Methods and compositions of non-PEG bioconjugates with ultra high drug to agent ratios

Abstract

The present invention provides novel non-PEG polymer bioconjugates having ultra high drug to agent ratios that bind to cancer-associated antigens. Non-PEG polymer bioconjugates with ultra-high DAR that bind to cancer-associated antigens can deliver at least one drug to cancer cells. Also provided are methods of using the bioconjugates, such as methods of inhibiting tumor growth, and methods of making the bioconjugates.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 540,322, filed September 25, 2023; U.S. Provisional Application No. 63 / 557,410, filed February 23, 2024; and U.S. Provisional Application No. 63 / 666,550, filed July 1, 2024, the contents of which are incorporated herein by reference in their entirety.

[0002] Refer to the sequence list submitted electronically The contents of the electronically submitted sequence listing (name: 5423_003PC03_Sequence listing_ST26.xml; size: 37168 bytes; creation date: September 25, 2024) are quoted in their entirety hereby for reference. Technical Field

[0003] The field of this invention generally relates to bioconjugates comprising reagents (e.g., antibodies, antigen-binding fragments thereof, cytokines, enzymes, or polynucleotides) that bind to cancer-associated antigens, the cancer-associated antigens being linked to a non-polyethylene glycol (PEG) polymer having a drug (e.g., a chemotherapeutic drug), methods of treating cancer using said bioconjugates, and / or methods of preparing said bioconjugates. Background Technology

[0004] Cancer is one of the leading causes of death in developed countries. In the United States alone, more than one million people are diagnosed with cancer each year, and 500,000 die from it. Overall, it is estimated that more than one-third of people will develop some form of cancer in their lifetime. There are more than 200 different types of cancer, four of which—breast cancer, lung cancer, colorectal cancer, and prostate cancer—account for more than half of all new cases (Jemal et al., 2003, Cancer J. Clin. 53:5-26).

[0005] Biological conjugates and antibody-drug conjugates (“ADCs”) offer a class of potent anticancer agents effective against a range of cancers. Approved ADCs typically consist of three distinct components: a reagent (e.g., an antibody); a linker; and a cytotoxic moiety (e.g., taxane). Antibodies or other reagents that specifically bind to cancer surface antigens are used to deliver cytotoxic drugs in the form of biological conjugates. The number of drug molecules conjugated to each antibody in an ADC is typically distributed, ranging from 0 to 8 per antibody. There is a need to develop new ADCs and biological conjugates with higher drug-to-reagent ratios (DARs). Summary of the Invention

[0006] This invention provides bioconjugates with ultra-high DAR, comprising antibodies, their antigen-binding fragments, or other targeting agents (e.g., cytokines, enzymes, polynucleotides), wherein the antibodies, their antigen-binding fragments, or other targeting agents bind to cancer-associated antigens and are conjugated to one or more drugs or drug classes, and methods of using the present invention. Compositions comprising bioconjugates (e.g., pharmaceutical compositions) are also provided. Furthermore, methods for preparing and using bioconjugates are provided, such as methods for using bioconjugates to inhibit tumor growth and / or treat cancer.

[0007] Therefore, in one aspect, the present invention provides a bioconjugate comprising a reagent linked to a non-polyethylene glycol (PEG) polymer, wherein the non-PEG polymer is conjugated to a first drug, the reagent is bound to a cancer-associated antigen, and wherein the bioconjugate has a drug-to-reagent ratio (DAR) of at least 20:1.

[0008] In another aspect, the present invention provides a bioconjugate comprising a reagent linked to a non-polyethylene glycol (PEG) polymer, wherein the non-PEG polymer is conjugated to a first drug and the reagent binds to a cancer-associated antigen, and wherein the bioconjugate has a drug-to-reagent ratio (DAR) of at least 100:1.

[0009] In some aspects, this disclosure provides a method for preparing bioconjugates, comprising: (a) Preparation of a non-PEG polymer backbone comprising a chain transfer agent (CTA) and one or more monomer units via reversible addition-fragmentation chain transfer (RAFT) polymerization. At least one of the monomer units contains an initiator molecule for atom transfer radical polymerization (ATRP); (b) Prepare a non-PEG polymer comprising the non-PEG polymer backbone of (a) and one or more monomer units by ATRP. At least one of the monomer units contains a functional group capable of performing a click response; (c) The first drug is coupled to the non-PEG polymer of (b) via a functional group capable of click reaction; and (d) The reagent that binds to cancer-associated antigens is coupled to the non-PEG polymer in (c).

[0010] In some aspects, the present invention provides a method for preparing bioconjugates, comprising: (a) Preparation of a non-PEG polymer backbone comprising a chain transfer agent (CTA) molecule and one or more monomer units via reversible addition-fragmentation chain transfer (RAFT) polymerization; At least one of the monomer units contains an initiator molecule for atom transfer radical polymerization (ATRP); (b) Couple a reagent that binds to cancer-associated antigens to the non-PEG polymer backbone of (a); (c) Prepare a non-PEG polymer comprising the non-PEG polymer backbone of (b) and one or more monomer units by ATRP. At least one of the monomeric units contains a functional group capable of performing a click reaction; and (d) The first drug is coupled to the non-PEG polymer of (c) by a functional group capable of click reaction.

[0011] In one respect, the DAR ratio is at least 30:1. In another respect, the DAR ratio is at least 40:1. In one respect, the DAR ratio is at least 50:1. In another respect, the DAR ratio is at least 60:1. In another respect, the DAR ratio is at least 70:1. In another respect, the DAR ratio is at least 80:1. In another respect, the DAR ratio is at least 90:1. In another respect, the DAR ratio is at least 100:1. In another respect, the DAR ratio is at least 110:1. In another respect, the DAR ratio is at least 120:1. In another respect, the DAR ratio is at least 130:1. In another respect, the DAR ratio is at least 140:1. In another respect, the DAR ratio is at least 150:1. In another respect, the DAR ratio is at least 160:1. In another respect, the DAR ratio is at least 170:1. In another respect, the DAR ratio is at least 180:1. In another respect, the DAR ratio is at least 190:1. In another respect, the DAR ratio is at least 200:1. In another respect, the DAR ratio is at least 210:1. In another respect, the DAR ratio is at least 220:1. On the other hand, the DAR ratio is at least 230:1. On the other hand, the DAR ratio is at least 240:1. On the other hand, the DAR ratio is at least 250:1. On the other hand, the DAR ratio is at least 260:1. On the other hand, the DAR ratio is at least 270:1. On the other hand, the DAR ratio is at least 280:1. On the other hand, the DAR ratio is at least 290:1. On the other hand, the DAR ratio is at least 300:1. On the other hand, the DAR ratio is at least 400:1. On the other hand, the DAR ratio is at least 500:1. On the other hand, the DAR ratio is at least 600:1. On the other hand, the DAR ratio is at least 700:1. On the other hand, the DAR ratio is at least 800:1. On the other hand, the DAR ratio is at least 900:1. On the other hand, the DAR ratio is at least 1,000:1. On the other hand, the DAR ratio is at least 1,100:1.

[0012] On the other hand, the reagents of the bioconjugates described herein are antibodies or antigen-binding fragments thereof. In some aspects, the antibodies or antigen-binding fragments thereof are full-length antibodies. In some aspects, the antibodies or antigen-binding fragments thereof are antigen-binding fragments. In some aspects, the antigen-binding fragments comprise Fab, Fab', F(ab')2, Fd, single-chain Fv or scFv, disulfide-linked Fv, V NAR domain, IgNar, intracellular antibody, IgG-CH2, microantibody, F(ab')3, tetraantibody, triantibody, biantibody, single-domain antibody (VHH), DVD-Ig, Fcab, mAb2, a(scFv)2, DARPin, or scFv-Fc. In some aspects, the antigen-binding fragments comprise single-chain Fv or scFv.

[0013] In some respects, the cancer-associated antigen of the bioconjugates described herein is HER2, EGFR, or phosphatidylserine (PS). In some respects, the antibody or antigen-binding fragment comprises a single-chain Fv or scFv and binds to HER2. In some respects, the antibody or antigen-binding fragment comprises a single-chain Fv or scFv and binds to EGFR. In some respects, the antibody or antigen-binding fragment comprises a single-chain Fv or scFv and binds to phosphatidylserine.

[0014] In some aspects, the antibody or antigen-binding fragment is coupled to a non-PEG polymer bottle brush via a non-cleavable band. In some aspects, the band contains an amino acid linker. In some aspects, the band contains a histidine tag (His tag). In some aspects, the antibody or antigen-binding fragment is coupled to a non-PEG polymer bottle brush via a glycine band, 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)5 (SEQ ID NO: 35). In some aspects, the antibody or antigen-binding fragment is coupled to a non-PEG polymer bottle brush via an ethylene glycol band, wherein the number of units is an integer between 5 and 25.

[0015] In some respects, the non-PEG polymers of the bioconjugates described herein are selected from the group consisting of: triethylene glycol methyl ether methacrylate (TEOMA), 2-(methylsulfinyl)ethyl methacrylate (MSEMA), 1-phenoxycarbonyl ethyl methacrylate, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate (PCMA), 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate (CBMA), 2-((2-bromo-2-methylpropionyl)oxy)ethyl methacrylate (BMPOEMA), 2-( 2-Bromo-2-methylpropionamido)ethyl methacrylate (BMPAEMA), polyethylene glycol monomethyl ether methacrylate (PEGMA) (where the number of ethylene glycol units is an integer from 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), PEGMA 300 EGMA 500 2-Dimethylaminoethyl methacrylate (DMAEMA), sulfobetaine methacrylate (SBMA), N,N-dimethylaminoethyl methacrylate, quaternary ammonium ethyl methacrylate, hydroxyethyl methacrylate, hydroxyethyl methacrylate (HEMA), 2-azidoethyl methacrylate, 2-azidotriethylene glycol methyl ether methacrylate (TEOMA-azido compound or TEOMA-N3), 2-azidotetraethylene glycol methyl ether methacrylate (PEGMA(n=4)-azido compound or PEGMA(n=4)-N3), 2-azidopentaethylene glycol methyl ether methacrylate (PEGMA(n=5)-N3). The non-PEG polymers include PEGMA (n=5)-N3, PEGMA (n=6)-N3, PEGMA (n=6)-N3, PEGMA (n=7)-N3, PEGMA (n=7)-N3, PEGMA (n=8)-N3, and PEGMA (n=9)-N3, or combinations thereof. In one aspect, the non-PEG polymer includes TEOMA. In one aspect, the non-PEG polymer comprises MSEMA. In one aspect, the non-PEG polymer comprises CBMA.

[0016] In some respects, the polymers of the bioconjugates described herein are conjugated to drugs via cleavable linkers. In some respects, the polymers are conjugated to drugs via amino acid linkers. In one respect, the polymers are conjugated to drugs via valine-citrulline-p-aminobenzylcarbamate, valine-citrulline, or alanine-alanine linkers.

[0017] In some aspects, the bioconjugates described herein further comprise a second drug. In some aspects, the ratio of the first drug to the second drug is from 1:1 to 20:1. In one aspect, the ratio of the first drug to the second drug is from 20:1 to 1:1.

[0018] In some respects, the bioconjugates described herein further comprise a second drug and a third drug. In one respect, the ratio of the first drug, the second drug, and the third drug is 1:1:1.

[0019] In some respects, the bioconjugates described herein further comprise a fourth drug. In one respect, the ratio of the first drug, the second drug, the third drug, and the fourth drug is 1:1:1:1.

[0020] In some respects, the bioconjugates described herein further comprise a fifth drug. In one respect, the ratio of the first drug, the second drug, the third drug, the fourth drug, and the fifth drug is 1:1:1:1:1.

[0021] In some respects, the bioconjugates described herein are chemotherapeutic agents. In some respects, the first, second, third, fourth, and / or fifth agents are chemotherapeutic agents. In some respects, the chemotherapeutic agents are selected from the group consisting of: MEK inhibitors, PI3K inhibitors, MAPK inhibitors, CDK inhibitors, CHK inhibitors, alkylating agents, cytoskeleton disruptors, histone deacetylase inhibitors, topoisomerase I or II inhibitors, their nucleotide analogs or precursors, peptide antibiotics, protein degrading agents, platinum-based reagents, retinoids, vinca alkaloids and their derivatives, and combinations thereof. In some respects, the chemotherapeutic agents include MEK inhibitors. In some respects, the MEK inhibitors are selected from the group consisting of: bemetinib, GDC-0973 (cobimetinib), selmetinib, trametinib. In some respects, the chemotherapeutic agents include PI3K inhibitors. In some respects, PI3K inhibitors are selected from the group consisting of: PI103; PI828; LY294002; Wollman penicillin; Demethoxychloric acid; IC486068; IC87114; GDC-0941; GDC-0980 (apitolisib); perifoxine; CAL101; PX-866; IPI-145; BAY 80-6946; BEZ235; P6503; TGR1202; SF1126; INK1117; BKM120; IL147; XL765; Palomida 529; GSK1059615; ZSTK474; PWT33597; TG100-115; CAL263; GNE-447; CUDC-907; and AEZS-136.

[0022] In one aspect, the present invention provides a bioconjugate comprising a reagent linked to a non-polyethylene glycol (PEG) polymer, wherein the reagent is an antibody or an antigen-binding fragment thereof, wherein the polymer is conjugated to a MEK inhibitor, a CDK inhibitor, and / or a PI3K inhibitor, and wherein the antibody or antigen-binding fragment thereof is bound to a HER2 protein or an EGFR protein or a phosphatidylserine, and wherein the bioconjugate has a drug-to-reagent ratio (DAR) of 60:1 to 200:1. In some aspects, the DAR of the bioconjugate is 60:1 to 150:1. In some aspects, the DAR of the bioconjugate is 60:1 to 110:1. In some aspects, the DAR of the bioconjugate is 60:1 to 300:1. In some aspects, the DAR of the bioconjugate is 150:1 to 300:1. In one aspect, this disclosure provides a bioconjugate comprising a reagent linked to a non-polyethylene glycol (PEG) polymer, wherein the reagent is an antibody or an antigen-binding fragment thereof, wherein the polymer is conjugated to a MEK inhibitor, a CDK inhibitor, and / or a PI3K inhibitor, and wherein the antibody or antigen-binding fragment thereof is bound to a HER2 protein or an EGFR protein or a phosphatidylserine, and wherein the bioconjugate has a drug-to-reagent ratio (DAR) of 100:1 to 1,100:1. In some aspects, the bioconjugate has a DAR of 200:1 to 1,100:1. In some aspects, the bioconjugate has a DAR of 400:1 to 1,100:1. In some aspects, the bioconjugate has a DAR of 600:1 to 1,100:1. In some aspects, the bioconjugate has a DAR of 800:1 to 1,100:1. In some aspects, the bioconjugate has a DAR of 900:1 to 1,100:1.

[0023] In some aspects, the MEK inhibitor is GDC-0973 and the PI3K inhibitor is GDC-0941. In another aspect, the MEK inhibitor is GDC-0973 and the PI3K inhibitor is GDC-0980 (apitolisib). In some aspects, the MEK inhibitor is GDC-0973 (cobimetinib), the CDK inhibitor is dinaciclib, and the PI3K inhibitor is GDC-0980 (apitolisib). In some aspects, the antibody or its antigen-binding fragment binds to the EGFR protein, the MEK inhibitor is GDC-0973 (cobimetinib), and the CDK inhibitor is dinaciclib. In some aspects, the antibody or its antigen-binding fragment binds to the EGFR protein, the PI3K inhibitor is GDC-0980 (apitolisib), and the CDK inhibitor is dinaciclib. In some cases, the antibody or its antigen-binding fragment binds to phosphatidylserine; the MEK inhibitor is GDC-0973 (cobitinib), and the CDK inhibitor is dinaciclib. In other cases, the antibody or its antigen-binding fragment binds to phosphatidylserine; the PI3K inhibitor is GDC-0980 (apirixe), and the CDK inhibitor is dinaciclib.

[0024] In one aspect, this disclosure also provides pharmaceutical compositions comprising the bioconjugates described herein and pharmaceutically acceptable carriers.

[0025] In some respects, this disclosure also provides kits containing the biological conjugates described herein.

[0026] In some aspects, the present invention also provides a method for inhibiting tumor growth in a subject, comprising administering a therapeutically effective amount of the biological conjugate or pharmaceutical composition described herein. In some aspects, the tumor is selected from ovarian tumors, brain tumors, breast tumors, uterine tumors, endometrial tumors, pancreatic tumors, kidney tumors, head and neck tumors, gastric tumors, and lung tumors. In some aspects, the tumor is a breast tumor, head and neck tumor, lung tumor, or gastric tumor.

[0027] In some aspects, the present invention provides a method of treating a subject with cancer, comprising administering to the subject a therapeutically effective amount of the biological conjugate or pharmaceutical composition described herein. In some aspects, the cancer is selected from ovarian cancer, brain cancer, breast cancer, uterine cancer, endometrial cancer, pancreatic cancer, kidney cancer, head and neck cancer, gastric cancer, and lung cancer. In some aspects, the cancer is breast cancer, head and neck cancer, lung cancer, or gastric cancer.

[0028] In some aspects, the present invention also provides a method for increasing the cytotoxicity of cancer cells, comprising contacting cancer cells with the biological conjugates or pharmaceutical compositions described herein.

[0029] In some aspects, the present invention also provides a method for inhibiting cancer proliferation in a subject with this need, comprising administering to the subject a therapeutically effective amount of the biological conjugate or pharmaceutical composition described herein.

[0030] In one aspect, this disclosure also provides isolated polynucleotides comprising a sequence having at least 90% identity with a sequence encoding one or more amino acid sequences of SEQ ID NO: 1-4, SEQ ID NO: 6-16, and SEQ ID NO: 20-31. In some aspects, the isolated polynucleotide has at least 95% identity with a sequence encoding one or more amino acid sequences of SEQ ID NO: 1-4, SEQ ID NO: 6-16, and SEQ ID NO: 20-31. In another aspect, the isolated polynucleotide has at least 99% identity with a sequence encoding one or more amino acid sequences of SEQ ID NO: 1-4, SEQ ID NO: 6-16, and SEQ ID NO: 20-31. In yet another aspect, the isolated polynucleotide is identical to a sequence encoding one or more amino acid sequences of SEQ ID NO: 1-4, SEQ ID NO: 6-16, and SEQ ID NO: 20-31.

[0031] In another respect, this disclosure also provides vectors comprising any one of the polynucleotides of SEQ ID NO: 1-4, SEQ ID NO: 6-16 and SEQ ID NO: 20-31.

[0032] In another aspect, this disclosure also provides a host cell comprising a vector containing polynucleotides of SEQ ID NO: 1-4, SEQ ID NO: 6-16, and SEQ ID NO: 20-31. In another aspect, the host cell is selected from the group consisting of: *Escherichia coli*, *Pseudomonas*, *Bacillus*, *Streptomyces*, yeast, *Pichia pastoris*, CHO, YB / 20, NSO, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, R1.1, BW, LM, COS 1, COS 7, BSC1, BSC40, BMT10 cells, plant cells, insect cells, and human cells in tissue culture.

[0033] In one aspect, this disclosure also provides a method for generating an antibody or an antigen-binding fragment thereof, comprising culturing host cells such that nucleic acid molecules are expressed and an antibody or an antigen-binding fragment thereof is generated, optionally wherein the method further comprises isolating the antibody or an antigen-binding fragment thereof from the culture.

[0034] In one respect, chain transfer agents (CTAs) are selected from... or In some respects, CTA is .

[0035] In some aspects, at least one monomer unit comprising an initiator molecule for atom transfer radical polymerization (ATRP) is selected from 2-((2-bromo-2-methylpropionyl)oxy)ethyl methacrylate (BMPOEMA) or 2-(2-bromo-2-methylpropionamido)ethyl methacrylate (BMPAEMA). In some aspects, at least one monomer unit comprising an initiator molecule for atom transfer radical polymerization (ATRP) is 2-(2-bromo-2-methylpropionamido)ethyl methacrylate (BMPAEMA). In some aspects, at least one monomer unit comprising an initiator molecule for atom transfer radical polymerization (ATRP) is selected from... or .

[0036] In some aspects, the non-PEG polymer backbone comprises at least a second monomer unit, wherein the second monomer unit is a non-PEG monomer containing methacrylate. In some aspects, the second monomer unit is selected from triethylene glycol methyl ether methacrylate (TEOMA), 2-(methylsulfinyl)ethyl methacrylate (MSEMA), 1-phenoxycarbonyl ethyl methacrylate, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate (PCMA), 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate (CBMA), 2-((2-bromo-2-methylpropionyl)oxy)ethyl methacrylate (BMPOEMA), 2-(2-bromo-2-methylpropionyl)oxy)ethyl methacrylate, etc. (Amide-based) Ethyl methacrylate (BMPAEMA), Polyethylene glycol monomethyl ether methacrylate (PEGMA) (where the number of ethylene glycol units is an integer from 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), PEGMA 300 PEGMA 5002-Dimethylaminoethyl methacrylate (DMAEMA), sulfobetaine methacrylate (SBMA), quaternary ammonium ethyl methacrylate, and hydroxyethyl methacrylate (HEMA) or combinations thereof. In some aspects, 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 from 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), PEGMA, etc. 300 or PEGMA 500 .

[0037] In some aspects, at least one monomeric unit containing a functional group capable of undergoing a click reaction is selected from 2-azidoethyl methacrylate, 2-azidotriethylene glycol methyl ether methacrylate (TEOMA-azido compound or TEOMA-N3), 2-azidotetraethylene glycol methyl ether methacrylate (PEGMA(n=4)-azido compound or PEGMA(n=4)-N3), 2-azidopentaneethylene glycol methyl ether methacrylate (PEGMA(n=5)-azido compound or PEGMA(n=5)-N3), 2-azidohexaethylene glycol methyl ether methacrylate. The monomeric unit comprising at least one functional group capable of undergoing a click reaction is 2-azidotriethylene glycol methyl ether methacrylate (PEGMA(n=6)-azido or PEGMA(n=6)-N3), 2-azidoheptaethylene glycol methyl ether methacrylate (PEGMA(n=7)-azido or PEGMA(n=7)-N3), 2-azidooctaethylene glycol methyl ether methacrylate (PEGMA(n=8)-azido or PEGMA(n=8)-N3), and 2-azidononethylene glycol methyl ether methacrylate (PEGMA(n=9)-azido or PEGMA(n=9)-N3), or combinations thereof. In some aspects, at least one monomeric unit comprising a functional group capable of undergoing a click reaction is 2-azidotriethylene glycol methyl ether methacrylate (TEOMA-azido or TEOMA-N3). In some aspects, at least one monomeric unit comprising a functional group capable of undergoing a click reaction is selected from... , ,or .

[0038] In some aspects, the non-PEG polymer comprises at least a second monomer unit, wherein the second monomer unit is a methacrylate containing a non-PEG monomer. In some aspects, the second monomer unit in the non-PEG polymer is selected from triethylene glycol methyl ether methacrylate (TEOMA), 2-(methylsulfinyl)ethyl methacrylate (MSEMA), 1-phenoxycarbonyl ethyl methacrylate, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate (PCMA), 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate (CBMA), 2-((2-bromo-2-methylpropionyl)oxy)ethyl methacrylate (BMPOEMA), 2-(2-bromo-2-methylpropionyl)oxy)ethyl methacrylate, etc. 1,3-methylpropionamide (MPAEMA), polyethylene glycol monomethyl ether methacrylate (PEGMA) (where the number of ethylene glycol units is an integer from 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), PEGMA 300 PEGMA 500 2-Dimethylaminoethyl methacrylate (DMAEMA), sulfobetaine methacrylate (SBMA), quaternary ammonium ethyl methacrylate, hydroxyethyl methacrylate (HEMA), 2-azidoethyl methacrylate, 2-azidotriethylene glycol methyl ether methacrylate (TEOMA-azido compound or TEOMA-N3), 2-azidotetraethylene glycol methyl ether methacrylate (PEGMA(n=4)-azido compound or PEGMA(n=4)-N3), 2-azidopentaethylene glycol methyl ether methacrylate (PEGMA(n=5)-azido compound or PEGMA(n... =5)-N3), 2-azidohexaethylene glycol methyl ether methacrylate (PEGMA(n=6)-azido or PEGMA(n=6)-N3), 2-azidoheptaethylene glycol methyl ether methacrylate (PEGMA(n=7)-azido or PEGMA(n=7)-N3), 2-azidooctaethylene glycol methyl ether methacrylate (PEGMA(n=8)-azido or PEGMA(n=8)-N3), and 2-azidononethylene glycol methyl ether methacrylate (PEGMA(n=9)-azido or PEGMA(n=9)-N3) or combinations thereof.

