250 mg / L, about 300 mg / L, about 350 mg / L, about 400 mg / L, about 450 mg / L, about 500 mg / L, about 550 mg / L, about 600 mg / L, about 650 mg / L, about 700 mg / L, In some embodiments, the potency of Compound (A1) in a C. vaccinii mutant is at least about 800 mg / L, e.g., about 800 mg / L to about 850 mg / L. In some embodiments, the potency of compound (A1) of the C. vaccinii promoter insertion mutant is at least about 600 mg / L, for example, about 600 mg / L to about 650 mg / L, when cultured, for example, in an animal component-free medium.

[0293] The C. vaccinii promoter insertion mutants described herein can be used for small-scale (e.g., research scale) or large-scale (e.g., production scale) production of compound (A1). In some embodiments, compound (A1) is produced in a fermentor or bioreactor. In some embodiments, compound (A1) is produced in a fermentation volume of about 50 L to about 250 L, e.g., about 50 L to about 100 L, about 100 L to about 150 L, or about 150 L to about 250 L, e.g., about 50 L, about 100 L, about 150 L, about 200 L, or about 250 L. In some embodiments, compound (A1) is produced in a fermentation volume of about 1,000 L to about 20,000 L, e.g., about 1,000 L to about 10,000 L or about 10,000 L to about 20,000 L, e.g., about 1,000 L, about 2,000 L, about 5,000 L, about 7,500 L, about 10,000 L, about 15,000 L, or about 20,000 L.

[0294] Destruction mutants Alternatively, or in combination, in some embodiments, the C. vaccinii mutant exhibits an increased isolated yield of compound (A1) compared to wild-type C. vaccinii when cultured under identical conditions. For example, the level or activity of another natural product produced by C. vaccinii may be reduced, e.g., the BGC of such a natural product may be disrupted. Without wishing to be bound by theory, it is believed that in addition to compound (A1) and its analogs, C. vaccinii produces at least two additional classes of natural products that may interfere to some extent with the purification of compound (A1). They belong to the violacein class and the cyclic lipodepsipeptide class, with compound J being the most abundant. In some embodiments, the isolated yield of compound (A1) may be increased by reducing the complexity of by-products that interfere with the purification of compound (A1).

[0295] In some embodiments, the BGC of violacein is altered, e.g., disrupted, or one or more genes of the violacein-BGC are knocked out. In some embodiments, the level or activity of violacein is reduced, e.g., eliminated.

[0296] Violacein is a purple pigment with a bis-indole structure common to most Chromobacteria, hence the name of the genus. Violacein has the following chemical structure: [ka]

[0297] Its biosynthesis has been elucidated, and the respective BGCs are described, for example, in August et al. (2000) J Mol Microbiol Biotechnol.;2(4):513-519. The extraction process is troublesome because the large amount of violacein present in the fermentation broth precipitates after each evaporation step, making subsequent solubilization time-consuming and labor-intensive. Therefore, size exclusion chromatography can be introduced as a first purification step to remove violacein and avoid further precipitation.

[0298] In some embodiments, the BGC of Compound J is altered, e.g., disrupted, or one or more genes of Compound J-BGC are knocked out. For example, production of one or more (e.g., two, three, four, or more) compounds from Compound J-BGC (e.g., any one, two, three, or all of Compound J, Compound F5, Compound F3, or Compound D) is reduced, e.g., eliminated. In some embodiments, production of Compound J, Compound F5, Compound F3, and Compound D is reduced, e.g., eliminated.

[0299] In some embodiments, the level or activity of Compound J is reduced, e.g., eliminated. In some embodiments, the BGC of Compound J is altered, e.g., disrupted, or one or more genes of the Compound J-BGC are knocked out.

[0300] Compound J has the following chemical structure: [ka]

[0301] Retrobiosynthetic analysis of compound J (a cyclic hexadepsipeptide with the biosynthetic sequence N-acyl-Ser-Val-His-Val-Leu-Val (SEQ ID NO: 318)) allowed the identification of its BGC from the sequenced genome of C. vaccinii. Upon size-exclusion chromatography, members of the compound J class co-elute with compound (A1)-rich fractions, which creates an extremely high viscosity (almost gel-like) upon solubilization for further purification by preparative HPLC, preventing injection onto the column. Consequently, fractions typically require further dilution, limiting the amount of compound processed in each run.

[0302] In some embodiments, the level or activity of compound F5 is reduced, eg, eliminated.

[0303] Compound F5 has the following chemical structure: [ka]

[0304] Compound F5 has the sequence N-(Z)-dec-3-enoyl- 1 Ser- 2 Val- 3 His- 4 Val- 5 Leu- 6 Val (SEQ ID NO: 321) is a linear lipopeptide. 1 Ser hydroxyl group and 6 The lactone between the C-terminal carboxyl group of Val is hydrolyzed.

[0305] In some embodiments, the level or activity of compound F3 is reduced, eg, eliminated.

[0306] Compound F3 has the following chemical structure: [ka]

[0307] Compound F3 has the sequence N-octanoyl- 1 Ser- 2 Val- 3 His- 4 Val- 5 Leu- 6 Val (SEQ ID NO: 322) cyclic lipodepsipeptide. 1 Ser hydroxyl group and 6 A hexapeptide lactone ring is formed between the C-terminal carboxyl group of Val and the hydroxyl group of Val.

[0308] In some embodiments, the level or activity of Compound D is reduced, eg, eliminated.

[0309] Compound D has the following chemical structure: [ka]

[0310] Compound D has the sequence N-octanyl- 1 Ser- 2 Val- 3 His- 4 Val- 5 Leu- 6 A linear lipopeptide of Val (SEQ ID NO: 323). Compared to compound F3 1 Ser hydroxyl group and 6 The lactone between the C-terminal carboxyl group of Val is hydrolyzed.

[0311] In some embodiments, the C. vaccinii disruption mutant has two or more (e.g., three or four) disruption mutations. For example, both the violacein-BGC and the compound J-BGC can be modified, e.g., disrupted.

[0312] In some embodiments, the isolated yield of compound (A1) from a C. vaccinii disruption mutant is increased by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%, or at least about 1-, 2-, 3-, 4-, or 5-fold compared to the isolated yield of a reference C. vaccinii, e.g., C. vaccinii not having the disruption mutation or wild-type C. vaccinii, when cultured under identical conditions. In some embodiments, the isolated yield of compound (A1) from a C. vaccinii disruption mutant is at least about 60%, e.g., about 65% to about 70%. In some embodiments, the isolated yield of compound (A1) from wild-type C. vaccinii is about 35% to about 45%, for example, about 40%.

[0313] In some embodiments, the potency of compound (A1) of a C. vaccinii disruption mutant does not differ by more than about 50%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, or about 10% compared to the potency of a reference C. vaccinii, e.g., a C. vaccinii that does not have a disruption mutation or a wild-type C. vaccinii, when cultured under identical conditions. In some embodiments, the potency of compound (A1) in a C. vaccinii disruption mutant is at least about 200 mg / L, about 250 mg / L, about 300 mg / L, about 350 mg / L, about 400 mg / L, about 450 mg / L, about 500 mg / L, about 550 mg / L, about 600 mg / L, about 650 mg / L, about 700 mg / L, about 7 ... 50 mg / L, about 800 mg / L, about 850 mg / L, about 900 mg / L, about 950 mg / L, about 1000 mg / L, about 1100 mg / L, about 1200 mg / L, about 1300 mg / L, about 1400 mg / L, about 1500 mg / L, about 1600 mg / L, about 1700 mg / L, about 1800 mg / L, about 1900 mg / L, or about 2000 mg / L. In some embodiments, the potency of compound (A1) in a C. vaccinii disruption mutant is at least about 800 mg / L, for example, from about 800 mg / L to about 850 mg / L. In some embodiments, the potency of compound (A1) in a C. vaccinii disruption mutant is at least about 600 mg / L, for example, about 600 mg / L to about 650 mg / L, when cultured, for example, in an animal component-free medium.

[0314] The C. vaccinii disruption mutants described herein can be used for small-scale (e.g., research scale) or large-scale (e.g., production scale) production of compound (A1). In some embodiments, compound (A1) is produced in a fermentor or bioreactor. In some embodiments, compound (A1) is produced in a fermentation volume of about 50 L to about 250 L, e.g., about 50 L to about 100 L, about 100 L to about 150 L, or about 150 L to about 250 L, e.g., about 50 L, about 100 L, about 150 L, about 200 L, or about 250 L. In some embodiments, compound (A1) is produced in a fermentation volume of about 1,000 L to about 20,000 L, e.g., about 1,000 L to about 10,000 L or about 10,000 L to about 20,000 L, e.g., about 1,000 L, about 2,000 L, about 5,000 L, about 7,500 L, about 10,000 L, about 15,000 L, or about 20,000 L.

[0315] Biosynthesis of compound (A1) The depsipeptide, compound A1, was described in Fujioka et al. (1988) J. Org. Chem.; 53(12) 2820-2825. It was isolated from a methanol extract of the whole plant of Ardisia crenata. Nearly 20 years later, it was discovered that compound A1 is actually produced by the strictly obligate bacterial endosymbiont of the plant, Ardisia crenata, Candidatus Burkholderia crenata (Carlier et al. (2016) Environ Microbiol.; 18(8):2507-22; GenBank accession number LGTG01000643.1 for the DNA sequence of the biosynthetic gene cluster (BGC) encoding the biosynthetic genes responsible for the biosynthesis of compound A1). The biosynthesis of compound (A1) by heterologous expression in Escherichia coli (E. coli) is described, for example, in Cruesemann et al. (2018) Angew. Chem. Int. Ed.; 57, 836-840.

[0316] As described in Example 1 herein, the translated amino acid sequence of the compound (A1)-BGC from B. crenata was used as a query sequence in genome mining of bacterial genomes from sequence databases and sequencing runs performed with PacBio sequencing technology. A cluster of highly homologous translated amino acid sequences and identical predicted protein function was discovered in the sequenced genome of C. vaccinii. Given the knowledge that C. vaccinii harbors the compound (A1)-BGC, meaningful BLAST hits could be retrieved from its published draft genome (Voeing et al. (2015) Genome Announcement 3, e00477-15) for frsC and frsH, but not for the full-length genes frsA, frsD, frsE, frsF, and frsG. A similar approach led Hermes et al. (2021) to identify C. vaccinii as the producer of A1. However, the cluster spanned six contigs, and extensive PCR-based gap closure was required to obtain the entire compound (A1)-BGC sequence (GenBank accession number: MT876545). The sequence generated in Example 1 herein demonstrates the advantages of PacBio sequencing for highly identical stretches of repeated sequences. The complete compound (A1)-BGC, including upstream and downstream regions encoding additional large NRPSs and several transposable elements, was identified (GenBank accession number: BankIt2437961 BSeq#1 MW732719). The gene organization of the transposable elements containing the BGCs of compound (A1) (frsA-H) and compound J (dis1-4) in C. vaccinii (76,974 bp), is shown in Figure 1. The organization of the genomic region containing Compound (A1)-BGC and NRPS A is further shown in Table 2-1.

[0317] The biosynthesis of compound (A1), including but not limited to the complete compound (A1)-BGC, is also disclosed, for example, in Pistorius et al. Genetic Engineering of Chromobacterium Vaccinii DSM 25150 for Improved Production of FR900359. ChemRxiv. Cambridge: Cambridge Open Engage; 2021, which is incorporated by reference in its entirety.

[0318] Cell Culture and Fermentation The microorganisms described herein can be cultured under conditions that allow the production of compound (A1). In general, conditions that can be optimized include the type and amount of carbon source, the type and amount of nitrogen source, the carbon-to-nitrogen ratio, oxygen level, growth temperature, pH, the length of the biomass production phase, the length of the target product accumulation phase, and the timing of cell harvest.

[0319] The culture medium generally contains a suitable carbon source. Carbon sources may include, but are not limited to, monosaccharides (e.g., glucose, fructose, xylose), disaccharides (e.g., lactose, sucrose), oligosaccharides, polysaccharides (e.g., starch, cellulose, hemicellulose, other lignocellulosic materials, or mixtures thereof), sugar alcohols (e.g., glycerol), and renewable feedstocks (e.g., cheese whey permeate, corn steep liquor, sugar beet molasses, barley malt). The carbon source may also be selected from one or more of the following non-limiting examples: linear or branched alkanes (e.g., hexane), linear or branched alcohols (e.g., hexanol), fatty acids (e.g., from about 10 carbons to about 22 carbons), esters of fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, and various commercially available fatty acid sources, including vegetable oils (e.g., soybean oil) and animal fats. Carbon sources can include single-carbon sources (e.g., carbon dioxide, methanol, formaldehyde, formate, and carbon-containing amines) from which metabolic conversion to key biochemical intermediates can occur. It is expected that the carbon source utilized can encompass a wide range of carbon-containing sources and will be limited only by the selection of the engineered microorganism(s). Non-limiting examples of acceptable growth media include Luria Broth (LB) agar, King's Medium B (KMB) agar, tryptic soy broth, yeast extract mannitol broth (YEM), glycerol yeast extract (GYEA), yeast extract-peptone-glycerol (YPG), peptone yeast extract glucose (PYG), MacConkey agar, or malt extract agar, or in flasks containing a suitable liquid medium such as tryptic soy broth, LB broth, YEM broth, KMB broth, GYEA broth, YPG broth, PYG broth, etc.

[0320] Nitrogen may be supplied from the inorganic phase (e.g., (NH4)2SO4) or the organic phase (e.g., urea or glutamate). In addition to suitable carbon and nitrogen sources, the culture medium also contains suitable minerals, salts, cofactors, buffers, vitamins, metal ions (e.g., Mn 2+ , Co2+ , Zn 2+ , Mg 2+ ) and other ingredients suitable for culturing microorganisms. Other chemicals may be added to the culture as needed. Non-limiting examples of such chemicals include tryptone peptone, calcium carbonate, propionic acid, sodium propionate, L-isoleucine, L-valine, L-phenylalanine, L-phenylbutyric acid, L-phenyllactic acid, sodium chloride, glycerol, Bacto soytone, Bacto peptone, Bacto soytone peptone, Bacto yeast extract, soybean peptone F, soybean peptone papain digest, vegetable peptone, vegetable yeast extract, fava bean peptone, casein peptone, HEPES, ammonium sulfate, magnesium sulfate, soyamine, Amicase®, Tubermine®, soy protein, salt-free yeast extract, Pharmamedia®, potato starch, L-phenyllactate, and L-phenylpyruvate.

[0321] Microorganisms are sometimes cultured in complex media (e.g., yeast extract-peptone-dextrose broth (YPD)). The culture medium, in some embodiments, is a common commercially prepared medium, such as LB medium. Other defined or synthetic growth media are known in the art and may also be used.

