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WO2025072355A1 - Procédés et compositions de bioconjugués non peg à rapport médicament/agent ultra-élevé - Google Patents

Procédés et compositions de bioconjugués non peg à rapport médicament/agent ultra-élevé Download PDF

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Publication number
WO2025072355A1
WO2025072355A1 PCT/US2024/048442 US2024048442W WO2025072355A1 WO 2025072355 A1 WO2025072355 A1 WO 2025072355A1 US 2024048442 W US2024048442 W US 2024048442W WO 2025072355 A1 WO2025072355 A1 WO 2025072355A1
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aspects
bioconjugate
drugs
dar
agent
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Inventor
Antonina SIMAKOVA
Krzysztof Matyjaszewski
Laura BENJAMIN
Adam VOELKER
Andrew KASSICK
Amanda MINCEY
Matthew Cummings
Cassandra CRIHFIELD
David Wilson
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Biohybrid Solutions Holdings Inc
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Biohybrid Solutions Holdings Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6883—Polymer-drug antibody conjugates, e.g. mitomycin-dextran-Ab; DNA-polylysine-antibody complex or conjugate used for therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the field of this invention generally relates to bioconjugates comprising agents (e.g., antibodies, antigen-binding fragments thereof, cytokines, enzymes, or polynucleotides) that bind to cancer-associated antigens linked to a non-polyethylene glycol (PEG) polymer with a drug (e.g., a chemotherapeutic drug), methods of using the bioconjugates for the treatment of cancer, and/or processes of making said bioconjugates.
  • agents e.g., antibodies, antigen-binding fragments thereof, cytokines, enzymes, or polynucleotides
  • PEG non-polyethylene glycol
  • Cancer is one of the leading causes of death in the developed world, with over one million people diagnosed with cancer and 500,000 deaths per year in the United States alone. Overall, it is estimated that more than 1 in 3 people will develop some form of cancer during their lifetime. There are more than 200 different types of cancer, four of which — breast, lung, colorectal, and prostate — account for over half of all new cases (Jemal et al., 2003, Cancer J. Clin. 53:5-26).
  • Bioconjugates and Antibody-drug conjugates provide a powerful class of anticancer agents with efficacy across a range of cancers.
  • Approved ADCs are commonly composed of three distinct elements: an agent (e.g., an antibody); a linker; and a cytotoxic moiety (e.g., a taxane).
  • Antibodies or other agents that specifically bind cancer surface antigens are used to deliver cytotoxic drugs in the form of bioconjugates.
  • the number of molecules of a drug conjugated per antibody in the ADC is typically a distribution of species ranging from 0-8 per antibody.
  • DAR drug-to- agent ratio
  • bioconjugates with ultra-high DAR comprising antibodies, antigen-binding fragments thereof, or other targeting agents (e.g., cytokines, enzymes, polynucleotides) that bind to cancer-associated antigens, and coupled to one or more drugs or more than one drug type, methods of their use.
  • Compositions e.g., pharmaceutical compositions
  • methods of making and using the bioconjugates are also provided, such as methods of using the bioconjugates to inhibit tumor growth and/or treat cancer.
  • the disclosure provides a bioconjugate comprising an agent linked to a non-polyethylene glycol (PEG) polymer, wherein the non-PEG polymer is coupled to a first drug and the agent binds to a cancera associated antigen, and wherein the bioconjugate has a drug-to-agent ratio (DAR) of at least 20: 1.
  • PEG polyethylene glycol
  • the disclosure provides a bioconjugate comprising an agent linked to a non-polyethylene glycol (PEG) polymer, wherein the non-PEG polymer is coupled to a first drug and the agent binds to a cancera associated antigen, and wherein the bioconjugate has a drug-to-agent ratio (DAR) of at least 100: 1.
  • PEG polyethylene glycol
  • the present disclosure provides a method of making a bioconjugate comprising:
  • the present disclosure provides a method of making a bioconjugate comprising:
  • the DAR is at least 30: 1. In a certain aspect, the DAR is at least 40: 1. In a certain aspect, the DAR is at least 50: 1. In another aspect, the DAR is at least 60: 1. In another aspect, the DAR is at least 70: 1. In another aspect, the DAR is at least 80: 1. In another aspect, the DAR is at least 90: 1. In another aspect, the DAR is at least 100: 1. In another aspect, the DAR is at least 110: 1. In another aspect, the DAR is at least 120: 1. In another aspect, the DAR is at least 130: 1. In another aspect, the DAR is at least 140: 1. In another aspect, the DAR is at least 150: 1. In another aspect, the DAR is at least 160: 1.
  • the DAR is at least 170: 1. In another aspect, the DAR is at least 180: 1. In another aspect, the DAR is at least 190: 1. In another aspect, the DAR is at least 200: 1. In another aspect, the DAR is at least 210: 1. In another aspect, the DAR is at least 220: 1. In another aspect, the DAR is at least 230: 1. In another aspect, the DAR is at least 240: 1. In another aspect, the DAR is at least 250: 1. In another aspect, the DAR is at least 260: 1. In another aspect, the DAR is at least 270: 1. In another aspect, the DAR is at least 280: 1. In another aspect, the DAR is at least 290: 1. In another aspect, the DAR is at least 300: 1.
  • the DAR is at least 400: 1. In another aspect, the DAR is at least 500: 1. In another aspect, the DAR is at least 600: 1. In another aspect, the DAR is at least 700: 1. In another aspect, the DAR is at least 800: 1. In another aspect, the DAR is at least 900: 1. In another aspect, the DAR is at least 1000: 1. In another aspect, the DAR is at least 1100: 1.
  • the agent of the bioconjugate described herein is an antibody or antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof is a full length antibody.
  • the antibody or antigen-binding fragment thereof is an antigen binding fragment.
  • the antigen binding fragment comprises a Fab, a Fab', a F(ab')2, a Fd, a single chain Fv or scFv, a disulfide linked Fv, a V NAR domain, a IgNar, an intrabody, an IgG-CH2, a minibody, a F(ab')3, a tetrabody, a triabody, a diabody, a single-domain antibody (VHH), DVD-Ig, Fcab, mAb2, a (scFv)2, DARPin, or a scFv-Fc.
  • the antigen binding fragment comprises a single chain Fv or scFv.
  • the cancer associated antigen of the bioconjugate described herein is HER2, EGFR, or phosphatidylserine (PS).
  • the antibody or antigen binding fragment comprises a single chain Fv or scFv and binds to HER2.
  • the antibody or antigen binding fragment comprises a single chain Fv or scFv and binds to EGFR.
  • the antibody or antigen binding fragment comprises a single chain Fv or scFv and binds to phosphatidylserine.
  • the antibody or antigen-binding fragment is coupled to the non-PEG polymer bottlebrush by a non-cleavable leash.
  • the leash comprises an amino acid linker.
  • the leash comprises a histidine tag (His-Tag).
  • the antibody or antigen-binding fragment is coupled to the non-PEG polymer bottlebrush by a glycine leash, GGGGS (SEQ ID NO: 32), (GGGGS) 2 (SEQ ID NO: 33), (GGGGS) 3 (SEQ ID NO: 5), (GGGGS) 4 (SEQ ID NO: 34), or (GGGGS)s (SEQ ID NO: 35).
  • the antibody or antigen-binding fragment is coupled to the non-PEG polymer bottlebrush by an ethylene glycol leash, wherein the number of units is an integer between 5-25.
  • the non-PEG polymer of the bioconjugate described herein is selected from the group consisting of: tri ethylene glycol methyl ether methacrylate (TEOMA), 2- (methylsulfmyl)ethyl methacrylate (MSEMA), 1 -phenoxy carbonyl ethyl methacrylate, 3-[[2- (methacryloyloxy)ethyl]dimethylammonio]propionate (PCMA), 3-[[2- (methacryloyloxy)ethyl]dimethylammonio]propionate (CBMA), 2-((2-bromo-2- methylpropanoyl)oxy)ethyl methacrylate (BMPOEMA), 2-(2-bromo-2-methylpropanamido)ethyl methacrylate (BMPAEMA), polyethylene glycol monomethyl ether methacrylate (PEGMA) wherein the number of ethylene glycol units is an integer between 3 to 9, tetraethylene glycol
  • the polymer of the bioconjugate described herein is coupled to the drug by a cleavable linker.
  • the polymer is coupled to the drug by an amino acid linker.
  • the polymer is coupled to the drug by a valine-citrulline-/?- aminobenzylcarbamate, valine-citrulline or alanine-alanine linker.
  • the bioconjugate described herein further comprises a second drug.
  • the ratio of the first drug to the second drug is 1 : 1 to 20: 1. In one aspect, the ratio of the first drug to the second drug is 20: 1 to 1 : 1.
  • the bioconjugate described herein further comprises a second drug and a third drug.
  • the ratio of the first drug to the second drug to the third drug is 1 : 1 : 1.
  • the bioconjugate described herein further comprises a fourth drug. In one aspect, the ratio of the first drug to the second drug to the third drug to the fourth drug is 1 : 1 : 1 : 1. [0020] In certain aspects, the bioconjugate described herein further comprises a fifth drug. In one aspect, the ratio of the first drug to the second drug to the third drug to the fourth drug to the fifth drug is 1 : 1 : 1 : 1 : 1.
  • the drugs of the bioconjugate described herein are chemotherapeutic drugs.
  • the first drug, the second drug, the third drug, the fourth drug, and/or the fifth drug is a chemotherapeutic drug.
  • the chemotherapeutic drugs are selected from the group consisting of: a MEK inhibitor, a PI3K inhibitor, a MAPK inhibitor, a CDK inhibitor, a CHK inhibitor, an alkylating agent, a cytoskeletal disruptor, a histone deacetylase inhibitor, a topoisomerase I or II inhibitor, nucleotide analogs or precursors thereof, peptide antibiotics, a protein degrader, platinum-based agents, retinoids, vinka alkaloids and derivatives thereof, and combinations thereof.
  • the chemotherapeutic drug comprises a MEK inhibitor.
  • the MEK inhibitor is selected from the group consisting of: binimetinib, GDC-0973 (cobimetinib), selumetinib, trametinib.
  • the chemotherapeutic drug comprises a PI3K inhibitor.
  • the PI3K inhibitor is selected from the group consisting of: PI103; PI828; LY294002; wortmannin; demethoxyviridin; IC486068; IC87114; GDC-0941; GDC-0980 (apitolisib); perifosine; CAL101; PX-866; IPI-145; BAY 80-6946; BEZ235; P6503; TGR1202; SF1126; INK1117; BKM120; IL147; XL765;
  • the disclosure provides a bioconjugate comprising an agent linked to a non-polyethylene glycol (PEG) polymer, wherein the agent is an antibody or antigen-binding fragment thereof, wherein the polymer is coupled to a MEK inhibitor, a CDK inhibitor, and/or a PI3K inhibitor, and the antibody or antigen-binding fragment thereof binds to a HER2 protein or EGFR protein or phosphatidylserine, and wherein the bioconjugate has a drug-to-agent ratio (DAR) of 60: 1 to 200:1. In a certain aspect, the bioconjugate has a DAR of 60: 1 to 150: 1.
  • DAR drug-to-agent ratio
  • the bioconjugate has a DAR of 60: 1 to 110: 1. In a certain aspect, the bioconjugate has a DAR of 60: 1 to 300: 1. In a certain aspect, the bioconjugate has a DAR of 150: 1 to 300: 1.
  • the disclosure provides a bioconjugate comprising an agent linked to a nonpolyethylene glycol (PEG) polymer, wherein the agent is an antibody or antigen-binding fragment thereof, wherein the polymer is coupled to a MEK inhibitor, a CDK inhibitor, and/or a PI3K inhibitor, and the antibody or antigen-binding fragment thereof binds to a HER2 protein or EGFR protein or phosphatidylserine, and wherein the bioconjugate has a drug-to-agent ratio (DAR) of 100:1 to 1,100: 1. In a certain aspect, the bioconjugate has a DAR of 200: 1 to 1,100: 1.
  • DAR drug-to-agent ratio
  • the bioconjugate has a DAR of 400: 1 to 1,100: 1. In a certain aspect, the bioconjugate has a DAR of 600: 1 to 1,100: 1. In a certain aspect, the bioconjugate has a DAR of 800: 1 to 1,100: 1. In a certain aspect, the bioconjugate has a DAR of 900: 1 to 1,100: 1.
  • the MEK inhibitor is GDC-0973 and the PI3K inhibitor is GDC- 0941.
  • the MEK inhibitor is GDC-0973 and the PI3K inhibitor is GDC-0980 (apitolisib).
  • the MEK inhibitor is GDC-0973 (cobimetinib)
  • the CDK inhibitor is dinaciclib
  • the PI3K inhibitor is GDC-0980 (apitolisib).
  • the antibody or antigen-binding fragment thereof binds to an EGFR protein
  • the MEK inhibitor is GDC-0973 (cobimetinib)
  • the CDK inhibitor is dinaciclib.
  • the antibody or antigenbinding fragment thereof binds to an EGFR protein
  • the PI3K inhibitor is GDC-0980 (apitolisib)
  • the CDK inhibitor is dinaciclib.
  • the antibody or antigen-binding fragment thereof binds to phosphatidylserine
  • the MEK inhibitor is GDC-0973 (cobimetinib)
  • the CDK inhibitor is dinaciclib.
  • the antibody or antigen-binding fragment thereof binds to phosphatidylserine
  • the PI3K inhibitor is GDC-0980 (apitolisib)
  • the CDK inhibitor is dinaciclib.
  • the disclosure also provides a pharmaceutical composition comprising a bioconjugate described herein and a pharmaceutically acceptable carrier. [0025] In a certain aspect, the disclosure also provides a kit comprising a bioconjugate described herein.
  • the disclosure also provides a method of inhibiting tumor growth in a subject, comprising administering a therapeutically effective amount of a bioconjugate described herein or a pharmaceutical composition described herein.
  • the tumor is a tumor selected from the group consisting of an ovarian tumor, a brain tumor, a breast tumor, a uterine tumor, an endometrial tumor, a pancreatic tumor, a renal tumor, a head and neck tumor, a gastric tumor, and a lung tumor.
  • the tumor is a breast tumor, a head and neck tumor, a lung tumor, or a gastric tumor.
  • the disclosure provides a method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of a bioconjugate described herein or a pharmaceutical composition described herein to the subject.
  • the cancer is a cancer selected from the group consisting of ovarian cancer, brain cancer, breast cancer, uterine cancer, endometrial cancer, pancreatic cancer, renal cancer, head and neck cancer, gastric cancer, and lung cancer.
  • the cancer is a breast cancer, head and neck cancer, lung cancer, or gastric cancer.
  • the disclosure also provides a method of increasing cancer cell cytotoxicity comprising contacting a cancer cell with a bioconjugate described herein or a pharmaceutical composition described herein.
  • the disclosure also provides a method of inhibiting proliferation of a cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the bioconjugate described herein or the pharmaceutical composition described herein to the subject.
  • the disclosure also provides an isolated polynucleotide comprising a sequence at least 90% identical to a sequence encoding the amino acid sequence of one or more of SEQ ID NOs: 1-4, 6-16, and 20-31.
  • the isolated polynucleotides is at least 95% identical to a sequence encoding the amino acid sequence of one or more of SEQ ID NOs: 1-4, 6-16, and 20-31.
  • the isolated polynucleotide is at least 99% identical to a sequence encoding the amino acid sequence of one or more of SEQ ID NOs: 1-4, 6-16, and 20- 31.
  • the isolated polynucleotide is identical to a sequence encoding the amino acid sequence of one or more of SEQ ID NOs: 1-4, 6-16, and 20-31.
  • the disclosure also provides a vector comprising any of the polynucleotides of SEQ ID NOs: 1-4, 6-16, and 20-31.
  • the disclosure also provides a host cell comprising the vector that contains a polynucleotide of SEQ ID NOs: 1-4, 6-16, and 20-31.
  • the host cell is selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, Pichia, CHO, YB/20, NS0, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2/0, Rl. l, B-W, L-M, COS 1, COS 7, BSC1, BSC40, BMT10 cell, plant cell, insect cell, and human cell in tissue culture.
  • the disclosure also provides a method of producing an antibody or antigen-binding fragment thereof comprising culturing the host cell so that the nucleic acid molecule is expressed and the antibody or antigen-binding fragment thereof is produced, optionally wherein the method further comprises isolating the antibody or antigen-binding fragment thereof from the culture.
  • the chain transfer agent (CTA) is selected from a ,
  • the at least one monomer units containing an initiator molecule for Atom Transfer Radical Polymerization is selected from 2-((2-bromo-2- methylpropanoyl)oxy)ethyl methacrylate (BMPOEMA) or 2-(2-bromo-2- methylpropanamido)ethyl methacrylate (BMPAEMA).