[0039] In some aspects, the coupling of drugs to polymers proceeds via azide-alkyne click reactions or anti-electron demanded Diels-Alder (IEDDA) reactions. In other aspects, the coupling of drugs to polymers proceeds via azide-alkyne click reactions. In some aspects, the azide-alkyne click reaction is selected from copper-catalyzed azide-alkyne cycloaddition (CuAAC), strain-promoted azide-alkyne cycloaddition (SPAAC), or strain-promoted alkyne-nitroketone cycloaddition (SPANC). In some aspects, the functional group capable of performing the click reaction is an azide (-N3). In some aspects, the coupling of drugs to polymers proceeds via anti-electron demanded Diels-Alder (IEDDA) reactions.

[0040] In one respect, coupling of reagents to polymers that bind cancer-associated antigens is achieved via azide-alkyne click reactions, strain-promoted azide-alkyne cycloaddition (SPAAC), or anti-electron-demand Diels-Alder (IEDDA) reactions.

[0041] In one aspect, the coupling of reagents to polymers that bind cancer-associated antigens is achieved via an azide-alkyne click reaction or strain-promoted azide-alkyne cycloaddition (SPAAC). In other aspects, the strain-promoted azide-alkyne cycloaddition (SPAAC) click reaction occurs between an azide functional group and a dibenzocyclooctyne (DBCO) functional group. In still other aspects, the dibenzocyclooctyne (DBCO) functional group is optionally substituted.

[0042] In some respects, the coupling of reagents to polymers containing cancer-associated antigens occurs via an inverse electron-demanding Diels-Alder (IEDDA) reaction. In other respects, the IEDDA reaction is a tetrazine-cyclooctene reaction. In still other respects, the functional group capable of click reactions is an azide (-N3).

[0043] In some respects, the components of the bioconjugate are selected from the components in Table A or their pharmaceutically acceptable salts.

[0044] In some respects, bioconjugates are compounds listed in Table B or their pharmaceutically acceptable salts. Attached Figure Description

[0045] Figure 1A An exemplary method for synthesizing macromonomers carrying loads is shown, which are then copolymerized to form a non-PEG polymer bottle brush.

[0046] Figure 1B An exemplary method for synthesizing bioconjugates with reagents and loads using a "grafting" approach for bioconjugation is demonstrated, wherein a fully formed non-PEG bottle brush polymer is attached to a targeting reagent.

[0047] Figure 2A An exemplary method for synthesizing bottle brushes is shown, wherein a non-PEG polymer backbone is first prepared, and then side chains are branched from the backbone.

[0048] Figure 2B An exemplary method is demonstrated for synthesizing bioconjugates with reagents and loads using a combination of “graft-to” and “graft-self” methods for bioconjugation, wherein a non-PEG polymer backbone is first attached to a reagent and then a non-PEG bottle brush polymer is formed in the presence of the reagent.

[0049] Figure 3A A diagram of an exemplary bioconjugate of this disclosure is provided, having a reagent (i.e., a full-length human IgG antibody) linked to a bottle-brush polymer, which is attached with a large payload (e.g., a drug) resulting in an extremely high DAR.

[0050] Figure 3B A diagram of an exemplary bioconjugate of the present disclosure is provided, having a reagent (i.e., scFv) linked to a bottle-brush polymer, which is attached with a large load (e.g., a drug) to produce a bioconjugate with ultra-high DAR.

[0051] Figure 4A The mass spectrum of naked trastuzumab is shown.

[0052] Figure 4B The mass spectrum of deglycosylated trastuzumab is shown.

[0053] Figure 4C The mass spectrum of trastuzumab-TCO(Int-23) is shown.

[0054] Figure 5 The mass spectrum of trastuzumab-DBCO(Int-30)(C) is shown.

[0055] Figure 6A The mass spectrum of naked cetuximab is shown.

[0056] Figure 6B The mass spectrum of deglycosylated cetuximab is shown.

[0057] Figure 6C This shows cetuximab-TCO ( Int-31 The mass spectrum line graph of ).

[0058] Figure 7 The purified scFv-TCO( Int-6 The mass spectrum at 28,190 Da.

[0059] Figure 8 Showing scFv-TCO( Int-6) and the combined scFv-main chain ( Int-7 The SDS-PAGE gel of the reagent demonstrated the connection between the reagent and the polymer backbone.

[0060] Figure 9 An SDS-PAGE gel showing the effect of different imidazole concentrations on products with various scFv backbone sizes, illustrating how the proportion of methacrylate polymers affects the polymerization of the polymer backbone.

[0061] Figure 10 Showing scFv-TCO( Int-6 ), polymer backbone and scFv-backbone ( Int-7 The size exclusion chromatography (SEC) line graph shows the connection between the reagent and the polymer backbone.

[0062] Figure 11 Showing the scFv-main chain ( Int-7 ) and scFv-bottle brush ( Int-8 A line graph of field flow separation (FFF) shows the polymerization of the bottle brush polymer from the polymer backbone.

[0063] Figure 12 The line plot of the UV-Vis absorption spectrum is shown, which shows the attachment of the apitolisib load by monitoring the 250-300 nm region, which corresponds to the apitolisib load.

[0064] Figure 13 Line graphs showing the ELISA assays of trastuzumab or trastuzumab scFv binding to HER2 at different concentrations are shown.

[0065] Figure 14 Line graphs showing the ELISA assays of scFv-bottle brush-loading (compound 1) and naked scFv binding to HER2 at different concentrations are presented.

[0066] Figure 15 Showing the main chain molecules of the bottle brush ( Int-15 SDS-PAGE gels of scFv-TCO(Int-6) bioconjugated with scFv-TCO(Int-6) at ratios of 1:1, 5:1, and 19:1 were obtained. The SDS-PAGE gels showed that the drug was linked to the non-PEG backbone by reducing scFv bands and enhancing the molar equivalent of the bottle brush backbone corresponding to scFv.

[0067] Figure 16 The image shows a protein blot of the first antibody against 6xHis mouse IgG and the second antibody against HRP mouse IgG, along with their connection to a bottle brush. No signal was observed in the polymer lanes, while the scFv-bottle brush polymer ( Int-18The lanes showed new bands at higher molecular weights (MW) at polymerization ratios of 1:1, 5:1, and 19:1.

[0068] Figure 17 The image shows a preparative size exclusion chromatography (SEC) line graph of the reaction product between trastuzumab-TCO and the polymer backbone pBMPAEMA-r-pNEOMA containing methyltetraazine functional groups.

[0069] Figure 18 Showing different monomer lengths ( Int-25 )and( Int-26 Trastuzumab-TCO ( Int-23 Linear plots of field flow separation (FFF) of the main chain of trastuzumab-pBMPAEMA-r-pNEOMA, collected by preparative size exclusion chromatography (SEC).

[0070] Figure 19 The trastuzumab-pBMPAEMA-r-pNEOMA backbone is shown. Int-26 ) and trastuzumab-vial brush ( Int-27 A line graph of field flow separation (FFF) shows the polymerization of the bottle brush polymer from the polymer backbone.

[0071] Figure 20 Line graphs showing ELISA assays of trastuzumab-bottle brush (Int-27), trastuzumab-main chain (Int-26), trastuzumab-TCO (Int-23), and naked trastuzumab binding to HER2 are shown.

[0072] Figure 21 This demonstrates the formation of cetuximab-polymer backbone pBMPAEMA-r-pNEOMA between cetuximab-TCO and the polymer backbone pBMPAEMA-r-pNEOMA. Int-32 Line graph of the reaction product size exclusion chromatography (SEC).

[0073] Figure 22 SDS-PAGE gel electrophoresis of the SEC fractions of the reaction between cetuximab-TCO and the polymer backbone pBMPAEMA-r-pNEOMA shows the product cetuximab-TCO-pBMPAEMA-r-pNEOMA. Int-32 )product.

[0074] Figure 23 Showing the trastuzumab-pCBMA vial brush (Int-33 Linear plot of field flow separation (FFF) of trastuzumab-pCBMA vial brush apiride (compound 7) and trastuzumab-pCBMA.

[0075] Figure 24 This shows the calculation of drug-loaded polymer brushes treated with cathepsin B ( Int-40 Linear graph of the amount of pyrolyzed apiride in the sample by reversed-phase liquid chromatography.

[0076] Figure 25 This shows a drug-loaded polymer brush treated with cathepsin B. Int-40 Linear graph of PI3K inhibitory activity of the piperidine-containing solution collected after the reaction.

[0077] Figure 26A A final molecular dynamics simulation framework diagram of the trastuzumab-bottle brush-cobibitinib loading bioconjugate is provided. The white area represents trastuzumab, and its antigen-binding region is dark gray. Except for the apilithe loading marked in black, the bottle brush is light gray. The gray in the middle represents the chain and main chain of the bottle brush, but is almost completely obscured by the bristles of the bottle brush.

[0078] Figure 26B A final molecular dynamics simulation framework diagram of the trastuzumab-bottle brush-cobibitinib loading bioconjugate is provided. The white area represents the trastuzumab scFv, with its antigen-binding region in dark gray. Except for the apilithe loading, which is marked in black, the bottle brush is light gray. The middle gray represents the chain and main chain of the bottle brush, but is almost completely obscured by the brush bristles.

[0079] Figure 27 A-27R shows confocal micrographs of the internalization control and test 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-pPEGMA with pHAb dye and a DAR of 55. 300 - Cobimetinib; letters D, J, and P represent trastuzumab-pCBMA-apirixe with pHAb dye, DAR 268; letters E, K, and Q represent trastuzumab-pPEGMA 300 - Dual-loaded apiritol + cobimetinib with pHAb dye, DAR 1081; letters F, L, and R represent anti-HER2 scFv pHAb dye. AF images are taken at 6 hours, GL images at 24 hours, and MR images at 48 hours.

[0080] Figure 28 A-28X shows confocal microscopy images of the internalized control and tested bioconjugates. Figure 28 A, 28I, and 28Q represent trastuzumab without pHAb dye; Figure 28 B, 28J, and 28R represent trastuzumab with pHAb dye; Figure 28C, 28K, and 28S represent trastuzumab-pPEGMA with a pHAb dye having a DAR of 55. 300 -Cobbitinib; Figure 28 D, 28L, and 28T represent trastuzumab-pCBMA-apirixe with a pHAb dye having a DAR of 268; Figure 28 E, 28M, and 28U represent trastuzumab-pPEGMA with a pHAb dye having a DAR of 1081. 300 - Dual-load apiritoxetine + cobimetinib; Figure 28 F, 28N, and 28V represent anti-HER2 scFv with pHAb dyes; Figure 28 G, 28O, and 28W represent trastuzumab-PEGMA with a pHAb dye having a DAR of 182. 300 - Apiriso; and Figure 28 H, 28P, and 28X represent trastuzumab-PEGMA with a pHAb dye having a DAR of 182. 300 - Appliese. Figure 28 A-28H shows BT474 cells. Figure 28 I-28P shows ZR751 cells. Figure 28 Q-28X displays MCF7 cells. Detailed Implementation

[0081] This invention provides bioconjugates and methods of using the same, the bioconjugates comprising reagents that bind to cancer-associated antigens, said reagents including antibodies or antigen-binding fragments thereof or other reagents (e.g., cytokines, enzymes, polynucleotides). Advantageously, the bioconjugates described herein provide high and ultra-high drug-to-reagent ratios.

[0082] I. Definition To facilitate understanding of this invention, some terms and phrases are defined below.

[0083] Units, prefixes, and symbols are represented in a form recognized by the International System of Units (SI). A range of values ​​includes the numerical value itself that defines the range. When listing ranges of values, it should be understood that each intermediate integer value between the said upper and lower limits of the range, and each fraction thereof, as well as each subrange between these values, is also specifically disclosed. The upper and lower limits of any range may be independently included in or excluded from the range, and each range including any endpoint, both endpoints, or neither endpoint is excluded is within the scope of this disclosure. Therefore, a range described herein should be understood as an abbreviation of all values ​​within that range, including the said endpoints. For example, the range 1 to 10 is understood to include any number, combination of numbers, or subrange derived from groups consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0084] Where values ​​are explicitly listed, it should be understood that values ​​of approximately the same number or quantity as the listed values ​​are also within the scope of this disclosure. Where combinations are disclosed, each sub-combination of the elements of that combination is also specifically disclosed and within the scope of this disclosure. Conversely, where different elements or groups of elements are disclosed individually, their combinations are also disclosed. When any disclosed element is disclosed as having multiple substitutes, examples of such disclosures where each substitute is individually excluded or any combination with other substitutes is excluded are also disclosed; more than one element may be disclosed with such exclusions, and all combinations of elements with such exclusions are thus disclosed.

[0085] As used in this disclosure and claims, the singular forms “a,” “an,” and “the” include the plural forms, unless the context clearly specifies otherwise.

[0086] It should be understood that if a word is capitalized when it should not be capitalized, then the word is understood as not capitalized. Conversely, if a word that should be capitalized is not capitalized, then it is understood as capitalized.

[0087] It should be understood that when the language “comprising” is used to describe aspects herein, similar aspects described by “comprising” and / or “substantially comprising” are also provided. In this disclosure, words such as “comprising,” “including,” “containing,” and “having” can mean “comprising,” “including,” etc.; “substantially consisting of” or “substantially consisting of” is open-ended and allows for more content than described, as long as the essential or novel features of the content are not altered by the presence of more content than described, but does not include prior art aspects.

[0088] The term "and / or" as used in phrases such as "A and / or B" is intended to include "A and B", "A or B", and "A and B". Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to include the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0089] As used in this article, the terms “drug” or “load” refer to any chemical or biological substance that causes physiological changes in an organism (e.g., a chemotherapy drug).

[0090] The term "polymer" or "synthetic polymer" refers to a polymer comprising one or more monomer units. Polymers or synthetic polymers can be produced by chemical methods. Synthetic polymers are not directly produced by living organisms. Synthetic polymers include homopolymers, heteropolymers, block polymers, copolymers, terpolymers, etc., as well as blends, compositions, and mixtures thereof. Examples of synthetic polymers include, but are not limited to, functionalized polymers, such as polymers comprising azides, triazole rings, alkynes, or trans-cyclooctene. Synthetic polymers can be or comprise one or more linear polymers, branched polymers, hyperbranched polymers, grafted polymers, block copolymers, brush polymers, and bottle-brush copolymers. Synthetic polymers include, but are not limited to, polyesters, poly(meth)acrylamide, poly(meth)acrylates, polyethers, and monomers having unsaturated bonds. For example, copolymers polymerized by ATRP are described in U.S. Patent Application Publication No. 2021 / 0316001 and U.S. Patent Application Publication No. 2021 / 0388337, the disclosures of which are incorporated herein by reference in their entirety. The bottle brush polymers described herein can exist in the form of copolymers, comprising a backbone formed of a hydrophobic, water-insoluble polymer and side chains formed of short, hydrophilic, non-cellularly bound polymers. Examples of other synthetic polymers include, but are not limited to: polyolefins (e.g., polyethylene and polypropylene), polychloroprene, polyethylene ethers (e.g., poly(vinyl acetate)), polyhalogenated polyethylene (e.g., poly(vinyl chloride)), poly(meth)acrylates (e.g., poly(methyl methacrylate)), poly(ethyl methacrylate), poly(triethylene glycol methyl ether methacrylate) (pTEOMA), poly(ethylene glycol (meth)acrylate) (pPEGMA) (wherein the average number of ethylene glycol units in the monomer unit is 3-9), 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), pPEGMA 300 pPEGMA 500Poly(2-(methylsulfinyl)ethyl methacrylate) (pMSEMA), poly(3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate) (pPCMA), poly(3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate) (CBMA), poly(2-((2-bromo-2-methylpropionyl)oxy)ethyl methacrylate, poly(2-((2-bromo-2-methylpropionyl)oxy)ethyl methacrylate) (pBMPOEMA), poly(2-(2-bromo-2-methylpropionamido)ethyl methacrylate, poly(2-(2-bromo-2-methylpropionamido)ethyl methacrylate) (pBMPAEMA), poly(2-dimethylaminoethyl methacrylate) (pDMAEMA), poly(sulfobetaine methacrylate) (pSBMA), poly(quaternary ammonium ethyl methacrylate), poly(hydroxyethyl methacrylate) (pHEMA), poly(2-azidoethyl methacrylate), poly(2-azidoethyl methacrylate), poly(2-azidotriethylene glycol methyl ether methacrylate) (pTEOMA-azido compound or pTEOMA-N3), poly(2-azidotetraethylene glycol methyl ether methacrylate) (pPEGMA(n=4)-azido compound or pPEGMA(n=4)-N3), poly(2-azidopentaneethylene glycol methyl ether methacrylate) (pPEGMA(n=5)-azido compound or pPEGMA(n=5)-N3), poly(2-azidohexaethylene glycol methyl ether methacrylate) (pPEGMA(n=6)-azido compound or pPEGMA(n=6)-N3), poly(2-azidoheptaethylene glycol methyl ether methacrylate) (pPEGMA(n=7)-azido compound or pPE GMA(n=7)-N3), poly(2-azido-octaethylene glycol methyl ether methacrylate) (pPEGMA(n=8)-azide or pPEGMA(n=8)-N3), poly(2-azido-nonethylene glycol methyl ether methacrylate) (pPEGMA(n=9)-azide or pPEGMA(n=9)-N3). These synthetic polymers may include useful derivatives, including synthetic polymers with substituted or added chemical groups, such as alkyl, alkylene, hydroxylated, oxidized, azidated, esterified, and other modifications conventionally performed by those skilled in the art. Synthetic polymers may include zwitterionic polymers, such as polyphosphorylcholine, polycarboxybetaine, and polysulfobetaine. Synthetic polymers may have side chains of betaine, carboxybetaine, sulfobetaine, or ethylene glycol, i.e., -(OCH2CH2)n- (n=3-9).

[0091] In this document, the terms "non-PEG polymer" or "non-PEG bottle brush polymer," which may be used interchangeably, refer to the following bottle brush polymers: (a) Does not contain ethylene glycol, i.e., the -OCH2CH2- unit; or (b) Includes 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 polymer brush is an integer from 3 to 9. Examples of non-PEG polymers or non-PEG bottle brush polymers include, but are not limited to, polymers made from monomers such as triethylene glycol methyl ether methacrylate (TEOMA), 2-(methylsulfinyl)ethyl methacrylate (MSEMA), 1-phenoxycarbonyl ethyl methacrylate, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate (PCMA), 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate (CBMA), and 2-((2-bromo-2-methylpropionyl)oxy)ethyl methacrylate (BMPOEMA). 2-(2-Bromo-2-methylpropionamido)ethyl methacrylate (BMPAEMA), polyethylene glycol monomethyl ether methacrylate (PEGMA) (where the number of ethylene glycol units is an integer from 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), PEGMA 300 PEGMA 500 2-Dimethylaminoethyl methacrylate (DMAEMA), sulfobetaine methacrylate (SBMA), quaternary ammonium ethyl methacrylate, hydroxyethyl methacrylate (HEMA), 2-azidoethyl methacrylate, 2-azidotriethylene glycol methyl ether methacrylate (TEOMA-azido compound or TEOMA-N3), 2-azidotetraethylene glycol methyl ether methacrylate (PEGMA(n=4)-azido compound or PEGMA(n=4)-N3), 2-azidopentaethylene glycol methyl ether methacrylate (PEGMA(n=5)-azido compound or PEG... PEGMA(n=5)-N3, 2-azidohexaethylene glycol methyl ether methacrylate (PEGMA(n=6)-azido or PEGMA(n=6)-N3), 2-azidoheptaethylene glycol methyl ether methacrylate (PEGMA(n=7)-azido or PEGMA(n=7)-N3), 2-azidooctaethylene glycol methyl ether methacrylate (PEGMA(n=8)-azido or PEGMA(n=8)-N3) and 2-azidononethylene glycol methyl ether methacrylate (PEGMA(n=9)-azido or PEGMA(n=9)-N3).

[0092] As used herein, the terms "initiator," "initiator molecule," "ATRP initiator," or "ATRP initiator molecule" refer to any molecule containing an atom that is a free radical transferable atom or group, such as a halogen. Standard organic synthesis techniques can be used to prepare ATRP initiators. ATRP initiators can be prepared by a variety of methods. This article will describe some general methods for preparing ATRP initiators. Typically, initiators can have the general formula: Y-(X). n In this molecule, Y is the core and X is an atom or group that can be transferred by free radicals. The number n can be any number greater than 1, depending on the functionality of the core group Y. For example, in β-alanine, n-(2-bromo-2-methyl-1-oxopropyl)-2,5-dioxo-1-pyrrolidine ester has the following structure: Y represents the N-succinimide ester of β-alanine in isobutyril, and X represents Br, where N is 1. See Murata et al., Biomacromolecules 15:2817-2823, 2014, which is incorporated herein by reference. In 2,5-dioxopyrrolidone-1-yl 4-(bis(2-(2-bromo-2-methylpropamido)ethyl)amino)-4-oxobutyrate, it has the following structure: Y represents the N-succinimide ester of a branched amide, X represents Br, and n is 2. Examples of initiators include, but are not limited to, bromine and other halogens.

[0093] 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 introduction of these functional groups can be accomplished as a single substitution, or the small molecule can have more than one initiation site for ATRP. For example, molecules containing more than one hydroxyl group can undergo esterification to generate α-haloesters, which can initiate ATRP. Other initiator residues can be introduced if desired. The small molecule to which the initiator is attached can be organic or inorganic; it can be used as long as the initiator does not poison the catalyst or interact adversely with the growing free radicals. Some examples of small molecules used as the basis for linking initiation sites are polydimethylsiloxane cubes, cyclotriphosphazene rings, 2-tris(hydroxyethyl)ethane, glucosyl compounds, etc. Furthermore, trichloromethyl isocyanate can be used to link initiator residues to any substance containing hydroxyl, thiol, amine, and / or amide groups.

[0094] As used herein, the term "macroinitiator" refers to any molecule of a synthetic polymer that contains atoms that are free radical transferable atoms or groups, such as halogens. For example, a prepolymer containing an initiator can be synthesized first via RAFT polymerization, and then a copolymer can be grafted onto the prepolymer via ATRP.

[0095] In some respects, macromolecular initiators can be synthesized to introduce ATRP initiating groups into different molecules, such as targeting molecules or bottle-brush backbones. Macromolecular initiators can take different forms and can be prepared by various methods known in the art. Macromolecular initiators can be soluble polymers, insoluble / crosslinked polymer supports, or surface or solid inorganic supports. Some common methods for preparing macromolecular initiators include modification of existing materials, and one or more ATRP / non-ATRP methods using ATRP. The polymerization of monomers, or the use of initiators containing ATRP initiator residues (for other types of polymerization). Those skilled in the art of materials / polymer modification understand the modification of macromolecular compounds / substrates to generate ATRP initiation sites. For example, cross-linked polystyrene with halomethyl groups on the benzene ring (for solid-phase peptide synthesis), functional molecules attached to silica surfaces, brominated soluble polymers (e.g., isoprene, styrene, and other monomers in copolymers), or attaching small molecules containing ATRP initiators to polymer chains, can all be used as macromolecular initiators. If one or more initiation sites are located at the ends of the polymer chain, a block (co)polymer is prepared; if the initiation sites are dispersed along the polymer chain, a graft (co)polymer is formed.

[0096] As used herein, the term "drug-to-reagent ratio" or "DAR" refers to the number of drugs (e.g., chemotherapeutic drugs) attached to a non-PEG polymer relative to a reagent (e.g., an antibody or its antigen-binding fragment, polynucleotide, cytokine, or enzyme) in the bioconjugates of this invention. For example, a DAR ratio of 100:1 means that 100 drugs are attached to a non-PEG polymer, which is attached to one reagent. One or more different drugs may be considered as drugs used to express a DAR ratio. Furthermore, one or more drugs may be attached to a non-PEG polymer associated with a reagent.