[0322] Microorganisms may be cultured on or in solid, semi-solid, or liquid media. In some embodiments, the media is drained from cells adhering to the plate. In certain embodiments, the liquid cell mixture is centrifuged, as known in the art, at a speed sufficient to pellet the cells but not disrupt them and allow for extraction of the media. The cells may then be resuspended in fresh media. The desired product may be purified from the culture media according to methods known in the art. In certain embodiments, the target product is extracted from the cultured microorganisms. Microbial cells may be concentrated by centrifugation at a speed sufficient to shear cell membranes. In some embodiments, the cells may be physically disrupted (e.g., shear force, sonication) or chemically disrupted (e.g., contact with detergents or other lysing agents). Phase separation by centrifugation or other methods known in the art may be performed, and the target product may be isolated by known methods.

[0323] Fermentation conditions can include any culture conditions suitable for maintaining a microorganism (e.g., in a stationary or growing state). For example, fermentation conditions can include several parameters, including, without limitation, temperature, oxygen content, nutrient content (e.g., glucose content), pH, agitation level (e.g., revolutions per minute), gas flow rate (e.g., air, oxygen, nitrogen gas), redox potential, cell density (e.g., optical density), cell viability, etc. Altering fermentation conditions (e.g., switching fermentation conditions) is the modification, alteration, or shift of one or more fermentation parameters. For example, fermentation conditions may be altered by increasing or decreasing temperature, increasing or decreasing pH (e.g., by adding or removing acid, base, or carbon dioxide), increasing or decreasing oxygen content (e.g., by introducing air, oxygen, carbon dioxide, nitrogen), increasing or decreasing air pressure (e.g., by introducing air, oxygen, carbon dioxide, nitrogen), increasing or decreasing agitation, and / or adding or removing nutrients (e.g., one or more sugars or sugar sources, biomass, vitamins, etc.), increasing or decreasing the culture-to-flask volume ratio, or a combination of the foregoing. Exemplary fermentation conditions are described herein. Aerobic conditions often include greater than about 50% dissolved oxygen (e.g., about 52%, about 54%, about 56%, about 58%, about 60%, about 62%, about 64%, about 66%, about 68%, about 70%, about 72%, about 74%, about 76%, about 78%, about 80%, about 82%, about 84%, about 86%, about 88%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, or a higher percentage of any one of the foregoing). Anaerobic conditions often include less than about 50% dissolved oxygen (e.g., about 1%, about 2%, about 4%, about 6%, about 8%, about 10%, about 12%, about 14%, about 16%, about 18%, about 20%, about 22%, about 24%, about 26%, about 28%, about 30%, about 32%, about 34%, about 36%, about 38%, about 40%, about 42%, about 44%, about 46%, about 48%, or a percentage lower than any one of the foregoing).

[0324] A variety of fermentation methods may be applied to produce a product of interest. In some embodiments, production of a target product from a recombinant microbial host is carried out using, for example, batch, fed-batch, or continuous fermentation methods.

[0325] Batch fermentation is often a closed system in which the medium composition is fixed at the beginning of the process and is not subjected to further additions during the process beyond those necessary to maintain pH and oxygen levels. At the beginning of the cultivation process, the medium is inoculated with the desired organism and growth or metabolic activity is allowed to occur without adding additional sources (e.g., carbon and nitrogen sources) to the medium. In batch processes, the metabolic and biomass composition of the system changes constantly until the point at which the cultivation is terminated. In a typical batch process, cells progress from a stationary lag phase to a high-growth logarithmic phase and finally to a stationary phase, where growth rate slows or stops. If left untreated, cells in the stationary phase will eventually die.

[0326] A variation of the standard batch method is the fed-batch method, in which a carbon source is constantly added to the fermenter over the course of the fermentation process. The fed-batch method is useful when catabolite repression tends to inhibit cellular metabolism or when it is desirable to have a repressed amount of carbon source in the medium at any given time. Measurement of the carbon source concentration in a fed-batch system can be estimated based on changes in measurable factors, such as pH, dissolved oxygen, and the partial pressure of waste gases (e.g., CO). Batch and fed-batch culture methods are known in the art. For examples of such methods, see Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, 2nd ed., (1989) Sinauer Associates, Sunderland, Mass., and Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227 (1992).

[0327] In continuous fermentation, a defined medium is often continuously added to a bioreactor while an equal amount of culture volume is simultaneously removed for product harvest. Continuous culture generally maintains a constant cell density of cells in the log phase of growth. Continuous or semi-continuous culture methods allow for the adjustment of one or more factors that affect cell growth or end-product concentration. For example, one approach allows the carbon source to be limiting while all other parameters are metabolically depressed. In some systems, several factors that affect growth may be continuously varied while the cell concentration, as measured by medium turbidity, remains constant. In continuous systems, steady-state growth is often maintained; thus, the cell growth rate is often balanced by cell losses due to culture withdrawal. Methods for adjusting nutrients and growth factors for continuous culture, as well as techniques for maximizing the rate of product formation, are known; various methods are discussed in detail in Brock, supra.

[0328] The product of interest may be provided in cultured microorganisms containing the target product; the cultured microorganisms may be supplied fresh, frozen in liquid medium, or dried. Fresh or frozen microorganisms may be contained in a suitable moisture-proof container, which may also be temperature-controlled, if necessary. The product of interest is sometimes provided in a substantially cell-free culture medium. In some embodiments, the product of interest is provided in a substantially pure form (e.g., 50% or more pure, 60% or more pure, 70% or more pure, 80% or more pure, 90% or more pure, 95% or more pure, 99% or more pure, or 99.5% or more pure). In some embodiments, the product of interest may be modified to become any one of a number of downstream products.

[0329] Drug Moiety (D) In some embodiments, the drug moiety (D) of the antibody drug conjugate of the present disclosure is a GNAQ inhibitor, a GNA11 inhibitor, or an inhibitor of GNAQ and GNA11 (GNAQ / GNA11 inhibitor). In some embodiments, the drug moiety (D) of the antibody drug conjugate of the present disclosure is a GNAQ inhibitor. In some embodiments, the drug moiety (D) of the antibody drug conjugate of the present disclosure is a GNA11 inhibitor. In some embodiments, the drug moiety (D) of the antibody drug conjugate of the present disclosure is an inhibitor of GNAQ and GNA11 (GNAQ / GNA11 inhibitor).

[0330] In some embodiments, the drug moiety (D) of the antibody drug conjugates of the present disclosure has the structure of formula (A): [ka] wherein Ro is methyl or ethyl, Ri is methyl or i-propyl, and R2 is methyl or ethyl.

[0331] In some embodiments, the drug moiety (D) of the antibody drug conjugates of the present disclosure has the following structure: [ka] The compound (A1) has the formula:

[0332] The synthesis of compound (A1) is also described in Example 3 of WO 2020 / 128612, which is incorporated by reference in its entirety.

[0333] The metabolic stability and PK properties of compound (A1) are also described in Examples 8 and 9, respectively, of WO 2020 / 128612, which is incorporated by reference in its entirety.

[0334] In some embodiments, the drug moiety (D) of the antibody drug conjugates of the present disclosure has the structure of formula (Ab): [ka] wherein R o is methyl or ethyl, and R i , R s , and R t are each independently methyl, i-propyl, or methylthiomethyl, and R 2 is methyl or ethyl.

[0335] In some embodiments, the drug moiety (D) of the antibody drug conjugates of the present disclosure has the structure: [ka] The compound (Ab1) has the formula:

[0336] In some embodiments, the drug moiety (D) of the antibody drug conjugates of the present disclosure has the structure: [ka] The compound (Ab2) has the formula:

[0337] In some embodiments, the drug moiety (D) of the antibody drug conjugates of the present disclosure has the structure: [ka] The compound (Ab3) has the formula:

[0338] In some embodiments, the drug moiety (D) of the antibody drug conjugates of the present disclosure has the structure of formula (Ac): [ka] wherein R 1 is methyl or isopropyl, and R 2 is methyl or ethyl.

[0339] In some embodiments, the drug moiety (D) of the antibody drug conjugates of the present disclosure has the following structure: [ka] The compound (Ac1) has the following structure:

[0340] In some embodiments, the drug moiety (D) of the antibody drug conjugates of the present disclosure has the following structure: [ka] The compound (Ac2) has the following structure:

[0341] In some embodiments, the drug moiety (D) of the antibody drug conjugates of the present disclosure has the following structure: [ka] The compound (Ac3) has the following structure:

[0342] In some embodiments, the drug moiety (D) of the antibody drug conjugates of the present disclosure has the following structure: [ka] Compound (Ac4) having the formula wherein R1 is methyl or =CH2, R2 is methyl or ethyl, R3 is methyl, ethyl, or isopropyl, and R4 is H, N-Ac-Hle, or N-Pr-Hle.

[0343] In some embodiments, the drug moiety (D) of the antibody drug conjugate of the present disclosure is compound (A1) and any of 2-15 as disclosed, for example, in Example 3.

[0344] Linker-drug moiety (L A -(D) n ) In some embodiments, the linker-drug moiety ((L A -(D) n ))) is the linker (L A), wherein the one or more drug moieties are each independently selected from a GNAQ inhibitor, a GNA11 inhibitor, or an inhibitor of GNAQ and GNA11 (GNAQ / GNA11 inhibitor).

[0345] In some embodiments, the linker-drug moiety ((L A -(D) n ))) is the linker (L A ), wherein the one or more drug moieties are each independently selected from a GNAQ inhibitor.

[0346] In some embodiments, the linker-drug moiety ((L A -(D) n ))) is the linker (L A ) attached to said antibody, wherein said one or more drug moieties are each independently selected from a GNA11 inhibitor.

[0347] In some embodiments, the linker-drug moiety ((L A -(D) n ))) is the linker (L A ), wherein the one or more drug moieties are each independently selected from inhibitors of GNAQ and GNA11 (GNAQ / GNA11 inhibitors).

[0348] In some embodiments, the linker-drug moiety ((L A -(D) n ))) is the linker (L A ), and one or more drug moieties covalently attached to a linker (L A ) is a cleavable linker, and the one or more drug moieties are each independently selected from a GNAQ inhibitor, a GNA11 inhibitor, or an inhibitor of GNAQ and GNA11 (GNAQ / GNA11 inhibitor).

[0349] In some embodiments, the linker-drug moiety ((L A -(D) n ))) is the linker (L A ), and one or more drug moieties covalently attached to a linker (L A ) is a cleavable linker and the one or more drug moieties are each independently selected from a GNAQ inhibitor.

[0350] In some embodiments, the linker-drug moiety ((L A -(D) n ))) is the linker (L A ), and one or more drug moieties covalently attached to a linker (L A ) is a cleavable linker, and the one or more drug moieties are each independently selected from a GNA11 inhibitor.

[0351] In some embodiments, the linker-drug moiety ((L A -(D) n ))) is the linker (L A ), and one or more drug moieties covalently attached to a linker (L A ) is a cleavable linker and the one or more drug moieties are each independently selected from inhibitors of GNAQ and GNA11 (GNAQ / GNA11 inhibitors).

[0352] In some embodiments, the linker-drug moiety ((L A -(D) n ))) is the linker (L A ), and one or more drug moieties covalently attached to a linker (L A ) is a non-cleavable linker, and the one or more drug moieties are each independently selected from a GNAQ inhibitor, a GNA11 inhibitor, or an inhibitor of GNAQ and GNA11 (GNAQ / GNA11 inhibitor).

[0353] In some embodiments, the linker-drug moiety ((L A -(D) n ))) is the linker (L A ), and one or more drug moieties covalently attached to a linker (L A ) is a non-cleavable linker and the one or more drug moieties are each independently selected from a GNAQ inhibitor.

[0354] In some embodiments, the linker-drug moiety ((L A -(D) n ))) is the linker (L A ), and one or more drug moieties covalently attached to a linker (L A ) is a non-cleavable linker, and the one or more drug moieties are each independently selected from a GNA11 inhibitor.

[0355] In some embodiments, the linker-drug moiety ((L A -(D) n ))) is the linker (L A ), and one or more drug moieties covalently attached to a linker (L A ) is a non-cleavable linker, and the one or more drug moieties are each independently selected from inhibitors of GNAQ and GNA11 (GNAQ / GNA11 inhibitors).

[0356] In some embodiments, the linker-drug moiety ((L A -(D) n ))) linker (L A ) is expressed as: [ka] (In the formula, X1 is a divalent linking group; X2 is a self-sacrificing spacer; Y1 is [ka] wherein Y1 * indicates the attachment point to X2, and Y1 ** indicates other attachment points; L1 is a bivalent peptide linker, and L2 is a bond or a linker It has.

[0357] In some embodiments, the linker-drug moiety, ((L A -(D) n ))) is the following formula: [ka] (In the formula, D is a GNAQ inhibitor, a GNA11 inhibitor or an inhibitor of GNAQ and GNA11; X1 is a divalent linking group; X2 is a self-sacrificing spacer; Y1 is [ka] wherein Y1 * indicates the attachment point to X2, and Y1 ** indicates the point of attachment to D; L1 is a bivalent peptide linker, L2 is a bond or a linker It has.

[0358] Linker-drug compound (L B -(D) n ) In some embodiments, the linker-drug of the present disclosure has the structure of Formula (B): R 8 -L B -(D) n (B) (In the formula, D is a GNAQ inhibitor, a GNA11 inhibitor or an inhibitor of GNAQ and GNA11; R 8is a reactive group; L B is a cleavable or non-cleavable linker, n is 1, 2, 3 or 4 It is a compound having the formula:

[0359] In some embodiments, D is a GNAQ inhibitor, a GNA11 inhibitor or an inhibitor of GNAQ and GNA11; R 8 is a reactive group; L B is a cleavable linker comprising one or more linker components selected from a self-immolative spacer, a phosphate group, a carbonate group, and a bivalent peptide linker; n is 1, 2, 3 or 4; A linker-drug of the present disclosure having the structure of Formula (B):

[0360] In some embodiments, the linker-drug of the present disclosure has the structure of formula (B-1): [ka] (In the formula, D is a GNAQ inhibitor, a GNA11 inhibitor or an inhibitor of GNAQ and GNA11; R 8 is a reactive group; X2 is a self-sacrificing spacer; Y1 is [ka] wherein Y1 * indicates the attachment point to X2, and Y1 ** indicates the point of attachment to D; L1 is a bivalent peptide linker, L2 is a bond or a linker It is a compound having the formula:

[0361] Specific embodiments and examples of linker-drug compounds of the present disclosure are provided below in the list of additional enumerated embodiments. It is recognized that the features specified in each embodiment can be combined with other specific features to provide further embodiments of the present disclosure.

[0362] Embodiment 1. A compound of Formula (B) or Formula (B-1), or a stereoisomer or pharmaceutically acceptable salt thereof, wherein D is a GNAQ inhibitor.

[0363] Embodiment 2. A compound of Formula (B) or Formula (B-1), or a stereoisomer or pharmaceutically acceptable salt thereof, wherein D is a GNA11 inhibitor.

[0364] Embodiment 3. A compound of Formula (B) or Formula (B-1), or a stereoisomer or pharmaceutically acceptable salt thereof, wherein D is an inhibitor of GNAQ and GNA11.