  • the at least one monomer units containing an initiator molecule for Atom Transfer Radical Polymerization is 2-(2-bromo-2-methylpropanamido)ethyl methacrylate (BMPAEMA).
  • the at least one monomer units containing an initiator molecule for Atom Transfer Radical Polymerization is selected from
  • the second monomer unit is selected from triethylene glycol methyl ether methacrylate (TEOMA), polyethylene glycol monomethyl ether methacrylate (PEGMA) wherein the number of ethylene glycol units is an integer between 3 to 9, tetraethylene glycol methyl ether methacrylate (TetEOMA), pentaethylene glycol methyl ether methacrylate (PEOMA), hexaethylene glycol methyl ether methacrylate (HEOMA), heptaethylene glycol methyl ether methacrylate (HPEOMA), octaethylene glycol methyl ether methacrylate (OEOMA), nonaethylene glycol methyl ether methacrylate (NEOMA), PEGMA300, or PEGMA500.
  • TEOMA triethylene glycol methyl ether methacrylate
  • PEGMA polyethylene glycol monomethyl ether methacrylate
  • TetEOMA tetraethylene glycol methyl ether methacrylate
  • POMA pent
  • the at least one monomer unit containing a functional group capable of undergoing a click reaction is 2-azido triethylene glycol methyl ether methacrylate (TEOMA- Azide or TEOMA-N3). In some aspects, the at least one monomer unit containing a functional group capable of undergoing a click reaction is selected
  • the non-PEG polymer comprises at least a second monomer unit, wherein the second monomer unit is a methacrylate containing non-PEG monomer.
  • the second monomer unit in the non-PEG polymer is selected from triethylene glycol methyl ether methacrylate (TEOMA), 2-(m ethyl sulfinyl)ethyl methacrylate (MSEMA), 1- phenoxycarbonyl ethyl methacrylate, 3-[[2- (methacryloyloxy)ethyl]dimethylammonio]propionate (PCMA), 3-[[2- (methacryloyloxy)ethyl]dimethylammonio]propionate (CBMA), 2-((2-bromo-2- methylpropanoyl)oxy)ethyl methacrylate (BMPOEMA), 2-(2-bromo-2-methylpropanamido)ethyl methacrylate (BMPA
  • the coupling of the drug to the polymer is performed by an azidealkyne click reaction or an Inverse electron-demand Diels-Alder (IEDDA) reaction.
  • the coupling of the drug to the polymer is performed by an azide-alkyne click reaction.
  • the azide-alkyne click reaction is selected from copper catalyzed azide-alkyne cycloaddition (CuAAC), strain-promoted azide-alkyne cycloaddition (SPAAC), or strain- promoted alkyne-nitrone cycloaddition (SPANC).
  • the functional group capable of undergoing a click reaction is an azide (-N3).
  • the coupling of the drug to the polymer is performed by an Inverse electron-demand Diels-Alder (IEDDA) reaction.
  • the coupling of the agent that binds to a cancer associated antigen to the polymer is performed by an azide-alkyne click reaction, a strain-promoted azide-alkyne cycloaddition (SPAAC), or an Inverse electron-demand Diels-Alder (IEDDA) reaction.
  • SPAAC strain-promoted azide-alkyne cycloaddition
  • IEDDA Inverse electron-demand Diels-Alder
  • the coupling of the agent that binds to a cancer associated antigen to the polymer is performed by an azide-alkyne click reaction or a strain-promoted azide-alkyne cycloaddition (SPAAC).
  • SPAAC strain-promoted azide-alkyne cycloaddition
  • DBCO dibenzocyclooctyne
  • the dibenzocyclooctyne (DBCO) functional group is optionally substituted.
  • the coupling of an agent that binds to a cancer associated antigen to the polymer is performed by an Inverse electron-demand Diels- Alder (IEDDA) reaction.
  • IEDDA Inverse electron-demand Diels-Alder
  • the Inverse electron-demand Diels-Alder (IEDDA) reaction is a tetrazine-cyclooctene reaction.
  • the functional group capable of undergoing a click reaction is an azide (-N3).
  • a component of the biocojugate is selected from a component in Table A, or a pharmaceutically acceptable salt thereof.
  • the biocojugate is a compound in Table B, or a pharmaceutically acceptable salt thereof.
  • Figure 1A demonstrates an exemplary process for synthesizing a macromonomer that carries payloads and is then copolymerized to form a non-PEG polymer bottlebrush.
  • Figure IB demonstrates an exemplary process for synthesizing a bioconjugate with an agent and a payload using a "grafting to" approach for bioconjugation where the fully formed non-PEG bottlebrush polymer is attached to a targeting agent.
  • Figure 2A demonstrates an exemplary process for synthesizing a bottlebrush where the non-PEG polymer backbone is made first and then side-chains are grafted from the backbone.
  • Figure 2B demonstrates an exemplary process for synthesizing a bioconjugate with an agent and a payload using a combination of "grafting to” and “grafting from” approach for bioconjugation where the non-PEG polymer backbone is attached to an agent first and then non- PEG bottlebrush polymer is formed in the presence of an agent.
  • Figure 3A provides a diagram of an exemplary bioconjugate of the present disclosure, which has an agent (i.e., full-length human IgG antibody) linked to a bottle brush polymer with a large number of payloads (e.g., drugs) attached resulting in an ultra-high DAR.
  • agent i.e., full-length human IgG antibody
  • payloads e.g., drugs
  • Figure 3B provides a diagram of an exemplary bioconjugate of the present disclosure, which has an agent (i.e., scFv) linked to a bottle brush polymer with a large number of payloads (e.g., drugs) attached resulting in an ultra-high DAR bioconjugate.
  • agent i.e., scFv
  • payloads e.g., drugs
  • Figure 4A shows a line graph of the mass spectrum of bare trastuzumab.
  • Figure 4B shows a line graph of the mass spectrum of deglycosylated trastuzumab.
  • Figure 4C shows a line graph of the mass spectrum of trastuzumab-TCO (Int-23).
  • Figure 5 shows a line graph of the mass spectrum of trastuzumab -DB CO (Int-30) (C).
  • Figure 6A shows a line graph of the mass spectrum of bare cetuximab.
  • Figure 6B shows a line graph of the mass spectrum of deglycosylated cetuximab.
  • Figure 6C shows a line graph of the mass spectrum of cetuximab-TCO (Int-31).
  • Figure 7 shows a graph of the mass spectrum of the purified scFv-TCO (Int-6) at 28,190 Da.
  • Figure 8 shows a SDS-PAGE gel of the scFv-TCO (Int-6) and the conjugated scFv- backbone (Int-7) demonstrating the attachment of the agent to the polymer backbone.
  • Figure 9 shows a SDS-PAGE gel of the effect of different imidazole concentrations on various scFv-backbone sized products, which shows how the ratios of the methyacrylate polymers affect the polymerization of the polymer backbone.
  • Figure 10 shows a line graph of the size exclusion chromatography (SEC) of the scFv- TCO (Int-6), the polymer backbone, and the scFv-backbone (Int-7), which shows the attachment of the agent to the polymer backbone.
  • Figure 11 shows a line graph of the Field-Flow Fractionation (FFF) of the scFv- backbone (Int-7) and the scFv-bottl ebrush (Int-8), which shows the polymerization of the bottlebrush polymers from the polymer backbone.
  • FFF Field-Flow Fractionation
  • Figure 12 shows a line graph of the UV-vis absorbance spectra showing the attachment of the apitolisib payload by monitoring the region from 250 - 300 nm, corresponding to the apitolisib payload.
  • Figure 13 shows a line graph of the ELISA assay of trastuzumab or the trastuzumab scFv binding to HER2 at various concentrations.
  • Figure 14 shows a line graph of the ELISA assay of the scFv-bottlebrush-payload (Compound 1) and bare scFv binding to HER2 at various concentrations.
  • Figure 15 shows a SDS-PAGE gel of the bottlebrush backbone molecule (Ini-15) bioconjugation to scFv-TCO (Int-6) at ratios of 1 : 1, 5: 1, and 19: 1.
  • the SDS-PAGE gel demonstrates the attachment of the agent to the non-PEG backbone by a reduction in the scFv band and with an increase in the bottlebrush backone molar equivalent to the scFv.
  • Figure 16 shows a Western blot of the primary antibody anti-6xHis mouse IgG and a secondary antibody HRP anti-mouse IgG and the attachment to the bottlebrush.
  • the polymer lane does not have any signal, while the scFv-bottlebrush polymer (Int-18) lanes show a new band at a higher MW for the 1 : 1, 5: 1, and 19:1 polymerization ratios.
  • Figure 17 shows a line graph of the preparatory size exclusion chromatography (SEC) of the products from the reaction between trastuzumab-TCO and the polymer backbone pBMPAEMA-r-pNEOMA that contains a methyl tetrazine functional group.
  • SEC preparatory size exclusion chromatography
  • Figure 18 shows a line graph of the Field-Flow Fractionation (FFF) of the trastuzumab- TCO (Int-23 and the trastuzumab -pBMPAEMA-r-pNEOMA backbones, with varying monomer lengths (Int-25) and (Int-26) ⁇ that were collected through preparatory size exclusion chromatography (SEC).
  • FFF Field-Flow Fractionation
  • Figure 19 shows a line graph of the Field-Flow Fractionation (FFF) of the trastuzumab- pBMPAEMA-r-pNEOMA backbone (Int-26) and the trastuzumab-bottlebrush (Ini-27) ⁇ which shows the polymerization of the bottlebrush polymers from the polymer backbone.
  • FFF Field-Flow Fractionation
  • Figure 20 shows a line graph of the ELISA assay of the trastuzumab-bottlebrush (Int- 27), trastuzumab-backbone (Int-26), trastuzumab-TCO (Int-23), and bare trastuzumab binding to HER2.
  • Figure 21 shows a line graph of the size exclusion chromatography (SEC) of the products from the reaction between cetuximab-TCO and the polymer backbone pBMPAEMA-r- pNEOMA to form cetuximab -polymer backbone pBMPAEMA-r-pNEOMA (Int-32) product.
  • SEC size exclusion chromatography
  • Figure 22 shows a SDS-PAGE gel electrophoresis of the SEC fractions of the reaction between cetuximab-TCO and the polymer backbone pBMPAEMA-r-pNEOMA and shows the product cetuximab-backbone pBMPAEMA-r-pNEOMA (Int-32) product.
  • Figure 23 shows a line graph of the Field-Flow Fractionation (FFF) of the trastuzumab- pCBMA bottlebrush (Int-33) and the trastuzumab-pCBMA bottlebrush apitolisib (Compound 7).
  • Figure 24 shows a line graph of the reverse-phase liquid chromatography used to calculate the amount of cleaved apitolisib in a sample of drug-loaded polymer brush (Int-40) treated with Cathepsin B.
  • FFF Field-Flow Fractionation
  • Figure 25 shows a line graph of the PI3K inhibition activity of apitolisib-containing solution collected after the reaction of drug-loaded polymer brush (Int-40 treated with Cathepsin B.
  • Figure 26A provides a diagram of the final molecular dynamics simulation frame of the trastuzumab-bottlebrush-cobimetinib payload bioconjugate.
  • White region is either trastuzumab with antigen-binding regions in dark grey.
  • the bottlebrush is light grey aside from the apitolisib payloads, which are black.
  • the midtone grey represents the leash and backbone of the bottlebrush but is almost completely obscured by the bottlebrush bristles.
  • Figure 26B provides a diagram of the final molecular dynamics simulation frame of the trastuzumab-bottlebrush-cobimetinib payload bioconjugate.
  • White region is either trastuzumab scFv with antigen-binding regions in dark grey.
  • the bottlebrush is light grey aside from the apitolisib payloads, which are black.
  • the midtone grey represents the leash and backbone of the bottlebrush but is almost completely obscured by the bottlebrush bristles.
  • Figures 27A-27R shows confocal microscopy images of internalized controls and tested bioconjugates.
  • Letters A, G, and M represent trastuzumab without pHAb Dye
  • Letters B, H, and N represent trastuzumab with pHAb Dye
  • Letters C, I, and O represent trastuzumab- pPEGMAsoo-cobimetinib with pHAb dye with a DAR of 55
  • Letters D, J, and P represent trastuzumab-pCBMA-apitolisib with pHAbdye with a DAR of 268
  • Letters E, K, and Q represent trastuzumab-pPEGMAsoo-dual-payload apitolisib+cobimetinib with pHAb dye with a DAR of 1081
  • Letters F, L, and R represent anti-HER2 scFv pHAb Dye.
  • Figures 28A-28X shows confocal microscopy images of internalized controls and tested bioconjugates.
  • Figures 28A, 281, and 28Q represent trastuzumab without pHAb Dye
  • Figures 28B, 28J, and 28R represent trastuzumab with pHAb Dye
  • Figures 28C, 28K, and 28S represent trastuzumab-pPEGMAsoo-cobimetinib with pHAb dye with a DAR of 55
  • Figures 28D, 28L, and 28T represent trastuzumab-pCBMA-apitolisib with pHAb dye with a DAR of 268
  • Figures 28E, 28M, and 28U represent trastuzumab-pPEGMAsoo-dual-payload apitolisib+cobimetinib with pHAb dye with a DAR of 1081;
  • Figures 28E, 28M, and 28U represent trastuzumab-pPEGMAs
  • bioconjugates comprising agents including antibodies or antigen-binding fragments thereof or other agents (e.g., cytokines, enzymes, polynucleotides) that bind to cancer-associated antigens, and methods of their use.
  • agents including antibodies or antigen-binding fragments thereof or other agents (e.g., cytokines, enzymes, polynucleotides) that bind to cancer-associated antigens, and methods of their use.
  • agents including antibodies or antigen-binding fragments thereof or other agents (e.g., cytokines, enzymes, polynucleotides) that bind to cancer-associated antigens, and methods of their use.
  • agents e.g., cytokines, enzymes, polynucleotides
  • a range of 1 to 10 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • a value is explicitly recited, it is to be understood that values that are about the same quantity or amount as the recited value are also within the scope of the disclosure.
  • a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed.
  • drug or “payload” as used herein means any chemical or biological substance that causes a change in an organism's physiology (e.g., a chemotherapeutic drug).
  • polymer or “synthetic polymer” refers to a polymer which comprises more than one monomer unit. A polymer or synthetic polymer can be produced by a chemical process. A synthetic polymer is not produced directly by a living organism. Synthetic polymers include a homopolymer, heteropolymer, block polymer, co-polymer, ter-polymer, etc., and blends, combinations, and mixtures thereof.
  • synthetic polymers include, but are not limited to, functionalized polymers, such as a polymer comprising an azide, a triazole ring, an alkyne, or a trans-cyclooctene.
  • a synthetic polymer may be or contain one or more of a linear polymer, a branched polymer, a hyperbranched polymer, a graft polymer, a block copolymer, a brush polymer, and a bottlebrush copolymer.
  • Synthetic polymers include, without limitation, polyesters, poly(meth)acrylamides, poly(meth)acrylates, polyethers, and monomers that have unsaturated bonds. For example, ATRP polymerized copolymers are described in U.S. Patent Application Publication No.
  • bottlebrush polymers described herein may be present in the form of copolymers, containing a backbone formed of hydrophobic, water-insoluble polymers and side chains formed of short, hydrophilic non-cell binding polymers.
  • Examples of other synthetic polymers include, but are not limited to, polyalkylenes such as polyethylene and polypropylene; polychloroprene; polyvinyl ethers such as poly(vinyl acetate); polyvinyl halides such as poly(vinyl chloride); poly(meth)acrylates such as poly(methyl (meth)acrylate), poly(ethyl (meth)acryate), poly(triethylene glycol methyl ether methacrylate) (pTEOMA), poly(polyethylene glycol (meth)acrylate) (pPEGMA) where the average number of ethylene glycol units in a monomer unit is 3 to 9, poly(tetraethylene glycol methyl ether methacrylate) (pTetEOMA), poly(pentaethylene glycol methyl ether methacrylate) (pPEOMA), poly(hexaethylene glycol methyl ether methacrylate) (pHEOMA), poly(heptaethylene glycol methyl ether methacrylate
  • These synthetic polymers may include useful derivatives, including synthetic polymers having substitutions, additions of chemical groups, for example, alkyl groups, alkylene groups, hydroxylations, oxidations, azidation, esterifications, and other modifications routinely made by those skilled in the art.
  • the synthetic polymers may include zwitterionic polymers such as, for example, polyphosphorylcholine, polycarboxybetaine, and polysulfobetaine.
  • non-PEG polymer or “non-PEG bottlebrush polymer”, which are used interchangeably herein, refer to a bottlebrush polymer that:
  • (a) does not comprise ethylene glycol, i.e., -OCH2CH2-, units; or
  • (b) comprises monomers containing ethylene glycol units, i.e., monomer side-chains, wherein the number of consecutive ethylene glycol units in the polymer backbone and/or any of the polymer brushes, is an integer between 3 to 9.