[0097] As used herein, the term "ultra-high drug-to-reagent ratio" refers to the amount of drug (e.g., a chemotherapeutic drug) attached to a non-PEG polymer relative to a reagent (e.g., an antibody or its antigen-binding fragment, polynucleotide, cytokine, or enzyme) in the bioconjugates of the present invention, wherein the ratio is at least 20 drugs to 1 reagent (20:1 DAR). In some aspects, 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 1,100:1. One or more different drugs can be considered as drugs used to express a DAR ratio. In addition, one or more drugs can be attached to a non-PEG polymer associated with the reagent.

[0098] The term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combination thereof, through at least one antigen recognition site within the variable region of an immunoglobulin molecule. As used herein, the term "antibody" includes complete polyclonal antibodies, complete 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 complete antibodies or other modified immunoglobulins), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing the antigenic determinant of an antibody, and any other modified immunoglobulin molecule containing an antigen recognition site, provided that the antibody exhibits the desired biological activity. Based on the identity of the heavy chain constant regions, referred to as α, δ, ε, γ, and μ, respectively, an antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Different types of immunoglobulins have different and well-known subunit structures and three-dimensional conformations. Antibodies can be naked or bound to other molecules such as toxins and radioactive isotopes.

[0099] The term "antibody fragment" refers to a portion of a complete antibody, specifically the antigenic determinant variable region of the complete antibody. Examples of 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, biantibodies, DARPins, DVD-Ig, Fcab, IgNAR, IgG-CH2, endoantibodies, linear antibodies, mAb2, microantibodies, single-chain antibodies, single-domain antibodies (VHH), tetraantibodies, triantibodies, V NAR domains, and multispecific antibodies formed from antibody fragments.

[0100] A biparatropic antibody is a bispecific antibody that recognizes and specifically binds to a target—such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of these substances—through two different antigen recognition sites within the variable region of an immunoglobulin molecule, binding to two different epitopes of the target. For example, a biparatropic anti-EGFR antibody or fragment binds to two different epitopes of EGFR. Biparatropic antibodies generally have stronger binding affinity and specificity than single-episode antibodies.

[0101] "Monoclonal antibody" refers to a homogeneous group of antibodies that involve highly specific recognition and binding to a single antigenic determinant or epitope. This contrasts with polyclonal antibodies, which typically consist of different antibodies targeting different antigenic determinants. The term "monoclonal antibody" includes complete and full-length monoclonal antibodies, as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, and other antibodies described herein), single-chain (scFv) mutants, fusion proteins containing antibody moieties, and any other modified immunoglobulin molecules containing antigen recognition sites. Furthermore, "monoclonal antibody" refers to antibodies prepared in a variety of ways, including but not limited to hybridoma, phage selection, recombinant expression, and transgenic animals.

[0102] The term "humanized antibody" refers to a form of non-human (e.g., mouse) antibody that is a specific immunoglobulin chain, chimeric immunoglobulin, or fragment thereof containing minimal non-human (e.g., mouse) sequence. Typically, humanized antibodies are human immunoglobulins in which residues of the complementarity-determining region (CDR) are replaced by residues of the CDR from a non-human species (e.g., mouse, rat, rabbit, hamster), possessing the desired specificity, affinity, and capability (Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536). In some cases, Fv framework region (FR) residues of human immunoglobulins are replaced by corresponding residues from antibodies derived from non-human species, possessing the desired specificity, affinity, and capability. Humanized antibodies can be further modified by replacing additional residues within the Fv framework region and / or substituted non-human residues to improve and optimize antibody specificity, affinity, and / or potency. Generally, humanized antibodies will contain substantially all, typically two or three, variable domains, which contain all or substantially all CDR regions corresponding to non-human immunoglobulins, while all or substantially all FR regions are common sequences of human immunoglobulins. Humanized antibodies may also contain at least a portion of immunoglobulin constant regions or domains (Fc), typically constant regions or domains of human immunoglobulins. Examples of methods for generating humanized antibodies are described in U.S. Patents 5,225,539 or 5,639,641.

[0103] The “variable region” of an antibody refers to the variable region of either the antibody light chain or the antibody heavy chain, either alone or in combination. Each variable region of the heavy and light chains consists of four frame regions (FRs), which are connected by three complementarity-determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are tightly bound together by the FRs and, together with the CDRs in the other chain, form the antigen-binding site of the antibody. At least two techniques exist for determining CDRs: (1) methods based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)); and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948). Furthermore, a combination of these two methods is sometimes used in the art to determine CDRs.

[0104] When dealing with residues in variable domains (approximately residues 1-107 in the light chain and approximately residues 1-113 in the heavy chain), the Kabat numbering system is typically used (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0105] The amino acid position numbering in Kabat refers to the numbering system used for heavy chain or light chain variable regions in antibody sequences, as described in Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) by Kabat et al. Using this numbering system, the actual linear amino acid sequence can contain fewer or more amino acids, corresponding to shortening or insertion of variable domains FR or CDR. For example, a heavy chain variable domain could include a single amino acid insertion after H2 residue 52 (according to Kabat residue 52a) and an inserted residue after heavy chain FR residue 82 (e.g., according to Kabat, residues 82a, 82b, and 82c, etc.). For a given antibody, the Kabat number of the residues can be determined by comparing homologous regions of the antibody sequence with a “standard” Kabat numbered sequence. Chothia refers to the position of the structural loop (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). When numbered using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34, depending on the loop length (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B exists, the loop ends at 32; if only 35A exists, the loop ends at 33; if both 35A and 35B exist, the loop ends at 34). The AbM hypervariable region represents a compromise between the Kabat CDR and the Chothia structural loop and is used by the AbM antibody modeling software from Oxford Molecular.

[0106]

[0107] The term "human antibody" refers to an antibody produced by a human or an antibody prepared using any technique known in the art that has an amino acid sequence corresponding to that of a human-produced antibody. This definition of human antibody includes full-length or complete antibodies, fragments thereof, and / or antibodies containing at least one human heavy chain and / or light chain polypeptide, such as antibodies containing mouse light chain and human heavy chain polypeptides.

[0108] The term "chimeric antibody" refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable regions of the light and heavy chains correspond to the variable regions of antibodies from one mammal (e.g., mouse, rat, rabbit, etc.), possessing the desired specificity, affinity, and capability, while the constant regions are sequence-homogeneous with antibodies from another mammal (usually human) to avoid triggering an immune response in that species.

[0109] The terms "epitope" or "antigenic determinant" are used interchangeably in this document and refer to an antigenic moiety that can be recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, an epitope can be formed by the juxtaposition of adjacent and non-adjacent amino acids through the ternary folding of the protein. Epitopes formed by consecutive amino acids are generally retained during protein denaturation, while epitopes formed by ternary folding are generally lost during protein denaturation. An epitope typically contains at least three, more commonly at least five or eight to ten amino acids, in a unique spatial conformation.

[0110] "Binding affinity" generally refers to the sum strength of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, "binding affinity" as used herein 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 molecule X for its partner Y is typically expressed as a dissociation constant (Kd). Affinity can be measured by methods commonly known in the art, including those described herein. Low-affinity antibodies typically bind to antigens more slowly and dissociate more readily, while high-affinity antibodies typically bind to antigens more quickly and for longer periods. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of this disclosure. Specific illustrative aspects are described below.

[0111] As used herein, the phrase "substantially similar" or "substantially identical" means that there is a sufficiently high degree of similarity between two values ​​(typically one related to the antibody of the present invention and the other to a reference / comparison antibody) such that a person skilled in the art would consider the difference between the two values ​​to be of little or no biological and / or statistical significance in the context of the biological characteristic measured by said value (e.g., Kd value). As a function of the reference / comparison antibody value, 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%.

[0112] As used in this article, “substantially pure” means material that is at least 50% pure (i.e. free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0113] As used in this article, the term "reagent" refers to a reagent that can target specific cells (e.g., cancer cells), including but not limited to reagents that target proteins, antibodies and antibody fragments, as well as reagents that include but are not limited to cytokines, enzymes and polynucleotides.

[0114] As used herein, the term "bioconjugate" refers to a compound or derivative thereof comprising a non-PEG polymer linked to a reagent and a drug, wherein the reagent is a target agent (e.g., an antibody, antigen-binding fragment, or target protein) or a reagent (e.g., a cytokine, enzyme, or polynucleotide), and is defined by the following formula: D z -L1-P-L2-A, where D z = Drug, z is 1-1,100, L1 = Connector, P = Non-PEG polymer, L2 = Chain (may include connector), A = Reagent.

[0115] A "linker" is any chemical part that can stably covalently link a compound (typically a drug) to a reagent. While the compound or reagent remains active, a linker can be sensitive to or substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage. In some aspects disclosed herein, the linker is a cleavable linker or a dissociable linker. In some aspects disclosed herein, the linker is an amino acid linker. In some aspects disclosed herein, the linker is a valine-citrulline or alanine-alanine linker. In some aspects disclosed herein, the linker is a peptide linker. In some aspects disclosed herein, the linker is a valine-citrulline-p-aminobenzylcarbamate (Val-Cit-Pab) linker. In some aspects disclosed herein, the valine-citrulline (Val-Cit) linker contains a p-aminobenzylcarbamate functional group. In some aspects disclosed herein, the linker is an alanine-alanine-p-aminobenzylcarbamate (Ala-Ala-Pab) linker. In some aspects disclosed herein, the alanine-alanine (Ala-Ala) linker contains a p-aminobenzylcarbamate functional group.

[0116] As used herein, the term "band" refers to any chemical portion of the polymer backbone of the bottle brush polymer described herein that can be stably covalently coupled to a compound (e.g., a reagent). The band is substantially resistant to one or more of acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, while the compound or reagent remains active. In some aspects disclosed herein, the band comprises a non-cleavable portion. In some aspects disclosed herein, the band is an amino acid sequence. In some aspects disclosed herein, the band comprises a histidine tag (His tag), a glycine unit sequence, (GGGGS)3 (SEQ ID NO: 5), ethylene glycol units (where the number of units is an integer between 5 and 25), or combinations thereof. In some aspects disclosed herein, the band comprises ethylene glycol units, where the number of units is an integer between 7 and 25. In some aspects disclosed herein, the band comprises ethylene glycol units, where the number of units is an integer between 8 and 25. In some aspects disclosed herein, the band comprises ethylene glycol units, where the number of units is an integer between 9 and 25. In some aspects disclosed herein, the chain includes ethylene glycol units, wherein the number of units is an integer between 10 and 25. In some aspects disclosed herein, the chain is ethylene glycol units, wherein the number of units is an integer between 15 and 25. In some aspects disclosed herein, the chain includes ethylene glycol units, wherein the number of units is an integer between 15 and 20. In some aspects disclosed herein, the chain includes a monomer that can be polymerized by a controlled radical polymerization reaction, wherein the number of repeating units is an integer between 5 and 80. In some aspects disclosed herein, the chain includes a monomer that can be polymerized by a controlled radical polymerization reaction, wherein the number of repeating units is an integer between 5 and 75. In some aspects disclosed herein, the chain includes a monomer that can be polymerized by a controlled radical polymerization reaction, wherein the number of repeating units is an integer between 5 and 70. In some aspects disclosed herein, the chain includes a monomer that can be polymerized by a controlled radical polymerization reaction, wherein the number of repeating units is an integer between 5 and 65. In some aspects disclosed herein, the chain includes a monomer that can be polymerized by a controlled radical polymerization reaction, wherein the number of repeating units is an integer between 5 and 60. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 5 and 55. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 5 and 50. In some aspects disclosed herein, the chain comprises monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 5 and 45. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 5 and 40.In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 5 and 35. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 5 and 30. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 5 and 25. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 5 and 20. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 5 and 15. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 5 and 10. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 7 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 8 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 9 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 15 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 20 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 25 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 30 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 35 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable via controlled radical polymerization, wherein the number of repeating units is an integer between 40 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable via controlled radical polymerization, wherein the number of repeating units is an integer between 45 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable via controlled radical polymerization, wherein the number of repeating units is an integer between 50 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable via controlled radical polymerization, wherein the number of repeating units is an integer between 55 and 80.In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 60 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 65 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 70 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 75 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 5 and 10. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 10 and 20. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 20 and 30. In some aspects disclosed herein, the chain includes monomers polymerizable via controlled radical polymerization, wherein the number of repeating units is an integer between 30 and 40. In some aspects disclosed herein, the chain includes monomers polymerizable via controlled radical polymerization, wherein the number of repeating units is an integer between 40 and 50. In some aspects disclosed herein, the chain includes monomers polymerizable via controlled radical polymerization, wherein the number of repeating units is an integer between 50 and 60. In some aspects disclosed herein, the chain includes monomers polymerizable via controlled radical polymerization, wherein the number of repeating units is an integer between 60 and 70. In some aspects disclosed herein, the chain includes monomers polymerizable via controlled radical polymerization, wherein the number of repeating units is an integer between 70 and 80.

[0117] The terms “cancer” and “cancerous” refer to or describe a physiological condition in mammals in which a population of cells is characterized by unregulated cell growth. Examples of cancer include, but are not limited to, tumors, lymphomas, blastomas, sarcomas, leukemias, multiple myeloma, myelodysplastic syndromes, and myeloproliferative disorders. More specific examples of these cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, various types of head and neck cancer, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndromes (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 precursor ALL (BCP-ALL), Ph+ ALL, Ph-like ALL, chronic lymphocytic leukemia (CLL), blast crisis / blast crisis chronic myeloid leukemia (BP-CML), or blast cell plasmacytoid dendritic cell tumor (BPDCN). Other examples of “cancer” include B-cell lymphomas (including NHL), precursor B-cell lymphoblastic leukemia / lymphoma and mature B-cell tumors (e.g., 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 follicular center lymphoma, marginal zone B-cell lymphoma (MALT type, nodular type, and splenic type), hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, plasmacytoma, plasmacytic myeloma, post-transplant lymphoproliferative disorders, Waldenström macroglobulinemia, and anaplastic large cell lymphoma (ALCL).

[0118] "Tumor" and "growth" refer to any mass of tissue resulting from excessive cell growth or proliferation, benign (non-cancerous) or malignant (cancerous), including precancerous lesions.

[0119] The terms “cancer cell,” “tumor cell,” and their grammatical equivalents refer to the total population of cells derived from a tumor or precancerous lesion, including non-tumorigenic cells and tumorigenic stem cells (cancer stem cells) that constitute the majority of the tumor cell population. As used herein, the term “tumor cell” will be modified by the term “non-tumorigenic” when referring only to those tumor cells that lack the capacity for renewal and differentiation to distinguish them from cancer stem cells.

[0120] The terms “anticancer drug,” “chemotherapy drug,” “cytotoxic fraction,” and “anticancer prodrug” refer to drugs (i.e., compounds) or prodrugs that are known or suspected of being able to treat cancer (i.e., kill cancer cells, inhibit the proliferation of cancer cells, or treat cancer-related symptoms).

[0121] The term "subject" refers to any animal (e.g., a mammal) that receives a particular treatment, including but not limited to humans, non-human primates, rodents, etc. Generally, the terms "subject" and "patient" are used interchangeably in this document for human subjects.

[0122] "Combined" administration with one or more other drugs includes simultaneous (parallel) and sequential administration in any order.

[0123] The term "pharmaceutical formulation" refers to a formulation in a form that enables the bioactivity of the active ingredient to be effective and does not contain any additional components (which may be determined based on the specific circumstances of the subject administering the formulation) that would have unacceptable toxicity to the subject. Such formulation may be sterile.

[0124] The “effective amount” of the bioconjugates disclosed herein is an amount sufficient to achieve a specific purpose. The “effective amount” can be determined empirically and in a conventional manner according to the stated purpose.

[0125] The term "therapeutic effective dose" refers to the amount of a biological conjugate that effectively "treats" a disease or disorder in a subject or mammal. In the case of cancer, a therapeutically effective dose of a biological conjugate may reduce the number of cancer cells; shrink the size of the tumor; inhibit (i.e., to some extent slow down and prevent, to some degree) the invasion of cancer cells into peripheral organs; inhibit (i.e., to some extent slow down and prevent, to some degree) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more cancer-related symptoms to some extent. In terms of the extent to which the biological conjugate can prevent the growth of existing cancer cells and / or kill existing cancer cells, it can be growth-inhibiting and / or cytotoxic. "Prophylactic effective dose" refers to an effective dose and time period that achieves the desired preventative effect. Typically, but not necessarily, prophylactic doses are used in subjects before or in the early stages of disease.

[0126] The terms "polynucleotide" or "nucleic acid" are used interchangeably herein to refer to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogues, or any substrate that can be incorporated into the polymer by DNA or RNA polymerases. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and their analogues. If present, the nucleotide structure can be modified before or after polymer assembly. The nucleotide sequence can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by binding to labeled components. Other types of modifications include, for example, “capping”, replacing one or more naturally occurring nucleotides with analogs, internucleotide modifications such as those with uncharged linkages (e.g., methylphosphonates, triphosphates, phosphatidyl esters, carbamates, etc.) and charged linkages (e.g., thiophosphates, dithiophosphates, etc.), those containing pendant moieties (e.g., proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.)), those with intercalating agents (e.g., acridine, psoralen, etc.), those containing chelating agents (e.g., metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those with modified linkages (e.g., α-anomeric nucleic acids, etc.), and unmodified forms of polynucleotides. Furthermore, any hydroxyl groups typically present in sugars can be substituted, for example, with phosphonate or phosphate groups, protected with standard protecting groups, or activated to prepare additional linkages with additional nucleotides, or can be conjugated to a solid support. The 5' and 3' terminal OH groups can be phosphorylated or partially substituted with amine or organic end-capping groups of 1-20 carbon atoms. Other hydroxyl groups can also be derived into standard protecting groups. Polynucleotides can also contain similar forms of ribose or deoxyribose known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xylose, or lythose, pyranose, furanose, sedoheptulose, acyclic analogs, and non-basic nucleoside analogs such as methylnucleosides. One or more phosphodiester bonds can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, those in which the phosphate group is substituted with P(O)S (“thioester”), P(S)S (“dithioester”), P(O)NR2 (“amid”), P(O)R, P(O)OR', CO, or CH2 (“methylacetal”), wherein each R or R' is independently H or optionally a substituted or unsubstituted alkyl (1-20C), aryl, alkenyl, cycloalkyl, cycloalkenyl, or aralkyl group containing an ether (-O-) bond. Not all linking bonds in a polynucleotide must be identical. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

[0127] As used herein, the terms “treatment,” “management,” or “curing” mean, for example, reducing the severity of a disease or condition; shortening the duration of a disease; improving or eliminating one or more symptoms associated with a disease or condition; providing a beneficial effect to a subject suffering from a disease or condition, but not necessarily curing the disease or condition. The term also includes prevention or avoidance of a disease or condition or its symptoms. In one aspect, the term “treatment” or “management” refers to reducing the number of tumor cells in a subject.

[0128] The term "vector" refers to a construct capable of delivering and expressing one or more target genes or sequences in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors bound to cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0129] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acids of any length. This polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acid components. The term also includes polymers of amino acids, either naturally occurring or modified through intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeled component. This definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids). It should be understood that, because the polypeptides of this disclosure are antibody-based, in some respects, polypeptides may exist in single-chain or related-chain form.

[0130] II. Reagents A. Antibodies, their antigen-binding fragments, or other target proteins This invention includes a bioconjugate comprising a targeting agent (e.g., a 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, wherein the polymer is conjugated to a chemotherapeutic drug, and the bioconjugate has a drug-to-agent ratio (DAR) of at least 20:1, at least 50:1, at least 100:1, at least 200:1, at least 300:1, at least 400:1, at least 500:1, at least 600:1, at least 700:1, at least 800:1, at least 900:1, at least 1000:1, or at least 1,100:1. The antibody and antigen-binding fragment allow the bioconjugate to bind to antigens on target cells (e.g., cancer cells) and be internalized by the target cells to exert a therapeutic effect. This conserves normal, healthy non-target cells. Methods that allow bioconjugates to be fully internalized by target cells include targeting receptors capable of internalizing bioconjugates or using peptide-mediated membrane permeation to deliver antibodies to their intracellular target proteins (see US Patent Application Publication No. 20080063633).

[0131] Furthermore, internalization aims to release non-PEG polymers and drugs from the reagent. This release then allows the free drug to be transported within cells, resulting in desired toxicity and cell death in the target cell population (e.g., cancer). For other applications, the reagent (e.g., antibodies and antigen-binding fragments) allows the bioconjugate to target extracellular targets, subsequently releasing free non-PEG polymers and drugs to the target and neighboring cells, resulting in cytotoxicity and cell death (see US Patent Application Publication No. 20190248887).

[0132] In addition to the targeted delivery of drugs mentioned above, antibody-antigen binding fragments possess other cytotoxic mechanisms. The Fc region of an antibody mediates several important effector functions, such as antibody-dependent cytotoxicity (ADCC), phagocytosis, and complement-dependent cytotoxicity (CDCC). Therefore, targeted delivery of antibody-antigen binding fragments can lead to target cell death by recruiting effector cells carrying Fc receptors, which can recognize and kill antibody-coated target cells; by phagocytosis and elimination of target cells by innate immune cells; and by recruiting complement factors, which lead to cell lysis and membrane attack complex (MAC) formation via the classical complement pathway. Thus, targeted delivery of bioconjugates via antibody-antigen binding fragments provides a higher level of control over the delivery of chemotherapeutic drugs and can mediate cell death through drug-induced or Fc-dependent immune pathways.

[0133] The effectiveness of the bioconjugates disclosed herein depends on the careful selection of appropriate reagents. Given the serious side effects and costs associated with cancer treatment, it is necessary to identify general factors that can be used to treat cancer and lead to a higher probability of treatment success. Cancer-associated antigens are an attractive target for bioconjugates because they can specifically target cancer cells while minimizing side effects. Cancer-associated antigens can be broadly categorized into the following groups: cancer-testis antigens (e.g., MAGE, NY-ESO-1), differentiation antigens (e.g., tyrosinase, PSMA), overexpression antigens (e.g., HER2, survivin, telomerase, WT1), cancer-specific antigens (e.g., β-catenin), aberrant post-translational modifications (e.g., MUC1), and tumor virus proteins (e.g., E6, E7). A broader term for pan-cancer antigens can be classified as any cell surface epitope expressed at higher levels in cancer cells than in normal tissues, or epitopes that can be induced to the cell surface at higher levels in cancer cells than in normal tissues. Targeting these cancer-associated antigens with antibodies allows for the delivery of drug payloads to cancer cells and subsequent cancer-specific reduction and / or elimination.

[0134] In some respects, reagents (e.g., antibodies or their antigen-binding fragments) allow bioconjugates to target and bind to cancer-associated antigens found on cancer cells, followed by internalization of the bioconjugates and release of the drug from non-PEG polymers.

[0135] In some respects, the reagent (e.g., an antibody or an antigen-binding fragment thereof) allows the bioconjugate to target and bind to cancer-associated antigens found on cancer cells and subsequently release the drug from the non-PEG polymer in the extracellular space.

[0136] In some respects, the reagent has one or more of the following functions: inhibiting the proliferation of tumor cells, reducing tumorigenicity by decreasing the frequency of cancer stem cells in the tumor, inhibiting tumor growth, increasing survival rate, inducing cell death of tumor cells, differentiating tumorigenic cells into a non-tumorigenic state, or preventing the metastasis of tumor cells.

[0137] In some respects, this reagent is specific to extracellular receptors, peptides, or lipids found on cancer cells. In other respects, it is specific to cancer-associated antigens originating from cancer.

[0138] In some respects, the cancers are selected from the group consisting of: tumors, lymphomas, blastomas, sarcomas, leukemias, multiple myeloma, myelodysplastic syndromes, and myeloproliferative disorders. In some respects, the cancers mentioned are selected from squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, various types of head and neck cancer, 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 precursor ALL (BCP-ALL), Ph+ ALL, Ph-like ALL, chronic lymphocytic leukemia (CLL), blast crisis / blast crisis chronic myeloid leukemia (BP-CML), or blast cell plasmacytoid dendritic cell tumor (BPDCN). Other examples of “cancer” include B-cell lymphomas (including NHL), precursor B-cell lymphoblastic leukemia / lymphoma, and mature B-cell tumors 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 follicular center lymphoma, marginal zone B-cell lymphoma (MALT type, nodular type, and splenic type), hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, plasmacytoma, plasmacytic myeloma, post-transplant lymphoproliferative disorders, Waldenström macroglobulinemia, and anaplastic large cell lymphoma (ALCL).

[0139] In some respects, the cancer-associated antigen is EGFR.