[0365] Embodiment 4. In the formula, D is [ka] (In the formula, R 0 is methyl or ethyl, and R 1 is methyl or isopropyl, and R 2 is methyl or ethyl, and *** L B or Y1) or a stereoisomer or a pharmaceutically acceptable salt thereof.

[0366] Embodiment 4a. Wherein D is [ka] (In the formula, R 0 is methyl or ethyl, and R 1 , R s , R tare each independently methyl, isopropyl, or methylthiomethyl; R 2 is methyl or ethyl, and *** L B or Y1) or a stereoisomer or a pharmaceutically acceptable salt thereof.

[0367] Embodiment 4b. wherein D is [ka] (In the formula, R 0 is methyl or ethyl, and R 1 is methyl or isopropyl, and R 2 is methyl or ethyl, and *** L B or Y1) or a stereoisomer or a pharmaceutically acceptable salt thereof.

[0368] Embodiment 5. In the formula, D is [ka] (In the formula, *** L B or Y1) or a stereoisomer or a pharmaceutically acceptable salt thereof.

[0369] Embodiment 6. A structure of formula (B-2), or a pharmaceutically acceptable salt thereof: [ka] (In the formula, R 0 is methyl or ethyl; R 1 is methyl or isopropyl; R 2 is methyl or ethyl, and X2, L1, L2 and R 8 is as defined in the compound of formula (B-1) above) or a stereoisomer or pharmaceutically acceptable salt thereof, wherein:

[0370] Embodiment 6a. A structure of formula (Bb-2), or a pharmaceutically acceptable salt thereof: [ka] (In the formula, R 0 is methyl or ethyl; R 1 , R s , and R t are each independently methyl, methylthiomethyl, or isopropyl; R 2 is methyl or ethyl, and X2, L1, L2 and R 8 is as defined in the compound of formula (Bb-1) above) or a stereoisomer or pharmaceutically acceptable salt thereof.

[0371] Embodiment 6b. A structure of formula (Bc-2), or a pharmaceutically acceptable salt thereof: [ka] (In the formula, R 0 is methyl or ethyl; R 1 is methyl or isopropyl; R 2 is methyl or ethyl, and X2, L1, L2 and R 8 is as defined in the compound of formula (Bc-1) above) or a stereoisomer or pharmaceutically acceptable salt thereof, wherein:

[0372] Embodiment 7. A structure of formula (B-2a), or a pharmaceutically acceptable salt thereof: [ka] (In the formula, X2, L1, L2 and R 8 is as defined in the compound of formula (B-1) above) or a stereoisomer or pharmaceutically acceptable salt thereof.

[0373] Embodiment 8. A structure of formula (B-3), or a pharmaceutically acceptable salt thereof: [ka] (In the formula, R 0 is methyl or ethyl; R 1 is methyl or isopropyl; R 2 is methyl or ethyl, and X2, L1, L2 and R 8 is as defined in the compound of formula (B-1) above) or a stereoisomer or pharmaceutically acceptable salt thereof, wherein:

[0374] Embodiment 8a. A structure of formula (Bb-3), or a pharmaceutically acceptable salt thereof: [ka] (In the formula, R 0 is methyl or ethyl; R 1 , R s , and R t are each independently methyl, methylthiomethyl, or isopropyl; R 2 is methyl or ethyl, and X2, L1, L2 and R8 is as defined in the compound of formula (Bb-1) above) or a stereoisomer or pharmaceutically acceptable salt thereof.

[0375] Embodiment 8b. A structure of formula (Bc-3), or a pharmaceutically acceptable salt thereof: [ka] (In the formula, R 0 is methyl or ethyl; R 1 is methyl or isopropyl; R 2 is methyl or ethyl, and X2, L1, L2 and R 8 is as defined in the compound of formula (Bc-1) above) or a stereoisomer or pharmaceutically acceptable salt thereof, wherein:

[0376] Embodiment 9. A structure of formula (B-3a), or a pharmaceutically acceptable salt thereof: [ka] (In the formula, X2, L1, L2 and R 8 is as defined in the compound of formula (B-1) above) or a stereoisomer or pharmaceutically acceptable salt thereof.

[0377] Embodiment 10. In the formula: R 0 is methyl or ethyl; R 1 is methyl or isopropyl; R 2 is methyl or ethyl; X2 is [ka] wherein X2 is a self-immolative spacer selected from * indicates the attachment point to L1, and X2 ** teeth, [ka] the point of attachment to the group, or [ka] indicates the point of attachment to the group; L1 is a bivalent peptide linker containing 2 to 4 amino acid residues; L2 is a linker, R 8 teeth, [ka] , -N3, -ONH2, -NR 4 C(=O)CH=CH2, SH, -SSR 13 , -S(=O)2(CH=CH2), -NR 4 S(=O)2(CH=CH2), -NR 4 C(=O)CH2Br, -NR 4 C(=O)CH2I, -NHC(=O)CH2Br, -NHC(=O)CH2I, -C(=O)NHNH2, [ka] is selected from: Each R 4 are independently selected from H and C1-C6 alkyl; Each R 5 are independently selected from H, C1-C6 alkyl, F, Cl, and —OH; Each R 6 are independently selected from H, C1-C6 alkyl, F, Cl, —NH2, —OCH3, —OCH2CH3, —N(CH3)2, —CN, —NO2, and —OH; and Each R 7is H, C 1~6 Alkyl, fluoro, benzyloxy substituted by -C(=O)OH, benzyl substituted by -C(=O)OH, C substituted by -C(=O)OH 1~4 C substituted by alkoxy and -C(=O)OH 1~4 independently selected from alkyl, A compound of formula (B-2) of embodiment 6, formula (B-3) of embodiment 8, or a pharmaceutically acceptable salt thereof.

[0378] Embodiment 11. In the formula, X2 is [ka] wherein X2 is a self-immolative spacer selected from * indicates the attachment point to L1, and X2 ** is the attachment point to Y1, [ka] the point of attachment to the group, or [ka] The compound of any one of embodiments 1 to 10, showing the point of attachment to the group.

[0379] Embodiment 12. In the formula, X2 is [ka] In the formula, X2 * indicates the attachment point to L1, and X2 ** is the attachment point to Y1, [ka] the point of attachment to the group, or [ka] 12. The compound of any one of embodiments 1 to 11, showing the point of attachment to the group.

[0380] Embodiment 13. The compound of any one of embodiments 1 to 12, wherein L1 is a bivalent peptide linker comprising 2 to 4 amino acid residues.

[0381] Embodiment 14. The compound of any one of embodiments 1-12, wherein L1 is a bivalent peptide linker comprising an amino acid residue selected from valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan, and tyrosine.

[0382] Embodiment 15. The compound of any one of embodiments 1 to 12, wherein L1 is a bivalent peptide linker comprising at least one valine (Val) or citrulline (Cit) residue.

[0383] Embodiment 16. The compound of any one of embodiments 1 to 12, wherein L1 is a bivalent dipeptide linker selected from ValCit, PheLys, ValAla, and ValLys.

[0384] Embodiment 17. In the formula, L1 is [ka] wherein L1 is a bivalent dipeptide linker selected from * indicates the attachment point to L2, and ** 13. The compound of any one of embodiments 1 to 12, wherein indicates the point of attachment to X2.

[0385] Embodiment 18. The compound of any one of embodiments 1 to 12, wherein L1 is ValCit.

[0386] Embodiment 19. In the formula, L1 is [ka] where L1 * indicates the attachment point to L2, and ** 13. The compound of any one of embodiments 1 to 12, wherein indicates the point of attachment to X2.

[0387] Embodiment 20. The compound of any one of embodiments 1 to 19, wherein L2 is a linker.

[0388] Embodiment 21. In the formula, L2 is - * C(=O)((CH2) m O) p (CH2) m ** -,- * C(=O)(CH2) m ** -,- * C(=O)(CH2) n NHC(=O)(CH2) m ** -,- * C(=O)(CH2) m NHC(=O)((CH2) m O) p (CH2) m ** -,- * ((CH2) m O) p (CH2) m ** -,- * ((CH2) m O) p (CH2) m ** -, -(CH2) m -,- * (CH2) m NHC(=O)(CH2) m ** -,- * (CH2) m NHC(=O)(CH2) m C(=O)NH(CH2) m ** -,- * ((CH2) m O) p (CH2) m NHC(=O)(CH2) m ** -,- ** ((CH2) m O) p CH2) mC(=O)NH(CH2) m ** -,- * (CH2) m C(R3)2 ** -and- * (CH2) m C(R3)2SS(CH2) m NHC(=O)(CH2) m ** -, wherein L2 is a linker selected from * indicates the attachment point to L1, and ** indicates the attachment point to R8; During the ceremony, each R3 is independently selected from H and C1-C6 alkyl; each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; 20. The compound of any one of embodiments 1 to 19, wherein each p is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14.

[0389] Embodiment 22. In the formula, L2 is - * C(=O)((CH2) m O) p (CH2) m ** -or- * C(=O)(CH2) m ** -, where L2 * indicates the attachment point to L1, and ** indicates the attachment point to R8, 20. The compound of any one of embodiments 1 to 19, wherein each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0390] Embodiment 23. In the formula, L2 is [ka] where L2 *indicates the attachment point to L1, and ** 20. The compound of any one of embodiments 1 to 19, wherein indicates the point of attachment to R8.

[0391] Embodiment 24. In the formula, R 8 teeth, [ka] , -N3, -ONH2, -NR 4 C(=O)CH=CH2, SH, -SSR 13 , -S(=O)2(CH=CH2), -NR 4 S(=O)2(CH=CH2), -NR 4 C(=O)CH2Br, -NR 4 C(=O)CH2I, -NHC(=O)CH2Br, -NHC(=O)CH2I, -C(=O)NHNH2, [ka] where: Each R 4 are independently selected from H and C1-C6 alkyl; Each R 5 are independently selected from H, C1-C6 alkyl, F, Cl, and —OH; Each R 6 are independently selected from H, C1-C6 alkyl, F, Cl, —NH2, —OCH3, —OCH2CH3, —N(CH3)2, —CN, —NO2, and —OH; and Each R 7 is H, C 1~6 Alkyl, fluoro, benzyloxy substituted by -C(=O)OH, benzyl substituted by -C(=O)OH, C substituted by -C(=O)OH 1~4 C substituted by alkoxy and -C(=O)OH 1~4 independently selected from alkyl, The compound of any one of embodiments 1 to 23.

[0392] Embodiment 25. In the formula, R8 teeth, [ka] The compound of any one of embodiments 1-23, wherein

[0393] Embodiment 26. In the formula, R 8 teeth, [ka] The compound of any one of embodiments 1-23, wherein

[0394] Embodiment 27. wherein: R 0 is methyl or ethyl; R 1 is methyl or isopropyl; R 2 is methyl or ethyl; X2 is [ka] In the formula, X2 * indicates the attachment point to L1, and X2 ** teeth, [ka] indicates the point of attachment to the group; L1 is [ka] where L1 * indicates the attachment point to L2, and ** indicates the attachment point to X2; L2 is [ka] where L2 * indicates the attachment point to L1, and ** indicates the attachment point to R8, and R 8 teeth, [ka] or a pharmaceutically acceptable salt thereof.

[0395] Embodiment 28. wherein: R 0 is methyl or ethyl; R 1 is methyl or isopropyl; R 2 is methyl or ethyl; X2 is [ka] In the formula, X2 * indicates the attachment point to L1, and X2 ** teeth, [ka] indicates the point of attachment to the group; L1 is [ka] where L1 * indicates the attachment point to L2, and ** indicates the attachment point to X2; L2 is [ka] where L2 * indicates the attachment point to L1, and ** indicates the attachment point to R8, and R 8 teeth, [ka] or a pharmaceutically acceptable salt thereof.

[0396] Embodiment 29. A compound of Formula (B), Formula (B-1) or Formula (B-2), [ka] A compound which is

[0397] Embodiment 30. A compound of Formula (B), Formula (B-1) or Formula (B-2), [ka] A compound which is

[0398] Embodiment 31. A compound of Formula (B), Formula (B-1) or Formula (B-3), [ka] A compound which is

[0399] Embodiment 32. A compound of Formula (B), Formula (B-1) or Formula (B-3), [ka] A compound which is

[0400] The synthesis of exemplary linker-drug compounds is also described in Example 1 of WO 2020 / 128612, which is incorporated by reference in its entirety.

[0401] antibody-drug conjugates In some embodiments, the antibody drug conjugate of the present disclosure is a conjugate of formula (C): Ab-(L A -(D) n ) y (C) (In the formula, D is a drug moiety; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; L A is a linker; n is 1, 2, 3 or 4, and y is 1, 2, 3 or 4 and where the linker-drug moiety (L A -(D) n ) is covalently attached to the antibody or antigen-binding fragment thereof.

[0402] In some embodiments, D is a GNAQ inhibitor, a GNA11 inhibitor or an inhibitor of GNAQ and GNA11; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; L A is a cleavable linker comprising one or more linker components selected from a self-immolative spacer, a phosphate group, a carbonate group, and a bivalent peptide linker; n is 1, 2, 3 or 4; y is 1, 2, 3 or 4; Linker-drug moiety (L A -(D) n ) is covalently attached to the antibody or antigen-binding fragment thereof, an antibody drug conjugate of the disclosure having the structure of formula (C):

[0403] In some embodiments, the antibody-drug conjugate of Formula (C) has the formula (C-1): [ka] (In the formula, D is a GNAQ inhibitor, a GNA11 inhibitor or an inhibitor of GNAQ and GNA11; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; X1 is a divalent linking group; X2 is a self-sacrificing spacer; Y1 is [ka] wherein Y1 * indicates the attachment point to X2, and Y1** indicates the point of attachment to D; L1 is a bivalent peptide linker; L2 is a bond or a linker; y is 1, 2, 3 or 4 It is a combination of

[0404] In the conjugate of formula (C), one or more linker-drug moieties (L B -(D) n ) can be covalently attached to an antibody or antigen-binding fragment thereof Ab, thereby attaching one or more drug moieties D to the antibody or antigen-binding fragment thereof Ab via a linker L A Covalently attached via L A is any chemical moiety capable of linking an antibody or antigen-binding fragment thereof Ab to one or more drug moieties D. Conjugates of formula (C), in which one or more drug moieties D are covalently attached to an antibody or antigen-binding fragment thereof Ab, can be formed using bifunctional or polyfunctional linker reagents having one or more reactive functional groups, which may be the same or different. One of the reactive functional groups of the bifunctional or polyfunctional linker reagent is used to react with a group on the antibody or antigen-binding fragment thereof Ab, for example, a thiol or amine (e.g., cysteine, the N-terminus, or an amino acid side chain, e.g., lysine), to form the linker L. A Such reactive functional groups of a bifunctional or polyfunctional linker reagent include, but are not limited to, maleimide, thiol, and NHS ester. One or more other reactive functional groups of a bifunctional or polyfunctional linker reagent may connect one or more drug moieties D to a linker L. A It is used to covalently attach to

[0405] In some embodiments, L A is a cleavable linker. In some embodiments, L A is a non-cleavable linker. Ais an acid labile linker, a photolabile linker, a peptidase cleavable linker, an esterase cleavable linker, a glycosidase cleavable linker, a phosphodiesterase cleavable linker, a disulfide bond reducible linker, a hydrophilic linker, or a dicarboxylic acid-based linker.

