WO2015015448A2 - Engineered polypeptide conjugates - Google Patents

Engineered polypeptide conjugates Download PDF

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Publication number
WO2015015448A2
WO2015015448A2 PCT/IB2014/063566 IB2014063566W WO2015015448A2 WO 2015015448 A2 WO2015015448 A2 WO 2015015448A2 IB 2014063566 W IB2014063566 W IB 2014063566W WO 2015015448 A2 WO2015015448 A2 WO 2015015448A2
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Prior art keywords
engineered
seq
polypeptide
antibody
polypeptide conjugate
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PCT/IB2014/063566
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French (fr)
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WO2015015448A3 (en
Inventor
Santiago Esteban FARIAS
Meritxell GALINDO CASAS
Pavel Strop
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Rinat Neuroscience Corp.
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Application filed by Rinat Neuroscience Corp. filed Critical Rinat Neuroscience Corp.
Priority to EP14777814.6A priority Critical patent/EP3027224B1/en
Priority to JP2016530656A priority patent/JP6521959B2/en
Priority to ES14777814T priority patent/ES2804614T3/en
Priority to US14/908,492 priority patent/US10195289B2/en
Priority to CA2919583A priority patent/CA2919583C/en
Publication of WO2015015448A2 publication Critical patent/WO2015015448A2/en
Publication of WO2015015448A3 publication Critical patent/WO2015015448A3/en
Priority to US16/231,242 priority patent/US10842881B2/en
Priority to US17/069,969 priority patent/US20210162058A1/en
Priority to US18/347,280 priority patent/US20240009319A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6805—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a vinca alkaloid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A61K47/6809—Antibiotics, e.g. antitumor antibiotics anthracyclins, adriamycin, doxorubicin or daunomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins

Definitions

  • the present invention relates generally to engineered polypeptide conjugates (e.g., antibody-drug conjugates) comprising specific acyl donor glutamine-containing tags and amine donor agents.
  • the invention also relates to methods for making such engineered polypeptide conjugates using transglutaminase and methods of using thereof.
  • Antibody therapy provides targeted therapeutic treatment in patients with various disorders, such as cancers and immunological diseases, and therefore has played an important role in biological research.
  • Different approaches of targeted antibody therapy including antibody-drug conjugates (ADC), have been explored. See, e.g., Doronian et al., Bioconj. Chem. 19: 1960-1963 (2008); and Junutula et al., Nature Biotechnology 26: 925-932 (2008).
  • ADC antibody-drug conjugates
  • cytotoxic small molecules are generally linked or conjugated to antibodies for targeted local delivery of the drug moieties to tumors.
  • Chemical modification has been widely used for conjugating drugs to antibodies either through lysine side chain amines or through cysteine sulfhydryl groups activated by reducing interchain disulfide bonds.
  • these types of "residue-specific" conjugation lead to a heterogeneous mixture of conjugates having different molar ratios of drug to antibody, different and non-specific conjugation sites, different efficiency, safety, and pharmacokinetics, and different clearance of antibody drug conjugates.
  • cysteine engineered antibodies and antibody-drug conjugates are complicated processes which require lengthy reaction procedures (e.g., reductions and oxidations). See, e.g., Gomez et al., Biotechnology and Bioengineering, 105(4): 748-760 (2009).
  • Antibody aggregates may also be generated during the process of making the cysteine engineered antibodies and the antibody-drug conjugates.
  • Transglutaminases (EC2.3.2.13; protein-glutamine:gamma-glutamyl transferee; protein- glutamine: amine ⁇ -glutamyltransferase; CAS 80146-85-6) belong to a family of enzymes that catalyze the acyl addition to a primary amine wherein the gamma-carboxamide group of peptide- bound ⁇ -glutanyl residue is the acyl donor and the primary amine is the acyl acceptor and the amine donor.