  • a non-PEG polymer or a non- PEG bottlebrush polymer include, but are not limited to, polymers made from monomers such as triethylene glycol methyl ether methacrylate (TEOMA), 2-(m ethyl sulfinyl)ethyl methacrylate (MSEMA), 1 -phenoxy carbonyl ethyl methacrylate, 3-[[2- (methacryloyloxy)ethyl]dimethylammonio]propionate (PCMA), 3-[[2- (methacryloyloxy)ethyl]dimethylammonio]propionate (CBMA), 2-((2-bromo-2- methylpropanoyl)oxy)ethyl methacrylate (
  • initiator refers to any molecule that contains an atom, which is a radically transferable atom or group, such as a halogen. Standard organic synthetic techniques can be applied to preparing ATRP initiators. Initiators for ATRP can be prepared by a variety of methods. Some general methods for preparing ATRP initiators will be described herein. In general, the initiators can have the general formula: Y — (X) n wherein Y is the core of the molecule and X is the radically transferable atom or group. The number n can be any number 1 or higher, depending on the functionality of the core group Y. For example, in beta-alanine, n-(2- bromo-2-methyl-l -oxopropyl)-, 2,5-dioxo-l-pyrrolidinyl ester, which has the following structure:
  • Y is represented by N-succinimide ester of beta-alanine of amide of isobutyril, and X is represented by Br with n of 1.
  • X is represented by Br with n of 1.
  • Y is represented by N-succinimide ester of the branched amide
  • X is represented by Br with n of 2.
  • initiators include, but are not limited to, bromine and other halogens.
  • Small molecule initiators are commercially available, such as alkyl-halides, 2- halopropionates and 2-haloisobutyrates, carbon tetrahalides, carbon trihalides, etc. These functional groups can be incorporated into other small molecules. The incorporation of these functional groups can be done as a single substitution, or the small molecule can have more than one initiating site for ATRP.
  • a molecule containing more than one hydroxyl group can undergo an esterification reaction to generate a-haloesters which can initiate ATRP.
  • Other initiator residues can be introduced as are desired.
  • the small molecules to which the initiators are attached can be organic or inorganic; so long as the initiator does not poison the catalyst or adversely interact with the propagating radical it can be used.
  • Some examples of small molecules that were used as a foundation for the attachment of initiating sites are polydimethylsiloxane cubes, cyclotriphosphazene rings, 2-tris(hydroxyethyl)ethane, glucose-based compounds, etc.
  • trichloromethyl isocyanate can be used to attach an initiator residue to any substance containing hydroxy, thiol, amine and/or amide groups.
  • microinitiator refers to any molecule that is a synthesized polymer that comprises an atom, which is a radically transferable atom or group, such as a halogen. For example, in first synthesizing a preformed polymer that comprises an initiator via RAFT polymerization, whereby a copolymer can then grafted to the preformed polymer via ATRP.
  • macroniniators can be synthesized to introduce ATRP initiating group(s) onto a different molecule such as targeting molecule or bottlebrush backbone.
  • the macroinitiators can take different forms, and can be prepared by different methods known in the art.
  • the macroinitiators can be soluble polymers, insoluble/crosslinked polymeric supports, surfaces, or solid inorganic supports.
  • Some general methods for the preparation of the macroinitiators include modification of an existing material, (co)polymerization of one or more an AB* monomers by ATRP/non-ATRP methods, or using initiators (for other types of polymerization) that contain an ATRP initiator residue.
  • Modification of macromolecular compounds/ substrates to generate an ATRP initiation site is understood to one skilled in the art of materials/polymer modification.
  • crosslinked polystyrene with halomethyl groups on the phenyl rings used in solid-phase peptide synthesis
  • attached functional molecules to silica surfaces brominated soluble polymers (such as (co)polymers of isoprene, styrene, and other monomers), or attached small molecules containing ATRP initiators to polymer chains can all be used as macromolecular initiators. If one or more initiating sites are at the polymer chain ends, then block (co)polymers are prepared; if the initiating sites are dispersed along the polymer chain, graft (co)polymers will be formed.
  • drug-to-agent ratio means the number of drugs (e.g., chemotherapeutic drugs) attached to a non-PEG polymer in relation to an agent (e.g., antibody or antigen-binding fragment thereof, polynucleotide, cytokine, or enzyme) in a bioconjugate of the present disclosure.
  • a DAR ratio of 100: 1 means that there are 100 drugs attached to a non-PEG polymer, which is attached to 1 agent.
  • One or more different drugs can be considered the drug for the purpose of expressing the DAR ratio.
  • one or more drugs can be attached to the non-PEG polymer in relation to an agent.
  • the term "ultra-high drug-to-agent ratio" as used herein means the number of drugs (e.g., chemotherapeutic drugs) attached to a non-PEG polymer in relation to an agent (e.g., antibody or antigen-binding fragment thereof, polynucleotide, cytokine, or enzyme) in a bioconjugate of the present disclosure, wherein the ratio is at least 20 drugs to 1 agent (20: 1 DAR).
  • drugs e.g., chemotherapeutic drugs
  • an agent e.g., antibody or antigen-binding fragment thereof, polynucleotide, cytokine, or enzyme
  • ultra-high DAR can be at least 50: 1, 100:1, 150:1, 200: 1, 250: 1, 300: 1, 350: 1, 400: 1, 450: 1, 500: 1, 550: 1, 600: 1, 650:1, 700:1, 750: 1, 800: 1, 850: 1, 900: 1, 950: 1, 1,000:1, 1,050: 1, or 1100: 1.
  • One or more different drugs can be considered the drug for the purpose of expressing the DAR ratio.
  • one or more drugs can be attached to the non- PEG polymer in relation to an agent.
  • antibody means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule.
  • antibody encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies or other modified immunoglobulins, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity.
  • An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively.
  • the different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations.
  • Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.
  • antibody fragment refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, F(ab')3, Fd, Fv, and disulfide linked Fv fragments, (scFv)2, scFv-Fc, diabodies, DARPins, DVD-Ig, Fcab, IgNAR, IgG-CH2, intrabodies, linear antibodies, mAb2, minibodies, single chain antibodies, single-domain antibodies (VHH), tetrabodies, triabodies, V NAR domains, and multispecific antibodies formed from antibody fragments.
  • a "biparatropic antibody” refers to a bispecific antibody that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through two distinct antigen recognition sites within the variable regions of the immunoglobulin molecule to bind two different epitopes of a target.
  • a biparatropic anti-EGFR antibody or fragment binds to two different epitopes of EGFR.
  • Biparatropic antibodies usually have stronger binding affinity and specificity than single epitope antibodies.
  • a “monoclonal antibody” refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants.
  • the term “monoclonal antibody” encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv, and other described herein), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site.
  • “monoclonal antibody” refers to such antibodies made in any number of manners including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals.
  • humanized antibody refers to forms of non-human (e.g. murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences.
  • humanized antibodies are human immunoglobulins in which residues from the complementary determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g.
  • the Fv framework region (FR) residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and capability.
  • the humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or capability.
  • the humanized antibody will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Pat. 5,225,539 or 5,639,641.
  • variable region of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination.
  • the variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) also known as hypervariable regions.
  • the CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies.
  • CDRs complementarity determining regions
  • the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g, Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • amino acid position numbering refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence can contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain.
  • a heavy chain variable domain can include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g.
  • residues 82a, 82b, and 82c, etc according to Kabat after heavy chain FR residue 82.
  • the Kabat numbering of residues can be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard” Kabat numbered sequence. Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
  • the end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35 A nor 35B is present, the loop ends at 32; if only 35 A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34).
  • the AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software.
  • human antibody means an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art. This definition of a human antibody includes intact or full- length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy and/or light chain polypeptide such as, for example, an antibody comprising murine light chain and human heavy chain polypeptides.
  • chimeric antibodies refers to antibodies wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species.
  • the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of mammals (e.g. mouse, rat, rabbit, etc) with the desired specificity, affinity, and capability while the constant regions are homologous to the sequences in antibodies derived from another (usually human) to avoid eliciting an immune response in that species.
  • epitopes or "antigenic determinant” are used interchangeably herein and refer to that portion of an antigen capable of being recognized and specifically bound by a particular antibody.
  • the antigen is a polypeptide
  • epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding are typically lost upon protein denaturing.
  • An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.
  • Binding affinity generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein.
  • Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer.
  • a variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure. Specific illustrative aspects are described in the following.
  • the phrase "substantially similar,” or “substantially the same”, as used herein, denotes a sufficiently high degree of similarity between two numeric values (generally one associated with an antibody of the disclosure and the other associated with a reference/comparator antibody) such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristics measured by said values (e.g., Kd values).
  • the difference between said two values is less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% as a function of the value for the reference/comparator antibody.
  • substantially pure refers to material which is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.
  • agent refers to agents that can target specific cells (e.g., cancer cells) including, but not limited to, targeting proteins, antibodies and antibody fragments, and agents including, but not limited to, cytokines, enzymes, and polynucleotides.
  • a targeting agent e.g., an antibody, antigen-binding fragment, or targeting protein
  • agent e.g., cytokine, enzyme, polynucleotide
  • a "linker” is any chemical moiety that is capable of linking a compound, usually a drug to an agent in a stable, covalent manner.
  • Linkers can be susceptible to or be substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the compound or the agent remains active.
  • the linker is a cleavable linker or dissociable linker.
  • the linker is an amino acid linker.
  • the linker is a valine-citrulline or alanine-alanine linker.
  • the linker is a peptide linker. In some aspects disclosed herein, the linker is a valine-citrulline-/?-aminobenzylcarbamate (Val-Cit-Pab) linker. In some aspects disclosed herein, the valine-citrulline (Val-Cit) linker contains a /?-aminobenzylcarbamate functional group. In some aspects disclosed herein, the linker is an alanine-alanine-/?-aminobenzylcarbamate (Ala- Ala-Pab) linker. In some aspects disclosed herein, the alanine-alanine (Ala-Ala) linker contains a /?-aminobenzylcarbamate functional group.
  • leash refers to any chemical moiety that is capable of coupling a compound, for example, an agent, to the polymer backbone of a bottlebrush polymer described herein in a stable, covalent manner. Leashes are substantially resistant to one or more of acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the compound or the agent remains active. In some aspects disclosed herein, the leash comprises a non-cleavable moiety. In some aspects disclosed herein, the leash is an amino acid sequence.
  • the leash comprises selected from a histidine tag (His-tag), a sequence of glycine units, (GGGGS)3 (SEQ ID NO: 5), ethylene glycol units, wherein the number of units is an integer between 5-25, or combinations thereof.
  • the leash comprises ethylene glycol units, wherein the number of units is an integer between 7-25.
  • the leash comprises ethylene glycol units, wherein the number of units is an integer between 8-25.
  • the leash comprises ethylene glycol units, wherein the number of units is an integer between 9-25.
  • the leash comprises ethylene glycol units, wherein the number of units is an integer between 10- 25.
  • the leash is ethylene glycol units, wherein the number of units is an integer between 15-25. In some aspects disclosed herein, the leash comprises ethylene glycol units, wherein the number of units is an integer between 15-20. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 5-80. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 5-75. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 5-70.
  • the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 5-65. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 5-60. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 5-55. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 5-50.
  • the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 5-45. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 5-40. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 5-35. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 5-30.
  • the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 5-25. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 5-20. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 5-15. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 5-10.
  • the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 7-80. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 8-80. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 9-80. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 15-80.
  • the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 20-80. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 25-80. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 30-80. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 35-80.
  • the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 40-80. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 45-80. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 50-80. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 55-80.
  • the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 60-80. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 65-80. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 70-80. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 75-80.
  • the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 5-10. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 10-20. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 20-30. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 30-40.
  • the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 40-50. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 50-60. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 60-70. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 70-80.
  • cancer refers to or describe the physiological condition in mammals in which a population of cells are characterized by unregulated cell growth.
  • examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, multiple myeloma, myelodysplastic syndromes, and myeloproliferative disorders.
  • cancers include squamous cell cancer, small-cell lung cancer, nonsmall cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, various types of head and neck cancers, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL) such as B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T ALL), mixed- lineage leukemia ALL (MLL-ALL), B-cell
  • ALL acute
  • B-cell lymphomas including NHL, precursor B-cell lymphoblastic leukemia/lymphoma and mature B-cell neoplasms, such as B-cell chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), including low-grade, intermediategrade and high-grade FL, cutaneous follicle center lymphoma, marginal zone B-cell lymphoma (MALT type, nodal and splenic type), hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia, and anaplastic large-cell lymphoma (ALCL).
  • NHL B-cell chronic lymphoc
  • cancer cell refers to the total population of cells derived from a tumor or a pre-cancerous lesion, including both non- tumorigenic cells, which comprise the bulk of the tumor cell population, and tumorigenic stem cells (cancer stem cells).
  • tumorigenic stem cells cancer stem cells.
  • tumorigenic stem cells cancer stem cells.
  • tumorigenic stem cells will be modified by the term “non-tumorigenic” when referring solely to those tumor cells lacking the capacity to renew and differentiate to distinguish those tumor cells from cancer stem cells.
  • anticancer drug refers to drugs (i.e., chemical compounds) or prodrugs known to, or suspected of being able to treat a cancer (i.e., to kill cancer cells, prohibit proliferation of cancer cells, or treat a symptom related to cancer).
  • subject refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment.
  • subject and patient are used interchangeably herein in reference to a human subject.
  • Administration "in combination with” one or more further drugs includes simultaneous (concurrent) and consecutive administration in any order.
  • composition refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject (which could be determined on a subject-by-subject basis) to which the formulation would be administered.
  • Such formulation can be sterile.
  • an “effective amount” of a bioconjugate as disclosed herein is an amount sufficient to carry out a specifically stated purpose.
  • An “effective amount” can be determined empirically and in a routine manner, in relation to the stated purpose.
  • the term “therapeutically effective amount” refers to an amount of a bioconjugate effective to "treat" a disease or disorder in a subject or mammal.
  • the therapeutically effective amount of the bioconjugate can reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and in a certain aspect, stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and in a certain aspect, stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer.
  • the bioconjugate can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic.
  • a "prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, a prophylactic dose is used in subjects prior to or at an earlier stage of disease.
  • Polynucleotide or “nucleic acid,” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.
  • a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure can be imparted before or after assembly of the polymer.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
  • Other types of modifications include, for example, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, cabamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g.,
  • any of the hydroxyl groups ordinarily present in the sugars can be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or can be conjugated to solid supports.
  • the 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms.
  • Other hydroxyls can also be derivatized to standard protecting groups.
  • Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, .a-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside.
  • One or more phosphodiester linkages can be replaced by alternative linking groups.
  • linking groups include, but are not limited to, aspects wherein phosphate is replaced by P(O)S ("thioate”), P(S)S ("dithioate”), "(0)NR2 ("amidate"), P(O)R, P(O)OR', CO or CH2 ("formacetal"), in which each R or R' is independently H or substituted or unsubstituted alkyl (1- 20 C) optionally containing an ether (—0—) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.
  • treat refers to, e.g., the reduction in severity of a disease or condition; the reduction in the duration of a disease course; the amelioration or elimination of one or more symptoms associated with a disease or condition; the provision of beneficial effects to a subject with a disease or condition, without necessarily curing the disease or condition.
  • the term also includes prophylaxis or prevention of a disease or condition or its symptoms thereof.
  • the term “treating” or “treatment” means reducing the number of tumor cells in a subject.
  • vector means a construct, which is capable of delivering, and expressing, one or more gene(s) or sequence(s) of interest in a host cell.
  • vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.
  • polypeptide polypeptide
  • peptide protein
  • the terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length.
  • the polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
  • the polypeptides of this disclosure are based upon antibodies, in certain aspects, the polypeptides can occur as single chains or associated chains.
  • the present disclosure comprises a bioconjugate comprising a targeting agent (e.g., protein, antibody, or antigen-binding fragment thereof) that binds to a cancer associated antigen or an agent linked to a non-polyethylene glycol (PEG) polymer, where the polymer is coupled to a chemotherapeutic drug, and the bioconjugate has an drug-to-agent ratio (DAR) of at least 20: 1, of at least 50: 1, of at least 100: 1, of at least 200:1, of at least 300: 1, of at least 400: 1, of at least 500: 1, of at least 600: 1, of at least 700:1, of at least 800: 1, of at least 900: 1, of at least 1,000: 1, or of at least 1, 100: 1.