[0406] In some embodiments, L A is a cleavable linker comprising one or more linker components selected from a self-immolative spacer, a phosphate group, a carbonate group, and a bivalent peptide linker.

[0407] In some embodiments, L A is a cleavable linker comprising one or more linker components selected from a self-immolative spacer, a phosphate group, a carbonate group, a bivalent peptide linker, and a bivalent linking group.

[0408] In some embodiments, L A is a cleavable linker comprising one or more linker components selected from a self-immolative spacer, a phosphate group, and a bivalent peptide linker.

[0409] In some embodiments, L A is a cleavable linker comprising one or more linker components selected from a self-immolative spacer, a phosphate group, a bivalent peptide linker, and a bivalent linking group.

[0410] In some embodiments, the linker (L A ) is expressed as: [ka] (In the formula, X1 is a divalent linking group; X2 is a self-sacrificing spacer; Y1 is [ka] wherein Y1 * indicates the attachment point to X2, and Y1** indicates other attachment points; L1 is a bivalent peptide linker, and L2 is a bond or a linker It has.

[0411] In some embodiments, the linker (L A ) is expressed as: [ka] (In the formula, X1 is a divalent linking group; X2 is a self-sacrificing spacer; Y1 is [ka] wherein Y1 * indicates the attachment point to X2; L1 is a bivalent peptide linker, and L2 is a bond or a linker It has.

[0412] In some embodiments, the linker (L A ) is expressed as: [ka] (In the formula, X1 is a divalent linking group; X2 is a self-sacrificing spacer; Y1 is [ka] wherein Y1 * indicates the attachment point to X2; L1 is a bivalent peptide linker, and L2 is a bond or a linker It has.

[0413] While the drug-antibody ratio has a precise integer value for a particular conjugate molecule (e.g., the product of n and y in Formula (C)), it is understood that when used to describe a sample containing many molecules, due to the degree of heterogeneity typically associated with the conjugation step, the value is often an average value. The average loading for a sample of conjugates is referred to herein as the drug-antibody ratio, or "DAR." In some embodiments, the DAR is from about 1 to about 5, and typically about 1, 2, 3, or 4. In some embodiments, at least 50% of the sample by weight is a compound having an average DAR plus or minus 2, and preferably at least 50% of the sample is a conjugate containing an average DAR plus or minus 1. Some embodiments include conjugates with a DAR of about 2. In some embodiments, a DAR of "about y" means that the measured value for DAR is within 20% of the product of n and y in Formula (I). In some embodiments, a DAR of "about n" means that the measured value for DAR is within 20% of n in Formula (II).

[0414] In some embodiments, the average molar ratio of drug to antibody in the conjugate of Formula (C) (i.e., the average value of the product of n and y, also known as the drug-antibody ratio (DAR)) is from about 1 to about 10, from about 1 to about 6 (e.g., 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0), about 1 to about 5, about 1.5 to about 4.5, or about 2 to about 4.

[0415] In some embodiments provided by the present disclosure, the conjugates have a substantially high degree of purity and have one or more of the following characteristics: (a) greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more, or about 100%) of the conjugate species are monomeric, preferably greater than about 95%; (b) the conjugate preparation has an unconjugated linker level of less than about 10% (e.g., about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or about 0% or less) (of total linker); (c) less than 10% of the conjugate species are crosslinked. (d) the level of free drug (ADP-induced platelet aggregation inhibitor, e.g., GNAQ inhibitor, GNA11 inhibitor, or GNAQ and GNA11 inhibitor) in the conjugate preparation is less than about 2% (e.g., about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, about 0.1%, or about 0% or less) (mol / mol of total drug).

[0416] Specific embodiments and examples of antibody drug conjugates of the present disclosure are provided in the list of additional enumerated embodiments below. It is recognized that the features specified in each embodiment can be combined with other specific features to provide further embodiments of the present disclosure.

[0417] Embodiment 33. A conjugate of Formula (C) or Formula (C-1), wherein D is a GNAQ inhibitor.

[0418] Embodiment 34. A conjugate of Formula (C) or Formula (C-1), wherein D is a GNA11 inhibitor.

[0419] Embodiment 35. A conjugate of Formula (C) or Formula (C-1), wherein D is an inhibitor of GNAQ and GNA11.

[0420] Embodiment 36. Wherein D is [ka] (In the formula, R 0 is methyl or ethyl, R1 is methyl or isopropyl, R2 is methyl or ethyl, and *** L A or Y1) A conjugate of formula (C) or formula (C-1):

[0421] Embodiment 36a. wherein D is [ka] (In the formula, R 0 is methyl or ethyl, and R 1 , R s , and R t are each independently methyl, methylthiomethyl, or isopropyl; R 2 is methyl or ethyl, and *** L A or Y1) A conjugate of formula (Cb) or formula (Cb-1):

[0422] Embodiment 36b. wherein D is [ka] (In the formula, R 0 is methyl or ethyl, R1 is methyl or isopropyl, R2 is methyl or ethyl, and *** L A or Y1) A bond of formula (Cc) or formula (Cc-1),

[0423] Embodiment 37. Wherein D is [ka] (In the formula, *** L A or Y1) A conjugate of formula (C) or formula (C-1):

[0424] Embodiment 38. Structure of formula (C-2): [ka] (In the formula, R 0 is methyl or ethyl; R 1 is methyl or isopropyl; R 2 is methyl or ethyl; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; X1 is a divalent linking group; X2 is a self-sacrificing spacer; L1 is a bivalent peptide linker; L2 is a bond or linker, and y is 1, 2, 3 or 4 A conjugate of formula (C) or formula (C-1) having the formula:

[0425] Embodiment 38a. Structure of formula (Cb-2): [ka] (In the formula, R 0 is methyl or ethyl; R 1 , R s , and R t are each independently methyl, methylthiomethyl, or isopropyl; R 2 is methyl or ethyl; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; X1 is a divalent linking group; X2 is a self-sacrificing spacer; L1 is a bivalent peptide linker; L2 is a bond or linker, and y is 1, 2, 3 or 4 A conjugate of formula (Cb) or formula (Cb-1):

[0426] Embodiment 38b. Structure of formula (Cc-2): [ka] (In the formula, R 0 is methyl or ethyl; R 1 is methyl or isopropyl; R 2 is methyl or ethyl; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; X1 is a divalent linking group; X2 is a self-sacrificing spacer; L1 is a bivalent peptide linker; L2 is a bond or linker, and y is 1, 2, 3 or 4 A conjugate of formula (Cc) or formula (Cc-1) having the formula:

[0427] Embodiment 39. Structure of Formula (C-2a): [ka] (In the formula, Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; X1 is a divalent linking group; X2 is a self-sacrificing spacer; L1 is a bivalent peptide linker; L2 is a bond or linker, and y is 1, 2, 3 or 4 A conjugate of formula (C) or formula (C-1) having the formula:

[0428] Embodiment 40. Structure of Formula (C-3): [ka] (In the formula, R 0 is methyl or ethyl; R 1 is methyl or isopropyl; R 2 is methyl or ethyl; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; X1 is a divalent linking group; X2 is a self-sacrificing spacer; L1 is a bivalent peptide linker; L2 is a bond or linker, and y is 1, 2, 3 or 4 A conjugate of formula (C) or formula (C-1) having the formula:

[0429] Embodiment 40a. Structure of Formula (Cb-3): [ka] (In the formula, R 0 is methyl or ethyl; R 1 , R s , and R t are each independently methyl, methylthiomethyl, or isopropyl; R 2 is methyl or ethyl; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; X1 is a divalent linking group; X2 is a self-sacrificing spacer; L1 is a bivalent peptide linker; L2 is a bond or linker, and y is 1, 2, 3 or 4 A conjugate of formula (Cb) or formula (Cb-1):

[0430] Embodiment 40b. Structure of Formula (Cc-3): [ka] (In the formula, R 0 is methyl or ethyl; R 1 is methyl or isopropyl; R 2 is methyl or ethyl; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; X1 is a divalent linking group; X2 is a self-sacrificing spacer; L1 is a bivalent peptide linker; L2 is a bond or linker, and y is 1, 2, 3 or 4 A conjugate of formula (Cc) or formula (Cc-1) having the formula:

[0431] Embodiment 41. Structure of formula (C-3a): [ka] (In the formula, Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; X1 is a divalent linking group; X2 is a self-sacrificing spacer; L1 is a bivalent peptide linker; L2 is a bond or linker, and y is 1, 2, 3 or 4 A conjugate of formula (C) or formula (C-1) having the formula:

[0432] Embodiment 42. wherein: R 0 is methyl or ethyl; R 1 is methyl or isopropyl; R 2 is methyl or ethyl; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; X2 is [ka] wherein X2 is a self-immolative spacer selected from * indicates the attachment point to L1, and X2 ** teeth, [ka] the point of attachment to the group, or [ka] indicates the point of attachment to the group; L1 is a bivalent peptide linker containing 2 to 4 amino acid residues; L2 is a linker; X1 is [ka] , ** -, wherein X1 is a divalent linking group selected from * indicates the attachment point to L2, and X1 ** indicates the point of attachment to Ab; During the ceremony, Each R 4 is independently selected from H and C1-C6 alkyl; Each R 5 are independently selected from H, C1-C6 alkyl, F, Cl, and —OH; Each R 6 are independently selected from H, C1-C6 alkyl, F, Cl, —NH2, —OCH3, —OCH2CH3, —N(CH3)2, —CN, —NO2, and —OH; Each R 7 are independently H, C 1~6 Alkyl, fluoro, benzyloxy substituted by -C(=O)OH, benzyl substituted by -C(=O)OH, C substituted by -C(=O)OH 1~4C substituted by alkoxy and -C(=O)OH 1~4 alkyl, y is 1, 2, 3 or 4; A conjugate of formula (C-2) of embodiment 54 or a conjugate of formula (C-3) of embodiment 58.

[0433] Embodiment 43. In the formula, X2 is [ka] wherein X2 is a self-immolative spacer selected from * indicates the attachment point to L1, and X2 ** is the attachment point to Y1, or [ka] the point of attachment to the group, or [ka] 43. The conjugate of any one of embodiments 38 to 42, showing the point of attachment to the group.

[0434] Embodiment 44. In the formula, X2 is [ka] In the formula, X2 * indicates the attachment point to L1, and X2 ** is the attachment point to Y1, or [ka] the point of attachment to the group, or [ka] 44. The conjugate of any one of embodiments 38 to 43, showing the point of attachment to the group.

[0435] Embodiment 45. The conjugate of any one of embodiments 38 to 44, wherein L1 is a bivalent peptide linker comprising 2 to 4 amino acid residues.

[0436] Embodiment 46. The conjugate of any one of embodiments 38 to 45, wherein L1 is a bivalent peptide linker comprising an amino acid residue selected from valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan, and tyrosine.

[0437] Embodiment 47. The conjugate of any one of embodiments 38 to 44, wherein L1 is a bivalent peptide linker comprising at least one valine (Val) or citrulline (Cit) residue.

[0438] Embodiment 48. The conjugate of any one of embodiments 38 to 44, wherein L1 is a bivalent dipeptide linker selected from ValCit, PheLys, ValAla, and ValLys.

[0439] Embodiment 49. In the formula, L1 is [ka] wherein L1 is a bivalent dipeptide linker selected from * indicates the attachment point to L2, and ** The conjugate of any one of embodiments 38 to 44, wherein indicates the point of attachment to X2.

[0440] Embodiment 50. The conjugate of any one of embodiments 38 to 44, wherein L1 is ValCit.

[0441] Embodiment 51. wherein: L1 is [ka] where L1 * indicates the attachment point to L2, and ** The conjugate of any one of embodiments 38 to 44, wherein indicates the point of attachment to X2.

[0442] Embodiment 52. The conjugate of any one of embodiments 38 to 51, wherein L2 is a linker.

[0443] Embodiment 53. In the formula, L2 is - * C(=O)((CH2) m O) p (CH2) m ** -,- * C(=O)(CH2) m ** -,- * C(=O)(CH2) n NHC(=O)(CH2) m ** -,- * C(=O)(CH2) m NHC(=O)((CH2) m O) p (CH2) m ** -,- * ((CH2) m O) p (CH2) m ** -,- * ((CH2) m O) p (CH2) m ** -, -(CH2) m -,- * (CH2) m NHC(=O)(CH2) m ** -,- * (CH2) m NHC(=O)(CH2) m C(=O)NH(CH2) m ** -,- * ((CH2) m O) p (CH2) m NHC(=O)(CH2) m ** -,- ** ((CH2) m O) p CH2) m C(=O)NH(CH2) m ** -,-* (CH2) m C(R3)2 ** -and- * (CH2) m C(R3)2SS(CH2) m NHC(=O)(CH2) m ** -, wherein L2 is a linker selected from * indicates the attachment point to L1, and ** indicates the attachment point to X1; During the ceremony, each R3 is independently selected from H and C1-C6 alkyl; each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; 52. The conjugate of any one of embodiments 38 to 51, wherein each p is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14.

[0444] Embodiment 54. In the formula, L2 is - * C(=O)((CH2) m O) p (CH2) m ** -or- * C(=O)(CH2) m ** -, where L2 * indicates the attachment point to L1, and ** indicates the attachment point to X1, 52. The conjugate of any one of embodiments 38 to 51, wherein each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0445] Embodiment 55. In the formula, L2 is [ka] and L2 * indicates the attachment point to L1, and **The conjugate of any one of embodiments 38 to 51, wherein indicates the point of attachment to X1.

[0446] Embodiment 56. In the formula, X1 is [ka] , ** -, wherein X1 is a divalent linking group selected from * indicates the attachment point to L2, and X1 ** indicates the point of attachment to Ab; During the ceremony, Each R 4 is independently selected from H and C1-C6 alkyl; Each R 5 are independently selected from H, C1-C6 alkyl, F, Cl, and —OH; Each R 6 are independently selected from H, C1-C6 alkyl, F, Cl, —NH2, —OCH3, —OCH2CH3, —N(CH3)2, —CN, —NO2, and —OH; Each R 7 are independently H, C 1~6 Alkyl, fluoro, benzyloxy substituted by -C(=O)OH, benzyl substituted by -C(=O)OH, C substituted by -C(=O)OH 1~4 C substituted by alkoxy and -C(=O)OH 1~4 selected from alkyl, The conjugate of any one of embodiments 38 to 55.