  • Transglutaminases have been used, for example, for the attachment of proteins to proteins. See, e.g., Tanaka et al, FEBS Letters 579:2092-2096 (2005). Enzymatic modification of antibodies using transglutaminases has also been reported. See Josten et al. J. of Immunological Methods 240:47-54 (2000); Takazawa et al., Biotechnology and Bioengineering 86(4): 399-404 (2004); Mindt et al., Bioconj. Chem 19:271-27 (2008), and Jeger et al., Angewandte Chemie, 49(51): 9995-9997 (2010). Protein conjugation or modification using transglutaminase provides the advantages of high selectivity, simplified reaction procedures, and mild reaction conditions. WO2012059882 describes site-specific conjugation of antibodies and small molecules mediated by a transglutaminase.
  • the present invention provides transglutaminase-mediated antibody drug conjugates comprising a specific acyl donor glutamine-containing tag engineered at a specific site of the antibody and an amine donor agent (e.g., linker-payload).
  • the invention also provides a homogenous site-specific transglutaminase-mediated antibody drug conjugate (e.g., at least about 99.8% site-specific) comprising the amino acid substitution from glutamine (Q) to asparagine (N) at position 295 of the antibody (Q295N; EU numbering scheme).
  • this invention provides an engineered polypeptide conjugate comprising the formula: polypeptide-T-A, wherein T is an acyl donor glutamine-containing tag engineered at a specific site, wherein A is an amine donor agent, wherein the amine donor agent is site- specifically conjugated to the acyl donor glutamine-containing tag at a carboxyl terminus, an amino terminus, or at an another site in the polypeptide, and wherein the acyl donor glutamine- containing tag comprises an amino acid sequence GGLLQGPP (SEQ ID NO: 13), LLQGPA (SEQ ID NO:4), or LLQGPP (SEQ ID NO: 11).
  • the invention provides an engineered Fc-containing polypeptide conjugate comprising the formula: (Fc-containing polypeptide-T-A), wherein T is an acyl donor glutamine-containing tag engineered at a specific site, wherein A is an amine donor agent, wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine- containing tag at a carboxyl terminus, an amino terminus, or at an another site in the Fc- containing polypeptide, wherein the acyl donor glutamine-containing tag comprises an amino acid sequence XXQX (SEQ ID NO:35), wherein X is any amino acid (e.g., X can be the same or different amino acid), and wherein the engineered Fc-containing polypeptide conjugate comprises an amino acid substitution from glutamine to asparagine at position 295 (Q295N; EU numbering scheme).
  • the acyl donor glutamine-containing tag is LLQGPA (SEQ ID NO:4), LLQGP (SEQ ID NO:5), LLQGPP (SEQ ID NO: 11) or GGLLQGPP (SEQ ID NO: 13).
  • the acyl donor glutamine-containing tag is not spatially adjacent to a reactive Lys (i.e., the ability to form a covalent bond as an amine donor in the presence of an acyl donor and a transglutaminase) in the polypeptide or the Fc-containing polypeptide.
  • the polypeptide or the Fc-containing polypeptide comprises an amino acid modification at the last amino acid position in the carboxyl terminus relative to a wild-type polypeptide at the same position.
  • the amino acid modification can be an amino acid deletion, insertion, substitution, mutation, or any combination thereof.
  • the polypeptide conjugate comprises a full length antibody heavy chain and an antibody light chain, wherein the acyl donor glutamine-containing tag is located at the carboxyl terminus of a heavy chain, a light chain, or both the heavy chain and the light chain.
  • the polypeptide conjugate comprises an antibody, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, a minibody, a diabody, or an antibody fragment.
  • the antibody is an IgG.
  • the amine donor agent comprises the formula: X-Y-Z, wherein X is an amine donor unit; Y is a linker; and Z is an agent moiety.
  • the amine donor unit-linker (X-Y) is a branched unit (e.g., at least 2 units) and the agent moiety comprises at least about 2 agent moieties.