  • a targeting agent e.g., protein, antibody, or antigen-binding fragment thereof
  • PEG non-polyethylene glycol
  • Antibodies and antigen-binding fragments allow for the bioconjugate to bind to an antigen on the target cell (e.g., cancer cells) and be internalized by the target cell to exert a therapeutic effect. This would spare normal, healthy non-target cells.
  • Approaches to allow bioconjugates to be internalized by the target cell intact include targeting receptors that internalize the bioconjugate or using peptide-mediated membrane penetration to deliver antibodies to their intracellular target proteins (see US Pat. App. Pub. 20080063633).
  • internalization is intended to release the non-PEG polymers and drug(s) from the agent. This release then allows free drug(s) to traffic inside the cell, resulting in desired toxicity and cell death of target cell populations (e.g., cancer).
  • target cell populations e.g., cancer
  • the agent e.g., antibody and antigen binding fragments
  • the agent allow for targeting of the bioconjugate to extracellular targets and subsequent release of free non-PEG polymers and drug(s) to the target and neighboring cells, resulting in cytotoxicity and cell death (see US Pat. App. Pub. 20190248887).
  • Antibodies and antigen-binding fragments have other mechanisms of cellular toxicity besides the targeted delivery of drugs as discussed above.
  • the Fc region of antibodies mediates several important effector functions, such as antibody-dependent cytotoxicity (ADCC), phagocytosis, and complement-dependent cytotoxicity (CDCC).
  • ADCC antibody-dependent cytotoxicity
  • CDC complement-dependent cytotoxicity
  • the targeted delivery of antibodies and antigen-binding fragments can result in targeted cell death via recruitment of Fc receptor-bearing effector cells that can recognize and kill antibody-coated target cells, engulfment and elimination of target cells by innate immune cells, and recruitment of complement factors that result in cell lysis via the classical complement pathway and formation of a membrane attack complex (MAC), respectfully. Therefore, targeted delivery of bioconjugates via antibodies and antigen-binding fragments afford a greater level of control for the delivery of chemotherapeutic drugs, and may mediate cellular death via drugs or induction of Fc-dependent immunological pathways.
  • ADCC antibody-
  • bioconjugates of the disclosure depend on the careful selection of an appropriate agent. Considering the severe side effects and expense associated with treating cancer, there is a need to identify factors that can be used in the treatment of cancer in general that lead to higher likelihood of treatment success. Cancer associated antigens are an attractive target for bioconjugates given their ability to specifically target cancer cells while minimizing side effects.
  • Cancer associated antigens can be classified into the following major groups: cancertestis antigens (e.g., MAGE, NY-ESO-1), differentiation antigens (e.g., tyrosinase, PSMA), overexpressed antigens (e.g., HER2, surviving, telomerase, WT1), cancer-specific antigens (e.g., P- catenin), abnormal post-translational modifications (e.g., MUC1), and oncoviral proteins (e.g., E6, E7).
  • a broader designation of pan-cancer antigens can be classified as any cell surface epitope expressed at higher levels in cancer cells compared to normal tissues, or epitopes that can be induced to the cell surface at higher levels in cancer cells compared to normal tissues.
  • the agent e.g., antibody or antigen-binding fragments thereof
  • the agent allows for the targeting and binding of the bioconjugate to cancer associated antigens found on cancer cells, subsequent internalization of the bioconjugate, and release of drugs from the non-PEG polymer.
  • the agent e.g., antibody or antigen-binding fragments thereof
  • the agent allows for the targeting and binding of the bioconjugate to cancer associated antigens found on cancer cells and subsequent release of drugs from the non-PEG polymer in the extracellular space.
  • the agent has one or more of the following effects: inhibit proliferation of tumor cells, reduce the tumorigenicity of a tumor by reducing the frequency of cancer stem cells in the tumor, inhibit tumor growth, increase survival, trigger cell death of tumor cells, differentiate tumorigenic cells to a non-tumorigenic state, or prevent metastasis of tumor cells.
  • the agent is specific to an extracellular receptor, polypeptide, or lipid found on cancer cells. In some aspects, the agent is specific to a cancer associated antigen derived from cancer.
  • the cancer is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, leukemia, multiple myeloma, myelodysplastic syndromes, and myeloproliferative disorders.
  • the cancer is selected from the group consisting of squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, various types of head and neck cancers, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL) such as B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T ALL), mixed-lineage leukemia ALL (ML
  • B-cell lymphomas including NHL, precursor B-cell lymphoblastic leukemia/lymphoma and mature B-cell neoplasms, such as B-cell chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), including low-grade, intermediate-grade and high-grade FL, cutaneous follicle center lymphoma, marginal zone B-cell lymphoma (MALT type, nodal and splenic type), hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia, and anaplastic large-cell lymphoma (ALCL).
  • NHL B-cell chronic lympho
  • the cancer associated antigen is EGFR.
  • the agent is an anti-EGFR antibody.
  • the antibody is a full-length anti-EGFR antibody.
  • the anti-EGFR antibody is a Fab, a Fab', a F(ab')2, a Fd, a single chain Fv or scFv, a disulfide linked Fv, a VNAR domain, a IgNar, an intrabody, an IgG-CH2, a minibody, a F(ab')3, a tetrabody, a triabody, a diabody, a singledomain antibody (VHH), DVD-Ig, Fcab, mAb2, a (scFv)2, DARPin, or a scFv-Fc antibody.
  • the anti-EGFR antibody is a VHH antibody.
  • the anti-EGFR antibody is a biparatropic antibody.
  • the anti-EGFR antibody is a scFv.
  • the anti-EGFR scFv antibody has a variable heavy chain of SEQ ID NO: 1, and/or a variable light chain of SEQ ID NO: 2.
  • the anti-EGFR scFv antibody has a variable heavy chain of SEQ ID NO: 3, and/or a variable light chain of SEQ ID NO: 4.
  • the anti-EGFR scFv antibody has a variable heavy chain of SEQ ID NO: 1 conjugated to a variable light chain of SEQ ID NO: 2 using a flexible glycine linker (SEQ ID NO: 5).
  • the anti-EGFR scFv antibody has a variable heavy chain of SEQ ID NO: 3 conjugated to a variable light chain of SEQ ID NO: 4 using a flexible glycine linker (SEQ ID NO: 5). In some aspects, the anti-EGFR scFv antibody has a full length sequence consisting of SEQ ID NO: 6. In some aspects, the anti-EGFR scFv antibody has a full length sequence consisting of SEQ ID NO: 7.
  • the anti-EGFR antibody or antigen-binding fragment in the bioconjugate comprises: (a) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 8; a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 9; and a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 10; and (b) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 11; a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 12; and a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 13.
  • the anti-EGFR antibody or antigen-binding fragment in the bioconjugate comprises: (a) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 14; a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 15; and a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and (b) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 11; a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 12; and a light chain variable region CDR3 comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 13.
  • the cancer associated antigen is HER2.
  • the antibody is a full length anti-HER2 antibody.
  • the anti-HER2 antibody is a Fab, a Fab', a F(ab')2, a Fd, a single chain Fv or scFv, a disulfide linked Fv, a VNAR domain, a IgNar, an intrabody, an IgG-CH2, a minibody, a F(ab')3, a tetrabody, a triabody, a diabody, a single-domain antibody (VHH), DVD-Ig, Fcab, mAb2, a (scFv)2, DARPin, or a scFv-Fc antibody.
  • the anti-HER2 antibody is a scFv. In some aspects, the anti-HER2 scFv antibody has a variable heavy chain of SEQ ID NO: 20, and/or a variable light chain of SEQ ID NO: 21. In some aspects, the anti-Her2 scFv antibody has a variable heavy chain of SEQ ID NO: 20 conjugated to a variable light chain of SEQ ID NO: 21 using a flexible glycine linker (SEQ ID NO: 5). In some aspects, the anti-HER2 scFv antibody has a full length sequence consisting of SEQ ID NO: 22. In some aspects, the anti-HER2 antibody is trastuzumab. In some aspects, trastuzumab has a heavy chain of SEQ ID NO: 23, and/or a light chain of SEQ ID NO: 24.
  • the anti-HER2 antibody or antigen-binding fragment in the bioconjugate comprises: (a) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 25; a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 26; and a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 27; and (b) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 28; a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 29; and a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 30.
  • the cancer-associated lipid is phosphatidylserine.
  • the agent is an anti-phosphatidylserine antibody.
  • the anti-phosphatidylserine antibody is bavituximab.
  • the anti-phosphatidylserine antibody bavituximab binding to phosphatidylserine is mediated by the serum protein p2-glycoprotein I (also known as Apolipoprotein H).
  • the entire family of PS-targeting antibodies includes antibodies that do bind directly to PS, i.e., direct PS-binding or PS-targeting antibodies.
  • Such a “direct PS-binding antibody” is an antibody that is not only functionally specific for PS, and targets and binds to PS in vitro and in vivo (as do the indirect binding antibodies), but that does not require a serum protein, such as P2-glycoprotein I, to form a tight binding complex with PS, even in in vitro binding assays.
  • the anti-phosphatidylserine antibody is a direct binder.
  • the direct binder is 9D2.
  • the 9D2 antibody is a mouse monoclonal antibody that has been shown to localize to tumor blood vessels and to exert anti-tumor effects in vivo (Ran et al., 2002).
  • the direct binder is 11.31.
  • the 11.31 antibody is a fully- human antibody that has also been shown to exert anti-tumor effects in vivo (e.g., in mice bearing MDA-MB-435 mammary carcinoma xenografts) and shows impressive anti-viral effects (Moody et al., 2010; U.S. Pat. No. 7,455,833).
  • the agent is a pan anti-cancer antigen.
  • the pan anticancer antigen is selected from the group consisting of MUC-1, epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen, ras, p53, P-catenin, CDK4, CDC27, a actinin-4, gangliosides, prostate-specific membrane antigen (PSMA), Wilms' tumor gene 1 (WT1), ephrin tyrosine kinase a3 (EphA3), CD20, B melanoma antigen (BAGE), GAGE, NY-ESO-1, and survivin.
  • MUC-1 epithelial tumor antigen
  • ETA epithelial tumor antigen
  • tyrosinase melanoma-associated antigen
  • ras p53
  • P-catenin CDK4, CDC27
  • actinin-4 gangliosides
  • the agents described herein can be in single-domain antibody (VHH) format. In some aspects, the agents described herein can be biparatropic antibodies.
  • the methods described herein are for modifying antibodies to generate bioconjugates.
  • the method involves deglycosylation of the antibody followed by a reaction with small molecules containing primary amines, catalyzed by bacterial transglutaminase. This approach allows for site-specific modification in the Fc region of an antibody without the need to incorporate non-canonical amino acids.
  • Cytokines are a critical mediator in both innate and acquired immune responses. They can alter the balance of cellular and humoral responses, alter class switching of B lymphocytes, and modify innate responses.
  • cytokines limit cancer growth by a direct anti-proliferative or pro-apoptotic activity, or indirectly stimulating the cytotoxic activity of immune cells against cancer cells.
  • Two cytokines, IL-2 and IFN-a demonstrated clinical benefit and consequently received The Food and Drug Administration (FDA) approval for the treatment of several malignant diseases.
  • FDA Food and Drug Administration
  • IL-2 was approved for the treatment of advanced renal cell carcinoma (RCC) and metastatic melanoma
  • IFN-a was approved for the treatment of hairy cell leukaemia, follicular non-Hodgkin lymphoma, melanoma and AIDS- related Kaposi's sarcoma.
  • the clinical use of these cytokines marked a milestone in cancer immunotherapy, as it was the first demonstration that immunotherapy could favorably tilt the balance between cancer and the anti-cancer immune response, leading to durable objective responses (Berraondo et al., 2018, British Journal of Cancer, 120(1): 6-15).
  • Enzymes can also play a role in mediating cell-directed toxicity and/or regenerating the potency of anti-cancer drugs or toxic small molecules within tumors (Deonarain et al., 1994, British Journal of Cancer, 70(5): 786-794). Furthermore, polynucleotides such as aptamers can be utilized to target common cancer associated antigens and signaling pathways, leading to reduction in cancer burden via payload delivery or inhibition, respectfully (Nimjee, et al., 2017, Annual Review of Pharmacology and Toxicology, 57:61-79).
  • the agent is an enzyme.
  • the agent is a cytokine.
  • the cytokine is interleukin (IL)-2, IL-7, IL-12, IL-15, IL-21, IL-23, interferon (IFN)-a, and IFN-y.
  • the agent is a polynucleotide.
  • the polynucleotide is an aptamer.
  • Cancer therapy may involve surgery, chemotherapy, hormonal therapy and/or radiation treatment to eradicate neoplastic cells in a subject (See, for example, Stockdale, 1998, "Principles of Cancer Patient Management", in Scientific American: Medicine, vol. 3, Rubenstein and Federman, eds., Chapter 12, Section IV). Cancer therapy can also involve biologies or immunotherapy. All of these approaches pose potential drawbacks for the subject. Surgery, for example, may be contraindicated due to the health of the subject or may be unacceptable to the subject. Additionally, surgery may not completely remove the neoplastic tissue. Radiation therapy is only effective when the neoplastic tissue exhibits a higher sensitivity to radiation than normal tissue, and radiation therapy can also often elicit serious side effects. Hormonal therapy is rarely given as a single drug and although it can be effective, is often used to prevent or delay recurrence of cancer after other treatments have removed the majority of the cancer cells.
  • Biological therapies/immunotherapies are limited in number and may produce side effects such as rashes or swellings, flu-like symptoms, including fever, chills and fatigue, digestive tract problems or allergic reactions.
  • chemotherapeutic drugs available for treatment of cancer.
  • a significant majority of cancer chemotherapeutics act by inhibiting DNA synthesis, either directly or indirectly by inhibiting the biosynthesis of the deoxyribonucleotide triphosphate precursors, to prevent DNA replication and concomitant cell division (see, for example, Gilman et al., Goodman and Gilman's: The Pharmacological Basis of Therapeutics, Eighth Ed. (Pergamom Press, New York, 1990)).
  • chemotherapeutic drugs have many drawbacks (see, for example, Stockdale, 1998, "Principles of Cancer Patient Management” in Scientific American Medicine, vol. 3, Rubenstein and Federman, eds., ch. 12, sect. 10). Almost all chemotherapeutic drugs are toxic, and chemotherapy causes significant, and often dangerous, side effects, including severe nausea, bone marrow depression, and immunosuppression. Methods for reducing the systemic toxicity and off-target effects of chemotherapeutic drugs are needed.
  • chemotherapeutic drugs are mitigated by coupling chemotherapeutic drugs to a non-PEG polymer that is conjugated to an agent (e.g., antibody, antigen binding fragment thereof, cytokine, enzyme, polynucleotide) in the form of a bioconjugate.
  • an agent e.g., antibody, antigen binding fragment thereof, cytokine, enzyme, polynucleotide
  • chemotherapeutic drugs as a bioconjugate allows for improved tumor targeting due to improved tissue penetration from the anti-PEG polymer, higher drug-to-agent ratio (e.g., high DAR (10+: 1) or ultra-high DAR (e.g., 100: 1 , 200: 1, 500: 1, or 1,100: 1)) that maintains potency with less toxic molecules, rescuing of otherwise systemically toxic payloads by inclusion in ultra-high DAR bioconjugates, and further increase in potency with two or more payloads that have nonoverlapping toxicities.
  • higher drug-to-agent ratio e.g., high DAR (10+: 1) or ultra-high DAR (e.g., 100: 1 , 200: 1, 500: 1, or 1,100: 1)
  • ultra-high DAR e.g., 100: 1 , 200: 1, 500: 1, or 1,100: 1
  • the chemotherapeutic refers to a non-nucleic acid molecule that is used to treat cancer and/or that has cytotoxic ability.
  • chemotherapeutic drugs can be described by mechanism of action or by chemical compound class.
  • the chemotherapeutic is selected from the group of alkylating drugs (e.g., melphalan, mitobronitol, cyclophosphamide, streptozotocin, triaziquone), anthracy clines (e.g., doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin), cytoskeletal disruptors (e.g., abaxrane, cabazitaxel, paclitaxel, taxotere, tesetaxel), epothilones (e.g, ixabepilone), histone deacetylase inhibitors (e.g., entinostat, romidepsin, vorinostat, zabadinostat), inhibitors of topoisomerase I (e.g., belotecan, camptothecin, irinotecan, topotecan) or topoisomerase II (
  • the chemotherapeutic drug is an AKT inhibitor.
  • the AKT inhibitor is an ATP-competitive inhibitor selected from the group consisting of isoquinoline-5-sulfonamides (e.g., H-8.