[0140] In some aspects, the reagent is an anti-EGFR antibody. In some aspects, the antibody is a full-length anti-EGFR antibody. In some aspects, the anti-EGFR antibody is Fab, Fab', F(ab')2, Fd, single-chain Fv or scFv, disulfide-linked Fv, VNAR domain, IgNar, intracellular antibody, IgG-CH2, microantibody, F(ab')3, tetraantibody, triantibody, biantibody, single-domain antibody (VHH), DVD-Ig, Fcab, mAb2, (scFv)2, DARPin, or scFv-Fc antibody. In some aspects, the anti-EGFR antibody is a VHH antibody. In some aspects, the anti-EGFR antibody is a bipara antibody.

[0141] In some aspects, the anti-EGFR antibody is an scFv. In some aspects, the anti-EGFR scFv antibody has the variable heavy chain of SEQ ID NO: 1 and / or the variable light chain of SEQ ID NO: 2. In some aspects, the anti-EGFR scFv antibody has the variable heavy chain of SEQ ID NO: 3 and / or the variable light chain of SEQ ID NO: 4. In some aspects, the anti-EGFR scFv antibody has the variable heavy chain of SEQ ID NO: 1, which is conjugated to the variable light chain of SEQ ID NO: 2 using a flexible glycine linker (SEQ ID NO: 5). In some aspects, the anti-EGFR scFv antibody has the variable heavy chain of SEQ ID NO: 3, which is conjugated to the 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 the full-length sequence consisting of SEQ ID NO: 6. In some aspects, the anti-EGFR scFv antibody has the full-length sequence consisting of SEQ ID NO: 7.

[0142] Table 1. Variable regions of heavy and light chains in C10KV3_LV1DE

[0143] Table 2. Variable regions of heavy and light chains in P2224KV3_LV1DE

[0144] Table 3. Flexible joints for connecting heavy and light variable zones

[0145] Table 4. Full length scFv of C10KV3_LV1DE

[0146] Table 5. P2224KV3_LV1DE full length scFv

[0147] Table 6. Complementarity Determination Regions of Variable Heavy Chain and Light Chain in C10KV3_LV1DE

[0148] In some respects, 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.

[0149] In some aspects, 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 an amino acid sequence selected from SEQ ID NO: 13.

[0150] In some respects, the cancer-associated antigen is HER2.

[0151] In some aspects, the antibody is a full-length anti-HER2 antibody. In some aspects, the anti-HER2 antibody is Fab, Fab', F(ab')2, Fd, single-chain Fv or scFv, disulfide-linked Fv, VNAR domain, IgNar, intracellular antibody, IgG-CH2, microantibody, F(ab')3, tetraantibody, triantibody, biantibody, single-domain antibody (VHH), DVD-Ig, Fcab, mAb2, (scFv)2, DARPin, or scFv-Fc antibody. In some aspects, the anti-HER2 antibody is scFv. In some aspects, the anti-HER2 scFv antibody has the variable heavy chain of SEQ ID NO: 20 and / or the variable light chain of SEQ ID NO: 21. In some aspects, the anti-HER2 scFv antibody has the variable heavy chain of SEQ ID NO: 20 conjugated to the variable light chain of SEQ ID NO: 21 using a flexible glycine linker (SEQ ID NO: 5). In some respects, the anti-HER2 scFv antibody has the full-length sequence consisting of SEQ ID NO: 22. In some respects, the anti-HER2 antibody is trastuzumab. In some respects, trastuzumab has the heavy chain of SEQ ID NO: 23 and / or the light chain of SEQ ID NO: 24.

[0152] In some respects, 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.

[0153] Table 7. Variable regions of anti-HER2 heavy and light chains

[0154] Table 8. Anti-HER2 full-length scFv

[0155] Table 9. Trastuzumab heavy and light chain regions

[0156] Table 10. Complementarity-determining regions of anti-HER2 variable heavy and light chains

[0157] In some respects, cancer-associated lipids are phosphatidylserine. In some respects, the agent is an antiphosphatidylserine antibody. In some respects, the antiphosphatidylserine antibody is bavismumab. The binding of the antiphosphatidylserine antibody bavismumab to phosphatidylserine is mediated by the serum protein β2-glycoprotein I (also known as apolipoprotein H). Besides indirect PS-binding or PS-targeting antibodies such as bavismumab, the entire family of PS-targeting antibodies includes antibodies that directly bind to PS, i.e., direct PS-binding or PS-targeting antibodies. Such "direct PS-binding antibodies" (or "direct PS-targeting antibodies") are antibodies that not only have functional specificity for PS, but also target and bind to PS in vitro and in vivo (as are indirect binding antibodies), and do not require a serum protein (such as β2-glycoprotein I) to form a tight-binding complex with PS, even in in vitro binding assays. In some respects, antiphosphatidylserine antibodies are direct binders. In some respects, the direct-binding antibody is 9D2. The 9D2 antibody is a mouse monoclonal antibody that has been shown to target tumor blood vessels and exert antitumor activity in vivo (Ran et al., 2002). In other respects, the direct-binding antibody is 11.31. The 11.31 antibody is a fully human antibody that has also been shown to exert antitumor activity in vivo (e.g., in mice carrying MDA-MB-435 breast cancer xenografts) and has shown impressive antiviral activity (Moody et al., 2010; U.S. Patent No. 7,455,833).

[0158] In some aspects, the reagent is a pan-cancer antigen. In some aspects, the pan-cancer antigen is selected from MUC-1, epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen, ras, p53, β-catenin, CDK4, CDC27, α-actin-4, gangliosides, prostate-specific membrane antigen (PSMA), nephroblastoma gene 1 (WT1), liver tyrosine kinase a3 (EphA3), CD20, B-melanoma antigen (BAGE), GAGE, NY-ESO-1, and survivin.

[0159] In some respects, the reagents described herein may be in the form of single-domain antibodies (VHH). In other respects, the reagents described herein may be bipara antibodies.

[0160] In some respects, the methods described herein are used to modify antibodies to produce bioconjugates. In some respects, the method involves deglycosylation of the antibody followed by reaction with a small molecule containing a primary amine, catalyzed by bacterial transglutaminase. This approach allows for site-specific modification of the Fc region of the antibody without the need for incorporation of non-standard amino acids.

[0161] B. Other reagents In this disclosure, agents other than antibodies or antibody fragments may be used in combination with chemotherapeutic agents conjugated with non-PEG polymers for beneficial therapeutic outcomes. Cytokines are key mediators of innate and acquired immune responses. They can alter the balance of cellular and humoral responses, change B lymphocyte class switching, and modify innate responses. Several cytokines limit cancer growth through direct antiproliferative or pro-apoptotic activity, or indirectly by stimulating the cytotoxic activity of immune cells against cancer cells. Two cytokines, IL-2 and IFN-α, have shown clinical benefit and have therefore been approved by the U.S. Food and Drug Administration (FDA) for the treatment of several malignancies. IL-2 is approved for the treatment of advanced renal cell carcinoma (RCC) and metastatic melanoma, while IFN-α is approved for the treatment of hairy cell leukemia, follicular non-Hodgkin lymphoma, melanoma, and HIV-related Kaposi's sarcoma. The clinical use of these cytokines marks a milestone in cancer immunotherapy, as it is the first time that immunotherapy has been demonstrated to favorably reverse the balance between cancer and anti-cancer immune responses, 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 the potency of anticancer 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 used to target common cancer-associated antigens and signaling pathways, reducing cancer burden through either load delivery or inhibitory effects (Nimjee, et al., 2017, Annual Review of Pharmacology and Toxicology, 57:61-79).

[0162] The efficacy of these agents is greatly enhanced by including non-PEG polymers that are coupled with chemotherapeutic drugs that enhance their activity.

[0163] In some respects, the reagent is an enzyme. In some respects, the reagent is a cytokine. In some respects, the cytokines are interleukin (IL)-2, IL-7, IL-12, IL-15, IL-21, IL-23, interferon (IFN)-α, and IFN-γ. In some respects, the reagent is a polynucleotide. In some respects, the polynucleotide is an aptamer.

[0164] III. Drugs A. Chemotherapy drugs Cancer treatment may include surgery, chemotherapy, hormone therapy, and / or radiation therapy to eradicate tumor 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 treatment may also involve biologics or immunotherapy. All of these approaches bring potential drawbacks to the subject. For example, surgery may be contraindicated or unacceptable to the subject due to their health condition. Furthermore, surgery may not completely remove the tumor tissue. Radiation therapy is only effective when tumor tissue is more sensitive to radiation than normal tissue, and it often causes serious side effects. Hormone therapy is rarely administered as a single agent, although it can be effective, but it is often used to prevent or delay cancer recurrence after other treatments have removed most of the cancer cells. Biologics / immunotherapy are limited in number and may produce side effects such as rash or swelling, flu-like symptoms including fever, chills and fatigue, gastrointestinal problems, or allergic reactions.

[0165] Regarding chemotherapy, there are various chemotherapy drugs that can be used to treat cancer. Most cancer chemotherapy drugs inhibit DNA synthesis by directly or indirectly inhibiting the biosynthesis of deoxyribonucleotide triphosphate precursors, thereby preventing DNA replication and the associated cell division (see, for example, Gilman et al., Goodman and Gilman's: The Pharmacological Basis of Therapeutics, Eighth Ed. (Pergamom Press, New York, 1990)).

[0166] Although a variety of chemotherapy drugs are available, chemotherapy has 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 chemotherapy drugs are toxic, and chemotherapy causes significant, often dangerous, side effects, including severe nausea, bone marrow suppression, and immunosuppression. Methods to reduce the systemic toxicity and off-target effects of chemotherapy drugs are needed.

[0167] In this invention, systemic toxicity and off-target effects of chemotherapeutic drugs are mitigated by conjugating them to non-PEG polymers, which are bound to reagents (e.g., antibodies, their antigen-binding fragments, cytokines, enzymes, polynucleotides) in the form of bioconjugates. Delivery of chemotherapeutic drugs as bioconjugates improves tumor targeting due to the following advantages: increased tissue penetration of anti-PEG polymers; higher drug-to-reagent ratios (e.g., high DAR (10+:1) or ultra-high DAR (e.g., 100:1, 200:1, 500:1, or 1,100:1)); retention of potency when using less toxic molecules; salvage of loads that would otherwise have systemic toxicity by incorporating them into ultra-high DAR bioconjugates; and further enhanced potency through the combined use of two or more loads with non-overlapping toxicities.

[0168] In some respects, chemotherapeutic agents refer to non-nucleic acid molecules used to treat cancer and / or possessing cytotoxic capabilities. These more traditional or conventional chemotherapeutic drugs can be described by their mechanism of action or class of chemical compounds. In some respects, chemotherapeutic agents are selected from the group consisting of: alkylating agents (e.g., melphalan, dibromomannitol, cyclophosphamide, streptozotocin, triamine), anthracyclines (e.g., doxorubicin, epirubicin, idarubicin, mitoxantrone, penoxorubicin), cytoskeleton disruptors (e.g., abaxrane, cabazitaxel, paclitaxel, docetaxel, tesetaxel), epomomycin (e.g., ixaprone), histone deacetylase inhibitors (e.g., entenotide, romidesin, vorinostat, zabadino), and topoisomerase I inhibitors (e.g., belotinib). The following are prohibited: tetan, camptothecin, irinotecan, topotecan) or topoisomerase II inhibitors (e.g., etoposide, teniposide, tafluposide), kinase inhibitors (e.g., bortezomib, erlotinib, gefitinib, imatinib), nucleoside analogs or their precursors (e.g., azacitidine, capecitabine, fluorouracil, methotrexate), peptide antibiotics (e.g., actinomycin, bleomycin), platinum-based drugs (e.g., cisplatin, nedaplatin, oxaliplatin, saplatin), retinoids (e.g., avitalic acid, bexarotin, tretinoin), and vinca alkaloids and their derivatives (e.g., vincristine, vinblastine, vindesine).

[0169] In some respects, chemotherapy drugs are AKT inhibitors. In some respects, AKT inhibitors are selected from the group consisting of ATP-competitive inhibitors: isoquinoline-5-sulfonamides (e.g., H-8, H-89), azaheptan derivatives (e.g., derived from (-)-balanol), aminofurazan (e.g., GSK690693), heterocyclic compounds (e.g., CCT128930, AZD5363, ipatasertib), phenylpyrazole derivatives (e.g., AT7867, AT13148), and thiophene carboxamide derivatives (e.g., GSK2110183, DC120, GSK2141795). In some aspects, the AKT inhibitor is an allosteric inhibitor selected from the group consisting of 2,3-diphenylquinoxaline analogs (e.g., MK-2206), alkyl phospholipids (e.g., edifoxine, mitifoxine, erufoxine), indole-3-methanol analogs (e.g., OSU-A9), sulfonamide derivatives (e.g., PH-316, PHT-427), thiourea derivatives (e.g., PIT-1, PIT-2, DM-PIT-1), and purine derivatives (e.g., tricerebroside, ARQ 092). In some aspects, the AKT inhibitor is an irreversible inhibitor selected from the group consisting of antibiotics, lactoquinomycin, frenolicin B, carafungin, mandelmycin, Boc-Phe-vinyl ketone, 4-hydroxynonenal (4-HNE), 1,6-naphthidone derivatives, and imidazo-1,2-pyridine derivatives. See Nitulescu, et al. Int JOncol. 48(3) 869-995, which is incorporated herein by reference.

[0170] In some respects, chemotherapy drugs are aurora kinase A, B, or C inhibitors. In some respects, aurora B kinase inhibitors are selected from Hesperadin, Barasertib, AZD1152, AT9283, Danusertib (PHA-739358), AMG900, CYC116, BI 811283, AZD2811, SP-96, GSK1070916, reversine, CCT129202, CCT137690, SNS-314, quercetin, and VX-680. See Borah, et al. Molecules. 26(7): 1981, which is incorporated herein by reference.

[0171] In some respects, chemotherapy drugs are ERK inhibitors. In some respects, ERK inhibitors are selected from the following group: 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 is incorporated herein by reference.

[0172] In some respects, chemotherapy drugs are PI3K inhibitors. In some respects, PI3K inhibitors are selected from PI103; PI828; LY294002; wortmannin; demethoxychloramycin; IC486068; IC87114; GDC-0941; GDC-0980 (apirixeil); perifosine; CAL101; PX-866; IPI-145; BAY 80-6946; BEZ235; P6503; TGR1202; SF1126; INK1117; BKM120; IL147; XL765; Palomid 529; GSK1059615; ZSTK474; PWT33597; TG 100-115; CAL263; GNE-447; CUDC-907; and AEZS-136.

[0173] In some respects, chemotherapy drugs are MEK inhibitors. In some respects, MEK inhibitors are selected from the following group: bemettinib, CC-90003, DEL-22379, GDC-0973 (cobitinib), selumetinib, and trametinib. See Han, et al. Journal of Hematology & Oncology. 14(1):1, which is incorporated herein by reference.

[0174] In some respects, chemotherapy drugs are MAPK inhibitors. In some respects, MAPK inhibitors are selected from the following group: farnesyltransferase inhibitors (FTI), sorafenib, vemurafenib, PLX8394, dabrafenib, ulixertinib, simvastatin, alisertib, and teriflunomide.

[0175] In some respects, chemotherapy drugs are B-RAF inhibitors. In some respects, B-RAF inhibitors are selected from the group consisting of vemurafenib, dabrafenib, cannefenib, and sorafenib. See Sanchez, et al. Drugs. 78(5): 549-566, which is incorporated herein by reference.

[0176] In some respects, chemotherapy drugs are RAS inhibitors. In some respects, RAS inhibitors are selected from the following group: Ganetespib, apatinib, Oncrasin-1, GDC-0449, ARS-1620, ARS-853, AMG510, MRTX849, BGB324, ABT-737, AZD6244, NVP-BEZ235, R115777, PPIN-1, PPIN-2, pan-RAS inhibitor 3144, Deltarasin, and sotorazib. See Shetu, et al. Int J Mol Sci. 23(7)3706, which is incorporated herein by reference.

[0177] In some respects, chemotherapy drugs are degrading agents. In some respects, degrading agents are selected from AKT and Myc degrading agents. In some respects, degrading agents are protein degrading agents. In some respects, protein degrading agents are selected from the following group: immunomodulatory drugs (iMiD), molecular glue (MG), selective estrogen receptor degrading agents (SERD), and proteolytic targeted chimeras (PROTAC). See Fang, et al. Trends Pharamcol Sci. 44(5):303-317; Xue, et al. Expert OpinDrug Discov. 18(4):467-483, which are incorporated herein by reference.

[0178] In some respects, chemotherapy drugs are cytotoxic agents. In other respects, cytotoxic agents are selected from alkylating agents, anthracycline antibiotics, cytoskeleton disruptors, epochymin, kinase inhibitors, histone deacetylase inhibitors, topoisomerase I or II inhibitors, their nucleotide analogs or precursors, peptide antibiotics, platinum-based drugs, retinoids, and vinca alkaloids and their derivatives as defined above.

[0179] In some respects, chemotherapy drugs are Wern syndrome helicases.

[0180] In some respects, chemotherapy drugs are cell cycle inhibitors. In other respects, cell cycle inhibitors are selected from the group consisting of: polo-like kinase inhibitors and cyclin-dependent kinase inhibitors.

[0181] In some respects, cell cycle inhibitors are aurora kinase inhibitors. In some respects, aurora kinase inhibitors are bariciceptor, aliceptor, (MLN8237), danuxertor (PHA-739358), AT9283, PF-03814735, or AMG900. In some respects, cell cycle inhibitors are Chk inhibitors. In some respects, Chk inhibitors are prexasertib (LY2606368) or AZD7762. In some respects, cell cycle inhibitors are Chk1 inhibitors. In some respects, Chk1 inhibitors are GDC-0575, rabusertib (LY2603618), VX-803 (M4344), CHIR-124, PF-477736, PD0166285, or SAR-020106. In some respects, cell cycle inhibitors are Chk2 inhibitors. In some respects, Chk2 inhibitors are BML-277. In some respects, polo-like kinase (PLK) inhibitors are BI 6727, BI2536, GSK461364A, rigosertib, or volasertib. In some respects, cell cycle inhibitors are cyclin-dependent kinase (CDK) inhibitors, such as palbociclib, abecilib, dinaciclib, ribociclib, TP-1287, vrapin, and NUV-422. In some respects, cyclin-dependent kinase inhibitors are combinations of CDK1, CDK2, CDK4, CDK6, CDK7, or some other CDK inhibitors. In some respects, cytoskeleton disruptors are taxanes (e.g., paclitaxel, docetaxel, or cabazitaxel), colchicine, vincristine, evodiamine, 6α-acetoxyanopterine, cytochalasin, lacucolin, astrocytin, sinularin, phalloidin, or eribulin.

[0182] In some respects, chemotherapy drugs are protein degraders. In other respects, protein degraders are protein hydrolysis-targeting chimeric (PROTAC) molecules.

[0183] IV. Non-PEG polymers, methods for producing non-PEG polymers, and methods for preparing bioconjugates containing non-PEG polymers. In some respects, non-PEG polymers are bottlebrush polymers. Bottlebrush polymers are copolymers comprising a master polymer chain (also called a polymer backbone) having different polymer side chains branching from the master polymer chain. Bottlebrush polymers can be, but are not limited to, block copolymers, block graft copolymers, random copolymers, brush-bead copolymers, gradient copolymers, or brush copolymers. Bottlebrush polymers are summarized below, and are incorporated herein by reference in, for example, Li, Z., et al., "Bottlebrushpolymers: From controlled synthesis, self-assembly, properties to applications," Progress in Polymer Science 116:101387 (2021) and Matyjaszewki, K., et al., "Macromolecular Engineering by Atom Transfer RadicalPolymerization," J. Am. Chem. Soc. 136:6513-6533 (2014).

[0184] Non-PEG bottle brush polymers can be synthesized using a variety of methods known in the art. In some respects, non-PEG bottle brush polymers are synthesized using reversible deactivating radical polymerization (RDRP) processes (formerly known as controlled radical polymerization (CRP)). RDRP methods include, but are not limited to, nitrile-oxygen mediated polymerization (NMP), atom transfer radical polymerization (ATRP), and reversible addition-fracture transfer (RAFT). Over the past two decades, these methods have been developed to yield (co)polymers with predetermined molecular weights, compositions, structures, and narrow / controllable molecular weight distributions. This approach has been used to bind proteins to polymers, for example, as described in Curr. Opin. Chem. Biol. 14(6): 818-827 (2010), which is incorporated herein by reference in its entirety. Functionalization of bioresponsive molecules with well-defined polymers can provide improved stability, customized solubility, predetermined transport pathways, and increase the therapeutic potential of already useful biomacromolecules, including peptides, proteins, nucleic acids, and polysaccharides, in a variety of applications.

[0185] Matyjaszewski and collaborators have disclosed a basic four-component ATRP method in numerous jointly assigned patents and patent applications, comprising the addition or in-situ formation of an initiator (e.g., a molecule having transferable atoms or groups fully incorporated into the final product), a transition metal, and a ligand forming a partially soluble transition metal complex that participates in a reversible redox reaction with the added initiator or inert polymer to form an active substance for copolymerization via free radical polymerization. The monomers of the composite, and numerous improvements to the basic ATRP method: U.S. Patents 5,763,546; 5,807,937; 5,789,487; 5,945,491; 6,111,022; 6,121,371; 6,124,411; 6,162,882; 6,624,262; 6,407,187; 6,512,060; 6,538,091; 6,541,580; 6,624,2 62; 6,627,314; 6,759,491; 6,790,919; 6,887,962; 7,019,082; 7,049,373; 7,064,166; 7,125,938; 7,157,530; 7,332,550; 7,407,995; 7,572,874; 7,678,869; 7,795,355; 7,825,199; 7,893,173; 7,893,174; 8,273,823; 8,252,880; 8,445,610; 8,962,764; 9,644,042; US Publication No. 20110060107A1; US20110065875A1; US20070155926A1; US20090171024A1 and International Patent Application PCT / US2011 / 051043, all of which are incorporated herein by reference.

[0186] The mechanism of the generally accepted ATRP reaction is shown in Scheme 1.

[0187]

[0188] In some respects, the ATRP method involves the regeneration of the activator via electron transfer (ARGET) ATRP. In ARGET ATRP, a reducing agent is used to regenerate the copper oxide catalyst, thereby reducing the loading of the copper catalyst. For example, ARGET ATRP is discussed in Pintauer, T, et al., "Atom transfer radical addition and polymerization reactions catalyzed by ppm amounts of copper complexes," Chemical Society Reviews 37(6):1087-1097 (2008), the entire contents of which are incorporated herein by reference.

[0189] Reversible addition-fracture transfer (RAFT) is another form of reversible deactivated radical polymerization that uses chain transfer agents, such as thiocarbonyl sulfides, to control the molecular weight and polydispersity of the resulting polymer. RAFT polymerization is exemplified in, for example, Chiefari, J., et al., "Living Free-Radical Polymerization by Reversible Addition". The Fragmentation Chain Transfer: The RAFT Process, as summarized in Macromolecules 31(16):5559-5562, is incorporated herein by reference in its entirety.

[0190] RDRP methods, such as ATRP, have been used to prepare polymers, such as bottle brush polymers bound to biomolecules, such as proteins. See, for example, US10,072,042, US9,539,338, US11,472,894, and US2021 / 0388337, all of which are incorporated herein by reference.

[0191] In some respects, bottle brush polymers are synthesized using “grafting to,” “grafting from,” or “grafting by…” methods. “Grafting to” involves combining a prepolymer with a main-chain polymer having reactive end groups. “Grafting from” involves growing a polymer from a functionalized polymer backbone with initiation sites. “Grafting by…” involves the synthesis of macromonomers that form the main-chain polymer after subsequent polymerization. These grafting techniques are summarized, for example, in Maity, N., et al., “Conducting Polymer Grafting: Recent and Key Developments,” Polymers 12(3):709 (2020), which is incorporated herein by reference in its entirety.

[0192] In some applications, bottle brush polymers are synthesized using a "grafting" method. The prepolymer (also known as a "macromonomer") is first synthesized, for example, via ATRP, and then conjugated / bonded with a drug, such as a chemotherapy drug, via click chemistry. The prepolymer is then attached to the polymer backbone. Figure 1A An exemplary process is shown. In some aspects, the macromonomer has the following structure:

[0193] Where n and m are positive integers. In some respects, the drug is conjugated to a macromonomer via an azide group. In some respects, the macromonomer has a molecular weight of about 5,000 to about 8,000. In some respects, the polymer backbone is formed by polymerizing a methacrylate-macromonomer by attaching methacrylate groups to the macromonomer. The methacrylate can be attached before or after the drug is conjugated to the macromonomer.