  • the amine donor unit-linker (X-Y) is selected from the group consisting of Ac-Lys-Gly, aminocaproic acid, Ac-Lys-P-Ala, amino-PEG2-C2, amino-PEG3-C2, amino-PEG6-C2, Ac-Lys-Val-Cit-PABC, amino-PEG6-C2-Val-Cit-PABC, aminocaproyl-Val- Cit-PABC, [(3R,5R)-l- ⁇ 3-[2-(2-aminoethoxy)ethoxy]propanoyl ⁇ piperidine-3,5-diyl]bis-Val-Cit- PABC, [(3S,5S)-l- ⁇ 3-[2-(2-aminoethoxy)ethoxy]propanoyl ⁇ piperidine-3,5-diyl]bis-Val-Cit- PABC, putrescine, and Ac-Lys-putrescine.
  • the agent moiety is a cytotoxic agent, including, but not limited to, an anthracycline, an auristatin, a camptothecin, a combretastatin, a dolastatin, a duocarmycin, an enediyne, a geldanamycin, an indolino-benzodiazepine dimer, a maytansine, a puromycin, a pyrrolobenzodiazepine dimer, a taxane, a vinca alkaloid, a tubulysin, a hemiasterlin, a spliceostatin, a pladienolide, and stereoisomers, isosteres, analogs, or derivatives thereof.
  • a cytotoxic agent including, but not limited to, an anthracycline, an auristatin, a camptothecin, a combretastatin, a dolastatin, a duocarmycin,
  • the cytotoxic agent is MMAD (Monomethyl Auristatin D) or 0101 (2-methylalanyl-N- [(3R, 4S, 5S)-3 -methoxy- 1 - ⁇ (2S)-2- [( ⁇ R, 2R)- 1 -methoxy-2-methyl-3 -oxo-3 - ⁇ [( 15)-2-phenyl- 1 -(1,3- thiazol-2-yl)ethyl]amino ⁇ propyl]pyrrolidin-l -yl ⁇ -5-methyl-l -oxoheptan-4-yl]-N-m ethyl -L- valinamide).
  • MMAD Monomethyl Auristatin D
  • 0101 (2-methylalanyl-N- [(3R, 4S, 5S)-3 -methoxy- 1 - ⁇ (2S)-2- [( ⁇ R, 2R)- 1 -methoxy-2-methyl-3 -oxo
  • the amine donor agent (X-Y-Z) includes, but is not limited to, Alexa 488 cadaverine, 5-FITC cadaverine, Alexa 647 cadaverine, Alexa 350 cadaverine, 5- TAMRA cadaverine, 5-FAM cadaverine, SRI 01 cadaverine, 5,6-TAMRA cadaverine, 5-FAM lysine, Ac-Lys-Gly (acetyl -lysine-glycine)-MM AD, amino-PEG3 -C2-MM AD, amino-PEG6-C2- MMAD, amino-PEG3-C2-amino-nonanoyl-MMAD, aminocaproyl-Val-Cit-PABC-MMAD, amino-PEG-C2-Val-Cit-PABC-MMAD, Ac-Lys-Val-Cit-PABC (acetyl-lysine-valine-citrulline- /?-aminobenzyloxycarbonyl
  • the invention provides a method for preparing an engineered Fc- containing polypeptide conjugate comprising the formula: (Fc-containing polypeptide-T-A), wherein T is an acyl donor glutamine-containing tag engineered at a specific site, wherein A is an amine donor agent, wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine-containing tag at a carboxyl terminus, an amino terminus, or at an another site in the Fc-containing polypeptide, wherein the acyl donor glutamine-containing tag comprises an amino acid sequence XXQX (SEQ ID NO:35), wherein X is any amino acid (e.g., X can be the same or a different amino acid), and wherein the engineered Fc-containing polypeptide conjugate comprises an amino acid substitution from glutamine to asparagine at position 295 (Q295N; EU numbering scheme), comprising the steps of: a) providing an engineered (Fc-containing polypeptide)-!
  • the engineered Fc-containing polypeptide-! is expressed in CHO cells
  • the invention provides a method for preparing an engineered polypeptide conjugate comprising the formula: polypeptide-!-A, wherein ! is an acyl donor glutamine-containing tag engineered at a specific site, wherein A is an amine donor agent, wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine- containing tag at a carboxyl terminus, an amino terminus, or at an another site in the polypeptide, and wherein the acyl donor glutamine-containing tag comprises an amino acid sequence LLQGPX, wherein X is A or P (SEQ ID NO: 14), or GGLLQGPP (SEQ ID NO: 13), comprising the steps of: a) providing an engineered polypeptide-!