[0447] Embodiment 57. In the formula, X1 is [ka] (Wherein, X1 * indicates the attachment point to L2, and X1 ** indicates the point of attachment to Ab) 56. The conjugate of any one of embodiments 38-55, wherein the divalent linking group is selected from:

[0448] Embodiment 58. In the formula, X1 is [ka] (Wherein, X1 * indicates the attachment point to L2, and X1 ** indicates the point of attachment to Ab) 56. The conjugate of any one of embodiments 38-55, wherein the divalent linking group is selected from:

[0449] Embodiment 59. wherein: Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; R 0 is methyl or ethyl; R 1 is methyl or isopropyl; R 2 is methyl or ethyl; X1 is [ka] wherein X1 is a divalent linking group selected from * indicates the attachment point to L2, and X1 ** indicates the attachment point to Ab, X2 is [ka] In the formula, X2 * indicates the attachment point to L1, and X2 ** teeth, [ka] indicates the point of attachment to the group; L1 is [ka] where L1 * indicates the attachment point to L2, and ** indicates the attachment point to X2; L2 is [ka] where L2 * indicates the attachment point to L1, and ** indicates the attachment point to R8, and y is 1, 2, 3 or 4; The conjugate of formula (C-2) of embodiment 38.

[0450] Embodiment 60. wherein: Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; R 0 is methyl or ethyl; R 1 is methyl or isopropyl; R 2 is methyl or ethyl; X1 is [ka] wherein X1 is a divalent linking group selected from * indicates the attachment point to L2, and X1 ** indicates the attachment point to Ab, X2 is [ka] In the formula, X2 * indicates the attachment point to L1, and X2 ** teeth, [ka] indicates the point of attachment to the group; L1 is [ka] where L1 * indicates the attachment point to L2, and ** indicates the attachment point to X2; L2 is [ka] where L2 * indicates the attachment point to L1, and ** indicates the attachment point to R8, and y is 1, 2, 3 or 4; The conjugate of formula (C-3) of embodiment 40.

[0451] Embodiment 61. Structure: [ka] (In the formula, y is 2 or 4, and Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein A conjugate of formula (C), formula (C-1) or formula (C-2) having the formula:

[0452] Embodiment 62. Structure: [ka] (In the formula, y is 2 or 4, and Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein A conjugate of formula (C), formula (C-1) or formula (C-2) having the formula:

[0453] Embodiment 63. Structure: [ka] (In the formula, y is 2 or 4, and Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein A conjugate of formula (C), formula (C-1) or formula (C-3) having the formula:

[0454] Embodiment 64. Structure: [ka] (In the formula, y is 2 or 4, and Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein A conjugate of formula (C), formula (C-1) or formula (C-3) having the formula:

[0455] Embodiment 65. Structure: [ka] (In the formula, y is 2; and Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein A conjugate of compound (E) having the formula:

[0456] Furthermore, the antibodies, antibody fragments (e.g., antigen-binding fragments), or functional equivalents of the present disclosure can be conjugated to a drug moiety that modifies a given biological response. The drug moiety should not be construed as limited to classical chemotherapeutic agents. For example, the drug moiety can be a protein, peptide, or polypeptide possessing a desired biological activity. Such proteins can include, for example, toxins such as abrin, ricin A, Pseudomonas exotoxin, cholera toxin, or diphtheria toxin; proteins such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, cytokines, apoptotic agents, anti-angiogenic agents, or biological response modifiers such as lymphokines.

[0457] In one embodiment, an antibody, antibody fragment (e.g., an antigen-binding fragment), or functional equivalent of the disclosure is conjugated to a drug moiety, such as a cytotoxin, a drug (e.g., an immunosuppressant), or a radiotoxin. Examples of cytotoxins include, but are not limited to, taxanes (see, e.g., WO 01 / 38318 and PCT / US03 / 02675), DNA alkylating agents (e.g., CC-1065 analogs), anthracyclines, tubulysin analogs, duocarmycin analogs, auristatin E, auristatin F, maytansinoids, pyrrolobenzodiazepines (PBDs), and cytotoxic agents containing reactive polyethylene glycol moieties (e.g., Sasse et al., J. Antibiot. (Tokyo), 53, 879-85 (2000); Suzawa et al., Bioorg. Med. Chem., 8, 2175-84 (2000); Ichimura et al., J. Antibiot. (Tokyo), 44, 1045-53 (1991); Francisco et al., J. Antibiot. (Tokyo), 44, 1045-53 (1991)). al., Blood (2003) (electronic publication ahead of print publication), U.S. Patent Nos. 5,475,092, 6,340,701, 6,372,738, and 6,436,931, U.S. Patent Application Publication No. 2001 / 0036923A1, pending U.S. Patent Applications Nos. 10 / 024,290 and 10 / 116,053, and WO 01 / 49698), taxon, cytochalasin B, gramicidin Examples of antihistamines include methadone D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof.Therapeutic agents include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine), ablating agents (e.g., mechlorethamine, thiotepa, chlorambucil, meiphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) Also included are cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine) (see, e.g., U.S. Patent Application Publication No. 20090304721 to Seattle Genetics).

[0458] Other examples of cytotoxins that can be conjugated to the antibodies, antibody fragments (antigen-binding fragments), or functional equivalents of the present disclosure include duocarmycins, calicheamicins, maytansines, and auristatins, and derivatives thereof.

[0459] Methods for conjugating various types of cytotoxins, linkers, and therapeutic agents to antibodies are known in the art; see, e.g., Saito et al., (2003) Adv. Drug Deliv. Rev. 55:199-215; Trail et al., (2003) Cancer Immunol. Immunother. 52:328-337; Payne, (2003) Cancer Cell 3:207-212; Allen, (2002) Nat. Rev. Cancer 2:750-763; Pastan and Kreitman, (2002) Curr. Opin. Investig. Drugs 3:1089-1091; Senter and Springer, (2001) Adv. Drug Deliv. Rev. 53:247-264.

[0460] The antibodies, antibody fragments (e.g., antigen-binding fragments), or functional equivalents of the present disclosure can also be conjugated to radioisotopes to produce cytotoxic radiopharmaceuticals, also referred to as radioimmunoconjugates. Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine-131, indium-111, yttrium-90, and lutetium-177. Methods for preparing radioimmunoconjugates are established in the art. Examples of radioimmunoconjugates are commercially available, e.g., Zevalin™ (DEC Pharmaceuticals) and Bexxar™ (Corixa Pharmaceuticals), and similar methods can be used to prepare radioimmunoconjugates using the antibodies of the present disclosure. In certain embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), which can be attached to the antibody via a linker molecule. Such linker molecules are generally known in the art and are described in Denardo et al., (1998) Clin Cancer Res. 4(10):2483-90; Peterson et al., (1999) Bioconjug. Chem. 10(4):553-7; and Zimmerman et al., (1999) Nucl. Med. Biol. 26(8):943-50, each of which is incorporated by reference in its entirety.

[0461] An antibody, antibody fragment (e.g., antigen-binding fragment), or functional equivalent of the present disclosure can also be conjugated to a heterologous protein or polypeptide (or a fragment thereof, preferably a polypeptide of at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 amino acids) to produce a fusion protein. In particular, the present disclosure provides fusion proteins comprising an antibody fragment (e.g., antigen-binding fragment) described herein (e.g., a Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment, VH domain, VH CDR, VL domain, or VL CDR) and a heterologous protein, polypeptide, or peptide.

[0462] Additional fusion proteins can be generated through the techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be used to alter the activity of an antibody or fragment thereof of the disclosure (e.g., an antibody or fragment thereof with higher affinity and lower dissociation rate). See generally U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al., (1997) Curr. Opinion Biotechnol. 8:724-33; Harayama, (1998) Trends Biotechnol. 16(2):76-82; Hansson et al., (1999) J. Mol. Biol. 287:265-76; and Lorenzo and Blasco, (1998) Biotechniques 24(2):308-313 (each of which patents and publications is incorporated herein by reference in its entirety). Antibodies or fragments thereof, or the encoded antibodies or fragments thereof, can be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion, or other methods prior to recombination. Polynucleotides encoding antibodies or fragments thereof that specifically bind to an antigen can be recombined with one or more components, motifs, sections, moieties, domains, fragments, etc., of one or more heterologous molecules.

[0463] Furthermore, the antibodies, antibody fragments (e.g., antigen-binding fragments), or functional equivalents of the present disclosure can be conjugated to a marker sequence, e.g., a peptide, to facilitate purification. In a preferred embodiment, the marker amino acid sequence is a hexahistidine peptide (SEQ ID NO: 267), such as the tag provided in pQE vectors (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA 91311), among others, many of which are commercially available. As described in Gentz ​​et al., (1989) Proc. Natl. Acad. Sci. USA 86:821-824, for example, hexahistidine (SEQ ID NO: 267) provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin ("HA") tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., (1984) Cell 37:767), and the "FLAG" tag (A. Einhauer et al., J. Biochem. Biophys. Methods 49:455-465, 2001). According to the present disclosure, antibodies or antigen-binding fragments can also be conjugated to tumor-penetrating peptides to enhance their effectiveness.

[0464] In other embodiments, the antibodies, antibody fragments (e.g., antigen-binding fragments), or functional equivalents of the present disclosure are conjugated to a diagnostic or detection agent. Such immunoconjugates may be useful for monitoring or prognosing the onset, development, progression, and / or severity of diseases and / or disorders as part of a clinical testing procedure, e.g., determining the effectiveness of a particular treatment. Such diagnosis and detection can be accomplished by coupling antibodies to detectable substances, including, but not limited to, various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; artificial groups, such as, but not limited to, streptavidin / biotin and avidin / biotin; fluorescent materials, such as, but not limited to, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 666, Alexa Fluor 688, Alexa Fluor 700, Alexa Fluor 668, Alexa Fluor 68 ... Fluor 750, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials, for example, but not limited to, luminol; bioluminescent materials, for example, but not limited to, luciferase, luciferin, and aequorin; radioactive materials, for example, but not limited to, iodine ( 131 I, 125 I, 123 I, and 121 I,), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 115 In, 113 In, 112 In, and 111 In,), technetium ( 99Tc), thallium ( 201 Ti), Gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F). 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh, 97 Ru, 68 Ge, 57 Co, 65 Zn, 85 Sr, 32 P, 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, 64 Cu, 113 Sn, and 117 This can be achieved by binding to Sn; as well as various positron-emitting metals using positron emission tomography, and non-radioactive paramagnetic metal ions.

[0465] The antibodies, antibody fragments (e.g., antigen-binding fragments), or functional equivalents of the present disclosure can also be attached to solid supports, which are particularly useful for immunoassays or purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0466] 3. ADC Conjugation and Preparation Methods for preparing antibody-drug conjugates of formula (C), formula (C-1) and formula (C-2) A general reaction scheme for the formation of conjugates of formula (C) is shown below in Scheme 1. [ka] where RG is a compatible reactive group R attached to the linker-drug compound. 8 A reactive group, by way of example only, a thiol, amine, or ketone, on the antibody or antigen-binding fragment thereof Ab that reacts with, thereby covalently linking the antibody or antigen-binding fragment thereof Ab to one or more linker-drug moieties. 8 A non-limiting example of such a reaction of a group is a maleimide (R 8 ), or hydroxylamine (R 8 ) is)

[0467] In one embodiment, D is a GNAQ inhibitor, a GNA11 inhibitor, or an inhibitor of GNAQ and GNA11 (GNAQ / GNA11 inhibitor), and L a are RG1 and R 8 wherein n is 1, 2, 3, or 4; and y is 1, 2, 3, or 4.

[0468] A general reaction scheme for the formation of conjugates of formula (C-1) is shown below in Scheme 2. [ka] where RG is a compatible reactive group R attached to the linker-drug moiety. 8 A reactive group, by way of example only, a thiol, amine, or ketone, on the antibody or antigen-binding fragment thereof Ab that reacts with, thereby covalently linking the antibody or antigen-binding fragment thereof Ab to one or more linker-drug moieties. 8 A non-limiting example of such a reaction of a group is a maleimide (R 8 ), or hydroxylamine (R 8) is)

[0469] In one embodiment, D is a GNAQ inhibitor, a GNA11 inhibitor, or an inhibitor of GNAQ and GNA11 (GNAQ / GNA11 inhibitor), and X1 is a combination of RG1 and R 8 reacts, Y is a phosphate group, X is a self-immolative spacer, L is a divalent peptide linker, L is a bond or linker, and y is 1, 2, 3, or 4.

[0470] A general reaction scheme for the formation of conjugates of formula (C-2) is shown below in Scheme 3. [ka] where RG is a compatible reactive group R attached to the linker-drug moiety. 8 A reactive group, by way of example only, a thiol, amine, or ketone, on the antibody or antigen-binding fragment thereof Ab that reacts with, thereby covalently linking the antibody or antigen-binding fragment thereof Ab to one or more linker-drug moieties. 8 A non-limiting example of such a reaction of a group is a maleimide (R 8 ), or hydroxylamine (R 8 ) is).

[0471] where R 0 is methyl and R 1 is isopropyl, and R 2 is ethyl, and X1 is R1 and R2 8 is a divalent linking group (e.g., a succinimide ring or oxime) formed when X reacts with X; X is a self-immolative spacer; L is a divalent peptide linker; L is a bond or linker; and y is 1, 2, 3, or 4.

[0472] A general reaction scheme for the formation of conjugates of formula (C-2) is shown below in Scheme 4. [ka] where RG is a compatible reactive group R attached to the linker-drug moiety. 8 A reactive group, by way of example only, a thiol, amine, or ketone, on the antibody or antigen-binding fragment thereof Ab that reacts with, thereby covalently linking the antibody or antigen-binding fragment thereof Ab to one or more linker-drug moieties. 8 A non-limiting example of such a reaction of a group is a maleimide (R 8 ), or hydroxylamine (R 8 ) is).

[0473] where R 0 is methyl or ethyl, and R 1 is methyl or isopropyl, and R 2 is methyl or ethyl, and X1 is R1 and R2 8 is a divalent linking group (e.g., a succinimide ring or oxime) formed when X reacts with X; X is a self-immolative spacer; L is a divalent peptide linker; L is a bond or linker; and y is 1, 2, 3, or 4.

[0474] Attachment methods for engineered cysteine ​​antibody residues Conjugates of the present disclosure can be prepared, for example, by using cysteine ​​residues engineered into antibodies by site-directed mutagenesis. Such site-directed conjugates are homogeneous and have improved properties (Junutula JR, Raab H, Clark S, Bhakta S, Leipold DD, Weir S, Chen Y, Simpson M, Tsai SP, Dennis MS, Lu Y, Meng YG, Ng C, Yang J, Lee CC, Duenas E, Gorrell J, Katta V, Kim A, McDorman K, Flagella K, Venook R, Ross S, Spencer SD, Lee Wong W, Lowman HB, Vandlen R, Sliwkowski MX, Scheller RH, Polakis P, Mallet W. (2008) Nature Biotechnology 26:925-932).