[0194] On the other hand, bottle brush polymers are synthesized using a "grafting" method. First, a prepolymer (also called a "macromonomer") is synthesized, for example via ATRP. Then, the macromonomer is attached to methacrylate groups, followed by conjugation with a drug, such as a chemotherapy drug, via click chemistry. The prepolymer is then attached to the polymer backbone. Figure 1A An exemplary process is shown. In some aspects, the macromonomer has the following structure:

[0195] Where n and m are positive integers. In some respects, the drug is conjugated to a macromonomer via an azide group. In some respects, the macromonomer has a molecular weight of about 5,000 to about 8,000 kDa.

[0196] On the other hand, bottle brush polymers are synthesized by first synthesizing a prepolymer containing an initiator (also known as a "macromolecule initiator") via RAFT polymerization, then grafting a copolymer onto the prepolymer via ATRP, and finally conjugating the copolymer formed via ATRP with a drug via click chemistry. Figure 2A-2B An exemplary process is illustrated. In some aspects, the macromolecular initiator has the following structure:

[0197] In some aspects, bottle brush polymers are synthesized by first synthesizing a prepolymer containing an initiator (also known as a "macromolecule initiator") via RAFT polymerization, wherein the prepolymer is synthesized by polymerizing one or more methacrylate monomers with a chain transfer agent (CTA). In some aspects, the CTA is 4-cyano-4-((phenylcarbonylthio)thio)pentanoic acid and has the following structure: .

[0198] In some respects, CTA is 2,5-dioxopyrrolidone-1-yl4-cyano-4-((phenylcarbonylthio)thio)valerate and has the following structure: .

[0199] In some respects, CTA is 2-(((butyrothio)carbonylthio)thio)propionic acid and has the following structure: .

[0200] In some respects, non-PEG prepolymers are first synthesized by polymerizing methacrylate monomers containing initiators with one or more other methacrylate monomers using CTA.

[0201] In some aspects, the ratio between one or more other methacrylate monomers and the methacrylate monomer containing the 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 The ratios are 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, 2.4:1.0, 2.5:1.0, 2.6:1.0, 2.7:1.0, 2.8:1.0, 2.9:1.0, or 3.0:1.0. In some aspects, the ratio between one or more other methacrylate monomers and the methacrylate monomer containing the 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 respects, the ratio between one or more other methacrylate monomers and the methacrylate monomer containing the initiator is 1.0:1.0, 2.0:1.0, or 3.0:1.0.

[0202] On the other hand, non-PEG prepolymers containing methacrylate monomers with initiators and one or more other methacrylate monomers have monomers with random "r" classification in the prepolymer. In some aspects, the random "r" classification of monomers in non-PEG prepolymers has the following structure: .

[0203] Macromolecular initiators with structures similar to the above, containing different integer numbers of repeating units, were also considered. In some aspects, the bottle brush polymer formed after grafting the copolymer onto the macromolecular initiator via ATRP has the following structure:

[0204] in n is an integer selected from 1 to 500; m is an integer selected from 1 to 500; k is an integer selected from 1 to 450; and l is an integer selected from 1 to 50.

[0205] In some respects, such as the compounds in this section, the ratio between one or more other methacrylate monomers (n) and the methacrylate monomer containing the initiator (m) 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, 2.4:1.0, 2.5:1.0, 2.6:1.0, 2.7:1.0, 2.8:1.0, 2.9:1.0, or 3.0:1.0. In some respects, such as the compounds in this section, the ratio between one or more other methacrylate monomers (n) and the methacrylate monomer (m) containing the 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 respects, such as the compounds in this section, the ratio between one or more other methacrylate monomers (n) and the methacrylate monomer (m) containing the initiator is 1.0:1.0, 2.0:1.0, or 3.0:1.0.

[0206] In some respects, such as the compounds in this section, the ratio between one or more other methacrylate monomers (k) and a methacrylate monomer (l) containing an azide group 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, 23.5∶ 1, 24:1, 24.5:1, 25:1, 25.5:1, 26:1, 26.5:1, 27:1, 27.5:1, 28:1, 28.5:1, 29:1, 29.5:1, 30:1, 30.5:1, 31:1, 31.5:1, 32:1, 32.5:1, 33:1, 33.5:1, 34:1, 34.5:1, 35.5:1, 36:1, 36.5:1, 37:1 37.5∶1, 38∶1, 38.5∶1, 39∶1, 39.5∶1, 40∶1, 40.5∶1, 41∶1, 41.5∶1, 42∶1, 42.5∶1, 43∶1, 43.5∶1, 44∶1, 44.5∶1, 45∶1, 45.5∶1, 46∶1, 46.5∶1, 47∶1, 47.5∶1, 48∶1, 48.5∶1, 49∶1, 49.5∶1 or 50∶1. In some respects, such as the compounds in this section, the ratio between one or more other methacrylate monomers (k) and methacrylate monomers (l) containing 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.In some aspects, such as the compounds of this section, the ratio between one or more other methacrylate monomers (k) and a methacrylate monomer (l) containing 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, such as the compounds of this section, the ratio between one or more other methacrylate monomers (k) and a methacrylate monomer (l) containing an azide group is 5:1, 10:1, 15:1, 20:1, 25:1, or 30:1. In some aspects, such as the compounds of this section, the ratio between one or more other methacrylate monomers (k) and a methacrylate monomer (l) containing an azide group is 10:1 or 15:1. In some aspects, such as the compounds of this section, the ratio between one or more other methacrylate monomers (k) and a methacrylate monomer (l) containing an azide group is 10:1.

[0207] In some respects, the non-PEG bottle brush polymers formed after grafting copolymers onto macromolecular initiators via ATRP are synthesized by polymerizing methacrylate monomers containing azide groups with one or more other methacrylate monomers.

[0208] In some aspects, the ratio between one or more other methacrylate monomers and methacrylate monomers containing azide groups is 1:1, 1.5:1, 2:1, 2.5:1, 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, 23.5∶1, 24∶1, 24.5∶ 1, 25:1, 25.5:1, 26:1, 26.5:1, 27:1, 27.5:1, 28:1, 28.5:1, 29:1, 29.5:1, 30:1, 30.5:1, 31:1, 31.5:1, 32:1, 32.5:1, 33:1, 33.5:1, 34:1, 34.5:1, 35:1, 35.5:1, 36:1, 36.5:1, 37:1, 37 0.5∶1, 38∶1, 38.5∶1, 39∶1, 39.5∶1, 40∶1, 40.5∶1, 41∶1, 41.5∶1, 42∶1, 42.5∶1, 43∶1, 43.5∶1, 44∶1, 44.5∶1, 45∶1, 45.5∶1, 46∶1, 46.5∶1, 47∶1, 47.5∶1, 48∶1, 48.5∶1, 49∶1, 49.5∶1 or 50∶1. In some aspects, the ratio between one or more other methacrylate monomers and methacrylate monomers containing azide groups 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. 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. In some aspects, the ratio between one or more other methacrylate monomers and the methacrylate monomer containing 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 one or more other methacrylate monomers and the methacrylate monomer containing an azide group is 5:1, 10:1, 15:1, 20:1, 25:1, or 30:1. In some aspects, the ratio between one or more other methacrylate monomers and the methacrylate monomer containing an azide group is 10:1 or 15:1. In some aspects, the ratio between one or more other methacrylate monomers and the methacrylate monomer containing an azide group is 10:1.

[0209] In some aspects, the ratio between one or more other methacrylate monomers and the methacrylate monomer containing an azide group is 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. In some aspects, the ratio between one or more other methacrylate monomers and the methacrylate monomer containing 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. In some aspects, the ratio between one or more other methacrylate monomers and methacrylate monomers containing azide groups 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. In some aspects, the ratio between one or more other methacrylate monomers and methacrylate monomers containing azide groups 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. In some aspects, the ratio between one or more other methacrylate monomers and the methacrylate monomer containing 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. In some aspects, the ratio between one or more other methacrylate monomers and the methacrylate monomer containing an azide group ranges from about 15:1 to 50:1, about 15:1 to 45:1, about 15:1 to 40:1, about 15:1 to 35:1, about 15:1 to 30:1, about 15:1 to 25:1, or about 15:1 to 20:1.

[0210] , in n is an integer selected from 1 to 500; m is an integer selected from 1 to 500; k is an integer selected from 1 to 450; and l is an integer selected from 1 to 50.

[0211] In some respects, the drug binds to the bottle brush polymer via the azide group in the structure described above. Bottle brush polymers with similar structures to those described above, containing different integer numbers of the repeating units shown, are also considered.

[0212] Click chemistry refers to the rapid and irreversible linkage of two reagents (e.g., polymers and drugs) in a reaction (typically a one-pot reaction). Various click chemical reactions, such as copper-catalyzed azide-alkyne cycloaddition (CuAAC), strain-promoted azide-alkyne cycloaddition (SPAAC), strain-promoted alkyne-nitroketone cycloaddition (SPANC), and other strained alkenes, have been used to link drugs to polymers. Click chemistry is discussed, for example, in Hein, CD, et al., Pharm Res25(10):2216-2230 (2008), Geng, Z., et al., J. Polymer Sci. 59(11):963-1042 (2021), which are incorporated herein by reference in their entirety.

[0213] In some respects, one or more drugs are coupled to non-PEG polymers via an azide-alkyne click reaction.

[0214] In some aspects, one or more drugs are coupled to non-PEG polymers via a strain-promoted azido-alkyne cycloaddition (SPAAC) click reaction. In some aspects, the strain-promoted azido-alkyne cycloaddition (SPAAC) click reaction occurs between an azide functional group and a dibenzocyclooctyne (DBCO) functional group. In some aspects, the dibenzocyclooctyne (DBCO) functional group is optionally substituted.

[0215] In some respects, the reagent is coupled to non-PEG bottle brush polymers via an azide-alkyne click reaction or an inverse electron-demanding Diels-Alder (IEDDA) reaction.

[0216] In some aspects, the azide-alkyne click reaction is a copper-catalyzed azide-alkyne cycloaddition (CuAAC), a strain-promoted azide-alkyne cycloaddition (SPAAC), or a strain-promoted alkyne-nitroketone cycloaddition (SPANC). In some aspects, the azide-alkyne click reaction is a copper-catalyzed azide-alkyne cycloaddition (CuAAC). In some aspects, the azide-alkyne click reaction is a strain-promoted azide-alkyne cycloaddition (SPAAC) or a strain-promoted alkyne-nitroketone cycloaddition (SPANC).

[0217] Reverse electron demand Diels-Alder (IEDDA) refers to a rapid and irreversible reaction that links two reagents, such as a polymer and a reagent. This reaction follows the typical Diels-Alder reaction, which utilizes the reaction between an electron-deficient diene and an electron-rich dienophile to form a new ring coupling the two reagents. Various IEDDA reactions, such as tetrazine and trans-cyclooctene, are used to link reagents to polymers. IEDDA reactions are further discussed, for example, in Oliveira et al., Chemical Society Reviews 46(16):4895-4950 (2017), and Zhang, et al., J. Org. Chem. 84(15): 9397-9445 (2019), the entire contents of which are incorporated herein by reference.

[0218] In some respects, the reagent is coupled to non-PEG brush polymers via an inverse electron demand Diels-Alder (IEDDA) reaction.

[0219] In some respects, the reverse electron-demanding Diels-Alder (IEDDA) reaction is tetrazine-cyclooctene.

[0220] "Non-PEG polymer" or "non-PEG bottle brush polymer" refers to the following bottle brush polymers: (a) Does not contain ethylene glycol, i.e., the -OCH2CH2- unit; or (b) Includes 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 polymer brush is an integer from 3 to 9. In some aspects, the non-PEG polymer comprises one or more monomers, including but not limited to triethylene glycol methyl ether methacrylate (TEOMA), 2-(methylsulfinyl)ethyl methacrylate (MSEMA), 1-phenoxycarbonyl ethyl methacrylate, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate (PCMA), 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate (CBMA), 2-((2-bromo-2-methylpropionyl)oxy)ethyl methacrylate (BMPOEMA), 2-(2- Bromo-2-methylpropionamido)ethyl methacrylate (BMPAEMA), polyethylene glycol monomethyl ether methacrylate (PEGMA) (where the number of ethylene glycol units is an integer from 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), PEGMA 300 PEGMA 500 2-Dimethylaminoethyl methacrylate (DMAEMA), sulfobetaine methacrylate (SBMA), quaternary ammonium ethyl methacrylate, hydroxyethyl methacrylate (HEMA), 2-azidoethyl methacrylate, 2-azidotriethylene glycol methyl ether methacrylate (TEOMA-azido compound or TEOMA-N3), 2-azidotetraethylene glycol methyl ether methacrylate (PEGMA(n=4)-azido compound or PEGMA(n=4)-N3), 2-azidopentaethylene glycol methyl ether methacrylate (PEGMA(n=5)-azido compound or PEGMA(n=5)-azido compound or PEGMA(n=4)-N3). PEGMA(n=5)-N3, PEGMA(n=6)-azido or PEGMA(n=6)-N3, PEGMA(n=7)-azido or PEGMA(n=7)-N3, PEGMA(n=8)-azido or PEGMA(n=8)-N3, and PEGMA(n=9)-N3, or combinations thereof.

[0221] In some aspects, non-PEG polymers include one or more polymers, including but not limited to poly(triethylene glycol methyl ether methacrylate) (pTEOMA), poly(polyethylene glycol (meth)acrylate) (pPEGMA) (wherein the number of glycol units is an integer from 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), and pPEGMA. 300 pPEGMA 500 Poly(2-(methylsulfinyl)ethyl methacrylate) (pMSEMA), poly(3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate (pPCMA), poly(3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate (pCBMA), poly(2-((2-bromo-2-methylpropionyl)oxy)ethyl methacrylate (pBMPOEMA), poly(2-(2-bromo-2-methylpropionamido)ethyl methacrylate) (pBMPAEMA), poly(2-dimethylaminoethyl methacrylate) (pDMAEMA), poly(sulfobetaine methacrylate) (pSBMA), poly(quaternary ammonium ethyl methacrylate), poly(hydroxyethyl methacrylate) (pHEMA), poly(2-azidoethyl methacrylate), poly(2-azidoethyl methacrylate), poly(2-azidotriethylene glycol methyl ether methacrylate) (pPEGMA-azido compound or pPEGMA-N3), poly(2-azidotetraethylene glycol methyl ether methacrylate) (pPEGMA(n=4)-azido compound or pPEGMA(n=4)-N3), poly(2-azidopentaneethylene glycol methyl ether methacrylate) (pPEGMA(n=5)-azido compound or pPEGMA(n=5)-N3), poly(2-azidohexaethylene glycol methyl ether Poly(2-azidoheptaethylene glycol methyl ether methacrylate) (pPEGMA(n=6)-azido or pPEGMA(n=6)-N3), poly(2-azidoheptaethylene glycol methyl ether methacrylate) (pPEGMA(n=7)-azido or pPEGMA(n=7)-N3), poly(2-azidooctaethylene glycol methyl ether methacrylate) (pPEGMA(n=8)-azido or pPEGMA(n=8)-N3) and poly(2-azidononethylene glycol methyl ether methacrylate) (pPEGMA(n=9)-azido or pPEGMA(n=9)-N3), or combinations thereof.

[0222] In some respects, non-PEG polymers may also refer to polymers that resist reactions with subject-derived PEG antibodies, thus resisting accelerated blood clearance and allergic reactions to bioconjugates induced by subject-derived PEG antibodies. In some respects, the compositions and methods described herein can be used to deliver antigenically reduced or eliminated molecules, thereby addressing pre-existing anti-PEG antibodies in mammals, including humans, that may compromise the safety and efficacy of PEGylated therapeutic agents. In some respects, the non-PEGylated bottle brush polymers and bioconjugates detailed herein comprise polymers resistant to accelerated blood clearance and allergic reactions to bioconjugates induced by subject-derived PEG antibodies.

[0223] In some respects, the non-PEG polymer resistant to subject-derived PEG antibodies is a polymer comprising one or more monomers, including but not limited to triethylene glycol methyl ether methacrylate (TEOMA), 2-(methylsulfinyl)ethyl methacrylate (MSEMA), 1-phenoxycarbonyl ethyl methacrylate, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate (PCMA), 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate (CBMA), and 2-((2-bromo-2-methylpropionyl)oxy)ethyl methacrylate (B... MPOEMA), 2-(2-bromo-2-methylpropionamido)ethyl methacrylate (BMPAEMA), polyethylene glycol monomethyl ether methacrylate (PEGMA) (where the number of ethylene glycol units is an integer from 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), PEGMA 300 PEGMA 5002-Dimethylaminoethyl methacrylate (DMAEMA), sulfobetaine methacrylate (SBMA), quaternary ammonium ethyl methacrylate, hydroxyethyl methacrylate (HEMA), 2-azidoethyl methacrylate, 2-azidotriethylene glycol methyl ether methacrylate (TEOMA-azido compound or TEOMA-N3), 2-azidotetraethylene glycol methyl ether methacrylate (PEGMA(n=4)-azido compound or PEGMA(n=4)-N3), 2-azidopentaethylene glycol methyl ether methacrylate (PEGMA(n=5)-azido compound or PEGMA(n=5)-azido compound or PEGMA(n=4)-N3). PEGMA(n=5)-N3, PEGMA(n=6)-azido or PEGMA(n=6)-N3, PEGMA(n=7)-azido or PEGMA(n=7)-N3, PEGMA(n=8)-azido or PEGMA(n=8)-N3, and PEGMA(n=9)-N3, or combinations thereof.

[0224] In some aspects, non-PEG polymers resistant to subject-derived PEG antibodies include polymers including, but 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 from 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), and pPEGMA. 300 pPEGMA 500Poly(2-(methylsulfinyl)ethyl methacrylate) (pMSEMA), poly(3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate) (pPCMA), poly(3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate) (pCBMA), poly(2-((2-bromo-2-methylpropionyl)oxy)ethyl methacrylate) (pBMPOEMA), poly(2-(2-bromo-2-methylpropionamido)ethyl methacrylate) (pBMPAEMA), poly(2-dimethylaminoethyl methacrylate) (pDMAEMA), poly(sulfobetaine methacrylate) (pSBMA), poly(quaternary ammonium ethyl methacrylate), poly(hydroxyethyl methacrylate) (pHEMA), poly(2-azidoethyl methacrylate), poly(2-azidoethyl methacrylate), poly(2-azidotriethylene glycol methyl ether methacrylate) (pBMPAEMA), poly(2-bromo-2-methylpropionyl)ethyl methacrylate, ...bromotriethylene glycol methyl ether methacrylate) (pBMPAEMA), poly(2-bromo-2-methylpropionyl)ethyl methacrylate, poly(2-bromotriethylene glycol methyl ether methacrylate) (pBMPAEMA), poly(2-bromo-2-methyl pPEGMA-azide pTEOMA-N3), poly(2-azidotetraethylene glycol methyl ether methacrylate) (pPEGMA(n=4)-azide or pPEGMA(n=4)-N3), poly(2-azidopentaethylene glycol methyl ether methacrylate) (pPEGMA(n=5)-azide or pPEGMA(n=5)-N3), poly(2-azidohexaethylene glycol methyl ether methacrylate) (pPEGMA(n=6)-azide or pPEGMA( (n=6)-N3), poly(2-azidoheptaethylene glycol methyl ether methacrylate) (pPEGMA(n=7)-azido or pPEGMA(n=7)-N3), poly(2-azidooctaethylene glycol methyl ether methacrylate) (pPEGMA(n=8)-azido or pPEGMA(n=8)-N3), and poly(2-azidononethylene glycol methyl ether methacrylate) (pPEGMA(n=9)-azido or pPEGMA(n=9)-N3), or combinations thereof.

[0225] In some respects, non-PEG bottle brush polymers are synthesized from monomers selected from TEOMA, MSEMA, PCMA, CBMA, and combinations thereof.

[0226] In some aspects, the non-PEG bottle brush polymer is composed of monomers selected from TEOMA, MSEMA, PCMA, CBMA, 2-((2-bromo-2-methylpropionyl)oxy)ethyl methacrylate (BMPOEMA), PEGMA (n=3-9), DMAEMA, SBMA, N,N-dimethylaminoethyl methacrylate, quaternary ammonium ethyl methacrylate, hydroxyethyl methacrylate, 2-azidoethyl methacrylate, 2-azidotriethylene glycol methyl ether methacrylate (TEOMA-azido compound or TEOMA-N3), 2-azidotetraethylene glycol methyl ether methacrylate (PEGMA(n=4)-azido compound or PEGMA(n=4)-N3), 2 2-Azide-pentaethylene glycol methyl ether methacrylate (PEGMA(n=5)-azido or PEGMA(n=5)-N3), 2-Azide-hexaethylene glycol methyl ether methacrylate (PEGMA(n=6)-azido or PEGMA(n=6)-N3), 2-Azide-heptaethylene glycol methyl ether methacrylate (PEGMA(n=7)-azido or PEGMA(n=7)-N3), 2-Azide-octaethylene glycol methyl ether methacrylate (PEGMA(n=8)-azido or PEGMA(n=8)-N3), and 2-Azide-nonaethylene glycol methyl ether methacrylate (PEGMA(n=9)-azido or PEGMA(n=9)-N3), or combinations thereof.

[0227] In some respects, bottle brush polymers are synthesized from zwitterionic monomers or zwitterionic polymers, which contain equal amounts of cationic and anionic charges along their monomeric or polymeric chains, respectively. The cationic charge typically arises from the presence of quaternary ammonium groups, while the anionic charge typically arises from the presence of sulfonate, carboxylate, 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 high dipole moments and highly charged groups, resulting in increased hydrophilicity compared to polymers containing PEG, an amphiphilic polymer with both hydrophilic and hydrophobic properties.

[0228] In some respects, the bottle brush polymer is synthesized from one or more zwitterionic monomers selected from 2-(methacryloyloxy)ethyl 2-(trimethylammonium)ethyl phosphate (PCMA), 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate (CBMA), sulfobetaine methacrylate (SBMA), or combinations thereof.

[0229] In some respects, the non-PEG polymer comprises one or more zwitterionic polymers selected from poly(2-(methacryloyloxy)ethyl 2-(trimethylammonium)ethyl phosphate) (PCMA), poly(3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate) (pCBMA), poly(sulfobetaine methacrylate) (pSBMA), or combinations thereof.

[0230] TEOMA is triethylene glycol methyl ether methacrylate, which has the following structure: .

[0231] In some respects, the structure of TEOMA can also be represented by the following structure: .

[0232] MSEMA is 2-(methylsulfinyl)ethyl methacrylate, which has the following structure: .

[0233] PCMA is 2-(methacryloyloxy)ethyl 2-(trimethylammonium)ethyl phosphate, which has the following structure: .

[0234] CBMA is 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate, which has the following structure: .

[0235] In some aspects, the non-PEG bottle brush polymer is synthesized by ATRP from a monomer comprising at least one initiator molecule for atom transfer radical polymerization (ATRP) and one or more monomer units. In some aspects, the monomer comprising at least one initiator molecule for atom transfer radical polymerization (ATRP) and one or more monomer units for ATRP is an initiator monomer. In some aspects, the initiator monomer is selected from 2-((2-bromo-2-methylpropionyl)oxy)ethyl methacrylate (BMPOEMA) and 2-(2-bromo-2-methylpropionamido)ethyl methacrylate (BMPAEMA) or combinations thereof.

[0236] BMPOEMA is 2-((2-bromo-2-methylpropionyl)oxy)ethyl methacrylate, which has the following structure: .

[0237] BMPAEMA is 2-(2-bromo-2-methylpropionamido)ethyl methacrylate, which has the following structure: .

[0238] PEGMA is polyethylene glycol monomethyl ether methacrylate, which has the following structure:

[0239] Where n is an integer between 3 and 9.

[0240] In some aspects, the monomer is PEGMA, and n is an integer from 3 to 9. In some aspects, the monomer is PEGMA, and n is an integer between 3 and 8. In some aspects, the monomer is PEGMA, and n is an integer between 3 and 7. In some aspects, the monomer is PEGMA, and n is an integer between 3 and 6. In some aspects, the monomer is PEGMA, and n is an integer between 3 and 5.