  • the engineered polypeptide-! molecule comprising the polypeptide and the acyl donor glutamine-containing tag; b) contacting the amine donor agent with the engineered polypeptide-! molecule in the presence of a transglutaminase; and c) allowing the engineered polypeptide-! to covalently link to the amine donor agent to form the engineered Fc-containing polypeptide conjugate.
  • the engineered polypeptide-! molecule is expressed in CHO cells.
  • the engineered polypeptide conjugate e.g., the engineered Fc- containing polypeptide conjugate, the engineered Fab-containing polypeptide conjugate, or the engineered antibody conjugate
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the engineered polypeptide conjugate as described herein (e.g., the engineered Fc-containing polypeptide conjugate, the engineered Fab-containing polypeptide conjugate, or the engineered antibody conjugate) and a pharmaceutically acceptable excipient.
  • the invention provides a method of treating a cancer in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition comprising the engineered polypeptide conjugate (e.g., the engineered Fc-containing polypeptide conjugate, the engineered Fab-containing polypeptide conjugate, or the engineered antibody conjugate) as described herein.
  • the engineered polypeptide conjugate e.g., the engineered Fc-containing polypeptide conjugate, the engineered Fab-containing polypeptide conjugate, or the engineered antibody conjugate
  • the invention provides a method of inhibiting tumor growth or progression in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition comprising the engineered polypeptide conjugate (e.g., the engineered Fc-containing polypeptide conjugate, the engineered Fab-containing polypeptide conjugate, or the engineered antibody conjugate) as described herein.
  • the engineered polypeptide conjugate e.g., the engineered Fc-containing polypeptide conjugate, the engineered Fab-containing polypeptide conjugate, or the engineered antibody conjugate
  • the invention provides a method of diagnosing cancer in a subject suspected of suffering from cancer, comprising a) contacting a sample of the subject with the engineered polypeptide conjugate (e.g., the engineered Fc-containing polypeptide conjugate, the engineered Fab-containing polypeptide conjugate, or the engineered antibody conjugate) as described herein under conditions that result in binding of the engineered polypeptide conjugate with a cancer-related protein, and b) determining binding of the engineered polypeptide conjugate to the cancer-related protein.
  • the engineered polypeptide conjugate e.g., the engineered Fc-containing polypeptide conjugate, the engineered Fab-containing polypeptide conjugate, or the engineered antibody conjugate
  • the invention provides an engineered polypeptide conjugate (e.g., the engineered Fc-containing polypeptide conjugate, the engineered Fab-containing polypeptide conjugate, or the engineered antibody conjugate) purified by the methods described herein.
  • an engineered polypeptide conjugate e.g., the engineered Fc-containing polypeptide conjugate, the engineered Fab-containing polypeptide conjugate, or the engineered antibody conjugate
  • Figures 1A-1D depict graphs showing different percentages of the clipping of four different acyl donor glutamine-containing tags (the "glutamine tags") expressed in CHO cells.
  • Figure 1A depicts the results of the glutamine tag TG6 (SEQ ID NO: 1) in Ab-TG6.
  • Figure IB depicts the results of the glutamine tag TG9 (SEQ ID NO:4) in Ab-TG9.
  • Figure 1C depicts the results of the glutamine tag TG10 (SEQ ID NO: 5) in Ab-TGIO.
  • Figure ID depicts the results for the glutamine tag TG17 (SEQ ID NO: 11) in Ab-TG17.
  • FIGS 2A-2B show that the glutamine tag LCQ04 (SEQ ID NO: 12) expressed in CHO cells was clipped from the C-terminus of an antibody light chain (2 A), whereas no clipping was observed in the glutamine tag LCQ05 (SEQ ID NO: 13) in Ab-LCQ05 (2B).