  • H-89 H-89
  • azepane derivatives e.g., derived from (-)- balanol
  • aminofurazans e.g., GSK690693
  • heterocyclic rings e.g., CCT128930, AZD5363, ipatasertib
  • phenylpyrazole derivatives e.g., AT7867, AT13148
  • thiophenecarboxamide derivatives e.g., GSK2110183, DC120, GSK2141795).
  • the AKT inhibitor is an allosteric inhibitor selected from the group consisting of 2,3 -diphenylquinoxaline analogues (e.g., MK-2206), alkylphospholipids (e.g., edelfosine, miltefosine, erufosine), indole-3 -carbinol analogues (e.g., OSU-A9), sulfonamide derivatives (e.g., PH-316, PHT-427), and thiourea derivatives (e.g., PIT-1, PIT-2, DM-PIT-1), purine derivatives (e.g., triciribine, ARQ 092).
  • 2,3 -diphenylquinoxaline analogues e.g., MK-2206
  • alkylphospholipids e.g., edelfosine, miltefosine, erufosine
  • the AKT inhibitor is an irreversible inhibitor selected from the group consisting of antibiotics, lactoquinomycin, Frenolicin B, kalafungin, medermycin, Boc-Phe-vinyl ketone, 4- hydroxynonenal (4-HNE), 1,6-naphthyridinone derivatives, and imidazo-l,2-pyridine derivatives. See, Nitulescu, et al. Int J Oncol. 48(3) 869-995, which has been incorporated by reference herein.
  • the chemotherapeutic drug is an Aurora kinase A, B, or C inhibitor.
  • the Aurora B kinase inhibitor is selected from the group consisting of Hesperadin, Barasertib, AZDI 152, AT9283, Danusertib (PHA-739358), AMG900, CYC 116, BI 811283, AZD2811, SP-96, GSK 1070916, reversine, CCT 129202, CCT137690, SNS-314, quercetin, and VX-680. See, Borah, et al. Molecules. 26(7): 1981, which has been incorporated by reference herein.
  • the chemotherapeutic drug is an ERK inhibitor.
  • the ERK inhibitor is selected from the group consisting of AZD0364, GDC-0994, MK-8353, BVD- 523, HH2710, KO-947, LTT462, LY3214996, and ONC201. See, Pan, et al. Acta Pharm Sin B. 12(5):2171 -2192, which has been incorporated by reference herein.
  • the chemotherapeutic drug is a PI3K inhibitor.
  • the PI3K inhibitor is selected from the group consisting of PI103; PI828; LY294002; wortmannin; demethoxyviridin; IC486068; IC87114; GDC-0941; GDC-0980 (apitolisib); perifosine; CAL101;
  • the chemotherapeutic drug is a MEK inhibitor.
  • the MEK inhibitor is selected from the group consisting of binimetinib, CC-90003, DEL-22379, GDC-0973 (cobimetinib), selumetinib, and tram etinib. See, Han, et al. Journal of Hematology & Oncology. 14(1): 1, which has been incorporated by reference herein.
  • the chemotherapeutic drug is a MAPK inhibitor.
  • the MAPK inhibitor is selected from the group consisting of Farnesyltransf erase inhibitors (FTIs), Sorafenib, Vemurafenib, PLX8394, Dabrafenib, Ulixertinib, Simvastatin, Alisertib, and Teriflunomide.
  • the chemotherapeutic drug is a B-RAF inhibitor.
  • the B-RAF inhibitor is selected from the group consisting of Vemurafenib, Dabrafenib, Encorafenib, and Sorafenib. See, Sanchez, et al. Drugs. 78(5): 549-566, which has been incorporated by reference herein.
  • the chemotherapeutic drug is a RAS inhibitor.
  • the RAS inhibitor is selected from the group consisting of Ganetespib, Apatinib, Oncrasin-1, GDC- 0449, ARS-1620, ARS-853, AMG 510, MRTX849, BGB324, ABT-737, AZD6244, NVP- BEZ235, R115777, PPIN-1, PPIN-2, pan-RAS Inhibitor 3144, Deltarasin, and Sotorasib. See, Shetu, et al. Int J Mol Sci. 23(7)3706, which has been incorporated by reference herein.
  • the chemotherapeutic drug is a degrader.
  • the degrader is selected from AKT and Myc degraders.
  • the degrader is a protein degrader.
  • the protein degrader is selected from the group consisting of immunomodulatory drugs (iMiDs), molecular glue (MG), selective estrogen receptor degraders (SERDs), and proteolysis-targeting chimeras (PROTACs). See, Fang, et al. Trends Pharamcol Sci. 44(5):303-317; Xue, et al. Expert Opin Drug Discov. 18(4):467-483, which have been incorporated by reference herein.
  • immunomodulatory drugs iMiDs
  • MG molecular glue
  • SESDs selective estrogen receptor degraders
  • PROTACs proteolysis-targeting chimeras
  • the chemotherapeutic drug is a cytotoxic agent.
  • the cytotoxic agent is selected from alkylating drugs, anthracyclines, cytoskeletal disruptors, epothilones, kinase inhibitors, histone deacetylase inhibitors, inhibitors of topoisomerase I or II, nucleotide analogs or precursors thereof, peptide antibiotics, platinum based drugs, retinoids, and vinka alkaloids and derivatives as previously defined.
  • the chemotherapeutic drug is a Wem syndrome helicase.
  • the chemotherapeutic drug is a cell cycle inhibitor.
  • the cell cycle inhibitor is selected from the group consisting of polo-like kinases inhibitors and cyclin-dependent kinase inhibitors.
  • the cell cycle inhibitor is an aurora kinase inhibitor.
  • the aurora kinase inhibitor is barasertib, alisertib (MLN8237), danusertib (PHA-739358), AT9283, PF-03814735, or AMG 900.
  • the cell cycle inhibitor is a Chk inhibitor.
  • the Chk inhibitor is prexasertib (LY2606368) or AZD7762.
  • the cell cycle inhibitor is a Chkl inhibitor.
  • the Chkl inhibitor is GDC-0575, rabusertib (LY2603618), VX-803 (M4344), CHIR-124, PF-477736, PD0166285, or SAR- 020106.
  • the cell cycle inhibitor is a Chk2 inhibitor.
  • the Chk2 inhibitor is BML-277.
  • the polo-like kinase (PLK) inhibitor is BI 6727, BI 2536, GSK461364A, rigosertib or volasertib.
  • the cell cycle inhibitor is a cyclin-dependent kinase (CDK) inhibitor such as palbociclib, abemaciclib, dinaciclib, ribociclib, TP- 1287, alvocidib, NUV-422.
  • CDK cyclin-dependent kinase
  • the cyclin-dependent kinase inhibitor is a CDK1, CDK2, CDK4, CDK6, CDK7 or some combination of CDK inhibitors.
  • the cytoskeletal disruptor is a taxane (e.g., paclitaxel, docetaxel, or cabazitaxel) colchicine, vinblastine, evodaimine, 6a-acetoxyanopterine, cytochalasin, latrunculin, staurosporine, sinularin, phalloidin, or eribulin.
  • taxane e.g., paclitaxel, docetaxel, or cabazitaxel
  • colchicine e.g., vinblastine, evodaimine, 6a-acetoxyanopterine, cytochalasin, latrunculin, staurosporine, sinularin, phalloidin, or eribulin.
  • the chemotherapeutic drug is a protein degrader.
  • the protein degrader is a proteolysis-targeting chimera (PROTAC) molecule.
  • Non-PEG Polymers Methods of Producing Non-PEG Polymers, and Methods of Making a Bioconjugate comprising Non-PEG Polymers
  • the non-PEG polymer is a bottlebrush polymer.
  • Bottlebrush polymers are copolymers comprising a main polymer chain (also referred to as a polymer backbone) having distinct polymer side chains branching from the main polymer chain.
  • Bottlebrush polymers can be, but are not limited to, block copolymers, blockgraft copolymers, statistical copolymers, brush-b-coil copolymers, gradient copolymers, or brush copolymers.
  • Bottlebrush polymers are summarized, e.g., in Li, Z., et al., "Bottlebrush polymers: From controlled synthesis, self-assembly, properties to applications," Progress in Polymer Science 776: 101387 (2021) and Matyjaszewki, K., et al. "Macromolecular Engineering by Atom Transfer Radical Polymerization,” J. Am. Chem. Soc. 736:6513-6533 (2014), which are hereby incorporated by reference. [0173] Non-PEG bottlebrush polymers may be synthesized using various methods known in the art.
  • the non-PEG bottlebrush polymer is synthesized using a reversible- deactivation radical polymerization (RDRP) process (formerly named controlled radical polymerization (CRP)).
  • RDRP procedures include, but are not limited to, Nitroxide Mediated Polymerization (NMP), Atom Transfer Radical Polymerization (ATRP), and Reversible Addition Fragmentation Transfer (RAFT). These procedures have evolved over the last two decades to provide access to (co)polymers with predefined molecular weights, compositions, architectures and narrow/controlled molecular weight distributions. Such processes have been used to conjugate proteins to polymers, e.g., as described in Curr. Opin. Chem. Biol.
  • bioresponsive molecules with well-defined polymers can provide improved stability, tailored solubility, predeterminable trafficking pathways, and increased therapeutic potential of already useful biomacromolecules including peptides, proteins, nucleic acids, and polysaccharides in a variety of applications.
  • Matyjaszewski and coworkers disclosed the fundamental four component ATRP process comprising the addition, or in situ formation, of an initiator, e.g., a molecule with a transferable atom or group that is completely incorporated into the final product, a transition metal and a ligand that form a partially soluble transition metal complex that participates in a reversible redox reaction with the added initiator or a dormant polymer to form the active species to copolymerize radically polymerizable monomers, and a number of improvements to the basic ATRP process, in a number of commonly assigned patents and patent applications: U.S. Pat. Nos.
  • the ATRP process is Activator Regenerated by Electron Transfer
  • ARGET ATRP oxidized copper catalysts are regenerated using reducing agents, enabling lower copper catalyst loadings.
  • ARGET ATRP is discussed, e.g., in Pintauer, T, et al., "Atom transfer radical addition and polymerization reactions catalyzed by ppm amounts of copper complexes," Chemical Society Reviews 57(6): 1087-1097 (2008), which is hereby incorporated by reference in its entirety.
  • Reversible Addition Fragmentation Transfer is another form of reversible- deactivation radical polymerization that uses a chain-transfer agent, e.g., in the form of a thiocarb onylthio compound, to control the molecular weight and poly dispersity of the resultant polymer.
  • RAFT polymerization is summarized, e.g., in Chiefari, J., et al, "Living Free-Radical Polymerization by Reversible Addition-Fragmentation Chain Transfer: The RAFT Process," Macromolecules 37(16): 5559-5562, which is hereby incorporated by reference in its entirety.
  • RDRP processes e.g., ATRP
  • polymers e.g., bottlebrush polymers
  • biomolecules e.g., proteins. See, e.g., U.S. 10,072,042, U.S. 9,539,338, U.S. 11,472,894, and U.S. 2021/0388337, all of which are herein incorporated by reference.
  • the bottlebrush polymer is synthesized using a "grafting to” approach, a “grafting from” approach, or a “grafting through” approach.
  • the "grafting to” approach involves conjugation of a preformed polymer to a backbone polymer having reactive end groups.
  • the “grafting from” approach involves growing a polymer from a polymer backbone functionalized with initiation sites.
  • the “grafting through” approach involves synthesis of macromonomers that form backbone polymer after subsequent polymerization.
  • the bottlebrush polymer is synthesized using a "grafting to" approach.
  • a preformed polymer (also referred to as a "macromonomer”) is first synthesized, e.g., via ATRP, then conjugated with a drug, e.g., a chemotherapeutic drug, via click chemistry.
  • the preformed polymer is then attached to a polymer backbone.
  • An exemplary process is illustrated in Figure 1A.
  • the macromonomer has the following structure: wherein n and m are positive integers.
  • a drug is conjugated to the macromonomer via the azide groups.
  • the macromonomer has a molecular weight of from about 5,000 to about 8,000.
  • the polymer backbone is formed by attaching a methacrylate group to the macromonomer and polymerizing the resulting methacrylate-macromonomer.
  • the methacrylate may be attached either before or after a drug is conjugated to the macromonomer.
  • the bottlebrush polymer is synthesized using a "grafting to" approach.
  • a preformed polymer (also referred to as a "macromonomer”) is first synthesized, e.g., via ATRP, then the macromonomer is attached to a methacrylate group, and then conjugated with a drug, e.g., a chemotherapeutic drug, via click chemistry.
  • the preformed polymer is then attached to a polymer backbone.
  • An exemplary process is illustrated in Figure 1A.
  • the macromonomer has the following structure: wherein n and m are positive integers.
  • a drug is conjugated to the macromonomer via the azide groups.
  • the macromonomer has a molecular weight of from about 5,000 to about 8,000 kDa.
  • the bottlebrush polymer is synthesized by first synthesizing a preformed polymer comprising an initiator (also referred to as a "macroinitiator") via RAFT polymerization, then grafting a copolymer to the preformed polymer via ATRP, then conjugating the copolymer formed via ATRP with a drug via click chemistry.
  • an initiator also referred to as a "macroinitiator”
  • ATRP grafting a copolymer to the preformed polymer via ATRP
  • conjugating the copolymer formed via ATRP with a drug via click chemistry An exemplary process is illustrated in Figure 2A-2B.
  • the macroinitiator has the following structure:
  • the bottlebrush polymer is synthesized by first synthesizing a preformed polymer comprising an initiator (also referred to as a "macroinitiator") via RAFT polymerization, where the preformed polymer is synthesized by polymerizing one or more methyacrylate monomers with a chain transfer agent (CTA).
  • the CTA is 4- cyano-4-((phenylcarbonothioyl)thio)pentanoic acid and has the following structure:
  • the CTA is 2,5-dioxopyrrolidin-l-yl 4-cyano-4- ((phenylcarbonothioyl)thio)pentanoate and has the following structure:
  • the CTA is 2-(((butylthio)carbonothioyl)thio)propanoic acid and has the following structure: [0186]
  • the non-PEG preformed polymer is first synthesized by polymerizing a methacrylate monomer comprising an initiator with one or more other methyacrylate monomers with a CT A.
  • the ratio between the one or more other methyacrylate monomers and the methacrylate monomer comprising an initiator is 0.1 : 1.0, 0.2: 1.0, 0.3: 1.0, 0.4: 1.0, 0.5: 1.0, 0.6:1.0, 0.7: 1.0, 0.8: 1.0, 0.9: 1.0, 1.0: 1.0, 1.1 : 1.0, 1.2: 1.0, 1.3: 1.0, 1.4: 1.0, 1.5: 1.0, 1.6: 1.0, 1.7: 1.0, 1.8:1.0, 1.9: 1.0, 2.0: 1.0, 2.1 : 1.0, 2.2: 1.0, 2.3: 1.0.
  • the ratio between the one or more other methyacrylate monomers and the methacrylate monomer comprising an initiator is 0.5:1.0, 1.0:1.0, 1.5: 1.0, 2.0: 1.0, 2.5: 1.0, or 3.0:1.0. In some aspects, the ratio between the one or more other methyacrylate monomers and the methacrylate monomer comprising an initiator is 1.0: 1.0, 2.0:1.0, or 3.0:1.0.
  • the non-PEG preformed polymer comprising a methacrylate monomer comprising an initiator and one or more other methyacrylate monomers has a random "r" assortment of the monomers in the preformed polymer.
  • the random "r" assortment of the monomers in the non-PEG preformed polymer has the following structure:
  • the bottlebrush polymer formed after grafting a copolymer to the macroinitiator via ATRP has the following structure:
  • 1 is an integer selected from 1 to 50.
  • the ratio between the one or more other methyacrylate monomers (n) and the methacrylate monomer comprising an initiator (m) is
  • the ratio between the one or more other methyacrylate monomers (n) and the methacrylate monomer comprising an initiator (m) is 0.5:1.0, 1.0:1.0, 1.5:1.0, 2.0:1.0, 2.5:1.0, or 3.0:1.0.
  • the ratio between the one or more other methyacrylate monomers (n) and the methacrylate monomer comprising an initiator (m) is 1.0:1.0, 2.0:1.0, or 3.0:1.0.
  • the ratio between the one or more other methyacrylate monomers (k) and the methacrylate monomer comprising an azide group (1) is 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1, 10.5:1, 11:1, 11.5:1, 12:1, 12.5:1, 13:1, 13.5:1, 14:1, 14.5:1, 15:1, 15.5:1, 16:1, 16.5:1, 17:1, 17.5:1, 18:1, 18.5:1, 19:1, 19.5:1, 20:1, 20.5:1, 21:1, 21.5:1, 22:1, 22.5:1, 23:1,
  • the ratio between the one or more other methyacrylate monomers (k) and the methacrylate monomer comprising an azide group (1) is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, or 50:1.