[0241] In some respects, the monomer is PEGMA, and n is an integer of 3. In some respects, the monomer is PEGMA, and n is an integer of 4. In some respects, the monomer is PEGMA, and n is an integer of 5. In some respects, the monomer is PEGMA, and n is an integer of 6. In some respects, the monomer is PEGMA, and n is an integer of 7. In some respects, the monomer is PEGMA, and n is an integer of 8. In some respects, the monomer is PEGMA, and n is an integer of 9.

[0242] TetEOMA is a tetraethylene glycol methyl ether methacrylate with the following structure: .

[0243] PEOMA is pentaethylene glycol methyl ether methacrylate, which has the following structure: .

[0244] HEOMA is hexaethylene glycol methyl ether methacrylate, which has the following structure: .

[0245] HPEOMA is heptaethylene glycol methyl ether methacrylate, which has the following structure: .

[0246] OEOMA is octaethylene glycol methyl ether methacrylate, which has the following structure: .

[0247] NEOMA is nonaethylene glycol methyl ether methacrylate, which has the following structure: .

[0248] In some aspects, the PEGMA monomer is selected from TEOMA, TetEOMA, PEOMA, HEOMA, HPEOMA, OEOMA, NEOMA, or combinations thereof. In some aspects, the PEG monomer is TEOMA. In some aspects, the PEG monomer is PEOMA. In some aspects, the PEG monomer is NEOMA.

[0249] DMAEMA is 2-dimethylaminoethyl methacrylate, which has the following structure: .

[0250] SBMA is sulfobetaine methacrylate, which has the following structure: .

[0251] Quaternary ammonium ethyl methacrylate has the following structure: .

[0252] In some respects, quaternary ammonium ethyl methacrylate possesses a balancing ion. In some respects, the balancing ion is a halogen. In some respects, the balancing ion is a bromide (Br). - ).

[0253] HEMA is hydroxyethyl methacrylate, which has the following structure: .

[0254] 2-Azide ethyl methacrylate has the following structure: .

[0255] In some respects, the monomer is replaced by a functional group that is reactive in click chemistry, such as an azide group (-N3).

[0256] In some aspects, the non-PEG bottle brush polymer backbone comprises one or more monomers selected from TEOMA, MSEMA, PCMA, CBMA, BMPOEMA, BMPAEMA, PEGMA (n=3-9), DMAEMA, SBMA, N,N-dimethylaminoethyl methacrylate, quaternary ammonium ethyl methacrylate, hydroxyethyl methacrylate, and 2-azidoethyl methacrylate, or combinations thereof. In some aspects, the non-PEG backbone of the bottle brush polymer comprises one or more monomers selected from BMPOEMA or BMPAEMA and at least one monomer selected from TEOMA, MSEMA, PCMA, CBMA, PEGMA (n=3-9), DMAEMA, SBMA, N,N-dimethylaminoethyl methacrylate, quaternary ammonium ethyl methacrylate, hydroxyethyl methacrylate, and 2-azidoethyl methacrylate, or combinations thereof. In some aspects, the non-PEG backbone of the bottle brush polymer comprises one or more monomers selected from BMPOEMA or BMPAEMA and at least one monomer selected from TEOMA, MSEMA, PCMA, CBMA, BMPOEMA, or BMPAEMA and PEGMA (n=3-9), or combinations thereof. In some aspects, the backbone of the non-PEG bottle brush polymer comprises one or more monomers selected from BMPOEMA or BMPAEMA and at least one other monomer selected from TEOMA, MSEMA, PCMA, CBMA, and PEGMA (n=3-9) or combinations thereof.

[0257] In some aspects, the non-PEG bottle brush polymer comprises one or more monomers containing an azide (-N3) functional group. In some aspects, the one or more monomers containing an azide group are selected from 2-azidoethyl methacrylate, 2-azidotriethylene glycol methyl ether methacrylate (TEOMA-azido compound or TEOMA-N3), 2-azidotetraethylene glycol methyl ether methacrylate (PEGMA(n=4)-azido compound or PEGMA(n=4)-N3), 2-azidopentaneethylene glycol methyl ether methacrylate (PEGMA(n=5)-azido compound or PEGMA(n=5)-N3), 2-azidohexaethylene glycol methyl ether methacrylate (PEGMA(n=6)-azido compound or PEGMA(n=6)-N3), and 2-azidoheptaethylene glycol methyl ether methacrylate (PEGMA(n=7)-azido compound or PEGMA(n=7)-N3). 2-Azide-octaethylene glycol methyl ether methacrylate (PEGMA(n=8)-azido or PEGMA(n=8)-N3), and 2-azido-nonethylene glycol methyl ether methacrylate (PEGMA(n=9)-azido or PEGMA(n=9)-N3), or combinations thereof. In some aspects, one or more monomers containing an azide group are selected from TEOMA-azido and PEGMA(n=5)-azido. In some aspects, the monomer containing an azide group is TEOMA-azido. In some aspects, the monomer containing an azide group is PEGMA(n=5)-azido.

[0258] TEOMA-azide (or TEOMA-N3) is 2-azidotriethylene glycol methyl ether methacrylate, which has the following structure: .

[0259] In some respects, the structure of TEOMA can also be represented by the following structure: .

[0260] PEGMA(n=4)-azide (or PEGMA(n=4)-N3) is 2-azidotetraethylene glycol methyl ether methacrylate, which has the following structure: .

[0261] PEGMA(n=5)-azide (or PEGMA(n=5)-N3) is 2-azidopentaethylene glycol methyl ether methacrylate, which has the following structure: .

[0262] PEGMA(n=6)-azide (or PEGMA(n=6)-N3) is 2-azidohexaethylene glycol methyl ether methacrylate, which has the following structure: .

[0263] PEGMA(n=7)-azide (or PEGMA(n=7)-N3) is 2-azidoheptaethylene glycol methyl ether methacrylate, which has the following structure: .

[0264] PEGMA(n=8)-azide (or PEGMA(n=8)-N3) is a 2-azido-octaethylene glycol methyl ether with the following structure: .

[0265] PEGMA(n=9)-azide (or PEGMA(n=9)-N3) is 2-azido-nonethylene glycol methyl ether methacrylate, which has the following structure: .

[0266] In some aspects, the non-PEG bottle brush polymers or bioconjugates described herein contain polymers resistant to the accelerated blood clearance of the bioconjugates and to allergic reactions caused by subject-derived PEG antibodies. In some aspects, the non-PEG bottle brush polymers or bioconjugates do not react with pre-existing anti-PEG antibodies in the subject. In some aspects, the non-PEG bottle brush polymers or bioconjugates comprise PEGMA, wherein the number of ethylene glycol units is an integer from 3 to 9, and the bottle brush polymers or bioconjugates do not react with pre-existing anti-PEG antibodies in the subject. In some aspects, the non-PEG bottle brush polymer or bioconjugate comprises one or more monomers selected from triethylene glycol methyl ether methacrylate (TEOMA), 2-(methylsulfinyl)ethyl methacrylate (MSEMA), 1-phenoxycarbonyl ethyl methacrylate, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate (PCMA), 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate (CBMA), 2-((2-bromo-2-methylpropionyl)oxy)ethyl methacrylate (BMPOEMA), 2 -(2-bromo-2-methylpropionamido)ethyl methacrylate (BMPAEMA), polyethylene glycol monomethyl ether methacrylate (PEGMA) (where the number of ethylene glycol units is an integer from 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 EGMA 500 2-Dimethylaminoethyl methacrylate (DMAEMA), sulfobetaine methacrylate (SBMA), N,N-dimethylaminoethyl methacrylate, quaternary ammonium ethyl methacrylate, hydroxyethyl methacrylate, hydroxyethyl methacrylate (HEMA), 2-azidoethyl methacrylate, 2-azidotriethylene glycol methyl ether methacrylate (TEOMA-azido compound or TEOMA-N3), 2-azidotetraethylene glycol methyl ether methacrylate (PEGMA(n=4)-azido compound or PEGMA(n=4)-N3), 2-azidopentaethylene glycol methyl ether methacrylate (PEGMA(n=5)-azido compound or PEGMA(n... =5)-N3), 2-azidohexaethylene glycol methyl ether methacrylate (PEGMA(n=6)-azido or PEGMA(n=6)-N3), 2-azidoheptaethylene glycol methyl ether methacrylate (PEGMA(n=7)-azido or PEGMA(n=7)-N3), 2-azidooctaethylene glycol methyl ether methacrylate (PEGMA(n=8)-azido or PEGMA(n=8)-N3) and 2-azidononethylene glycol methyl ether methacrylate (PEGMA(n=9)-azido or PEGMA(n=9)-N3), and the bottle brush polymer or bioconjugate does not react with the pre-existing anti-PEG antibody in the subject.

[0267] In some aspects, non-PEG bottle brush polymers or bioconjugates contain ethylene glycol units and still retain resistance to the accelerated blood clearance of the bioconjugate and allergic reactions caused by subject-derived PEG antibodies. In some aspects, non-PEG bottle brush polymers include polymers containing ethylene glycol units, and such polymers are selected from poly(triethylene glycol methyl ether methacrylate) (pTEOMA), poly(ethylene glycol (meth)acrylate) (pPEGMA), wherein the number of ethylene glycol units is an integer from 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), and pPEGMA. 300 pPEGMA 500Poly(2-(methylsulfinyl)ethyl methacrylate) (pMSEMA), poly(3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate) (pPCMA), poly(3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate) (pCBMA), poly(2-((2-bromo-2-methylpropionyl)oxy)ethyl methacrylate) (pBMPOEMA), poly(2-(2-bromo-2-methylpropionamido)ethyl methacrylate) (pBMPAEMA), poly(2-dimethylaminoethyl methacrylate) (pDMAEMA), poly(sulfobetaine methacrylate) (pSBMA), poly(quaternary ammonium ethyl methacrylate), poly(hydroxyethyl methacrylate) (pHEMA), poly(2-azidoethyl methacrylate), poly(2-azidoethyl methacrylate), poly(2-azidotriethylene glycol methyl ether methacrylate) (pTEOMA) -Azide or pTEOMA-N3), poly(2-azidotetraethylene glycol methyl ether methacrylate) (pPEGMA(n=4)-Azide or pPEGMA(n=4)-N3), poly(2-azidopentaethylene glycol methyl ether methacrylate) (pPEGMA(n=5)-Azide or pPEGMA(n=5)-N3), poly(2-azidohexaethylene glycol methyl ether methacrylate) (pPEGMA(n=6)-Azide or pPEGMA(n=6) Poly(2-azidoheptaethylene glycol methyl ether methacrylate) (pPEGMA(n=7)-azido or pPEGMA(n=7)-N3), poly(2-azidooctaethylene glycol methyl ether methacrylate) (pPEGMA(n=8)-azido or pPEGMA(n=8)-N3), and poly(2-azidononethylene glycol methyl ether methacrylate) (pPEGMA(n=9)-azido or pPEGMA(n=9)-N3)) or combinations thereof.

[0268] This disclosure provides a method for preparing bioconjugates comprising the non-PEG polymers described herein.

[0269] On one hand, the present invention provides a method for preparing bioconjugates, comprising: (a) Preparation of a non-PEG polymer backbone comprising a chain transfer agent (CTA) and one or more monomer units via reversible addition-fragmentation chain transfer (RAFT) polymerization. At least one of the monomer units contains an initiator molecule for atom transfer radical polymerization (ATRP); (b) Prepare a non-PEG polymer comprising the non-PEG polymer backbone of (a) and one or more monomer units by ATRP. At least one of the monomer units contains a functional group capable of performing a click response; (c) The first drug is coupled to the non-PEG polymer of (b) via a functional group capable of click reaction; and (d) The reagent that binds to cancer-associated antigens is coupled to the non-PEG polymer in (c).

[0270] On the one hand, this disclosure provides a method for preparing biological conjugates, comprising: (a) Preparation of a non-PEG polymer backbone comprising a chain transfer agent (CTA) molecule and one or more monomer units via reversible addition-fragmentation chain transfer (RAFT) polymerization; At least one of the monomer units contains an initiator molecule for atom transfer radical polymerization (ATRP); (b) Couple a reagent that binds to cancer-associated antigens to the non-PEG polymer backbone of (a); (c) Prepare a non-PEG polymer comprising the non-PEG polymer backbone of (b) and one or more monomer units by ATRP. At least one of the monomeric units contains a functional group capable of performing a click reaction; and (d) The first drug is coupled to the non-PEG polymer of (c) by a functional group capable of click reaction.

[0271] On the one hand, this disclosure provides a method for preparing biological conjugates, comprising: (a) Preparation of a non-PEG polymer by ATRP, the polymer comprising a molecule containing at least one initiator molecule for atom transfer radical polymerization (ATRP) and one or more monomer units. At least one of the monomer units contains a functional group capable of performing a click response; (b) Prepare a non-PEG polymer bottle brush by ATRP comprising (a) a non-PEG polymer, a molecule containing at least one initiator molecule for ATRP and one or more monomer units; (c) The first drug is coupled to the non-PEG polymer of (b) via a functional group capable of click reaction; and (d) The reagent that binds to cancer-associated antigens is coupled to the non-PEG polymer in (c).

[0272] In some aspects of the above methods, the non-PEG polymer of (a) is coupled to a molecule containing a methacrylate functional group. In some aspects of the above methods, the non-PEG polymer of (a) contains a methacrylate functional group.

[0273] On one hand, the present invention provides a method for preparing bioconjugates, comprising: (a) Preparation of a non-PEG polymer by ATRP, the polymer comprising a molecule containing at least one initiator molecule for atom transfer radical polymerization (ATRP) and one or more monomer units. At least one of the monomer units contains a functional group capable of performing a click response; (b) The first drug is coupled to the non-PEG polymer of (a) by a functional group capable of click reaction; (c) Preparing a non-PEG polymer bottle brush comprising (b) the non-PEG polymer, a molecule containing at least one initiator molecule for ATRP, and one or more monomer units via ATRP; and (d) The reagent that binds to cancer-associated antigens is coupled to the non-PEG polymer in (c).

[0274] In some aspects, the non-PEG polymer of (a) is coupled to a molecule containing a methacrylate functional group. In some aspects, the non-PEG polymer of (a) contains a methacrylate functional group. In some aspects, the non-PEG polymer of (b) is coupled to a molecule containing a methacrylate functional group. In some aspects, the non-PEG polymer of (b) contains a methacrylate functional group.

[0275] V. Connector Typically, the bioconjugates described herein comprise a non-PEG polymer having a linker for attaching the drug to the non-PEG polymer and a band containing a linker for attaching the reagent to the non-PEG polymer. The linker may be a protease-cleavable linker, an acid-cleavable linker, a disulfide linker, or a self-stabilizing linker. In some respects, the linker is cleavable under intracellular conditions, thus cleavage of the linker releases the drug from the reagent in the intracellular environment.

[0276] In some respects, the linker can be cleaved by cleaving agents present in the intracellular environment (e.g., in lysosomes, endosomes, or cisterns). The linker can be, for example, a peptide linker cleaved by intracellular peptidases or proteases, including but not limited to lysosomal or endosomal proteases. Typically, the peptide linker is at least two amino acids long or at least three amino acids long. Cleavage agents can include cathepsins B and D, as well as plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of the active drug into the target cell (see, for example, Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). In specific respects, peptide linkers cleavable by intracellular proteases are Val-Cit linkers or Phe-Lys linkers (see, for example, US Patent 6,214,345, which describes a method for synthesizing doxorubicin using a Val-Cit linker, which is incorporated herein by reference). One advantage of using intracellular proteolytic release of the drug is that drug activity is generally reduced when conjugated, and the serum stability of the conjugate is generally high. See also U.S. Patent No. 9,345,785, which is incorporated herein by reference.

[0277] In some respects, cleavable adapters are pH-sensitive, meaning they are sensitive to hydrolysis at certain pH values. Typically, pH-sensitive adapters are hydrolyzable under acidic conditions. For example, acid-labile adapters that are hydrolyzable in lysosomes (e.g., hydrazones, thioureas, cis-aconitamides, orthoesters, acetals, ketals, etc.) can be used. (See, for example, US Patent Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, PharmaTherapeutics 83:67-123; Neville et at, 1989, Biol. Chem. 264:14653-14661, all of which are incorporated herein by reference.) Such adapters are relatively stable under neutral pH conditions, such as in blood, but unstable below pH 5.5 or 5.0 (the approximate pH of lysosomes). In some respects, the hydrolyzable linker is a thioether linker (e.g., a thioether linked to a drug via an acylhydrazone bond (see, for example, U.S. Patent 5,622,929, which is incorporated herein by reference).

[0278] In some respects, the linker can be cleaved under reducing conditions (e.g., disulfide linkers). A variety of disulfide linkers are known, including those that can be formed using, for example, SATA (N-succinimidyl-5-acetylthioacetate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene), or a combination of SPDB and SMPT (see, for example, Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW Vogel ed., Oxford U. Press, 1987. See also US Patent No. 4,880,935; all of which are incorporated herein by reference).

[0279] In some respects, the linker unit is non-cleavable, and the drug is released through reagent degradation. (See US Publication 2005 / 0238649, which is incorporated herein by reference).

[0280] In some respects, the adapter is largely insensitive to the extracellular environment.

[0281] In some respects, adapters promote cell internalization. In some respects, adapters promote cell internalization when bound to reagents (e.g., antibodies, target proteins, therapeutic proteins). In other respects, adapters promote cell internalization when bound to drugs and reagents.

[0282] In some aspects, the drug is coupled to the polymer via a cleavable linker. In some aspects, the drug is coupled to the polymer via an amino acid linker. In some aspects, the chemotherapeutic drug is coupled to the polymer via a valine-citrulline (Val-Cit) linker. In some aspects, the chemotherapeutic drug is coupled to the polymer via a valine-citrulline-p-aminobenzylcarbamate (Val-Cit-Pab) linker. In some aspects, the valine-citrulline (Val-Cit) linker contains a p-aminobenzylcarbamate functional group. In some aspects, the chemotherapeutic drug is coupled to the polymer via an alanine-alanine (Ala-Ala) linker. In some aspects, the chemotherapeutic drug is coupled to the polymer via an alanine-alanine-p-aminobenzylcarbamate (Ala-Ala-Pab) linker. In some aspects, the alanine-alanine (Ala-Ala) linker contains a p-aminobenzylcarbamate functional group.

[0283] VI. Chain Typically, the bioconjugates described herein comprise a non-PEG polymer having a band that links the reagent to the non-PEG polymer. The band can be an incised band, an amino acid-incised band, an acid-incised band, a light-incised band, a peptidase-incised band, an esterase-incised band, or a disulfide-incised band, provided the compound or reagent remains active. Furthermore, the incised band can be a series of ethylene glycol repeating units, wherein the number of units is an integer between 5 and 25. In some respects, the band is incised under intracellular conditions.

[0284] In some respects, the chain band is substantially resistant to one or more acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, or disulfide bond cleavage, provided that the compound or reagent remains active. In some respects disclosed herein, the chain band contains cleavable portions.

[0285] In some aspects, the band contains a histidine tag (His tag), a glycine repeat unit sequence, GGGGS (SEQ ID NO: 32), glycine repeat unit sequence (GGGGS)2 (SEQ ID NO: 33), glycine repeat unit sequence (GGGGS)3 (SEQ ID NO: 5), glycine repeat unit sequence (GGGGS)4 (SEQ ID NO: 34), glycine repeat unit sequence (GGGGS)5 (SEQ ID NO: 35), and an ethylene glycol repeat unit, wherein the number of units is an integer between 5 and 25. In some aspects disclosed herein, the band contains an ethylene glycol repeat unit, wherein the number of units is an integer between 5 and 25. In some aspects disclosed herein, the band contains an ethylene glycol repeat unit, wherein the number of units is an integer between 7 and 25. In some aspects disclosed herein, the band contains an ethylene glycol repeat unit, wherein the number of units is an integer between 8 and 25. In some aspects disclosed herein, the band contains an ethylene glycol repeat unit, wherein the number of units is an integer between 9 and 25. In some aspects disclosed herein, the chain includes repeating ethylene glycol units, wherein the number of units is an integer between 5 and 20. In some aspects disclosed herein, the chain includes repeating ethylene glycol units, wherein the number of units is an integer between 5 and 15. In some aspects disclosed herein, the chain includes repeating ethylene glycol units, wherein the number of units is an integer between 10 and 25. In some aspects disclosed herein, the chain includes repeating ethylene glycol units, wherein the number of units is an integer between 15 and 25. In some aspects disclosed herein, the chain includes repeating ethylene glycol units, wherein the number of units is an integer between 15 and 20.

[0286] In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 5 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable by a controlled radical polymerization reaction, wherein the number of repeating units is an integer between 5 and 75. In some aspects disclosed herein, the chain includes monomers polymerizable by a controlled radical polymerization reaction, wherein the number of repeating units is an integer between 5 and 70. In some aspects disclosed herein, the chain includes monomers polymerizable by a controlled radical polymerization reaction, wherein the number of repeating units is an integer between 5 and 65. In some aspects disclosed herein, the chain includes monomers polymerizable by a controlled radical polymerization reaction, wherein the number of repeating units is an integer between 5 and 60. In some aspects disclosed herein, the chain includes monomers polymerizable by a controlled radical polymerization reaction, wherein the number of repeating units is an integer between 5 and 55. In some aspects disclosed herein, the chain includes monomers polymerizable by a controlled radical polymerization reaction, wherein the number of repeating units is an integer between 5 and 50. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 5 and 45. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 5 and 40. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 5 and 35. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 5 and 30. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 5 and 25. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 5 and 20. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 5 and 15. In some aspects disclosed herein, the chain includes monomers polymerizable via controlled radical polymerization, wherein the number of repeating units is an integer between 5 and 10. In some aspects disclosed herein, the chain includes monomers polymerizable via controlled radical polymerization, wherein the number of repeating units is an integer between 10 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable via controlled radical polymerization, wherein the number of repeating units is an integer between 15 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable via controlled radical polymerization, wherein the number of repeating units is an integer between 20 and 80.In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 25 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 30 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 35 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 40 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 45 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 50 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 55 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 60 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 65 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 70 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 75 and 80. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 10 and 20. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 20 and 30. In some aspects disclosed herein, the chain includes monomers polymerizable by controlled radical polymerization, wherein the number of repeating units is an integer between 30 and 40. In some aspects disclosed herein, the chain includes monomers polymerizable via controlled radical polymerization, wherein the number of repeating units is an integer between 40 and 50. In some aspects disclosed herein, the chain includes monomers polymerizable via controlled radical polymerization, wherein the number of repeating units is an integer between 50 and 60. In some aspects disclosed herein, the chain includes monomers polymerizable via controlled radical polymerization, wherein the number of repeating units is an integer between 60 and 70. In some aspects disclosed herein, the chain includes monomers polymerizable via controlled radical polymerization, wherein the number of repeating units is an integer between 70 and 80.

[0287] VII. Biological conjugates The bioconjugates of this disclosure (i.e., those comprising non-PEG polymers) can increase the effectiveness of the reagent by reducing anti-drug antibodies (ADAs) found when the PEGylation reagent is administered to a subject, improving biological stability, improving physicochemical properties, allowing for flexible routes of administration, and / or enhancing the manufacturability of the bioconjugates for treating a subject or patient.

[0288] This invention also relates to bioconjugates comprising reagents linked or conjugated to a non-polyethylene glycol (PEG) polymer, said reagents including antibodies, antibody fragments, functional equivalents, modified antibodies, other protein or polynucleotide reagents, and aspects thereof disclosed herein, wherein said non-PEG polymer is linked or conjugated to a drug. The non-PEG polymer consists of a backbone and hydrophilic side chains, the hydrophilic side chains containing reactive moieties suitable for drug conjugation (e.g., ...). Figure 1A Non-PEG polymers allow drug loadings to be combined at ultra-high drug-to-reagent ratios (DARs) and provide the use of hybrid loadings, high-efficiency loadings, and loadings with problematic properties (e.g., systemic toxicity, poor pharmacokinetics). Figures 3A-3B These non-PEG polymers, when conjugated with various drugs (e.g., loads), increase the therapeutic index of the bioconjugates. Specifically, the ultra-high DAR (100+ loads per agent) is suitable for low-expression targets (e.g., cancer), provides the ability to use mixed drug loads for synthetic lethal strategies, and the ability to use highly effective and precise drugs (e.g., loads) that are intolerable as systemic therapy, and improves tissue penetration compared to other bioconjugates in the art.