  • Figures 3A-3B show that similar low levels of aggregates were observed for the glutamine tags TG6 (SEQ ID NO: l) and TG17 (SEQ ID NO: 11) in Ab-TG6-AcLys-VC-PABC- 0101 ( Figure 3 A) and Ab-TG17-AcLys-VC-PABC-0101 ( Figure 3B) using analytical size exclusion chromatography (SEC).
  • SEC analytical size exclusion chromatography
  • Figures 4A-4B show that similar Drug-Antibody-Ratio (DAR) was observed for the glutamine tags TG6 (SEQ ID NO: l) and TG17 (SEQ ID NO: 11) in Ab-TG6 and Ab-TG6- AcLys-VC-PABC-0101 ( Figure 4A) and Ab-TG17 and Ab-TG17-AcLys-VC-PABC-0101 ( Figure 4B) using Hydrophobic Interaction Chromatography (HIC).
  • DAR Drug-Antibody-Ratio
  • Figures 5A-5B show that similar Drug-Antibody-Ratio (DAR) was observed for the glutamine tags TG6 (SEQ ID NO: l) and TG17 (SEQ ID NO: 11) in Ab-TG6 and Ab-TG17 ( Figure 5 A) and Ab-TG6-AcLys-VC-PABC-0101 and Ab-TG17-AcLys-VC-PABC-0101 ( Figure 5B) using Mass Spectrometry (MS).
  • DAR Drug-Antibody-Ratio
  • Figures 6A-6B show that antibody conjugates Ab-TG6-AcLys-VC-PABC-0101 and Antibody-TG17-AcLys-VC-PABC-0101 had the similar in vitro efficacy for cytotoxicity in BxPC3 cell line ( Figure 6A) and OVCAR3 cell line ( Figure 6B).
  • Figures 7A-7B show that antibody conjugate (Ab-TG17-AcLys-PABC-0101) having the glutamine tag TG17 (SEQ ID NO: 11) had similar activity in in vivo tumor models (% body weight change in Figure 7A; and tumor volume in Figure 7B) in comparison to the antibody conjugate (Ab-TG6-AcLys-PABC-0101) having the glutamine tag TG6 (SEQ ID NO: 1).
  • Figure 8 shows the pharmacokinetics for antibody conjugates Ab-TG6-AcLys-VC- PABC-0101, Antibody-TG17-AcLys-VC-PABC-0101, Ab-TG6, and Ab-TG17.
  • Figure 9A shows that transglutaminase can recognize Q295 in aglycosylated IgGs, and conjugation can be achieved at this site (conjugation of Amino-PEG6-MMAD to a tryptic peptide (SEQ ID NO: 15)).
  • Figure 9B shows that 1.3% of the total injected Q295 peptide was conjugated with amino- PEG6-MMAD in the heavy chain of the antibody, assuming 100% digestion efficiency.
  • the present invention provides transglutaminase-mediated antibody-drug conjugates comprising a specific acyl donor glutamine-containing tag engineered at a specific site of the antibody and an amine donor agent (e.g., linker-payload).
  • the invention also provides a homogenous site-specific transglutaminase-mediated antibody-drug conjugate comprising the amino acid substitution from glutamine (Q) to asparagine (N) at position 295 (Q295N; EU numbering scheme).
  • the inventors have discovered that proteolysis of an acyl donor glutamine- containing tag engineered at C-terminus of an antibody's heavy chain or light chain, when expressed in CHO cells, can be prevented by using several newly designed glutamine-containing tags having unique sequences (e.g., LLQGPA (SEQ ID NO:4), LLQGPP (SEQ ID NO: 11), and GGLLQGPP (SEQ ID NO: 13)).
  • mutation at position 295 i.e., Q295N; EU numbering scheme
  • the antibody drug conjugate eliminates the off- target conjugation of a small percentage of aglycosylated antibody at position 295 and yields highly homogenous conjugates that are better than 99.8% site-specific.