[0475] Because engineered cysteines in antibodies expressed in mammalian cells are modified with adducts (disulfides), such as glutathione (GSH) and / or cysteine, during their biosynthesis (Chen et al. 2009), the engineered cysteine ​​residues in the initially expressed product are unreactive with thiol-reactive reagents, such as maleimides or bromo- or iodoacetamide groups. To conjugate a payload to the engineered cysteine ​​after expression, the glutathione or cysteine ​​adducts must be removed by reducing their disulfide adducts, which generally also involves reducing native disulfides in the expressed protein. Deprotection of the adducted engineered cysteines can be achieved by first exposing the antibody to a reducing agent, such as dithiothreitol (DTT), TCEP, or reduced cysteine, followed by a procedure that allows for the reoxidation of all native disulfide bonds in the antibody to restore and / or stabilize functional antibody structure.

[0476] Several methods can be used to reduce and reoxidize antibodies with engineered cysteine ​​residues for the preparation of antibody-drug conjugates. Attempts to follow previously described reoxidation protocols using high concentrations of CuSO4 resulted in protein precipitation (Junutula JR, Raab H, Clark S, Bhakta S, Leipold DD, Weir S, Chen Y, Simpson M, Tsai SP, Dennis MS, Lu Y, Meng YG, Ng C, Yang J, Lee CC, Duenas E, Gorrell J, Katta V, Kim A, McDorman K, Flagella K, Venook R, Ross S, Spencer SD, Lee Wong W, Lowman HB, Vandlen R, Sliwkowski MX, Scheller RH, Polakis P, Mallet W. (2008) Nature Biotechnology 26:925). The inventors have successfully prepared and obtained antibody drug conjugates using several different methods of reduction and antibody reoxidation.

[0477] The following describes a method for reducing and reoxidizing an antibody with engineered cysteine ​​residues for the preparation of antibody-drug conjugates. Freshly prepared DTT is added to the purified Cys mutant antibody to a final concentration of 10 mM. After incubation with DTT at room temperature for 1 hour, the mixture is dialyzed against PBS at 4°C for 3 days, with daily buffer exchanges to remove DTT and reoxidize the antibody's native disulfide bonds. An alternative method is to remove the reducing reagent through a desalting column, such as Sephadex G-25 equilibrated with PBS. Once the protein is fully reduced, 1 mM oxidized ascorbate (dehydroascorbic acid) is optionally added to the desalted sample, and the reoxidation incubation is carried out for 20–24 hours.

[0478] In another exemplary method, deprotection of engineered Cys residues is achieved by adding fully reduced cysteine ​​at a concentration of 20 mM to antibody bound to Protein A-Sepharose resin. Reduction of the Cys adduct is achieved by incubation for approximately 30-60 minutes at room temperature, followed by rapid removal of the reducing agent by washing the resin with 50 beds of PBS. Reoxidation of the reduced antibody is achieved by incubating the washed slurry at room temperature with or without the addition of 50-2000 nM CuCl2 as a promoter. Except for the use of copper sulfate, examples herein use each of the protocols described herein with similar results. Reoxidation restores intrachain disulfides, while dialysis, desalting, or Protein A chromatography removes the reducing agent and glutathione originally linked to the cysteine ​​and engineered cysteine ​​of the antibody. Typically, the reoxidation process is monitored using HPLC reverse-phase chromatography. The antibody is loaded onto a PLRP-S column (4000 Å, 50 mm × 2.1 mm, Agilent) heated to 80 °C and eluted using a linear gradient of 30-45% CH3CN in water containing 0.1% TFA at 1.5 mL / min with peak detection at 215, 254, and 280 nm.

[0479] After reoxidation, the antibody is conjugated to a linker-drug compound, e.g., a compound of Formula (B), Formula (B-1), Formula (B-2), or Formula (B-3) (see Schemes 1-4). For example, a compound of Formula (B), Formula (B-1), Formula (B-2), or Formula (B-3) is added to the reoxidized Cys mutant antibody in PBS buffer (pH 7.2) at 5-10 molar equivalents relative to the antibody. Incubation is carried out for 1-2 hours. The conjugation process is monitored by reverse-phase HPLC, which allows separation of conjugated from unconjugated antibody. The conjugation reaction mixture is analyzed on a PRLP-S column (4000 Å, 50 mm × 2.1 mm, Agilent) heated to 80°C, and the column is eluted with a linear gradient of 30-60% acetonitrile in water containing 0.1% TFA at a flow rate of 1.5 ml / min. Elution of the protein from the column is monitored at 280 nm, 254 nm and 215 nm.

[0480] Alternatively, for antibodies bound to Protein A resin, once the antibody is reoxidized, the resin is washed with 10 column volumes of PBS, then the resin is resuspended in an equal volume of PBS and an 8-fold excess of a compound of Formula (B), Formula (B-1), Formula (B-2) or Formula (B-3) (in DMSO) is added and incubated for 2 hours at room temperature. The resin is then washed with 50 column volumes of PBS and the resulting antibody-drug conjugate is eluted from the Protein A resin, neutralized with 1 / 10 volume of 1 M Tris pH 9.0, buffer exchanged into an appropriate buffer and subjected to preparative size exclusion chromatography (if required).

[0481] Immunoconjugates are also characterized in terms of the average loading of drug moieties relative to antibody-binding moieties, commonly referred to as the drug-antibody ratio (DAR). DAR values ​​are estimated, for example, from LC-MS data on reduced and deglycosylated samples. LC / MS allows for quantification of the average number of payload (drug moiety) molecules attached to the antibody in an ADC. HPLC separates antibodies into light and heavy chains, and also separates heavy chains (HC) and light chains (LC) according to the number of linker-payload groups per chain. Mass spectrometry data allows for the identification of component species in the mixture, such as LC, LC+1, LC+2, HC, HC+1, HC+2, etc. From the average loading of LC and HC chains, the average DAR for the ADC can be calculated. The DAR for a given immunoconjugate sample represents the average number of drug (payload) molecules attached to a tetrameric antibody containing two light chains and two heavy chains.

[0482] Throughout the text of this application, in the event of any conflict between the text of the specification and the sequence listing, the text of the specification shall control.

[0483] [Table 2]

[0484] [Table 3]

[0485] Table 4

[0486] Table 5

[0487] Table 6

[0488] Table 7

[0489] Table 8

[0490] Table 9

[0491] Table 10

[0492] Table 11

[0493] Table 12

[0494] Table 13

[0495] Table 14

[0496] Table 15

[0497] Table 16

[0498] Table 17

[0499] Table 18

[0500] Table 19

[0501] Table 20

[0502] Table 21

[0503] Table 22

[0504] Table 23

[0505] Table 24

[0506] Table 25

[0507] Table 26

[0508] Table 27

[0509] Table 28

[0510] Table 29

[0511] Table 30

[0512] Table 31

[0513] Table 32

[0514] Table 33

[0515] Table 34

[0516] Table 35

[0517] Table 36

[0518] Table 37

[0519] Table 38

[0520] Table 39

[0521] Table 40

[0522] Table 41

[0523] Table 42

[0524] Table 43

[0525] Table 44

[0526] Table 45

[0527] Table 46

[0528] Table 47

[0529] Table 48

[0530] Table 49

[0531] Table 50

[0532] Table 51

[0533] Table 52

[0534] Table 53

[0535] Table 54

[0536] Table 55

[0537] [Table 56]

[0538] [Table 57]

[0539] The generation of anti-PMEL17 antibodies is also described in Example 2 of WO 2020 / 128612, which is incorporated by reference in its entirety.

[0540] Other antibodies of the present disclosure include those in which the amino acids or nucleic acids encoding the amino acids have been mutated, but which have at least about 60, about 70, about 80, about 90, or about 95 percent identity to the sequences set forth in Table 2. In some embodiments, this includes mutant amino acid sequences in which about 1, about 2, about 3, about 4, or about 5 amino acids have been mutated in the variable region when compared to the variable region set forth in the sequences set forth in Table 2, while retaining substantially the same therapeutic activity as the antibodies set forth in Table 2.

[0541] Because each of these antibodies can bind to PMEL17, the VH, VL, full-length light chain, and full-length heavy chain sequences (amino acid sequences and nucleotide sequences encoding the amino acid sequences) can be "mixed and matched" to generate other PMEL17-binding antibodies of the present disclosure. Such "mixed and matched" PMEL17-binding antibodies can be tested using binding assays known in the art (e.g., ELISAs and other assays described in the Examples section). When these chains are mixed and matched, the VH sequence from a particular VH / VL pairing should be replaced with a structurally similar VH sequence. Similarly, the full-length heavy chain sequence from a particular full-length heavy chain / full-length light chain pairing should be replaced with a structurally similar full-length heavy chain sequence. Similarly, the VL sequence from a particular VH / VL pairing should be replaced with a structurally similar VL sequence. Similarly, the full-length light chain sequence from a particular full-length heavy chain / full-length light chain pairing should be replaced with a structurally similar full-length light chain sequence. Thus, in some embodiments, the disclosure provides an isolated antibody or antibody fragment (e.g., Fab or Fab') having: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 42, 64, 88, 112, 132, 149, 165, 184, 196, 215, 227, 239, or 254; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 25, 29, 53, 75, 99, 119, 143, 159, 171, 190, 202, 221, 233, 248, or 260; wherein the antibody specifically binds to PMEL17.

[0542] In some embodiments, the disclosure provides (i) an isolated monoclonal antibody having a full-length heavy chain comprising an amino acid sequence optimized for expression in a cell of a mammalian expression system selected from the group consisting of SEQ ID NOs: 12, 44, 66, 90, 114, 134, 151, 167, 186, 198, 217, 229, 241, 256, or 315; and a full-length light chain comprising an amino acid sequence optimized for expression in a mammalian cell selected from the group consisting of SEQ ID NOs: 23, 27, 31, 55, 77, 101, 121, 145, 161, 173, 192, 204, 223, 235, 250, or 262; or (ii) a functional protein comprising an antigen-binding portion thereof.

[0543] In some embodiments, the disclosure provides PMEL17-binding antibodies comprising the heavy and light chain CDR1, CDR2, and CDR3, or a combination thereof, set forth in Table 2. The amino acid sequences of the VH CDR1 of the antibodies are set forth in, for example, SEQ ID NOs: 1, 4, 5, 7, 33, 36, 37, 39, 57, 60, 79, 82, 83, 85, 103, 106, 107, 109, 123, 126, 127, 129, 175, 178, 179, 181, 206, 209, 210, and 212. The amino acid sequences of antibody VH CDR2 are shown, for example, in SEQ ID NOs: 2, 6, 8, 34, 38, 40, 58, 61, 62, 80, 84, 86, 104, 108, 110, 124, 128, 130, 176, 180, 182, 207, 211, and 213. The amino acid sequences of antibody VH CDR3 are shown, for example, in SEQ ID NOs: 3, 9, 35, 41, 59, 63, 81, 87, 105, 111, 125, 131, 147, 148, 163, 164, 177, 183, 194, 195, 208, 214, 225, 226, 237, 238, 252, and 253. The amino acid sequences of VL CDR1 of the antibody are shown, for example, in SEQ ID NOs: 14, 17, 20, 46, 49, 52, 68, 71, 74, 92, 95, 98, 116, 136, 139, 142, 153, 156, 158, 243, 245, and 247. The amino acid sequences of VL CDR2 of the antibody are shown, for example, in SEQ ID NOs: 15, 18, 47, 50, 69, 72, 93, 96, 137, 140, and 154. The amino acid sequences of the VL CDR3 of the antibodies are shown, for example, in SEQ ID NOs: 16, 19, 48, 51, 70, 73, 94, 97, 117, 118, 138, 141, 155, 157, 169, 170, 188, 189, 200, 201, 219, 220, 231, 232, 244, 246, 258, and 259.

[0544] Each of these antibodies can bind to PMEL17, and provided that its antigen-binding specificity is primarily provided by the CDR1, 2, and 3 regions, the VH CDR1, CDR2, and CDR3 sequences and the VL CDR1, CDR2, and CDR3 sequences can be "mixed and matched" (i.e., CDRs from different antibodies can be mixed and matched). Such "mixed and matched" PMEL17-binding antibodies can be tested using binding assays known in the art and those described in the Examples (e.g., ELISAs). When VH CDR sequences are mixed and matched, the CDR1, CDR2, and / or CDR3 sequence from a particular VH sequence should be replaced with structurally similar CDR sequences. Similarly, when VL CDR sequences are mixed and matched, the CDR1, CDR2, and / or CDR3 sequence from a particular VL sequence should be replaced with structurally similar CDR sequences. It will be readily apparent to one skilled in the art that novel VH and VL sequences may be generated by replacing the sequences of one or more VH and / or VL CDR regions with structurally similar sequences derived from the CDR sequences set forth herein for the monoclonal antibodies of the present disclosure.

[0545] Thus, in some embodiments, the present disclosure provides a heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 4, 5, 7, 33, 36, 37, 39, 57, 60, 79, 82, 83, 85, 103, 106, 107, 109, 123, 126, 127, 129, 175, 178, 179, 181, 206, 209, 210, and 212; , 104, 108, 110, 124, 128, 130, 176, 180, 182, 207, 211, and 213; heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 35, 41, 59, 63, 81, 87, 105, 111, 125, 131, 147, 148, 163, 164, 177, 183, 194, 195, 208, 214, 225, 226, 237, 238, 252, and 253; a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 17, 20, 46, 49, 52, 68, 71, 74, 92, 95, 98, 116, 136, 139, 142, 153, 156, 158, 243, 245, and 247; and a light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 18, 47, 50, 69, 72, 93, 96, 137, 140, and 154. and a light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 19, 48, 51, 70, 73, 94, 97, 117, 118, 138, 141, 155, 157, 169, 170, 188, 189, 200, 201, 219, 220, 231, 232, 244, 246, 258, and 259; wherein the antibody specifically binds to PMEL17.

[0546] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain CDR1 of SEQ ID NO: 1, 4, 5, or 7; a heavy chain CDR2 of SEQ ID NO: 2, 6, or 8; a heavy chain CDR3 of SEQ ID NO: 3 or 9; a light chain CDR1 of SEQ ID NO: 14, 17, or 20; a light chain CDR2 of SEQ ID NO: 15 or 18; and a light chain CDR3 of SEQ ID NO: 16 or 19.

[0547] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain CDR1 of SEQ ID NO: 33, 36, 37, or 39; a heavy chain CDR2 of SEQ ID NO: 34, 38, or 40; a heavy chain CDR3 of SEQ ID NO: 35 or 41; a light chain CDR1 of SEQ ID NO: 46, 49, or 52; a light chain CDR2 of SEQ ID NO: 47 or 50; and a light chain CDR3 of SEQ ID NO: 48 or 51.

[0548] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain CDR1 of SEQ ID NO: 5, 7, 57, or 60; a heavy chain CDR2 of SEQ ID NO: 58, 61, or 62; a heavy chain CDR3 of SEQ ID NO: 59 or 63; a light chain CDR1 of SEQ ID NO: 68, 71, or 74; a light chain CDR2 of SEQ ID NO: 69 or 72; and a light chain CDR3 of SEQ ID NO: 70 or 73.