  • the ratio between the one or more other methyacrylate monomers (k) and the methacrylate monomer comprising an azide group (1) is 1:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, or 50:1. In some aspects, e.g., the compounds of this section, the ratio between the one or more other methyacrylate monomers (k) and the methacrylate monomer comprising an azide group (1) is 5:1. 10:1, 15:1, 20:1, 25:1, or 30:1.
  • the ratio between the one or more other methyacrylate monomers (k) and the methacrylate monomer comprising an azide group (1) is 10:1 or 15:1. In some aspects, e.g., the compounds of this section, the ratio between the one or more other methyacrylate monomers (k) and the methacrylate monomer comprising an azide group (1) is 10:1.
  • the non-PEG bottlebrush polymer formed after grafting a copolymer to the macroinitiator via ATRP is synthesized by polymerizing a methacrylate monomer comprising an azide group with one or more other methyacrylate monomers.
  • the ratio between the one or more other methyacrylate monomers and the methacrylate monomer comprising an azide group is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, or 50:1.
  • the ratio between the one or more other methyacrylate monomers and the methacrylate monomer comprising an azide group is 1:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, or 50:1. In some aspects, the ratio between the one or more other methyacrylate monomers and the methacrylate monomer comprising an azide group is 5:1. 10:1, 15:1, 20:1, 25:1, or 30:1. In some aspects, the ratio between the one or more other methyacrylate monomers and the methacrylate monomer comprising an azide group is 10:1 or 15:1. In some aspects, the ratio between the one or more other methyacrylate monomers and the methacrylate monomer comprising an azide group is 10:1.
  • the ratio between the one or more other methyacrylate monomers and the methacrylate monomer comprising an azide group ranges from about 1 : 1 to 50: 1, about 1 : 1 to 45:1, about 1:1 to 40:1, about 1:1 to 35:1, about 1:1 to 30:1, about 1:1 to 25:1, about 1:1 to 20:1, about 1:1 to 15:1, or about 1:1 to 10:1.
  • the ratio between the one or more other methyacrylate monomers and the methacrylate monomer comprising an azide group ranges from about 2:1 to 50:1, about 2:1 to 45:1, about 2:1 to 40:1, about 2:1 to 35:1, about 2:1 to 30:1, about 2:1 to 25:1, about 2:1 to 20:1, about 2:1 to 15:1, or about 2:1 to 10:1.
  • the ratio between the one or more other methyacrylate monomers and the methacrylate monomer comprising an azide group ranges from about 5: 1 to 50: 1, about 5: 1 to 45: 1, about 5: 1 to 40: 1, about 5:1 to 35:1, about 5:1 to 30:1, about 5:1 to 25:1, about 5:1 to 20:1, about 5:1 to 15:1, or about 5:1 to 10:1.
  • the ratio between the one or more other methyacrylate monomers and the methacrylate monomer comprising an azide group ranges from about 8:1 to 50:1, about 8:1 to 45:1, about 8:1 to 40:1, about 8:1 to 35:1, about 8:1 to 30:1, about 8:1 to 25:1, about 8:1 to 20 : 1 , about 8 : 1 to 15 : 1 , or about 8 : 1 to 10 : 1.
  • the ratio between the one or more other methyacrylate monomers and the methacrylate monomer comprising an azide group ranges from about 10:1 to 50:1, about 10:1 to 45:1, about 10:1 to 40:1, about 10:1 to 35:1, about 10:1 to 30:1, about 10:1 to 25:1, about 10:1 to 20:1, or about 10:1 to 15:1.
  • 1 is an integer selected from 1 to 50.
  • Click chemistry refers to a reaction (typically a one pot reaction) that quickly and irreversibly connects two reagents, e.g., a polymer and a drug.
  • Various click chemistry reactions e.g., copper catalyzed azide-alkyne cycloaddition (CuAAC), strain-promoted azide-alkyne cycloaddition (SPAAC), strain-promoted alkyne-nitrone cycloaddition (SPANC), and other reactions of strained alkenes, are used to attach drugs to polymers.
  • CuAAC copper catalyzed azide-alkyne cycloaddition
  • SPAAC strain-promoted azide-alkyne cycloaddition
  • SPANC strain-promoted alkyne-nitrone cycloaddition
  • other reactions of strained alkenes are used to attach drugs to polymers.
  • the agent is coupled to the non-PEG bottlebrush polymer by an azide- alkyne click reaction or an Inverse electron-demand Diels-Alder (IEDDA) reaction.
  • IEDDA Inverse electron-demand Diels-Alder
  • the azide-alkyne click reaction is copper catalyzed azide-alkyne cycloaddition (CuAAC) or strain-promoted azide-alkyne cycloaddition (SPAAC), or strain- promoted alkyne-nitrone cycloaddition (SPANC).
  • the azide-alkyne click reaction is copper catalyzed azide-alkyne cycloaddition (CuAAC).
  • the azide- alkyne click reaction is strain-promoted azide-alkyne cycloaddition (SPAAC), strain-promoted alkyne-nitrone cycloaddition (SPANC).
  • IEDDA Inverse electron-demand Diels- Alder
  • a reaction that quickly and irreversibly connects two reagents, e.g., a polymer and an agent.
  • the reaction follows the prototypical Diels-Alder reaction that utilizes a reaction between an electron-poor diene and an electron-rich dienophile to form a new ring that couples the two reagents.
  • Various Inverse electron-demand Diels-Alder (IEDDA) reactions e.g., tetrazine and trans-cycloctene, are used to attach agents to polymers.
  • IEDDA Inverse electron-demand Diels- Alder
  • the agent is coupled to the non-PEG bottlebrush polymer by an Inverse electron-demand Diels-Alder (IEDDA) reaction.
  • IEDDA Inverse electron-demand Diels-Alder
  • the Inverse electron-demand Diels- Alder (IEDDA) reaction is tetrazine-cyclooctene.
  • non-PEG polymer or “non-PEG bottlebrush polymer” refers to a bottlebrush polymer that:
  • (a) does not comprise ethylene glycol, i.e., -OCH2CH2-, units; or
  • the non-PEG polymer comprises one or more monomers that include, but are not limited to, triethylene glycol methyl ether methacrylate (TEOMA), 2-(methylsulfmyl)ethyl methacrylate (MSEMA), 1- phenoxycarbonyl ethyl methacrylate, 3-[[2- (methacryloyloxy)ethyl]dimethylammonio]propionate (PCMA), 3-[[2- (methacryloyloxy)ethyl]dimethylammonio]propionate (CBMA), 2-((2-bromo-2- methylpropanoyl)oxy)ethyl methacrylate (BMPOEMA), 2-(2-bromo-2-methylpropanamido)e
  • TEOMA triethylene glycol methyl ether methacrylate
  • MSEMA 2-(methylsulfmyl)ethyl methacrylate
  • PCMA 3-[[2- (methacryloyloxy)ethyl]dimethyl
  • the non-PEG polymer comprises one or more polymers that include, but are not limited to, poly(triethylene glycol methyl ether methacrylate) (pTEOMA), poly(polyethylene glycol (meth)acrylate) (pPEGMA) wherein the number of ethylene glycol units is an integer between 3 to 9, poly(tetraethylene glycol methyl ether methacrylate) (pTetEOMA), poly(pentaethylene glycol methyl ether methacrylate) (pPEOMA), poly(hexaethylene glycol methyl ether methacrylate) (pHEOMA), poly(heptaethylene glycol methyl ether methacrylate) (pHPEOMA), poly(octaethylene glycol methyl ether methacrylate) (pOEOMA), poly(nonaethylene glycol methyl ether methacrylate) (NEOMA), pPEGMAsoo, pPEGMAsoo, poly(2-(methylsulfin
  • the non-PEG polymer can also refer to a polymer that is resistant to reacting with subject-derived PEG antibodies and are therefore resistant to accelerated blood clearance of the bioconjugate and hypersensitivity reactions caused by subject-derived PEG antibodies.
  • the compositions and methods described herein may be used to deliver molecules with reduced or eliminated antigenicity, and thereby address pre-existing anti- PEG antibodies in mammals, including humans, that may undermine the safety and efficacy of PEGylated therapeutics.
  • the non-PEG bottlebrush polymers and bioconjugates detailed herein contain polymers that are resistant to accelerated blood clearance of the bioconjugate and hypersensitivity reactions caused by subject-derived PEG antibodies.
  • the non-PEG polymer that is resistant to subject-derived PEG antibodies is a polymer that comprises one or more monomers that include, but are not limited to, triethylene glycol methyl ether methacrylate (TEOMA), 2-(methylsulfmyl)ethyl methacrylate (MSEMA), 1 -phenoxy carbonyl ethyl methacrylate, 3-[[2- (methacryloyloxy)ethyl]dimethylammonio]propionate (PCMA), 3-[[2- (methacryloyloxy)ethyl]dimethylammonio]propionate (CBMA), 2-((2-bromo-2- methylpropanoyl)oxy)ethyl methacrylate (BMPOEMA), 2-(2-bromo-2-methylpropanamido)ethyl methacrylate (BMPAEMA), polyethylene glycol monomethyl ether methacrylate (PEGMA) wherein the number of ethylene glycol units is
  • OEOMA nonaethylene glycol methyl ether methacrylate
  • NEOMA 2-dimethylaminoethyl methacrylate
  • DMAEMA 2-dimethylaminoethyl methacrylate
  • SBMA sulfobetaine methacrylate
  • HEMA quaternary ammonium ethyl methacrylate
  • HEMA 2-azidoethyl methacrylate
  • TEOMA-Azide or TEOMA-N3 2-azido tetraethylene glycol methyl ether methacrylate
  • the non-PEG polymers that are resistant to subject-derived PEG antibodies comprises polymers that include, but are not limited to, poly(triethylene glycol methyl ether methacrylate) (pTEOMA), poly(polyethylene glycol (meth)acrylate) (pPEGMA) wherein the number of ethylene glycol units is an integer between 3 to 9, poly(tetraethylene glycol methyl ether methacrylate) (pTetEOMA), poly(pentaethylene glycol methyl ether methacrylate) (pPEOMA), poly(hexaethylene glycol methyl ether methacrylate) (pHEOMA), poly(heptaethylene glycol methyl ether methacrylate) (pHPEOMA), poly(octaethylene glycol methyl ether methacrylate) (pOEOMA), poly(nonaethylene glycol methyl ether methacrylate) (NEOMA), pPEGMAsoo, pPEGMAsoo, poly(nonaethylene glycol
  • the bottlebrush polymer is synthesized from zwitterionic monomers or zwitterionic polymers, which contain the same number of cationic and anioinic charges along their monomer or polymer chains, respectively.
  • the cationic charge is often contributed due to the presence of quaternized ammonium groups, while the anionic charge is often contributed to the presence of sulfonate, caboxylate, and/or phosphonate groups, (see Zheng, Lichuen et al. Reactive and Functional Polymers, 118: 51-61 (2017).
  • Zwitterionic polymers or polymers made from zwitterionic monomers contain both high dipole moments and highly charged groups, which leads to polymers with increased hydrophilicity, as compared to polymers containing PEG, which are amphiphilic with both hydrophilic and hydrophobic properties.
  • the bottlebrush polymer is synthesized from one or more zwitterionic monomers selected from 2-(methacryloyloxy)ethyl 2-(trimethylammonio)ethyl Phosphate (PCMA), 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propionate (CBMA), sulfobetaine methacrylate (SBMA), or combinations thereof.
  • PCMA 2-(methacryloyloxy)ethyl 2-(trimethylammonio)ethyl Phosphate
  • CBMA 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propionate
  • SBMA sulfobetaine methacrylate
  • the non-PEG polymer comprises one or more zwitterionic polymers selected from poly(2-(methacryloyloxy)ethyl 2-(trimethylammonio)ethyl Phosphate) (PCMA), poly(3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propionate) (pCBMA), poly(sulfobetaine methacrylate) (pSBMA), or combinations thereof.
  • PCMA poly(2-(methacryloyloxy)ethyl 2-(trimethylammonio)ethyl Phosphate)
  • pCBMA poly(3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propionate)
  • pSBMA poly(sulfobetaine methacrylate)
  • TEOMA is triethylene glycol methyl ether methacrylate, which has the following structure:
  • the structure of TEOMA can also be represented by the following structure:
  • MSEMA is 2-(methylsulfmyl)ethyl methacrylate, which has the following structure:
  • PCMA is 2-(methacry!oyloxy)ethyl 2-(trimethylammonio)ethyl phosphate, which has the following structure:
  • CBMA is 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propionate, which has the following structure:
  • the non-PEG bottlebrush polymer is synthesized from a monomer containing at least one initiator molecule for Atom Transfer Radical Polymerization (ATRP) and one or more monomer units by ATRP.
  • the monomer containing at least one initiator molecule for Atom Transfer Radical Polymerization (ATRP) and one or more monomer units by ATRP is an inimer.
  • the inimer is selected from 2-((2-bromo-2- methylpropanoyl)oxy)ethyl methacrylate (BMPOEMA) and 2-(2-bromo-2- methylpropanamido)ethyl methacrylate (BMPAEMA), or combinations thereof.
  • BMPOEMA is 2-((2-bromo-2-methylpropanoyl)oxy)ethyl methacrylate, which has the following structure:
  • BMPAEMA is 2-(2-bromo-2-methylpropanamido)ethyl methacrylate, which has the following structure:
  • PEGMA is polyethylene glycol monomethyl ether methacrylate, which has the following structure: wherein, n is an integer between 3 to 9.
  • the monomer is PEGMA and n is an integer between 3 to 9. In some aspects, the monomer is PEGMA and n is an integer between 3 to 8. In some aspects, the monomer is PEGMA and n is an integer between 3 to 7. In some aspects, the monomer is PEGMA and n is an integer between 3 to 6. In some aspects, the monomer is PEGMA and n is an integer between 3 to 5.
  • the monomer is PEGMA and n is an integer of 3. In some aspects, the monomer is PEGMA and n is an integer of 4. In some aspects, the monomer is PEGMA and n is an integer of 5. In some aspects, the monomer is PEGMA and n is an integer of 6. In some aspects, the monomer is PEGMA and n is an integer of 7. In some aspects, the monomer is PEGMA and n is an integer of 8. In some aspects, the monomer is PEGMA and n is an integer of 9. [0225] TetEOMA is tetraethylene glycol methyl ether methacrylate, which has the following structure:
  • PEOMA is pentaethylene glycol methyl ether methacrylate, which has the following structure:
  • HEOMA is hexaethylene glycol methyl ether methacrylate, which has the following structure:
  • HPEOMA is heptaethylene glycol methyl ether methacrylate, which has the following structure:
  • OEOMA is octaethylene glycol methyl ether methacrylate, which has the following structure:
  • NEOMA is nonaethylene glycol methyl ether methacrylate, which has the following structure:
  • the PEGMA monomer is selected from TEOMA, TetEOMA, PEOMA, HEOMA, HPEOMA, OEOMA, NEOMA, or a combination thereof. In some aspects, the PEGMA monomer is selected from TEOMA, PEOMA, NEOMA, or a combination thereof. In some aspects, the PEGMA monomer is TEOMA. In some aspects, the PEGMA monomer is PEOMA. In some aspects, the PEGMA monomer is NEOMA.
  • DMAEMA is 2-dimethylaminoethyl methacrylate, which has the following structure:
  • the non-PEG bottlebrush polymer comprises one or more monomers containing an azide group (-N3) functional group.
  • the non-PEG bottlebrush polymers or bioconjugates described herein contain polymers that are resistant to accelerated blood clearance of the bioconjugate and hypersensitivity by subject-derived PEG antibodies.
  • the non-PEG bottlebrush polymer or bioconjugate is not reactive with pre-existing anti-PEG antibodies in a subject.
  • the non-PEG bottlebrush polymer or bioconjugate comprises PEGMA, wherein the number of ethylene glycol units is an integer between 3 to 9 and the bottlebrush polymer or bioconjugate is not reactive with pre-existing anti-PEG antibodies in a subject.
  • the non-PEG bottlebrush polymer or bioconjugate comprises one or more monomers selected from triethylene glycol methyl ether methacrylate (TEOMA), 2-(m ethyl sulfinyljethyl methacrylate (MSEMA), 1 -phenoxy carbonyl ethyl methacrylate, 3-[[2- (methacryloyloxy)ethyl]dimethylammonio]propionate (PCMA), 3-[[2- (methacryloyloxy)ethyl]dimethylammonio]propionate (CBMA), 2-((2-bromo-2- methylpropanoyl)oxy)ethyl methacrylate (BMPOEMA), 2-(2-bromo-2-methylpropanamido)ethyl methacrylate (BMPAEMA), polyethylene glycol monomethyl ether methacrylate (PEGMA) wherein the number of ethylene glycol units is an integer between 3 to 9, tetraethylene glycol
  • the non-PEG bottlebrush polymer or bioconjugate comprises polymers that contain ethylene glycol units and still remain resistant to accelerated blood clearance of the bioconjugate and hypersensitivity reactions caused by subject-derived PEG antibodies.