[0289] In some aspects, the bioconjugates of the present invention have formula D z -L1-P-L2-A, where D z =Drug, z is 1-1,100, L1 = Connector, P = Non-PEG polymer, L2 = Chain (which may include a connector), A = Reagent. In some respects, D z This can be any drug described herein. In some aspects, L1 can be any connector described herein. In some aspects, P can be any connector described herein. In some aspects, L2 can be any connector described herein. In some aspects, A can be any connector described herein.

[0290] In some aspects, the reagent (e.g., an antibody) is linked to or conjugated to a drug, as described herein, 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, 1,000:1, 1,050:1, or 1,100:1. In some aspects, the drug is a chemotherapeutic agent. In some aspects, the chemotherapeutic agent is a MEK inhibitor. In some aspects, the chemotherapeutic agent is a PI3K inhibitor. In some aspects, the chemotherapeutic agent is a CDK inhibitor. In some aspects, the chemotherapeutic agent is a CHK inhibitor. In some aspects, the chemotherapeutic agent is apitolisib. In some aspects, the chemotherapeutic agent is cobimetinib. In some cases, the chemotherapy drug is dinaciclib. In others, the chemotherapy drug is prexasertib.

[0291] In some respects, the reagent is linked or combined with two different drugs. In some respects, the reagent is linked or combined with two different drugs in a 1:1 ratio. In some respects, both different drugs are chemotherapy drugs.

[0292] In some aspects, the reagent is linked or conjugated with two different drugs, and the DAR of the two drug combinations is 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. In some aspects, the reagent is linked or conjugated with two different drugs (e.g., including one or more chemotherapeutic drugs), and the DAR of the two drug combinations is between 50 and 150:1. In some aspects, the reagent is linked or conjugated with two different drugs (e.g., including one or more chemotherapeutic drugs), and the DAR of the two drug combinations is between 150 and 250:1. In some aspects, the reagent is linked or conjugated with two different drugs (e.g., including one or more chemotherapy drugs), and the DAR of the two drug combinations is between 250 and 350:1. In some aspects, the reagent is linked or conjugated with two different drugs (e.g., including one or more chemotherapy drugs), and the DAR of the two drug combinations is 350 to 450:1. In some aspects, the reagent is linked or conjugated with two different drugs (e.g., including one or more chemotherapy drugs), and the DAR of the two drug combinations is between 450 and 550:1. In some aspects, the reagent is linked or conjugated with two different drugs (e.g., including one or more chemotherapy drugs), and the DAR of the two drug combinations is between 550 and 650:1. In some aspects, the reagent is linked or conjugated with two different drugs (e.g., including one or more chemotherapy drugs), and the DAR of the two drug combinations is between 650 and 750:1. In some aspects, the reagent is linked or conjugated with two different drugs (e.g., including one or more chemotherapy drugs), and the DAR of the two drug combinations is between 750 and 850:1. In some aspects, the reagent is linked or conjugated with two different drugs (e.g., including one or more chemotherapeutic drugs), and the DAR of the two drug combinations is between 850 and 950:1. In some aspects, the reagent is linked or conjugated with two different drugs (e.g., including one or more chemotherapeutic drugs), and the DAR of the two drug combinations is between 950 and 1,050:1. In some aspects, the reagent is linked or conjugated with two different drugs (e.g., including one or more chemotherapeutic drugs), and the DAR of the two drug combinations is between 1,050 and 1,150:1.

[0293] In some aspects, the reagent (e.g., an antibody) is linked or conjugated to two drugs. In some aspects, the reagent (e.g., an antibody) is linked or conjugated to two drugs in a 1:1 ratio. In some aspects, the reagent is linked or conjugated to two drugs in a 2:1 ratio. In some aspects, the reagent is linked or conjugated to two drugs in a 3:1 ratio. In some aspects, the reagent is linked or conjugated to two drugs in a 4:1 ratio. In some aspects, the reagent is linked or conjugated to two drugs in a 5:1 ratio. In some aspects, the reagent is linked or conjugated to two drugs in a 6:1 ratio. In some aspects, the reagent is linked or conjugated to two drugs in a 7:1 ratio. In some aspects, the reagent is linked or conjugated to two drugs in an 8:1 ratio. In some aspects, the reagent is linked or conjugated to two drugs in a 9:1 ratio. In some aspects, the reagent is linked or combined with two drugs, wherein the ratio of the two different drugs is 10:1. In some aspects, the reagent is linked or combined with two drugs, wherein the ratio of the two different drugs is 11:1. In some aspects, the reagent is linked or combined with two drugs, wherein the ratio of the two different drugs is 12:1. In some aspects, the reagent is linked or combined with two drugs, wherein the ratio of the two different drugs is 13:1. In some aspects, the reagent is linked or combined with two drugs, wherein the ratio of the two different drugs is 14:1. In some aspects, the reagent is linked or combined with two drugs, wherein the ratio of the two different drugs is 15:1. In some aspects, the reagent is linked or combined with two drugs, wherein the ratio of the two different drugs is 16:1. In some aspects, the reagent is linked or combined with two drugs, wherein the ratio of the two different drugs is 17:1. In some aspects, the reagent is linked or combined with two drugs, wherein the ratio of the two different drugs is 18:1. In some aspects, the reagent is linked or conjugated with two drugs, wherein the ratio of the two different drugs is 19:1. In some aspects, the reagent is linked or conjugated with two drugs, wherein the ratio of the two different drugs is 20:1. In some aspects, the reagent is linked or conjugated with two drugs, wherein the ratio of the two different drugs is from 1:1 to 5:1. In some aspects, the reagent is linked or conjugated with two drugs, wherein the ratio of the two different drugs is from 5:1 to 10:1. In some aspects, the reagent is linked or conjugated with two drugs, wherein the ratio of the two different drugs is from 10:1 to 15:1. In some aspects, the reagent is linked or conjugated with two drugs, wherein the ratio of the two different drugs is from 15:1 to 20:1.In some aspects, the reagent is linked or conjugated with two drugs, wherein the ratio of the two different drugs is from 1:1 to 10:1. In some aspects, the reagent is linked or conjugated with two drugs, wherein the ratio of the two different drugs is from 10:1 to 20:1. In some aspects, the reagent is linked or conjugated with two drugs, wherein the ratio of the two different drugs is from 1:1 to 20:1. In some aspects, both different drugs are chemotherapeutic agents. In some aspects, the chemotherapeutic agent is a MEK inhibitor and a PI3K inhibitor. In some aspects, the chemotherapeutic agent is a CDK inhibitor and a PI3K inhibitor. In some aspects, the chemotherapeutic agent is a CHK inhibitor and a PI3K inhibitor. In some aspects, the chemotherapeutic agent is a MEK inhibitor. In some aspects, the chemotherapeutic agent is a PI3K inhibitor. In some aspects, the chemotherapeutic agent is a CDK inhibitor. In some aspects, the chemotherapeutic agent is a CHK inhibitor. In some aspects, the chemotherapeutic agent is apitolisib. In some aspects, the chemotherapeutic agent is cobimetinib. In some aspects, the chemotherapeutic agent is dinaciclib. In some respects, the chemotherapy drug is presertin.

[0294] In some respects, the reagent is linked or combined with three different drugs. In some respects, the reagent is linked or combined with three different drugs in a ratio of 1:1:1. In some respects, all three different drugs are chemotherapy drugs.

[0295] In some aspects, the reagent is linked or conjugated with three different drugs, and the DAR of all combined drugs is 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. In some aspects, the reagent is linked or conjugated with three different drugs (e.g., including one or more chemotherapeutic drugs) in a drug combination DAR of 50-150:1. In some aspects, the reagent is linked or conjugated with three different drugs (e.g., including one or more chemotherapeutic drugs) in a drug combination DAR of 150-250:1. In some aspects, the reagent is linked or conjugated with three different drugs (e.g., including one or more chemotherapy drugs) in a drug combination DAR of 250-350:1. In some aspects, the reagent is linked or conjugated with three different drugs (e.g., including one or more chemotherapy drugs) in a drug combination DAR of 350-450:1. In some aspects, the reagent is linked or conjugated with three different drugs (e.g., including one or more chemotherapy drugs) in a drug combination DAR of 450-550:1. In some aspects, the reagent is linked or conjugated with three different drugs (e.g., including one or more chemotherapy drugs) in a drug combination DAR of 550-650:1. In some aspects, the reagent is linked or conjugated with three different drugs (e.g., including one or more chemotherapy drugs) in a drug combination DAR between 650-750:1. In some aspects, the reagent is linked or conjugated with three different drugs (e.g., including one or more chemotherapy drugs) in a drug combination DAR between 750-850:1. In some aspects, the reagent is linked or conjugated with three different drugs (e.g., including one or more chemotherapeutic drugs), and the drug combination has a DAR between 850 and 950:1. In some aspects, the reagent is linked or conjugated with three different drugs (e.g., including one or more chemotherapeutic drugs), and the drug combination has a DAR between 950 and 1,050:1. In some aspects, the reagent is linked or conjugated with three different drugs (e.g., including one or more chemotherapeutic drugs), and the drug combination has a DAR between 1,050 and 1,150:1.

[0296] In some respects, the ratio of the three different drugs is 5:1:1. In some respects, the ratio of the three different drugs is 10:1:1. In some respects, the ratio of the three different drugs is 15:1:1. In some respects, the ratio of the three different drugs is 20:1:1. In some respects, the ratio of the three different drugs is 1:5:1. In some respects, the ratio of the three different drugs is 1:10:1. In some respects, the ratio of the three different drugs is 1:15:1. In some respects, the ratio of the three different drugs is 1:20:1. In some respects, the ratio of the three different drugs is 1:1:5. In some respects, the ratio of the three different drugs is 1:1:10. In some respects, the ratio of the three different drugs is 1:1:15. In some respects, the ratio of the three different drugs is 1:1:20.

[0297] In some respects, the reagent is linked to or conjugated with four different drugs. In some respects, all four different drugs are chemotherapy drugs.

[0298] In some aspects, the reagent is linked or conjugated with four different drugs, and the DAR of all drug combinations is 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. In some aspects, the reagent is linked or conjugated with four different drugs (e.g., including one or more chemotherapeutic drugs) in a drug combination DAR of 50-150:1. In some aspects, the reagent is linked or conjugated with four different drugs (e.g., including one or more chemotherapeutic drugs) in a drug combination DAR of 150-250:1. In some aspects, the reagent is linked or conjugated with four different drugs (e.g., including one or more chemotherapy drugs) in a drug combination DAR of 250-350:1. In some aspects, the reagent is linked or conjugated with four different drugs (e.g., including one or more chemotherapy drugs) in a DAR of 350-450:1. In some aspects, the reagent is linked or conjugated with four different drugs (e.g., including one or more chemotherapy drugs) in a DAR of 450-550:1. In some aspects, the reagent is linked or conjugated with four different drugs (e.g., including one or more chemotherapy drugs) in a DAR of 550-650:1. In some aspects, the reagent is linked or conjugated with four different drugs (e.g., including one or more chemotherapy drugs) in a DAR of 650-750:1. In some aspects, the reagent is linked or conjugated with four different drugs (e.g., including one or more chemotherapy drugs) in a DAR of 750-850:1. In some aspects, the reagent is linked or conjugated to a DAR of four different drugs (e.g., including one or more chemotherapeutic drugs) in a drug combination of 850-950:1. In some aspects, the reagent is linked or conjugated to a DAR of four different drugs (e.g., including one or more chemotherapeutic drugs) in a drug combination of 950-1,050:1. In some aspects, the reagent is linked or conjugated to a DAR of four different drugs (e.g., including one or more chemotherapeutic drugs) in a drug combination of 1,050-1,150:1.

[0299] In some aspects, the ratio of the four different drugs is 5:1:1:1. In some aspects, the ratio of the four different drugs is 10:1:1:1. In some aspects, the ratio of the four different drugs is 15:1:1:1. In some aspects, the ratio of the four different drugs is 20:1:1:1. In some aspects, the ratio of the four different drugs is 1:5:1:1. In some aspects, the ratio of the four different drugs is 1:10:1:1. In some aspects, the ratio of the four different drugs is 1:15:1:1. In some aspects, the ratio of the four different drugs is 1:20:1:1. In some aspects, the ratio of the four different drugs is 1:1:5:1. In some aspects, the ratio of the four different drugs is 1:1:10:1. In some aspects, the ratio of the four different drugs is 1:1:15:1. In some aspects, the ratio of the four different drugs is 1:1:20:1. In some aspects, the ratio of the four different drugs is 1:1:1:5. In some respects, the ratio of the four different drugs is 1:1:1:10. In some respects, the ratio of the four different drugs is 1:1:1:15. In some respects, the ratio of the four different drugs is 1:1:1:20.

[0300] In some respects, the reagent is linked to or conjugated with five different drugs. In some respects, these five different drugs are all chemotherapy drugs.

[0301] In some aspects, the reagent is linked or conjugated with five different drugs, all drug combinations having a DAR of 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. In some aspects, the reagent is linked or conjugated with five different drugs (e.g., including one or more chemotherapeutic drugs) at a DAR of 50-150:1. In some aspects, the reagent is linked or conjugated with five different drugs (e.g., including one or more chemotherapeutic drugs) at a DAR of 150-250:1. In some aspects, the reagent is linked or conjugated with a DAR of five different drugs (e.g., including one or more chemotherapy drugs) in a drug combination of 250-350:1. In some aspects, the reagent is linked or conjugated with a DAR of five different drugs (e.g., including one or more chemotherapy drugs) in a drug combination of 350-450:1. In some aspects, the reagent is linked or conjugated with a DAR of five different drugs (e.g., including one or more chemotherapy drugs) in a drug combination of 450-550:1. In some aspects, the reagent is linked or conjugated with a DAR of five different drugs (e.g., including one or more chemotherapy drugs) in a drug combination of 550-650:1. In some aspects, the reagent is linked or conjugated with a DAR of five different drugs (e.g., including one or more chemotherapy drugs) in a drug combination of 650-750:1. In some aspects, the reagent is linked or conjugated with a DAR of five different drugs (e.g., including one or more chemotherapy drugs) in a drug combination of 750-850:1. In some aspects, the reagent is linked or conjugated with five different drugs (e.g., including one or more chemotherapeutic drugs) at a drug combination DAR of 850-950:1. In some aspects, the reagent is linked or conjugated with five different drugs (e.g., including one or more chemotherapeutic drugs) at a drug combination DAR between 950-1,050:1. In some aspects, the reagent is linked or conjugated with five different drugs (e.g., including one or more chemotherapeutic drugs) at a drug combination DAR between 1,050-1,150:1.

[0302] In some respects, the ratio of the five different drugs is 5:1:1:1:1. In some respects, the ratio of the five different drugs is 10:1:1:1:1. In some respects, the ratio of the five different drugs is 15:1:1:1:1. In some respects, the ratio of the five different drugs is 20:1:1:1:1. In some respects, the ratio of the five different drugs is 1:5:1:1:1. In some respects, the ratio of the five different drugs is 1:10:1:1:1. In some respects, the ratio of the five different drugs is 1:15:1:1:1. In some respects, the ratio of the five different drugs is 1:20:1:1:1. In some respects, the ratio of the five different drugs is 1:1:5:1:1. In some respects, the ratio of the five different drugs is 1:1:10:1:1. In some respects, the ratio of the five different drugs is 1:1:15:1:1. In some respects, the ratio of the five different drugs is 1:1:20:1:1. In some respects, the ratio of the five different drugs is 1:1:1:5:1. In some respects, the ratio of the five different drugs is 1:1:1:10:1. In some respects, the ratio of the five different drugs is 1:1:1:15:1. In some respects, the ratio of the five different drugs is 1:1:1:20:1. In some respects, the ratio of the five different drugs is 1:1:1:1:5. In some respects, the ratio of the five different drugs is 1:1:1:1:10. In some respects, the ratio of the five different drugs is 1:1:1:1:15. In some respects, the ratio of the five different drugs is 1:1:1:1:20.

[0303] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 10:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 20:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 30:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 40:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 50:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 60:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 70:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 80:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 90:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 100:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 110:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 120:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 130:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 140:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 150:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of at least 160:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of at least 170:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of at least 180:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of at least 190:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of at least 200:1.

[0304] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of at least 210:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of at least 220:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of at least 230:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of at least 240:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of at least 250:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of at least 260:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of at least 270:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 280:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 290:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 300:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 350:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 400:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 450:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 500:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of at least 550:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of at least 600:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of at least 650:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of at least 700:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of at least 750:1.In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 800:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 850:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 900:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 950:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 1,000:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 1,050:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of at least 1,100:1.

[0305] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 10-20:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 10-30:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 10-40:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 10-50:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 10-60:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 10-70:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 10-80:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 10-90:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 10-100:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 10-110:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 10-120:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 10-130:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 10-140:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 10-150:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 10-160:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 10-170:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 10-180:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 10-190:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 10-200:1.

[0306] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 10-210:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 10-220:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 10-230:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 10-240:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 10-250:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 10-260:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 10-270:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 10-280:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 10-290:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 10-300:1.

[0307] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 10-350:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 10-400:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 10-450:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 10-500:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 10-550:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 10-550:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 10-600:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 10-650:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 10-700:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 10-750:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 10-800:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 10-850:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 10-900:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 10-950:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 10-1,000:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 10-1,050:1. In other aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 10-1,100:1.

[0308] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-30:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-40:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-50:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-60:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-70:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-80:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-90:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 20-100:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 20-110:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 20-120:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 20-130:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 20-140:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 20-150:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 20-160:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 20-170:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 20-180:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 20-190:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 20-200:1.

[0309] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-210:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-220:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-230:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-240:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-250:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-260:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-270:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-280:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-290:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-300:1.

[0310] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-350:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-400:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-450:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-500:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-550:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-550:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-600:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-650:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-700:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-750:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-800:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-850:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-900:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-950:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-1,000:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-1,050:1. In other aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 20-1,100:1.

[0311] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-40:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-50:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-60:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-70:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-80:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-90:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-100:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 30-110:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 30-120:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 30-130:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 30-140:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 30-150:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 30-160:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 30-170:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-180:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-190:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-200:1.

[0312] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-210:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-220:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-230:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-240:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-250:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-260:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-270:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-280:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-290:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-300:1.

[0313] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-350:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-400:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-450:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-500:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-550:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-600:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-650:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 30-700:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 30-750:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 30-800:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 30-850:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 30-900:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 30-950:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 30-1,000:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-1,050:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 30-1,100:1.

[0314] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-50:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-60:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-70:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-80:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-90:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-100:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-110:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-120:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-130:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-140:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-150:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-160:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-170:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-180:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-190:1. In other aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-200:1.

[0315] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-210:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-220:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-230:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-240:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-250:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-260:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-270:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-280:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-290:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-300:1.

[0316] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-350:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-400:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-450:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-500:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-550:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-600:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-650:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-700:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-750:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-800:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-850:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-900:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-950:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 40-1,000:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 40-1,050:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 40-1,100:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-60:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-70:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-80:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-90:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-100:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-110:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-120:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-130:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-140:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-150:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-160:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-170:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-180:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-190:1. In some respects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a ratio of 50 to 200:1 DAR.

[0317] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-210:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-220:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-230:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-240:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-250:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-260:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-270:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-280:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-290:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-300:1.

[0318] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-350:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-400:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-450:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-500:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-550:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-600:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-650:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-700:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-750:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-800:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-850:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-900:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-950:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-1,000:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-1,050:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 50-1,100:1.

[0319] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-70:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-80:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-90:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-100:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-110:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-120:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-130:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-140:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-150:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-160:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-170:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-180:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-190:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-200:1.

[0320] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-210:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-220:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-230:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-240:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-250:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-260:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-270:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-280:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-290:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-300:1.

[0321] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-350:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-400:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-450:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-500:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-550:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-600:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-650:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-700:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-750:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-800:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-850:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-900:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-950:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-1,000:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-1,050:1. In other aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 60-1,100:1.

[0322] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-80:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-90:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-100:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-110:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-120:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-130:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-140:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-150:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-160:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-170:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-180:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-190:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-200:1.

[0323] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-210:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-220:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-230:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-240:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-250:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-260:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-270:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-280:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-290:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-300:1.

[0324] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-350:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-400:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-450:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-500:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-550:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-600:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-650:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-700:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-750:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-800:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-850:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-900:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-950:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-1,000:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-1,050:1. In other aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 70-1,100:1.

[0325] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-90:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-100:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-110:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-120:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-130:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-140:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-150:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-160:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-170:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-180:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-190:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-200:1.

[0326] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-210:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-220:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-230:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-240:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-250:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-260:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-270:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-280:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-290:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-300:1.

[0327] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-350:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-400:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-450:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-500:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-550:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-600:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-650:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-700:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-750:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-800:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-850:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-900:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-950:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 80-1,000:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 80-1,050:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 80-1,100:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-100:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-110:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-120:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-130:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-140:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-150:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-160:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-170:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-180:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-190:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-200:1.

[0328] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-210:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-220:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-230:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-240:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-250:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-260:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-270:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-280:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-290:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-300:1.

[0329] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-350:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-400:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-450:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-500:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-550:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-600:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-650:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-700:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-750:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-800:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-850:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-900:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-950:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-1,000:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-1,050:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 90-1,100:1.

[0330] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-110:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-120:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-130:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-140:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-150:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-160:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-170:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-180:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-190:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-200:1.

[0331] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-210:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-220:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-230:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-240:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-250:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-260:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-270:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-280:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-290:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-300:1.

[0332] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-350:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-400:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-450:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-500:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-550:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-600:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-650:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-700:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-750:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-800:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-850:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-900:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-950:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 100-1,000:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 100-1,050:1. In other aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 100-1,100:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-120:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-130:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-140:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-150:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-160:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-170:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-180:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-190:1. In other aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-200:1.

[0333] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-210:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-220:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-230:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-240:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-250:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-260:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-270:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-280:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-290:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-300:1.

[0334] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-350:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-400:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-450:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-500:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-550:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-600:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-650:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 110-700:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 110-750:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 110-800:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 110-850:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 110-900:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 110-950:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 110-1,000:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-1,050:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 110-1,100:1.

[0335] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-130:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-140:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-150:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-160:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-170:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-180:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-190:1. In some respects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-200:1.

[0336] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-210:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-220:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-230:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-240:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-250:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-260:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-270:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-280:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-290:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-300:1.

[0337] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-350:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-400:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-450:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-500:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-550:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-600:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-650:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-700:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-750:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-800:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-850:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-900:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-950:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-1,000:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-1,050:1. In other aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 120-1,100:1.

[0338] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-140:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-150:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-160:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-170:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-180:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-190:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-200:1.

[0339] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-210:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-220:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-230:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-240:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-250:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-260:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-270:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-280:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-290:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-300:1.

[0340] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-350:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-400:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-450:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-500:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-550:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-600:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-650:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-700:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-750:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-800:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-850:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-900:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-950:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-1,000:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-1,050:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 130-1,100:1.

[0341] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 140-150:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 140-160:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 140-170:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 140-180:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 140-190:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 140-200:1.

[0342] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 140-210:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 140-220:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 140-230:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 140-240:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 140-250:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 140-260:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 140-270:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 140-280:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 140-290:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 140-300:1.

[0343] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 140-350:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 140-400:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 140-450:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 140-500:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 140-550:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 140-600:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 140-650:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 140-700:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 140-750:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 140-800:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 140-850:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 140-900:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 140-950:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapy drugs) at a DAR ratio of 140-1,000:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 140-1,050:1. In other aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 140-1,100:1.

[0344] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 150-160:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 150-170:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 150-180:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 150-190:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 150-200:1.

[0345] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 150-210:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 150-220:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 150-230:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 150-240:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 150-250:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 150-260:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 150-270:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 150-280:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 150-290:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 150-300:1.

[0346] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 150-350:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 150-400:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 150-450:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 150-500:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 150-550:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 150-600:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 150-650:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 150-700:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 150-750:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 150-800:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 150-850:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 150-900:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 150-950:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic agents) at a DAR ratio of 150-1,000:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 150-1,050:1. In other aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 150-1,100:1.

[0347] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 160-170:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 160-180:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 160-190:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 160-200:1.