[0549] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain CDR1 of SEQ ID NO: 79, 82, 83, or 85; a heavy chain CDR2 of SEQ ID NO: 80, 84, or 86; a heavy chain CDR3 of SEQ ID NO: 81 or 87; a light chain CDR1 of SEQ ID NO: 92, 95, or 98; a light chain CDR2 of SEQ ID NO: 93 or 96; and a light chain CDR3 of SEQ ID NO: 94 or 97.

[0550] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain CDR1 of SEQ ID NO: 103, 106, 107, or 109; a heavy chain CDR2 of SEQ ID NO: 104, 108, or 110; a heavy chain CDR3 of SEQ ID NO: 105 or 111; a light chain CDR1 of SEQ ID NO: 49, 52, or 116; a light chain CDR2 of SEQ ID NO: 47 or 50; and a light chain CDR3 of SEQ ID NO: 117 or 118.

[0551] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain CDR1 of SEQ ID NO: 123, 126, 127, or 129; a heavy chain CDR2 of SEQ ID NO: 124, 128, or 130; a heavy chain CDR3 of SEQ ID NO: 125 or 131; a light chain CDR1 of SEQ ID NO: 136, 139, or 142; a light chain CDR2 of SEQ ID NO: 137 or 140; and a light chain CDR3 of SEQ ID NO: 138 or 141.

[0552] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain CDR1 of SEQ ID NO: 123, 126, 127, or 129, a heavy chain CDR2 of SEQ ID NO: 124, 128, or 130; a heavy chain CDR3 of SEQ ID NO: 147 or 148; a light chain CDR1 of SEQ ID NO: 153, 156, or 158; a light chain CDR2 of SEQ ID NO: 50 or 154; and a light chain CDR3 of SEQ ID NO: 155 or 157.

[0553] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain CDR1 of SEQ ID NO: 103, 106, 107, or 109, a heavy chain CDR2 of SEQ ID NO: 104, 108, or 110; a heavy chain CDR3 of SEQ ID NO: 163 or 164; a light chain CDR1 of SEQ ID NO: 49, 52, or 116; a light chain CDR2 of SEQ ID NO: 47 or 50; and a light chain CDR3 of SEQ ID NO: 169 or 170.

[0554] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain CDR1 of SEQ ID NO: 175, 178, 179, or 181; a heavy chain CDR2 of SEQ ID NO: 176, 180, or 182; a heavy chain CDR3 of SEQ ID NO: 177 or 183; a light chain CDR1 of SEQ ID NO: 49, 52, or 116; a light chain CDR2 of SEQ ID NO: 47 or 50; and a light chain CDR3 of SEQ ID NO: 188 or 189.

[0555] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain CDR1 of SEQ ID NO: 103, 106, 107, or 109, a heavy chain CDR2 of SEQ ID NO: 104, 108, or 110; a heavy chain CDR3 of SEQ ID NO: 194 or 195; a light chain CDR1 of SEQ ID NO: 49, 52, or 116; a light chain CDR2 of SEQ ID NO: 47 or 50; and a light chain CDR3 of SEQ ID NO: 200 or 201.

[0556] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain CDR1 of SEQ ID NO: 206, 209, 210, or 212; a heavy chain CDR2 of SEQ ID NO: 207, 211, or 213; a heavy chain CDR3 of SEQ ID NO: 208 or 214; a light chain CDR1 of SEQ ID NO: 153, 156, or 158; a light chain CDR2 of SEQ ID NO: 50 or 154; and a light chain CDR3 of SEQ ID NO: 219 or 220.

[0557] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain CDR1 of SEQ ID NO: 206, 209, 210, or 212, a heavy chain CDR2 of SEQ ID NO: 207, 211, or 213; a heavy chain CDR3 of SEQ ID NO: 225 or 226; a light chain CDR1 of SEQ ID NO: 136, 139, or 142; a light chain CDR2 of SEQ ID NO: 137 or 140; and a light chain CDR3 of SEQ ID NO: 231 or 232.

[0558] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 206, 209, 210 or 212, an HCDR2 of SEQ ID NO: 207, 211 or 213, and an HCDR3 of SEQ ID NO: 237 or 238; and a light chain variable region comprising an LCDR1 of SEQ ID NO: 243, 245 or 247, an LCDR2 of SEQ ID NO: 47 or 50, and an LCDR3 of SEQ ID NO: 244 or 246.

[0559] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 206, 209, 210 or 212, an HCDR2 of SEQ ID NO: 207, 211 or 213, and an HCDR3 of SEQ ID NO: 252 or 253; and a light chain variable region comprising an LCDR1 of SEQ ID NO: 153, 156 or 158, an LCDR2 of SEQ ID NO: 50 or 154, and an LCDR3 of SEQ ID NO: 258 or 259.

[0560] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is heavy chain CDR1 of SEQ ID NO: 1, heavy chain CDR2 of SEQ ID NO: 2, heavy chain CDR3 of SEQ ID NO: 3, light chain CDR1 of SEQ ID NO: 14, light chain CDR2 of SEQ ID NO: 15, and light chain CDR3 of SEQ ID NO: 16; a heavy chain CDR1 of SEQ ID NO: 4, a heavy chain CDR2 of SEQ ID NO: 2, a heavy chain CDR3 of SEQ ID NO: 3, a light chain CDR1 of SEQ ID NO: 14, a light chain CDR2 of SEQ ID NO: 15, and a light chain CDR3 of SEQ ID NO: 16; a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:6, a heavy chain CDR3 of SEQ ID NO:3, a light chain CDR1 of SEQ ID NO:17, a light chain CDR2 of SEQ ID NO:18, and a light chain CDR3 of SEQ ID NO:19; or Heavy chain CDR1 of SEQ ID NO: 7, heavy chain CDR2 of SEQ ID NO: 8, heavy chain CDR3 of SEQ ID NO: 9, light chain CDR1 of SEQ ID NO: 20, light chain CDR2 of SEQ ID NO: 18, and light chain CDR3 of SEQ ID NO: 16 The CDR sequences are selected from:

[0561] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is a heavy chain CDR1 of SEQ ID NO: 33, a heavy chain CDR2 of SEQ ID NO: 34, a heavy chain CDR3 of SEQ ID NO: 35, a light chain CDR1 of SEQ ID NO: 46, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 48; a heavy chain CDR1 of SEQ ID NO: 36, a heavy chain CDR2 of SEQ ID NO: 34, a heavy chain CDR3 of SEQ ID NO: 35, a light chain CDR1 of SEQ ID NO: 46, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 48; a heavy chain CDR1 of SEQ ID NO: 37, a heavy chain CDR2 of SEQ ID NO: 38, a heavy chain CDR3 of SEQ ID NO: 35, a light chain CDR1 of SEQ ID NO: 49, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 51; or Heavy chain CDR1 of SEQ ID NO: 39, heavy chain CDR2 of SEQ ID NO: 40, heavy chain CDR3 of SEQ ID NO: 41, light chain CDR1 of SEQ ID NO: 52, light chain CDR2 of SEQ ID NO: 50, and light chain CDR3 of SEQ ID NO: 48 The CDR sequences are selected from:

[0562] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is a heavy chain CDR1 of SEQ ID NO: 57, a heavy chain CDR2 of SEQ ID NO: 58, a heavy chain CDR3 of SEQ ID NO: 59, a light chain CDR1 of SEQ ID NO: 68, a light chain CDR2 of SEQ ID NO: 69, and a light chain CDR3 of SEQ ID NO: 70; a heavy chain CDR1 of SEQ ID NO: 60, a heavy chain CDR2 of SEQ ID NO: 58, a heavy chain CDR3 of SEQ ID NO: 59, a light chain CDR1 of SEQ ID NO: 68, a light chain CDR2 of SEQ ID NO: 69, and a light chain CDR3 of SEQ ID NO: 70; a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:61, a heavy chain CDR3 of SEQ ID NO:59, a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:72, and a light chain CDR3 of SEQ ID NO:73; or Heavy chain CDR1 of SEQ ID NO: 7, heavy chain CDR2 of SEQ ID NO: 62, heavy chain CDR3 of SEQ ID NO: 63, light chain CDR1 of SEQ ID NO: 74, light chain CDR2 of SEQ ID NO: 72, and light chain CDR3 of SEQ ID NO: 70 The CDR sequences are selected from:

[0563] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is a heavy chain CDR1 of SEQ ID NO: 79, a heavy chain CDR2 of SEQ ID NO: 80, a heavy chain CDR3 of SEQ ID NO: 81, a light chain CDR1 of SEQ ID NO: 92, a light chain CDR2 of SEQ ID NO: 93, and a light chain CDR3 of SEQ ID NO: 94; a heavy chain CDR1 of SEQ ID NO: 82, a heavy chain CDR2 of SEQ ID NO: 80, a heavy chain CDR3 of SEQ ID NO: 81, a light chain CDR1 of SEQ ID NO: 92, a light chain CDR2 of SEQ ID NO: 93, and a light chain CDR3 of SEQ ID NO: 94; a heavy chain CDR1 of SEQ ID NO: 83, a heavy chain CDR2 of SEQ ID NO: 84, a heavy chain CDR3 of SEQ ID NO: 81, a light chain CDR1 of SEQ ID NO: 95, a light chain CDR2 of SEQ ID NO: 96, and a light chain CDR3 of SEQ ID NO: 97; or Heavy chain CDR1 of SEQ ID NO: 85, heavy chain CDR2 of SEQ ID NO: 86, heavy chain CDR3 of SEQ ID NO: 87, light chain CDR1 of SEQ ID NO: 98, light chain CDR2 of SEQ ID NO: 96, and light chain CDR3 of SEQ ID NO: 94 The CDR sequences are selected from:

[0564] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is heavy chain CDR1 of SEQ ID NO: 103, heavy chain CDR2 of SEQ ID NO: 104, heavy chain CDR3 of SEQ ID NO: 105, light chain CDR1 of SEQ ID NO: 116; light chain CDR2 of SEQ ID NO: 47; and light chain CDR3 of SEQ ID NO: 117; heavy chain CDR1 of SEQ ID NO: 106, heavy chain CDR2 of SEQ ID NO: 104, heavy chain CDR3 of SEQ ID NO: 105, light chain CDR1 of SEQ ID NO: 116, light chain CDR2 of SEQ ID NO: 47, and light chain CDR3 of SEQ ID NO: 117; a heavy chain CDR1 of SEQ ID NO: 107, a heavy chain CDR2 of SEQ ID NO: 108, a heavy chain CDR3 of SEQ ID NO: 105, a light chain CDR1 of SEQ ID NO: 49, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 118; or Heavy chain CDR1 of SEQ ID NO: 109, heavy chain CDR2 of SEQ ID NO: 110, heavy chain CDR3 of SEQ ID NO: 111, light chain CDR1 of SEQ ID NO: 52, light chain CDR2 of SEQ ID NO: 50, and light chain CDR3 of SEQ ID NO: 117 The CDR sequences are selected from:

[0565] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is a heavy chain CDR1 of SEQ ID NO: 123, a heavy chain CDR2 of SEQ ID NO: 124, a heavy chain CDR3 of SEQ ID NO: 125, a light chain CDR1 of SEQ ID NO: 136, a light chain CDR2 of SEQ ID NO: 137, and a light chain CDR3 of SEQ ID NO: 138; a heavy chain CDR1 of SEQ ID NO: 126, a heavy chain CDR2 of SEQ ID NO: 124, a heavy chain CDR3 of SEQ ID NO: 125, a light chain CDR1 of SEQ ID NO: 136, a light chain CDR2 of SEQ ID NO: 137, and a light chain CDR3 of SEQ ID NO: 138; a heavy chain CDR1 of SEQ ID NO: 127, a heavy chain CDR2 of SEQ ID NO: 128, a heavy chain CDR3 of SEQ ID NO: 125, a light chain CDR1 of SEQ ID NO: 139, a light chain CDR2 of SEQ ID NO: 140, and a light chain CDR3 of SEQ ID NO: 141; or Heavy chain CDR1 of SEQ ID NO: 129, heavy chain CDR2 of SEQ ID NO: 130, heavy chain CDR3 of SEQ ID NO: 131, light chain CDR1 of SEQ ID NO: 142, light chain CDR2 of SEQ ID NO: 140, and light chain CDR3 of SEQ ID NO: 138 The CDR sequences are selected from:

[0566] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is a heavy chain CDR1 of SEQ ID NO: 123, a heavy chain CDR2 of SEQ ID NO: 124, a heavy chain CDR3 of SEQ ID NO: 147, a light chain CDR1 of SEQ ID NO: 153, a light chain CDR2 of SEQ ID NO: 154, and a light chain CDR3 of SEQ ID NO: 155; a heavy chain CDR1 of SEQ ID NO: 126, a heavy chain CDR2 of SEQ ID NO: 124, a heavy chain CDR3 of SEQ ID NO: 147, a light chain CDR1 of SEQ ID NO: 153, a light chain CDR2 of SEQ ID NO: 154, and a light chain CDR3 of SEQ ID NO: 155; a heavy chain CDR1 of SEQ ID NO: 127, a heavy chain CDR2 of SEQ ID NO: 128, a heavy chain CDR3 of SEQ ID NO: 147, a light chain CDR1 of SEQ ID NO: 156, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 157; or Heavy chain CDR1 of SEQ ID NO: 129, heavy chain CDR2 of SEQ ID NO: 130, heavy chain CDR3 of SEQ ID NO: 148, light chain CDR1 of SEQ ID NO: 158, light chain CDR2 of SEQ ID NO: 50, and light chain CDR3 of SEQ ID NO: 155 The CDR sequences are selected from:

[0567] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is a heavy chain CDR1 of SEQ ID NO: 103, a heavy chain CDR2 of SEQ ID NO: 104, a heavy chain CDR3 of SEQ ID NO: 163, a light chain CDR1 of SEQ ID NO: 116, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 169; a heavy chain CDR1 of SEQ ID NO: 106, a heavy chain CDR2 of SEQ ID NO: 104, a heavy chain CDR3 of SEQ ID NO: 163, a light chain CDR1 of SEQ ID NO: 116, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 169; a heavy chain CDR1 of SEQ ID NO: 107, a heavy chain CDR2 of SEQ ID NO: 108, a heavy chain CDR3 of SEQ ID NO: 163, a light chain CDR1 of SEQ ID NO: 49, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 170; or Heavy chain CDR1 of SEQ ID NO: 109, heavy chain CDR2 of SEQ ID NO: 110, heavy chain CDR3 of SEQ ID NO: 164, light chain CDR1 of SEQ ID NO: 52, light chain CDR2 of SEQ ID NO: 50, and light chain CDR3 of SEQ ID NO: 169 The CDR sequences are selected from:

[0568] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is a heavy chain CDR1 of SEQ ID NO: 175, a heavy chain CDR2 of SEQ ID NO: 176, a heavy chain CDR3 of SEQ ID NO: 177, a light chain CDR1 of SEQ ID NO: 116, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 188; a heavy chain CDR1 of SEQ ID NO: 178, a heavy chain CDR2 of SEQ ID NO: 176, a heavy chain CDR3 of SEQ ID NO: 177, a light chain CDR1 of SEQ ID NO: 116, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 188; a heavy chain CDR1 of SEQ ID NO: 179, a heavy chain CDR2 of SEQ ID NO: 180, a heavy chain CDR3 of SEQ ID NO: 177, a light chain CDR1 of SEQ ID NO: 49, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 189; or Heavy chain CDR1 of SEQ ID NO: 181, heavy chain CDR2 of SEQ ID NO: 182; heavy chain CDR3 of SEQ ID NO: 183, light chain CDR1 of SEQ ID NO: 52, light chain CDR2 of SEQ ID NO: 50, and light chain CDR3 of SEQ ID NO: 188 The CDR sequences are selected from:

[0569] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is a heavy chain CDR1 of SEQ ID NO: 103, a heavy chain CDR2 of SEQ ID NO: 104, a heavy chain CDR3 of SEQ ID NO: 194, a light chain CDR1 of SEQ ID NO: 116, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 200; a heavy chain CDR1 of SEQ ID NO: 106, a heavy chain CDR2 of SEQ ID NO: 104, a heavy chain CDR3 of SEQ ID NO: 194, a light chain CDR1 of SEQ ID NO: 116, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 200; a heavy chain CDR1 of SEQ ID NO: 107, a heavy chain CDR2 of SEQ ID NO: 108, a heavy chain CDR3 of SEQ ID NO: 194, a light chain CDR1 of SEQ ID NO: 49, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 201; or Heavy chain CDR1 of SEQ ID NO: 109, heavy chain CDR2 of SEQ ID NO: 110, heavy chain CDR3 of SEQ ID NO: 195, light chain CDR1 of SEQ ID NO: 52, light chain CDR2 of SEQ ID NO: 50, and light chain CDR3 of SEQ ID NO: 200 The CDR sequences are selected from:

[0570] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is a heavy chain CDR1 of SEQ ID NO: 206, a heavy chain CDR2 of SEQ ID NO: 207, a heavy chain CDR3 of SEQ ID NO: 208, a light chain CDR1 of SEQ ID NO: 153, a light chain CDR2 of SEQ ID NO: 154, and a light chain CDR3 of SEQ ID NO: 219; a heavy chain CDR1 of SEQ ID NO: 209, a heavy chain CDR2 of SEQ ID NO: 207, a heavy chain CDR3 of SEQ ID NO: 208, a light chain CDR1 of SEQ ID NO: 153, a light chain CDR2 of SEQ ID NO: 154, and a light chain CDR3 of SEQ ID NO: 219; a heavy chain CDR1 of SEQ ID NO: 210, a heavy chain CDR2 of SEQ ID NO: 211, a heavy chain CDR3 of SEQ ID NO: 208, a light chain CDR1 of SEQ ID NO: 156, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 220; or Heavy chain CDR1 of SEQ ID NO: 212, heavy chain CDR2 of SEQ ID NO: 213, heavy chain CDR3 of SEQ ID NO: 214, light chain CDR1 of SEQ ID NO: 158, light chain CDR2 of SEQ ID NO: 50, and light chain CDR3 of SEQ ID NO: 219 The CDR sequences are selected from:

[0571] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is a heavy chain CDR1 of SEQ ID NO: 206, a heavy chain CDR2 of SEQ ID NO: 207, a heavy chain CDR3 of SEQ ID NO: 225, a light chain CDR1 of SEQ ID NO: 136, a light chain CDR2 of SEQ ID NO: 137, and a light chain CDR3 of SEQ ID NO: 231; a heavy chain CDR1 of SEQ ID NO: 209, a heavy chain CDR2 of SEQ ID NO: 207, a heavy chain CDR3 of SEQ ID NO: 225, a light chain CDR1 of SEQ ID NO: 136, a light chain CDR2 of SEQ ID NO: 137, and a light chain CDR3 of SEQ ID NO: 231; a heavy chain CDR1 of SEQ ID NO: 210, a heavy chain CDR2 of SEQ ID NO: 211, a heavy chain CDR3 of SEQ ID NO: 225, a light chain CDR1 of SEQ ID NO: 139, a light chain CDR2 of SEQ ID NO: 140, and a light chain CDR3 of SEQ ID NO: 232; or Heavy chain CDR1 of SEQ ID NO: 212, heavy chain CDR2 of SEQ ID NO: 213, heavy chain CDR3 of SEQ ID NO: 226, light chain CDR1 of SEQ ID NO: 142; light chain CDR2 of SEQ ID NO: 140; and light chain CDR3 of SEQ ID NO: 231 The CDR sequences are selected from:

[0572] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 206, an HCDR2 of SEQ ID NO: 207, and an HCDR3 of SEQ ID NO: 237, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 243, an LCDR2 of SEQ ID NO: 47, and an LCDR3 of SEQ ID NO: 244; a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 209, an HCDR2 of SEQ ID NO: 207, and an HCDR3 of SEQ ID NO: 237, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 243, an LCDR2 of SEQ ID NO: 47, and an LCDR3 of SEQ ID NO: 244; a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 210, an HCDR2 of SEQ ID NO: 211, and an HCDR3 of SEQ ID NO: 237, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 245, an LCDR2 of SEQ ID NO: 50, and an LCDR3 of SEQ ID NO: 246; or a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 212, an HCDR2 of SEQ ID NO: 213, and an HCDR3 of SEQ ID NO: 238; and a light chain variable region comprising an LCDR1 of SEQ ID NO: 247, an LCDR2 of SEQ ID NO: 50, and an LCDR3 of SEQ ID NO: 244. The CDR sequences are selected from:

[0573] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 206, an HCDR2 of SEQ ID NO: 207, and an HCDR3 of SEQ...

Claims

1. 1. A method for producing a partially reoxidized antibody or antigen-binding fragment thereof, comprising: providing an antibody or antigen-binding fragment thereof comprising one or more capped cysteine ​​residues; reducing the one or more capped cysteine ​​residues, thereby providing an antibody or antigen-binding fragment thereof comprising one or more decapped cysteine ​​residues; and storing the antibody or antigen-binding fragment thereof comprising the one or more decapped cysteine ​​residues under oxidizing conditions that allow the one or more off-target cysteine ​​residues to be recapped. Including, thereby producing said partially reoxidized antibody or antigen-binding fragment thereof.

2. The method of claim 1 , wherein the one or more capped cysteine ​​residues are located in a constant domain heavy chain of the antibody or antigen-binding fragment thereof.

3. The method of claim 1 , wherein the antibody or antigen-binding fragment thereof comprises two or four capped cysteine ​​residues.

4. 2. The method of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a capped cysteine ​​residue located in the CH1 region of the antibody or antigen-binding fragment thereof, optionally wherein the antibody or antigen-binding fragment thereof comprises a capped cysteine ​​residue located in each CH1 region of the antibody or antigen-binding fragment thereof.

5. 2. The method of claim 1, wherein the one or more capped cysteine ​​residues are one or more engineered surface-exposed cysteine ​​residues.

6. 2. The method of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a capped cysteine ​​residue located at residue 152 (EU numbering) in the CH1 region of the antibody or antigen-binding fragment thereof.

7. 2. The method of claim 1, wherein the one or more capped cysteine ​​residues are capped with cysteine ​​or glutathione.

8. The method of claim 1 , wherein the one or more decapped cysteine ​​residues are located in a constant domain heavy chain of the antibody or antigen-binding fragment thereof.

9. The method of claim 1 , wherein the antibody or antigen-binding fragment thereof comprises two or four decapped cysteine ​​residues.

10. 2. The method of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a decapped cysteine ​​residue located in the CH1 region of the antibody or antigen-binding fragment thereof, optionally wherein the antibody or antigen-binding fragment thereof comprises a decapped cysteine ​​residue located in each CH1 region of the antibody or antigen-binding fragment thereof.

11. 2. The method of claim 1, wherein the one or more decapped cysteine ​​residues are one or more engineered surface-exposed cysteine ​​residues.

12. 2. The method of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a decapped cysteine ​​residue located at residue 152 (EU numbering) of the CH1 region of the antibody or antigen-binding fragment thereof.

13. 2. The method of claim 1, comprising reducing the one or more capped cysteine ​​residues by contacting the antibody or antigen-binding fragment thereof containing one or more capped cysteine ​​residues with a reducing wash buffer, thereby providing an antibody or antigen-binding fragment thereof containing one or more decapped cysteine ​​residues.

14. The method of claim 1, wherein the reducing and washing buffer comprises 5 mM to 50 mM cysteine. Optionally, the reducing and washing buffer comprises 10 mM to 40 mM, 20 mM to 30 mM, 5 mM to 15 mM, 5 mM to 25 mM, 5 mM to 35 mM, 5 mM to 45 mM, 40 mM to 50 mM, 30 mM to 50 mM, 20 mM to 50 mM, 10 mM to 50 mM, cysteine ​​at 10 mM, 10 mM to 20 mM, 15 mM to 25 mM, 25 mM to 35 mM, 30 mM to 40 mM, 35 mM to 45 mM, or 5 mM to 15 mM, optionally wherein the reducing wash buffer comprises 10 mM, 12 mM, 14 mM, 16 mM, 20 mM, 25 mM, or 30 mM cysteine; and / or (b) the reduction wash buffer has a pH of 6.0 to 7.5, optionally the pH is 6.5 to 7.2 or 6.8 to 7.0, optionally the pH is 6.9; The method of claim 13.

15. 14. The method of claim 13, wherein contacting the antibody or antigen-binding fragment thereof comprising one or more capped cysteine ​​residues with the reducing wash buffer is performed on a column, optionally wherein the column is a cation exchange chromatography column.

16. The method of claim 1, further comprising recovering the antibody or antigen-binding fragment thereof comprising the one or more decapped cysteine ​​residues in the eluate after contact with a reducing wash buffer. (a) the elution solution has a pH of 5.5 to 6.0, optionally the pH is 5.8; and / or (b) the concentration of the antibody or antigen-binding fragment thereof comprising the one or more decapped cysteine ​​residues in the eluate is between 5 g / L and 25 g / L, optionally the concentration of the antibody or antigen-binding fragment thereof comprising the one or more decapped cysteine ​​residues in the eluate is between 8 g / L and 20 g / L, 10 g / L and 18 g / L, 12 g / L and 15 g / L, 5 g / L and 20 g / L, 5 g / L and 15 g / L, 5 g / L and 10 g / L, 20 g / L and 25 g / L, 15 g / L and 25 g / L, 10 g / L and 25 g / L. 10 g / L to 20 g / L, 13 g / L to 14 g / L, 12 g / L to 15 g / L, and optionally the concentration of the antibody or antigen-binding fragment thereof comprising the one or more decapped cysteine ​​residues in the eluate is 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 13.5 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, or 25 g / L.

17. The method of claim 16.

18. 17. The method of claim 16, wherein the antibody or antigen-binding fragment thereof comprising the one or more decapped cysteine ​​residues is stored in the elution solution with agitation.

19. 17. The method of claim 16, wherein the antibody or antigen-binding fragment thereof comprising the one or more decapped cysteine ​​residues is stored in the elution solution without stirring.

20. The method of claim 20, wherein the antibody or antigen-binding fragment thereof comprising the one or more decapped cysteine ​​residues is stored in a container that is up to 50%-70% or 60% filled by volume, optionally the container is up to 60% filled by volume. (b) the antibody or antigen-binding fragment thereof comprising one or more decapped cysteine ​​residues is stored for between 12 hours and 96 hours, optionally the antibody or antigen-binding fragment thereof comprising one or more decapped cysteine ​​residues is stored for between 12 hours and 84 hours, between 24 hours and 84 hours, between 36 hours and 72 hours, between 48 hours and 60 hours, between 24 hours and 72 hours, between 24 hours and 60 hours, between 24 hours and 48 hours, between 24 hours and 36 hours, between 72 hours and 84 hours, 60 hours to 84 hours, 48 ​​hours to 84 hours, 36 hours to 84 hours, 12 hours to 36 hours, 24 hours to 48 hours, 36 hours to 60 hours, 48 ​​hours to 72 hours, 72 hours to 96 hours, or 48 hours to 96 hours, optionally the antibody or antigen-binding fragment thereof comprising one or more decapped cysteine ​​residues is stored for 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, or 96 hours. (c) the antibody or antigen-binding fragment thereof comprising the one or more decapped cysteine ​​residues is stored at room temperature or at 2°C to 8°C or 4°C; and / or (d) storing the antibody or antigen-binding fragment thereof containing the one or more decapped cysteine ​​residues under an air gap; The method of claim 1.

21. The method of claim 1, further comprising purifying the partially reoxidized antibody or antigen-binding fragment thereof. (a) the antibody or antigen-binding fragment thereof comprises a substitution of one or more amino acids by cysteines on its constant region at positions selected from the group consisting of positions 117, 119, 121, 124, 139, 152, 153, 155, 157, 164, 169, 171, 174, 189, 191, 195, 197, 205, 207, 246, 258, 269, 274, 286, 288, 290, 292, 293, 320, 322, 326, 333, 334, 335, 337, 344, 355, 360, 375, 382, ​​390, 392, 398, 400 and 422 of the heavy chain of the antibody or antigen-binding fragment thereof, and the positions are numbered according to the EU system; and / or (b) the antibody or antigen-binding fragment thereof comprises a substitution of one or more amino acids by cysteine ​​on its constant region at a position selected from the group consisting of positions 107, 108, 109, 114, 129, 142, 143, 145, 152, 154, 156, 159, 161, 165, 168, 169, 170, 182, 183, 197, 199, and 203 of the light chain of the antibody or antigen-binding fragment thereof, wherein the positions are numbered according to the EU system, and the light chain is a human kappa light chain; The method of claim 1.

23. 2. The method of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a substitution of an amino acid with a cysteine ​​on its constant region at positions 152, 375, or both 152 and 375 of the heavy chain of the antibody or antigen-binding fragment thereof, positions being numbered according to the EU system.

24. The method of claim 1, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody, an isolated antibody, a synthetic antibody, or an engineered antibody.

25. 25. A method of producing an antibody drug conjugate comprising attaching a linker-drug moiety to the partially reoxidized antibody or antigen-binding fragment thereof produced by the method of any one of claims 1 to 24, thereby producing an antibody drug conjugate.

26. 26. The method of claim 25, further comprising purifying the antibody-drug conjugate.

27. (a) the method results in at least 75% on-site coupling or one linker-drug moiety ("HC1") per heavy chain; optionally, the method results in at least 80%, 85%, 90%, or 95% on-site coupling or one linker-drug moiety ("HC1") per heavy chain; (b) the process results in less than 25% off-site coupling or one linker-drug moiety ("LC1") per light chain and / or two linker-drug moieties ("HC2") per heavy chain; optionally, the process results in less than 20%, 15%, 10%, or 5% off-site coupling or one linker-drug moiety ("LC1") per light chain and / or two linker-drug moieties ("HC2") per heavy chain; and / or (c) the process results in a purity of at least 70%, optionally the process results in a purity of at least 75%, 80%, 85%, 90%, or 95%, optionally the purity being determined by non-reducing CE-SDS.

26. The method of claim 25.