  • the non-PEG bottlebrush polymer comprises polymers that contain ethylene glycol units and the polymer is selected from poly(triethylene glycol methyl ether methacrylate) (pTEOMA), poly(polyethylene glycol (meth)acrylate) (pPEGMA) wherein the number of ethylene glycol units is an integer between 3 to 9, poly(tetraethylene glycol methyl ether methacrylate) (pTetEOMA), poly(pentaethylene glycol methyl ether methacrylate) (pPEOMA), poly(hexaethylene glycol methyl ether methacrylate) (pHEOMA), poly(heptaethylene glycol methyl ether methacrylate) (pHPEOMA), poly(octaethylene glycol methyl ether
  • the present disclosure provides methods of making the bioconjugates comprising non- PEG polymers described herein.
  • the present disclosure provides a method of making a bioconjugate comprising: (a) preparing a non-PEG polymer backbone comprising a chain transfer agent (CTA) and one or more monomer units by Reversible Addition Fragmentation chain Transfer (RAFT) polymerization, wherein at least one of the monomer units contains an initiator molecule for Atom Transfer Radical Polymerization (ATRP);
  • CTA chain transfer agent
  • RAFT Reversible Addition Fragmentation chain Transfer
  • ATRP Atom Transfer Radical Polymerization
  • the present disclosure provides a method of making a bioconjugate comprising:
  • the present disclosure provides a method of making a bioconjugate comprising:
  • the non-PEG polymer of (a) is coupled to a molecule containing a methacrylate functional group. In some aspects, of the methods described above the non-PEG polymer of (a) comprises a methacrylate functional group.
  • the present disclosure provides a method of making a bioconjugate comprising:
  • the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 5-20. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 5-15. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 5-10. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 10-80.
  • the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 15-80. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 20-80. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 25-80. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 30-80.
  • the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 75-80. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 10-20. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 20-30. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 30-40.
  • the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 40-50. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 50-60. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 60-70. In some aspects disclosed herein, the leash comprises a monomer polymerizable by controlled radical polymerization, wherein the number of repeat units is an integer between 70-80. VII. Bioconjugates
  • Bioconjugates of the present disclosure can increase the effectiveness of agents by reducing anti-drug antibodies (AD As) found when PEGylated agents are administered to a subject, improving biologic stability, improving physicochemical properties, allowing for utilization of flexible administration routes, and/or enhancing the manufacturability of biological conjugates that are used for treatment of a subject or patient.
  • AD As anti-drug antibodies
  • bioconjugates comprising agents including antibodies, antibody fragments, functional equivalents, improved antibodies, other protein or polynucleotide agents, and their aspects as disclosed herein, linked or conjugated to a nonpolyethylene glycol (PEG) polymer that is linked or conjugated to a drug.
  • PEG polyethylene glycol
  • the non-PEG polymer is comprised of a backbone chain with hydrophilic side chains containing reactive moieties that can be utilized for conjugation of drugs (for example, Figure 1A).
  • the non-PEG polymer allows for the conjugation of drug payloads at an ultra-high drug-to-agent ratio (DAR), and affords use of mixed payloads, high potency payloads, and payloads with problematic properties (e.g., systemic toxicity, poor pharmacokinetics) (for example, Figure 3A-3B).
  • DAR drug-to-agent ratio
  • the conjugation of these non-PEG polymers with various drugs (e.g., payloads) allows for the increase in the therapeutic index of the bioconjugate.
  • the ultra-high DAR (100+ payloads per agent) is suitable for low expression targets (e.g., cancer), affords the ability to use mixed drug payloads for synthetic lethal strategies and use of highly potent precision-medicine drugs (e.g., payloads) that were intolerable as systemic therapies, and improved tissue penetration compared to other bioconjugates in the art.
  • D z can be any drug described herein.
  • LI can be any linker described herein.
  • P can be any linker described herein.
  • L2 can be any linker described herein.
  • A can be any linker described herein.
  • the agent e.g., antibody
  • the agent is linked or conjugated to one drug with a DAR of at least 100: 1, 150: 1, 200: 1, 250:1, 300: 1, 350: 1, 400: 1, 450: 1, 500: 1, 550: 1, 600: 1, 650: 1, 700: 1, 750: 1, 800: 1, 850:1, 900: 1, 950: 1, 1000: 1, 1050: 1, or 1100: 1 as disclosed herein.
  • the drug is a chemotherapeutic drug.
  • the chemotherapeutic drug is a MEK inhibitor.
  • the chemotherapeutic drug is a PI3K inhibitor.
  • the chemotherapeutic drug is a CDK inhibitor.
  • the chemotherapeutic drug is a CHK inhibitor. In some aspects, the chemotherapeutic drug is apitolisib. In some aspects, the chemotherapeutic drug is cobimetinib. In some aspects, the chemotherapeutic drug is dinaciclib. In some aspects, the chemotherapeutic drug is prexasertib.
  • the agent is linked or conjugated to two distinct drugs. In some aspects, the agent is linked or conjugated to two distinct drugs, wherein the ratio between the two distinct drugs is 1 : 1. In some aspects, the two distinct drugs are all chemotherapeutic drugs.
  • the agent is linked or conjugated to two distinct drugs with a DAR for both drugs combined being at least 50: 1, 100: 1, 150: 1, 200: 1, 250:1, 300: 1, 350: 1, 400: 1, 450: 1, 500: 1, 550: 1, 600: 1, 650: 1, 700: 1, 750: 1, 800:1, 850:1, 900: 1, 950: 1, 1,000: 1, 1050:1, or 1,100: 1.
  • the agent is linked or conjugated to two distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for both drugs combined being between 50-150: 1.
  • the agent is linked or conjugated to two distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for both drugs combined being between 150-250: 1 In some aspects, the agent is linked or conjugated to two distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for both drugs combined being between 250-350: 1. In some aspects, the agent is linked or conjugated to two distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between for both drugs combined being 350-450: 1.
  • the agent is linked or conjugated to two distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for both drugs combined being between 450-550: 1. In some aspects, the agent is linked or conjugated to two distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for both drugs combined being between 550-650: 1. In some aspects, the agent is linked or conjugated to two distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for both drugs combined being between 650-750: 1.
  • the agent is linked or conjugated to two distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for both drugs combined being between 750-850: 1. In some aspects, the agent is linked or conjugated to two distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for both drugs combined being between 850-950: 1. In some aspects, the agent is linked or conjugated to two distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for both drugs combined being between 950-1,050: 1.
  • the agent is linked or conjugated to two distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for both drugs combined being between 1,050-1,150: 1.
  • the agent e.g., antibody
  • the agent is linked or conjugated to two drugs.
  • the agent e.g., antibody
  • the ratio between the two distinct drugs is 1 : 1.
  • the agent is linked or conjugated to two drugs wherein the ratio between the two distinct drugs is 2: 1.
  • the agent is linked or conjugated to two drugs wherein the ratio between the two distinct drugs is 3 : 1.
  • the agent is linked or conjugated to two drugs wherein the ratio between the two distinct drugs is 4: 1. In some aspects, the agent is linked or conjugated to two drugs wherein the ratio between the two distinct drugs is 5:1. In some aspects, the agent is linked or conjugated to two drugs wherein the ratio between the two distinct drugs is 6: 1. In some aspects, the agent is linked or conjugated to two drugs wherein the ratio between the two distinct drugs is 7: 1. In some aspects, the agent is linked or conjugated to two drugs wherein the ratio between the two distinct drugs is 8: 1. In some aspects, the agent is linked or conjugated to two drugs wherein the ratio between the two distinct drugs is 9: 1. In some aspects, the agent is linked or conjugated to two drugs wherein the ratio between the two distinct drugs is 10: 1.
  • the agent is linked or conjugated to two drugs wherein the ratio between the two distinct drugs is 11 : 1. In some aspects, the agent is linked or conjugated to two drugs wherein the ratio between the two distinct drugs is 12: 1. In some aspects, the agent is linked or conjugated to two drugs wherein the ratio between the two distinct drugs is 13 : 1. In some aspects, the agent is linked or conjugated to two drugs wherein the ratio between the two distinct drugs is 14: 1. In some aspects, the agent is linked or conjugated to two drugs wherein the ratio between the two distinct drugs is 15: 1. In some aspects, the agent is linked or conjugated to two drugs wherein the ratio between the two distinct drugs is 16: 1. In some aspects, the agent is linked or conjugated to two drugs wherein the ratio between the two distinct drugs is 17: 1.
  • the agent is linked or conjugated to two drugs wherein the ratio between the two distinct drugs is 18: 1. In some aspects, the agent is linked or conjugated to two drugs wherein the ratio between the two distinct drugs is 19: 1. In some aspects, the agent is linked or conjugated to two drugs wherein the ratio between the two distinct drugs is 20: 1. In some aspects, the agent is linked or conjugated to two drugs wherein the ratio between the two distinct drugs is from 1 : 1 to 5 : 1. In some aspects, the agent is linked or conjugated to two drugs wherein the ratio between the two distinct drugs is from 5: 1 to 10: 1. In some aspects, the agent is linked or conjugated to two drugs wherein the ratio between the two distinct drugs is from 10:1 to 15: 1.
  • the agent is linked or conjugated to two drugs wherein the ratio between the two distinct drugs is from 15: 1 to 20: 1. In some aspects, the agent is linked or conjugated to two drugs wherein the ratio between the two distinct drugs is from 1 : 1 to 10: 1. In some aspects, the agent is linked or conjugated to two drugs wherein the ratio between the two distinct drugs is from 10: 1 to 20: 1. In some aspects, the agent is linked or conjugated to two drugs wherein the ratio between the two distinct drugs is from 1 : 1 to 20: 1. In some aspects, the two distinct drugs are both chemotherapeutic drugs. In some aspects, the chemotherapeutic drugs are a MEK inhibitor and a PI3K inhibitor.
  • the chemotherapeutic drugs are a CDK inhibitor and a PI3K inhibitor. In some aspects, the chemotherapeutic drugs are a CHK inhibitor and a PI3K inhibitor. In some aspects, the chemotherapeutic drugs are MEK inhibitors. In some aspects, the chemotherapeutic drugs are PI3K inhibitors. In some aspects, the chemotherapeutic drugs are CDK inhibitors. In some aspects, the chemotherapeutic drugs are CHK inhibitors. In some aspects, the chemotherapeutic drugs are apitolisib. In some aspects, the chemotherapeutic drugs are cobimetinib. In some aspects, the chemotherapeutic drugs are dinaciclib. In some aspects, the chemotherapeutic drugs are prexasertib.
  • the agent is linked or conjugated to three distinct drugs. In some aspects, the agent is linked or conjugated to three distinct drugs, wherein the ratio between the three distinct drugs is 1 : 1 : 1. In some aspects, the three distinct drugs are all chemotherapeutic drugs.
  • the agent is linked or conjugated to three distinct drugs with a DAR for all drugs combined being at least 50: 1, 100: 1, 150: 1, 200: 1, 250:1, 300: 1, 350: 1, 400: 1, 450: 1, 500: 1, 550: 1, 600: 1, 650: 1, 700: 1, 750: 1, 800:1, 850: 1, 900: 1, 950: 1, 1,000: 1, 1,050: 1, or 1,100:1.
  • the agent is linked or conjugated to three distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for the drugs combined being between 50-150: 1.
  • the agent is linked or conjugated to three distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for the drugs combined being between 450-550: 1. In some aspects, the agent is linked or conjugated to three distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for the drugs combined being between 550-650: 1. In some aspects, the agent is linked or conjugated to three distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for drugs combined being between 650-750: 1.
  • the agent is linked or conjugated to three distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for drugs combined being between 750-850: 1. In some aspects, the agent is linked or conjugated to three distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for drugs combined being between 850-950: 1. In some aspects, the agent is linked or conjugated to three distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for the drugs combined being between 950-1,050: 1. In some aspects, the agent is linked or conjugated to three distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for the drugs combined being between 1,050-1,150: 1.
  • three distinct drugs e.g., including one or more chemotherapeutic drugs
  • the ratio between the three distinct drugs is 5 : 1 : 1. In some aspects, the ratio between the three distinct drugs is 10: 1 : 1. In some aspects, the ratio between the three distinct drugs is 15 : 1 : 1. In some aspects, the ratio between the three distinct drugs is 20: 1 : 1. In some aspects, the ratio between the three distinct drugs is 1 :5: 1. In some aspects, the ratio between the three distinct drugs is 1 : 10: 1. In some aspects, the ratio between the three distinct drugs is 1 : 15 : 1. In some aspects, the ratio between the three distinct drugs is 1 :20: 1. In some aspects, the ratio between the three distinct drugs is 1 : 1 :5. In some aspects, the ratio between the three distinct drugs is 1 : 1 : 10. In some aspects, the ratio between the three distinct drugs is 1 : 1 : 15. In some aspects, the ratio between the three distinct drugs is 1 : 1 :20.
  • the agent is linked or conjugated to four distinct drugs.
  • the four distinct drugs are all chemotherapeutic drugs.
  • the agent is linked or conjugated to four distinct drugs with a DAR for all drugs combined being at least 50: 1, 100: 1, 150: 1, 200: 1, 250:1, 300: 1, 350: 1, 400: 1, 450: 1, 500: 1, 550: 1, 600: 1, 650: 1, 700: 1, 750: 1, 800:1, 850: 1, 900: 1, 950: 1, 1,000: 1, 1,050: 1, or 1,100:1.
  • the agent is linked or conjugated to four distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for the drugs combined being between 50-150: 1.
  • the agent is linked or conjugated to four distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for the drugs combined being between 150-250: 1 In some aspects, the agent is linked or conjugated to four distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for the drugs combined being between 250-350: 1. In some aspects, the agent is linked or conjugated to four distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between for the drugs combined being 350-450: 1.
  • the agent is linked or conjugated to four distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for the drugs combined being between 450-550: 1. In some aspects, the agent is linked or conjugated to four distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for the drugs combined being between 550-650: 1. In some aspects, the agent is linked or conjugated to four distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for the drugs combined being between 650-750: 1.
  • the agent is linked or conjugated to four distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for the drugs combined being between 750-850: 1. In some aspects, the agent is linked or conjugated to four distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for the drugs combined being between 850-950: 1. In some aspects, the agent is linked or conjugated to four distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for the drugs combined being between 950-1,050: 1. In some aspects, the agent is linked or conjugated to four distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for the drugs combined being between 1,050-1,150: 1.
  • four distinct drugs e.g., including one or more chemotherapeutic drugs
  • the ratio between the four distinct drugs is 5 : 1 : 1 : 1. In some aspects, the ratio between the four distinct drugs is 10: 1 : 1 : 1. In some aspects, the ratio between the four distinct drugs is 15 : 1 : 1 : 1. In some aspects, the ratio between the four distinct drugs is 20: 1 : 1 : 1. In some aspects, the ratio between the four distinct drugs is 1 :5: 1 :1. In some aspects, the ratio between the four distinct drugs is 1 : 10: 1 : 1. In some aspects, the ratio between the four distinct drugs is 1 : 15 : 1 : 1. In some aspects, the ratio between the four distinct drugs is 1 :20: 1 : 1.
  • the ratio between the four distinct drugs is 1 : 1 : 5 : 1. In some aspects, the ratio between the three distinct drugs is 1 : 1 : 10: 1. In some aspects, the ratio between the four distinct drugs is 1 : 1 : 15 : 1. In some aspects, the ratio between the four distinct drugs is 1 : 1 :20: 1. In some aspects, the ratio between the four distinct drugs is 1 : 1 : 1 :5. In some aspects, the ratio between the four distinct drugs is 1 : 1 : 1 : 10. In some aspects, the ratio between the four distinct drugs is 1 : 1 : 1 : 15. In some aspects, the ratio between the four distinct drugs is 1 :1 : 1 :20.
  • the agent is linked or conjugated to five distinct drugs.
  • the five distinct drugs are all chemotherapeutic drugs.
  • the agent is linked or conjugated to five distinct drugs with a DAR for all drugs combined being at least 50:1, 100: 1, 150: 1, 200: 1, 250: 1, 300: 1, 350: 1, 400: 1, 450:1, 500: 1, 550: 1, 600: 1, 650: 1, 700: 1, 750: 1, 800:1, 850: 1, 900: 1, 950: 1, 1,000: 1, 1,050: 1, or 1,100:1.
  • the agent is linked or conjugated to five distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for the drugs combined being between 50-150: 1.