[0348] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 160-210:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 160-220:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 160-230:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 160-240:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 160-250:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 160-260:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 160-270:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 160-280:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 160-290:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 160-300:1.

[0349] In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 160-350:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 160-400:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 160-450:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 160-500:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 160-550:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 160-600:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 160-650:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 160-700:1. In some aspects, the reagent is linked or conjugated with one or more drugs (e.g., including one or more chemotherapeutic drugs) at a DAR ratio of 160-750:...

Claims

1. A bioconjugate comprising a reagent linked to a non-polyethylene glycol (PEG) polymer, wherein the non-PEG polymer is conjugated to a first drug and the reagent binds to a cancer-associated antigen, and wherein the bioconjugate has a drug-to-reagent ratio (DAR) of at least 100:

1.

2. A method for preparing the bioconjugate according to claim 1, comprising: (a) Preparation of a non-PEG polymer backbone comprising a chain transfer agent (CTA) and one or more monomer units via reversible addition-fragmentation chain transfer (RAFT) polymerization. At least one of the monomer units contains an initiator molecule for atom transfer radical polymerization (ATRP); (b) Prepare a non-PEG polymer comprising the non-PEG polymer backbone of (a) and one or more monomer units by ATRP. At least one of the monomer units contains a functional group capable of performing a click response; (c) coupling the first drug to the non-PEG polymer of (b) via a functional group capable of click reaction; and (d) The reagent that binds to the cancer-associated antigen is coupled to the non-PEG polymer described in (c).

3. A method for preparing the bioconjugate according to claim 1, comprising: (a) Preparation of a non-PEG polymer backbone comprising a chain transfer agent (CTA) molecule and one or more monomer units via reversible addition-fragmentation chain transfer (RAFT) polymerization; At least one of the monomer units contains an initiator molecule for atom transfer radical polymerization (ATRP); (b) Coupling the reagent that binds to cancer-associated antigens with the non-PEG polymer backbone of (a); (c) A non-PEG polymer comprising the non-PEG polymer backbone and one or more monomer units described in (b) is prepared by ATRP. At least one of the monomeric units contains a functional group capable of performing a click reaction; and (d) The first drug is coupled to the non-PEG polymer of (c) via the functional group capable of click reaction.

4. The bioconjugate according to any one of claims 1-3, wherein the DAR ratio is at least 110:

1.

5. The bioconjugate according to any one of claims 1-4, wherein the DAR ratio is at least 120:

1.

6. The bioconjugate according to any one of claims 1-5, wherein the DAR ratio is at least 130:

1.

7. The bioconjugate according to any one of claims 1-6, wherein the DAR ratio is at least 140:

1.

8. The bioconjugate according to any one of claims 1-7, wherein the DAR ratio is at least 150:

1.

9. The bioconjugate according to any one of claims 1-8, wherein the DAR ratio is at least 160:

1.

10. The bioconjugate according to any one of claims 1-9, wherein the DAR ratio is at least 170:

1.

11. The bioconjugate according to any one of claims 1-10, wherein the DAR ratio is at least 180:

1.

12. The bioconjugate according to any one of claims 1-11, wherein the DAR is at least 190:

1.

13. The bioconjugate according to any one of claims 1-12, wherein the DAR ratio is at least 200:

1.

14. The bioconjugate according to any one of claims 1-13, wherein the DAR is at least 210:

1.

15. The bioconjugate according to any one of claims 1-14, wherein the DAR is at least 220:

1.

16. The bioconjugate according to any one of claims 1-15, wherein the DAR ratio is at least 230:

1.

17. The bioconjugate according to any one of claims 1-16, wherein the DAR ratio is at least 240:

1.

18. The bioconjugate according to any one of claims 1-17, wherein the DAR ratio is at least 250:

1.

19. The bioconjugate according to any one of claims 1-18, wherein the DAR is at least 260:

1.

20. The bioconjugate according to any one of claims 1-19, wherein the DAR ratio is at least 270:

1.

21. The bioconjugate according to any one of claims 1-20, wherein the DAR is at least 280:

1.

22. The bioconjugate according to any one of claims 1-21, wherein the DAR is at least 290:

1.

23. The bioconjugate according to any one of claims 1-22, wherein the DAR ratio is at least 300:

1.

24. The bioconjugate according to any one of claims 1-23, wherein the DAR ratio is at least 400:

1.

25. The bioconjugate according to any one of claims 1-24, wherein the DAR ratio is at least 500:

1.

26. The bioconjugate according to any one of claims 1-25, wherein the DAR ratio is at least 600:

1.

27. The bioconjugate according to any one of claims 1-26, wherein the DAR ratio is at least 700:

1.

28. The bioconjugate according to any one of claims 1-27, wherein the DAR ratio is at least 800:

1.

29. The bioconjugate according to any one of claims 1-28, wherein the DAR ratio is at least 900:

1.

30. The bioconjugate according to any one of claims 1-29, wherein the DAR is at least 1000:

1.

31. The bioconjugate according to any one of claims 1-30, wherein the DAR is at least 1100:

1.

32. The bioconjugate according to any one of claims 1-31, wherein the reagent is an antibody or an antigen-binding fragment thereof.

33. The bioconjugate according to any one of claims 1-32, wherein the antibody or its antigen-binding fragment is a full-length antibody.

34. The bioconjugate according to any one of claims 1-32, wherein the antibody or its antigen-binding fragment is an antigen-binding fragment.

35. The bioconjugate of claim 34, wherein the antigen-binding fragment comprises Fab, Fab', F(ab')2, Fd, single-chain Fv or scFv, disulfide-linked Fv, V NAR domain, IgNar, intracellular antibody, IgG-CH2, microantibody, F(ab')3, tetraantibody, triantibody, biantibody, single-domain antibody, DVD-Ig, Fcab, mAb2, a(scFv)2, DARPin, or scFv-Fc.

36. The bioconjugate of claim 35, wherein the antigen-binding fragment comprises a single-chain Fv or scFv or a single-domain antibody.

37. The bioconjugate according to any one of claims 1-36, wherein the cancer-associated antigen is HER2, EGFR, or phosphatidylserine.

38. The bioconjugate according to any one of claims 1-32 and 34-37, wherein the antibody or antigen-binding fragment comprises a single-chain Fv or scFv or a single-domain antibody and binds to HER2.

39. The bioconjugate according to any one of claims 1-32 and 34-37, wherein the antibody or antigen-binding fragment comprises a single-chain Fv or scFv or a single-domain antibody and binds to EGFR.

40. The bioconjugate according to any one of claims 1-32 and 34-37, wherein the antibody or antigen-binding fragment comprises a single-chain Fv or scFv or a single-domain antibody and is bound to phosphatidylserine.

41. The bioconjugate according to any one of claims 1-40, wherein the antibody or antigen-binding fragment is coupled to the non-PEG polymer bottle brush via an indivisible chain.

42. The bioconjugate according to any one of claims 1-41, wherein the chain comprises an amino acid linker.

43. The bioconjugate according to any one of claims 1-42, wherein the chain band comprises a histidine tag (His tag).

44. The bioconjugate according to any one of claims 1-43, wherein the antibody or antigen-binding fragment is coupled to the non-PEG polymer bottle brush via a glycine chain comprising 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)5 (SEQ ID NO: 35).

45. The bioconjugate according to any one of claims 1-44, wherein the antibody or antigen-binding fragment is coupled to the non-PEG polymer bottle brush via an ethylene glycol chain, wherein the number of units is an integer between 5 and 25.

46. ​​The bioconjugate according to any one of claims 1-45, wherein the non-PEG polymer comprises one or more monomers selected from the group consisting of: triethylene glycol methyl ether methacrylate (TEOMA), 2-(methylsulfinyl)ethyl methacrylate (MSEMA), 1-phenoxycarbonyl ethyl methacrylate, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate (PCMA), 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate (CBMA), 2-((2-bromo-2-methylpropionyl)oxy)ethyl methacrylate (… BMPOEMA), 2-(2-bromo-2-methylpropionamido)ethyl methacrylate (BMPAEMA), polyethylene glycol monomethyl ether methacrylate (PEGMA), wherein the number of ethylene glycol units is an integer from 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), PEGMA 300 PEGMA 500 2-Dimethylaminoethyl methacrylate (DMAEMA), sulfobetaine methacrylate (SBMA), N,N-dimethylaminoethyl methacrylate, quaternary ammonium ethyl methacrylate, hydroxyethyl methacrylate, hydroxyethyl methacrylate (HEMA), 2-azidoethyl methacrylate, 2-azidotriethylene glycol methyl ether methacrylate (TEOMA-azido compound or TEOMA-N3), 2-azidotetraethylene glycol methyl ether methacrylate (PEGMA(n=4)-azido compound or PEGMA(n=4)-N3), 2-azidopentaethylene glycol methyl ether methacrylate (PEGMA(n=5)-N3). PEGMA(n=5)-N3, PEGMA(n=6)-N3, PEGMA(n=6)-N3, PEGMA(n=7)-N3, PEGMA(n=8)-N3, and PEGMA(n=9)-N3, and combinations thereof.

47. The bioconjugate according to any one of claims 1-46, wherein the non-PEG polymer comprises TEOMA.

48. The bioconjugate according to any one of claims 1-46, wherein the non-PEG polymer comprises MSEMA.

49. The bioconjugate according to any one of claims 1-46, wherein the non-PEG polymer comprises CBMA.

50. The bioconjugate according to any one of claims 1-49, wherein the non-PEG polymer is conjugated to the drug via a cleavable connector.

51. The bioconjugate according to any one of claims 1-50, wherein the non-PEG polymer is conjugated to the first drug via an amino acid linker.

52. The bioconjugate according to any one of claims 1-51, wherein the non-PEG polymer is coupled to the first drug via a valine-citrulline-p-aminobenzylcarbamate, valine-citrulline, or alanine-alanine linker.

53. The bioconjugate according to any one of claims 1-52, wherein the bioconjugate further comprises a second drug.

54. The bioconjugate according to claim 53, wherein the ratio of the first drug to the second drug is from 1:1 to 20:

1.

55. The bioconjugate according to claim 53, wherein the ratio of the first drug to the second drug is 20:1 to 1:

1.

56. The bioconjugate according to any one of claims 1-55, wherein the bioconjugate further comprises a third drug.

57. The bioconjugate according to claim 56, wherein the ratio of the first drug, the second drug, and the third drug is 1:1:

1.

58. The bioconjugate according to any one of claims 1-57, wherein the bioconjugate further comprises a fourth drug.

59. The bioconjugate according to claim 58, wherein the ratio of the first drug, the second drug, the third drug, and the fourth drug is 1:1:1:

1.

60. The bioconjugate according to any one of claims 1-59, wherein the bioconjugate further comprises a fifth drug.

61. The bioconjugate according to claim 60, wherein the ratio of the first drug, the second drug, the third drug, the fourth drug, and the fifth drug is 1:1:1:1:

1.

62. The bioconjugate according to any one of claims 1-61, wherein the first drug, the second drug, the third drug, the fourth drug and / or the fifth drug are chemotherapeutic drugs.

63. The bioconjugate according to claim 62, wherein the chemotherapeutic agent is selected from the group consisting of: MEK inhibitors, PI3K inhibitors, MAPK inhibitors, alkylating agents, cytoskeleton disruptors, histone deacetylase inhibitors, topoisomerase I or II inhibitors, their nucleotide analogs or precursors, peptide antibiotics, protein degrading agents, platinum-based reagents, retinoids, vinca alkaloids and their derivatives, and combinations thereof.

64. The bioconjugate of claim 63, wherein the chemotherapeutic agent comprises a MEK inhibitor.

65. The bioconjugate according to claim 64, wherein the MEK inhibitor is selected from the group consisting of: bemetinib, GDC-0973 (cobitinib), selmetinib, trametinib.

66. The bioconjugate of claim 63, wherein the chemotherapeutic agent comprises a PI3K inhibitor.

67. The bioconjugate according to claim 66, wherein the PI3K inhibitor is selected from the group consisting of: PI103; PI828; LY294002; Wollman penicillin; Demethoxychloramphenicol; IC486068; IC87114; GDC-0941; GDC-0980 (apirixol); perifoxine; CAL101; PX-866; IPI-145; BAY 80-6946; BEZ235; P6503; TGR1202; SF1126; INK1117; BKM120; IL147; XL765; Palomida 529; GSK1059615; ZSTK474; PWT33597; TG 100-115; CAL263; GNE-447; CUDC-907; and AEZS-136.

68. A bioconjugate comprising a reagent linked to a non-polyethylene glycol (PEG) polymer, wherein the reagent is an antibody or an antigen-binding fragment thereof, wherein the polymer is coupled to a MEK inhibitor, a CDK inhibitor, and / or a PI3K inhibitor, and wherein the antibody or the antigen-binding fragment thereof is bound to a HER2 protein, a rEGFR protein, or a phosphatidylserine, and wherein the bioconjugate has a drug-to-reagent ratio (DAR) of 100:1 to 1,100:

1.

69. The bioconjugate according to claim 68, wherein the DAR of the bioconjugate is from 200:1 to 1,100:

1.

70. The bioconjugate according to claim 68, wherein the DAR of the bioconjugate is from 400:1 to 1,100:

1.

71. The bioconjugate according to claim 68, wherein the DAR of the bioconjugate is 600:1 to 1,100:

1.

72. The bioconjugate according to claim 68, wherein the DAR of the bioconjugate is from 800:1 to 1,100:

1.

73. The bioconjugate according to claim 68, wherein the DAR of the bioconjugate is 900:1 to 1,100:

1.

74. The bioconjugate according to any one of claims 68-73, wherein the MEK inhibitor is GDC-0973 (cobimetinib) and the PI3K inhibitor is GDC-0980 (apirixe).

75. The bioconjugate according to any one of claims 68-73, wherein the MEK inhibitor is GDC-0973 (cobimetinib), the CDK inhibitor is denasinib, and the PI3K inhibitor is GDC-0980 (apirixe).

76. The bioconjugate according to any one of claims 68-75, wherein the antibody or its antigen-binding fragment binds to the EGFR protein, the MEK inhibitor is GDC-0973 (cobibitinib), and the CDK inhibitor is denasinib.

77. The bioconjugate according to any one of claims 68-75, wherein the antibody or its antigen-binding fragment binds to the EGFR protein, the PI3K inhibitor is GDC-0980 (apirixate), and the CDK inhibitor is denasinib.

78. The bioconjugate according to any one of claims 68-75, wherein the antibody or its antigen-binding fragment binds to phosphatidylserine, the MEK inhibitor is GDC-0973 (cobibitinib), and the CDK inhibitor is denasinib.

79. The bioconjugate according to any one of claims 68-75, wherein the antibody or its antigen-binding fragment binds to phosphatidylserine, the PI3K inhibitor is GDC-0980 (apirixe), and the CDK inhibitor is denasinib.

80. A pharmaceutical composition comprising the bioconjugate of any one of claims 1-79 and a pharmaceutically acceptable carrier.

81. A kit comprising the bioconjugate of any one of claims 1-79.

82. A method for inhibiting tumor growth in a subject, comprising administering to the subject a therapeutically effective amount of the bioconjugate of any one of claims 1-79 or the pharmaceutical composition of claim 80.

83. The method of claim 82, wherein the tumor is selected from the group consisting of: ovarian tumors, brain tumors, breast tumors, uterine tumors, endometrial tumors, pancreatic tumors, kidney tumors, head and neck tumors, gastric tumors, and lung tumors.

84. The method according to claim 82, wherein the tumor is a breast tumor, a head and neck tumor, a lung tumor, or a gastric tumor.

85. A method of treating cancer in a subject in need, comprising administering to the subject a therapeutically effective amount of any one of claims 1-79 or the pharmaceutical composition of claim 80.

86. The method of claim 85, wherein the cancer is selected from the group consisting of: ovarian cancer, brain cancer, breast cancer, uterine cancer, endometrial cancer, pancreatic cancer, kidney cancer, head and neck cancer, stomach cancer, and lung cancer.

87. The method of claim 86, wherein the cancer is breast cancer, head and neck cancer, lung cancer, or stomach cancer.

88. A method for increasing the cytotoxicity of cancer cells, comprising contacting cancer cells with a bioconjugate of any one of claims 1-79 or a pharmaceutical composition of claim 80.

89. A method for inhibiting cancer proliferation in a subject in need, comprising administering to the subject a therapeutically effective amount of any one of claims 1-79 or the pharmaceutical composition of claim 80.

90. An isolated polynucleotide comprising a sequence having at least 90% sequence identity with one or more amino acid sequences encoding SEQ ID NO: 1-4, SEQ ID NO: 6-16 and SEQ ID NO: 20-31.

91. The isolated polynucleotide according to claim 90, wherein the sequence has at least 95% sequence identity with the sequence encoding one or more amino acid sequences of SEQ ID NO: 1-4, SEQ ID NO: 6-16 and SEQ ID NO: 20-31.

92. The isolated polynucleotide according to claim 91, wherein the sequence has at least 99% sequence identity with one or more amino acid sequences encoding SEQ ID NO: 1-4, SEQ ID NO: 6-16 and SEQ ID NO: 20-31.

93. The isolated polynucleotide according to claim 92, wherein the sequence is identical to the sequence encoding one or more amino acid sequences in SEQ ID NO: 1-4, SEQ ID NO: 6-16 and SEQ ID NO: 20-31.

94. A vector comprising any one of the polynucleotides of claims 90-93.

95. A host cell comprising the vector of claim 94.

96. The host cell according to claim 95, selected from the group consisting of: *Escherichia coli*, *Pseudomonas*, *Bacillus*, *Streptomyces*, yeast, CHO, YB / 20, NSO, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, R1.1, BW, LM, COS 1, COS 7, BSC1, BSC40, BMT10 cells, plant cells, insect cells, and human cells from tissue cultures.

97. A method for producing an antibody or an antigen-binding fragment thereof, comprising culturing a host cell as claimed in claim 95 or 96 such that a nucleic acid molecule is expressed and an antibody or an antigen-binding fragment thereof is produced, optionally, wherein the method further comprises isolating the antibody or an antigen-binding fragment thereof from the culture.

98. The method according to claim 2 or 3, wherein the chain transfer agent (CTA) is selected from... or .

99. The method of claim 98, wherein the CTA is .

100. The method according to claim 2 or 3, wherein the monomer unit containing the initiator molecule for atom transfer radical polymerization (ATRP) is selected from 2-((2-bromo-2-methylpropionyl)oxy)ethyl methacrylate (BMPOEMA) or 2-(2-bromo-2-methylpropionamido)ethyl methacrylate (BMPAEMA).

101. The method of claim 100, wherein the at least one monomer unit comprising an initiator molecule (ATRP) for atom transfer radical polymerization is 2-(2-bromo-2-methylpropionamido)ethyl methacrylate (BMPAEMA).

102. The method of claim 100, wherein the at least one monomer unit comprising an initiator molecule for atom transfer radical polymerization (ATRP) is selected from... or .

103. The method according to claim 2 or 3, wherein the non-PEG polymer backbone comprises at least one second monomer unit, wherein the second monomer unit is a non-PEG monomer containing methacrylate.

104. The method according to claim 103, wherein the second monomer unit is selected from triethylene glycol methyl ether methacrylate (TEOMA), 2-(methylsulfinyl)ethyl methacrylate (MSEMA), 1-phenoxycarbonyl ethyl methacrylate, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate (PCMA), 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate (CBMA), 2-((2-bromo-2-methylpropionyl)oxy)ethyl methacrylate (BMPOEMA), 2-(2-... 1,2-bromo-2-methylpropionamido)ethyl methacrylate (BMPAEMA), polyethylene glycol monomethyl ether methacrylate (PEGMA), wherein the number of ethylene glycol units is an integer from 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), PEGMA 300 PEGMA 500 2-Dimethylaminoethyl methacrylate (DMAEMA), sulfobetaine methacrylate (SBMA), quaternary ammonium ethyl methacrylate and hydroxyethyl methacrylate (HEMA), or combinations thereof.

105. The method according to claim 104, wherein the second monomer unit is selected from triethylene glycol methyl ether methacrylate (TEOMA), polyethylene glycol monomethyl ether methacrylate (PEGMA), wherein the number of glycol units is an integer from 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), and PEGMA. 300 or PEGMA 500 .

106. The method according to claim 2 or 3, wherein the at least one monomeric unit containing a functional group capable of performing a click reaction is selected from 2-azidoethyl methacrylate, 2-azidotriethylene glycol methyl ether methacrylate (TEOMA-azido compound or TEOMA-N3), 2-azidotetraethylene glycol methyl ether methacrylate (PEGMA(n=4)-azido compound or PEGMA(n=4)-N3), 2-azidopentaneethylene glycol methyl ether methacrylate (PEGMA(n=5)-azido compound or PEGMA(n=5)-N3), 2- PEGMA(n=6)-azido or PEGMA(n=6)-N3, PEGMA(n=7)-azido or PEGMA(n=7)-N3, PEGMA(n=8)-azido or PEGMA(n=8)-N3, and PEGMA(n=9)-N3 or combinations thereof.

107. The method according to claim 106, wherein the at least one monomeric unit containing a functional group capable of performing a click reaction is 2-azidotriethylene glycol methyl ether methacrylate (TEOMA-azide or TEOMA-N3).

108. The method of claim 106, wherein the at least one monomeric unit comprising a functional group capable of performing a click response is selected from... , or .

109. The method of claim 2 or 3, wherein the non-PEG polymer comprises at least a second monomer unit, wherein the second monomer unit is a methacrylate containing a non-PEG monomer.

110. The method of claim 109, wherein the second monomer unit in the non-PEG polymer is selected from triethylene glycol methyl ether methacrylate (TEOMA), 2-(methylsulfinyl)ethyl methacrylate (MSEMA), 1-phenoxycarbonyl ethyl methacrylate, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate (PCMA), 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate (CBMA), 2-((2-bromo-2-methylpropionyl)oxy)ethyl methacrylate (BMPOEMA), 2 -(2-bromo-2-methylpropionamido)ethyl methacrylate (BMPAEMA), polyethylene glycol monomethyl ether methacrylate (PEGMA, wherein the number of ethylene glycol units is an integer from 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), PEGMA 300 EGMA 500 2-Dimethylaminoethyl methacrylate (DMAEMA), sulfobetaine methacrylate (SBMA), quaternary ammonium ethyl methacrylate, hydroxyethyl methacrylate (HEMA), 2-azidoethyl methacrylate, 2-azidotriethylene glycol methyl ether methacrylate (TEOMA-azido compound or TEOMA-N3), 2-azidotetraethylene glycol methyl ether methacrylate (PEGMA(n=4)-azido compound or PEGMA(n=4)-N3), 2-azidopentaethylene glycol methyl ether methacrylate (PEGMA(n=5)-azido compound or ...----------------------------------------------------------------------------------- PEGMA(n=5)-N3, PEGMA(n=6)-azido or PEGMA(n=6)-N3, PEGMA(n=7)-azido or PEGMA(n=7)-N3, PEGMA(n=8)-azido or PEGMA(n=8)-N3, and PEGMA(n=9)-N3, or combinations thereof.

111. The method according to claim 2 or 3, wherein the coupling of the drug with the polymer is carried out by an azide-alkyne click reaction or an inverse electron demand Diels-Alder (IEDDA) reaction.

112. The method according to claim 111, wherein the azide-alkyne click reaction is selected from copper-catalyzed azide-alkyne cycloaddition (CuAAC), strain-promoted azide-alkyne cycloaddition (SPAAC), or strain-promoted alkyne-nitroketone cycloaddition (SPANC).

113. The method according to claim 2 or 3, wherein the coupling of the reagent binding the cancer-associated antigen to the polymer is carried out by an azide-alkyne click reaction or an inverse electron demand Diels-Alder (IEDDA) reaction.

114. The method of claim 113, wherein the coupling of the reagent binding the cancer-associated antigen to the polymer is carried out via an inverse electron demand Diels-Alder (IEDDA) reaction.

115. The method according to claim 114, wherein the reverse electron demand Diels-Alder (IEDDA) reaction is a tetrazine-cyclooctene reaction.

116. The method according to claim 2 or 3, wherein the functional group capable of performing the click reaction is an azide (-N3).

117. The bioconjugate according to claim 1, wherein the components of the bioconjugate are selected from the components in Table A or their pharmaceutically acceptable salts.

118. The bioconjugate according to claim 1, wherein the bioconjugate is a compound in Table B or a pharmaceutically acceptable salt thereof.

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