  • the agent is linked or conjugated to five distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for the drugs combined being between 150-250: 1 In some aspects, the agent is linked or conjugated to five distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for the drugs combined being between 250-350: 1. In some aspects, the agent is linked or conjugated to five distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between for the drugs combined being 350-450: 1.
  • the agent is linked or conjugated to five distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for the drugs combined being between 450-550: 1. In some aspects, the agent is linked or conjugated to five distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for the drugs combined being between 550-650: 1. In some aspects, the agent is linked or conjugated to five distinct drugs (e.g., including one or more chemotherapeutic drugs) at a DAR for the drugs combined being between 650-750: 1.
  • the ratio between the five distinct drugs is 1 : 1 : 1 : 15:1. In some aspects, the ratio between the five distinct drugs is 1 : 1 : 1 :20: 1. In some aspects, the ratio between the five distinct drugs is 1 : 1 : 1 :5. In some aspects, the ratio between the five distinct drugs is 1 : 1 : 1 : 1 : 10. In some aspects, the ratio between the five distinct drugs is 1 : 1 : 1 : 1 :15. In some aspects, the ratio between the five distinct drugs is 1 : 1 : 1 : 1 :20.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR of at least 10: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR of at least 20: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR of at least 30: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR of at least 40:1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-100:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-110: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-120: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-130: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-140: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-150:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-160: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-170: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-210: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-220: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-230: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-240: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-250: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-260: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-270: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-280: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-290:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-300: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-350: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-400: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-450: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-500: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-550: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-600: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-650: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-700: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-750:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-800: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-850: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-900: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-950: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-1,000: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-1,050: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 10-1,100:1
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-30: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-40: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-50: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-60: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-70: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-80: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-90: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-100: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-110:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-120: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-130: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-140: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-150: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-160:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-170: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-180: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-190: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-200: 1. [0292] In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-210: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-220: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-230: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-240: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-250: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-260: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-270: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-280: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-290:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-300: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-350: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-400: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-450: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-500: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-550: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-600: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-650: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-700: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-750:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-800: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-850: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-900: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-950: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-1,000: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-1,050: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 20-1,100:1
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-40: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-50: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-60: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-70: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-80: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-90: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-100: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-110: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-120:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-130: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-140: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-150: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-160: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-170:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-180: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-190: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-200: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-210: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-220: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-230: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-240: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-250: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-260: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-270: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-280: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-950: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-1,000: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-1,050: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 30-1,100:1
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 170-950:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 170-1,000: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 170-1,050: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 170-1,100: 1
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 190-950:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 190-1,000: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 190-1,050: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 190-1,100: 1
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 200-210:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 200-220: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 200-230: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 200-240:1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 200-250: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 200-260: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 200-270:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 200-280: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 200-290: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 200-300: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 200-350:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 200-400: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 200-450: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 200-500:1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 200-550: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 200-600: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 200-650:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 200-700: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 200-750: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 200-800:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 200-850: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 200-900: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 200-950:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 200-1,000: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 200-1,050: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 200-1, 100: 1
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 210-220:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 210-230: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 210-240: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 210-250:1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 210-260: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 210-270: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 210-280:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 210-290: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 210-300: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 210-350:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 210-400: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 210-450: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 210-500:1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 210-550: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 210-600: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 210-650:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 210-700: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 210-750: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 210-800:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 210-850: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 210-900: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 210-950:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 210-1,000: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 210-1,050: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 210-1, 100: 1
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 220-230:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 220-240: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 220-250: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 220-260:1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 220-270: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 220-280: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 220-290:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 220-300: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 220-350:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 220-400: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 220-450: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 220-500:1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 220-550: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 220-600: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 220-650:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 220-700: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 220-750: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 220-800:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 220-850: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 220-900: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 220-950:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 220-1,000: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 220-1,050: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 220-1, 100: 1
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 230-240:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 230-250: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 230-260: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 230-270: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 230-280: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 230-290: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 230-300: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 230-350:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 230-400: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 230-450: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 230-500:1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 230-550: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 230-600: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 230-650:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 230-700: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 230-750: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 230-800:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 230-850: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 230-900: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 230-950:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 230-1,000: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 230-1,050: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 230-1, 100: 1 [0353] In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 240-250:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 240-260: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 240-270: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 240-280:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 240-290: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 240-300: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 240-350:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 240-400: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 240-450: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 240-500:1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 240-550: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 240-600: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 240-650:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 240-700: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 240-750: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 240-800:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 240-850: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 240-900: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 240-950:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 240-1,000: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 240-1,050: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 240-1, 100: 1
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 250-260:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 250-270: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 250-280: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 250-290:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 250-300: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 250-350:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 250-400: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 250-450: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 250-500:1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 250-550: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 250-600: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 250-650:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 250-700: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 250-750: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 250-800:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 250-850: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 250-900: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 250-950:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 250-1,000: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 250-1,050: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 250-1, 100: 1
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 260-270:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 260-280: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 260-290: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 260-300: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 260-350:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 260-400: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 260-450: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 260-500:1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 260-550: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 260-600: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 260-650:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 260-700: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 260-750: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 260-800:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 260-850: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 260-900: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 260-950:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 260-1,000: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 260-1,050: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 260-1, 100: 1
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 270-280:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 270-290: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 270-300:1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 270-350:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 270-400: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 270-450: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 270-500:1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 270-550: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 270-600: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 270-650:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 270-700: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 270-750: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 270-800:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 270-850: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 270-900: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 270-950:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 270-1,000: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 270-1,050: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 270-1, 100: 1
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 280-290:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 280-300: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 280-350:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 280-400: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 280-450: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 280-500:1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 280-550: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 280-600: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 280-650:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 280-700: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 280-750: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 280-800:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 280-850: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 280-900: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 280-950:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 280-1,000: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 280-1,050: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 280-1, 100: 1
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 290-300:1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 290-350:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 290-400: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 290-450: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 290-500:1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 290-550: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 290-600: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 290-650:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 290-700: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 290-750: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 290-800:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 290-850: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 290-900: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 290-950:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 290-1,000: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 290-1,050: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 290-1, 100: 1
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 300-350:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 300-400: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 300-450: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 300-500:1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 300-550: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 300-600: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 300-650:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 300-700: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 300-750: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 300-800:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 300-850: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 300-900: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 300-950:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 300-1,000: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 300-1,050: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 300-1, 100: 1
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 350-400:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 350-450: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 350-500: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 350-550:1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 350-600: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 350-650: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 350-700:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 350-750: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 350-800: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 350-850:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 350-900: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 350-950: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 350-1,000: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 350-1,050: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 350-1,100:1
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 400-450:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 400-500: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 400-550: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 400-600:1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 400-650: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 400-700: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 400-750:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 400-800: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 400-850: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 400-900:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 400-950: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 400-1,000: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 400-1,050: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 400-1,100: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 450-500:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 450-550: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 450-600: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 450-650:1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 450-700: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 450-750: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 450-800:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 450-850: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 450-900: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 450-950:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 450-1,000: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 450-1,050: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 450-1, 100: 1.
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 500-550:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 500-600: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 500-650: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 500-700:1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 500-750: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 500-800: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 500-850:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 500-900: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 500-950: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 500-1,000: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 500-1,050: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 500-1, 100: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 550-600:1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 550-650: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 550-700: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 550-750:1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 550-800: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 550-850: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 550-900: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 550-950: 1.
  • drugs e.g., including one or more chemotherapeutic drugs
  • the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 550-1,000: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 550-1,050: 1. In some aspects, the agent is linked or conjugated to one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR between 550-1,100: 1.
  • the method of inhibiting proliferation of cancer or cancer cells comprises contacting the cancer or cancer cells with the anti-phosphatidylserine bioconjugate in vivo.
  • contacting a cancer or cancer cell with an anti-phosphatidylserine bioconjugate is undertaken in an animal model.
  • an anti-phosphatidylserine bioconjugate can be administered to xenografts externalizing phosphatidylserine that have been grown in immunocompromised mice (e.g. NOD/SCID mice) to inhibit cancer growth or metastasis or surival.
  • HisPurTM Ni-NTA resins are used for purification of the His-tagged agents and bioconjugates. Step-wise imidazole elution at different concentrations is utilized to purify different structures. Small Molecule Chromatography:
  • the synthesized organic crude product-containing residues were purified by passing the crude material or concentrate through either a Biotage brand silica gel column pre-packed with flash silica (SiCh) or reverse phase flash silica (Cl 8) and eluting the product off the column with a solvent gradient as indicated.
  • a description of silica gel (0-40% EtOAc/hexane) means the product was obtained by elution from the column packed with silica using a solvent gradient of 0% to 40% EtOAc in hexanes.
  • Biological products for example, proteins or protein fragments, were not purified by small molecule chromatography.
  • samples were diluted directly 1 in 1000 to approximately 0.01 mg/mL in water containing 0.1% formic acid.
  • salt was removed through molecular weight cut off using a 10 kDa or 50 kDa filter for scFv or trastuzumab, respectively, and the final samples were left in deionized water from a milliQ water system equipped with a 0.2 pm filter at concentrations above 5 mg/mL. Samples were then diluted to 0.01 mg/mL in LC-MS grade water containing 0.1% formic acid.
  • the mobile phase was increased to 40% mobile phase B, where it was held until 7.5 minutes.
  • the mobile phase was again increased to 90% mobile phase B between 7.5 and 9.0 minutes.
  • the mobile phase was held at 90% B for 1 minute before returning the column back to 10% B from 10.0 to 11.0 minutes, where the mobile phase was held until the next injection.
  • a table depicting this gradient is included below.
  • FFF-MALS Field-Flow Fractionation-Multi-Angle Light Scattering
  • UV absorbance data was collected at 280 nm, the refractive index signal was monitored using a 658 nm LED at a temperature of 25 °C, and the multi-angle light scattering detector with inline dynamic light scattering was operated at 662 nm. Data were processed using ASTRA software (Wyatt Technology /Waters Corporation). Molecular weights were calculated using the refractive index increment (dn/dc) and/or ultraviolet absorbance extinction coefficients for the protein and/or polymer components or combinations thereof, depending on the composition of the analyte.
  • Refractive index increments (dn/dc) for analytes were calculated using ASTRA software's "100% mass recovery" method.
  • Corresponding experimental UV extinction coefficients were determined by using the input dn/dc to calculate analyte concentration from the refractive index signal, then using that concentration to determine the extinction coefficient based on the UV absorbance signal.
  • ELISA enzyme linked immunosorbent assay
  • the biological molecule selected was an anti-EGFR bivalent variable domain of heavy chain of heavy-chain antibody (VHH) construct.
  • the CHARMM36-JUL2022 forcefield was extended to incorporate additional chemical structures as necessary to compile the complete ADC structure. Chemical structures boundaries for individual parameterization are demarcated with dotted lines. The associated 3-4 letter residue names for each chemical structure defined in the extended forcefield are provided.
  • the charge penalties provided by the CGenFF server were used as weights to adjust individual atom charges such that the residue had net 0 charge. Exceptions were hydrogen atoms; any minor charge penalties assigned to these were set to 0 before the weighted adjustments were made.
  • residue names were added to the residuetypes.dat file of the forcefield and categorized as Protein type.
  • AlphaFol d2 was used to predict the structure of the VHH based on its sequence and obtain a representative pdb file.
  • Structural files for the binding molecules, the extended forcefield, and the appropriate mdp files were uploaded to one A10 instance of LambdaCloud installed with the 2022 software version of GROMACS for each biologic and the files were converted to GROMACS specific files along with the files for the topology and the positional restraints.
  • Selected forcefield was the local, extended forcefield, the tip3p water model was used, termini were manually defined to be charged, and all chains were merged into a single topology.
  • a dodecahedral water box was defined with a 1 nm buffer between the edge of the structure and the periodic boundaries of the water. The water box was solvated using the spc216.gro structure and the overall system charge was neutralized utilizing sodium and chloride ions.
  • NVT, N (number of particles), V (volume), and T (temperature), equilibration was conducted with a molecular dynamics integrator over 10,000 steps, with a time step of 2 femtosecond (fs). Positional restraints were applied to all heavy atoms, and the LINCS algorithm was used to constrain hydrogen bonds with a single iteration and an order of 4.
  • the simulation utilized a Verlet cutoff scheme with a neighbor list updated every 10 steps. Electrostatic interactions were handled using the Particle Mesh Ewald (PME) method, with a PME order of 4 and a Fourier grid spacing of 0.16 nm. Both Coulomb and van der Waals interactions had a cutoff radius of 1.0 nm.
  • PME Particle Mesh Ewald
  • a dispersion correction for energy and pressure was applied.
  • the system's temperature was controlled using the V-rescale thermostat, with separate coupling groups for protein and non-protein components, each maintained at 310.15 K with a coupling constant of 0.1 ps.
  • Periodic boundary conditions were applied in all three dimensions. Initial velocities were generated corresponding to the target temperature of 310.15 K using a random seed.
  • NPT, N number of particles
  • P pressure
  • T temperature
  • equilibration was performed similarly to the previous NVT equilibration, excepting the inclusion of pressure coupling.
  • the simulation continued from the previous run with positional restraints in place.
  • the Parrinello-Rahman barostat was used for isotropic pressure coupling, maintaining a reference pressure of 1.0 bar with a compressibility of 4.5e-5 bar 1 and a coupling constant of 2.0 picosecond (ps).
  • Temperature control was consistent with the NVT settings, with the same thermostat and temperature reference. Additionally, no new velocities were generated, and reference coordinate scaling was applied to the center of mass.
  • the trajectory was cleaned by removing solvent and ion atoms while centering coordinates on the biologic via the tijconv command.
  • the last frame of the trajectory was extracted to represent the relaxed trastuzumab antibody or the relaxed scFv structure.
  • the average of the top 1% summed solvent-exposed surface areas across all time points of each analysis trajectory was taken as the maximum possible surface area of the enzyme-cleavable linker and used to calculate the percentage exposure of each linker.
  • the solvent exposure of the cytotoxic payloads was calculated in a similar way to the solvent exposure of the enzyme-cleavable linkers, except that the atom indices for the /?-aminobenzylcarbamate (PAB) portion of the enzyme-cleavable linker was removed from the payload (PAY) residue index groups before calculation.
  • PAB atom indices for the /?-aminobenzylcarbamate
  • Table 12 shows the ratios of nonaethylene glycol methyl ether methacrylate to inimer to RAFT CTA to azobisisobutyronitrile (AIBN) for the synthesis of three other non-PEG polymer backbones, using the same method as Int-2.
  • Table 12 demonstrates that different compositions can be prepared by varying reaction conditions.

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Abstract

La présente invention concerne de nouveaux bioconjugués polymères non PEG ayant un rapport médicament-agent ultra-élevé qui se lient à des antigènes associés au cancer. Les bioconjugués polymères non PEG ayant un DAR ultra-élevé qui se lient à des antigènes associés au cancer peuvent administrer au moins un médicament aux cellules cancéreuses. L'invention concerne en outre des procédés d'utilisation des bioconjugués, tels que des procédés d'inhibition de la croissance tumorale, et des procédés de fabrication des bioconjugués.
PCT/US2024/048442 2023-09-25 2024-09-25 Procédés et compositions de bioconjugués non peg à rapport médicament/agent ultra-élevé Pending WO2025072355A1 (fr)

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US63/557,410 2024-02-23
US202463666550P 2024-07-01 2024-07-01
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009140423A2 (fr) * 2008-05-13 2009-11-19 University Of Washington Bioconjugués polymères ciblés
WO2014093640A1 (fr) * 2012-12-12 2014-06-19 Mersana Therapeutics,Inc. Conjugués hydroxy-polymère-médicament-protéine
US9156913B2 (en) * 2010-08-13 2015-10-13 Roche Glycart Ag Anti-tenascin-C A2 antibodies and methods of use
US9872853B2 (en) * 2016-06-10 2018-01-23 Eli Lilly And Company 2,3-dihydro-1H-indole compounds
US11241485B2 (en) * 2017-06-12 2022-02-08 Obsidian Therapeutics, Inc. PDE5 compositions and methods for immunotherapy

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009140423A2 (fr) * 2008-05-13 2009-11-19 University Of Washington Bioconjugués polymères ciblés
US9156913B2 (en) * 2010-08-13 2015-10-13 Roche Glycart Ag Anti-tenascin-C A2 antibodies and methods of use
WO2014093640A1 (fr) * 2012-12-12 2014-06-19 Mersana Therapeutics,Inc. Conjugués hydroxy-polymère-médicament-protéine
US9872853B2 (en) * 2016-06-10 2018-01-23 Eli Lilly And Company 2,3-dihydro-1H-indole compounds
US11241485B2 (en) * 2017-06-12 2022-02-08 Obsidian Therapeutics, Inc. PDE5 compositions and methods for immunotherapy

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