  • the invention provides an engineered polypeptide conjugate comprising the formula: polypeptide-T-A, wherein T is an acyl donor glutamine-containing tag engineered at a specific site; wherein A is an amine donor agent; wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine-containing tag at a carboxyl terminus, an amino terminus, or at an another site in the polypeptide, and wherein the acyl donor glutamine-containing tag comprises an amino acid sequence LLQGPX, wherein X is A or P (SEQ ID NO: 14), or GGLLQGPP (SEQ ID NO: 13).
  • the polypeptide is an Fc-containing polypeptide or a Fab-containing polypeptide.
  • the invention provides an engineered Fc-containing polypeptide conjugate comprising the formula: (Fc-containing polypeptide)-T-A, wherein T is an acyl donor glutamine-containing tag engineered at a specific site; wherein A is an amine donor agent; wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine- containing tag at a carboxyl terminus, an amino terminus, or at an another site in the Fc- containing polypeptide, wherein the acyl donor glutamine-containing tag comprises an amino acid sequence XXQX (SEQ ID NO:35), wherein X is any amino acid, and wherein the engineered Fc-containing polypeptide conjugate comprises an amino acid substitution from glutamine to asparagine at position 295 (Q295N; EU numbering scheme).
  • the invention provides a method for preparing an engineered polypeptide conjugate comprising the formula: polypeptide-T-A, wherein T is an acyl donor glutamine-containing tag engineered at a specific site, wherein A is an amine donor agent; wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine- containing tag at a carboxyl terminus, an amino terminus, or at an another site in the polypeptide, and wherein the acyl donor glutamine-containing tag comprises an amino acid sequence LLQGPX, wherein X is A or P (SEQ ID NO: 14), or GGLLQGPP (SEQ ID NO: 13), comprising the steps of: a) providing an engineered polypeptide-T molecule comprising the polypeptide and the acyl donor glutamine-containing tag; b) contacting the amine donor agent with the engineered polypeptide-T molecule in the presence of a transglutaminase; and c) allowing the engineered polypeptide-
  • polypeptide oligopeptide
  • peptide protein
  • the terms “polypeptide”, “oligopeptide”, “peptide” and “protein” are used interchangeably herein to refer to chains of amino acids of any length, preferably, relatively short (e.g., 10-100 amino acids).
  • the chain may be linear or branched, it may comprise modified amino acids, and/or may be interrupted by non-amino acids.
  • the terms also encompass an amino acid chain that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
  • polypeptides can occur as single chains or associated chains.
  • Fc-containing polypeptide refers to a polypeptide (e.g., an antibody or an immunoadhesin) comprising the carboxyl terminal polypeptide sequences of an immunoglobulin heavy chain.
  • the Fc-containing polypeptide may comprise native or variant Fc regions (i.e., sequences).
  • the Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain.
  • An Fc- containing polypeptide may comprise part or all of a wild-type hinge sequence (generally at amino terminus of the Fc-containing polypeptide).
  • An Fc-containing polypeptide may also be a dimer.
  • An Fc-containing polypeptide may be obtained or derived from any suitable immunoglobulin, such as from at least one of the various IgGl, IgG2, IgG3, or IgG4 subtypes, or from IgA, IgE, IgD or IgM.
  • suitable immunoglobulin such as from at least one of the various IgGl, IgG2, IgG3, or IgG4 subtypes, or from IgA, IgE, IgD or IgM.
  • the boundaries of the Fc region of an immunoglobulin heavy chain might vary, for example, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Glu216, or from Ala231, to the carboxyl -terminus thereof.
  • An Fc-containing polypeptide may be an Fc-containing fusion polypeptide, wherein one or more polypeptides are operably linked to an Fc-containing polypeptide.
  • An Fc fusion combines the Fc polypeptide of an immunoglobulin with a fusion partner, which in general may be any protein, polypeptide, or small molecule. Virtually any protein or small molecule may be linked to the Fc region to generate an Fc-containing fusion polypeptide.
  • Fc-containing fusion partners may include, but are not limited, the target-binding region of a receptor, an adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine, or some other protein or protein domain.
  • acyl donor glutamine-containing tag refers to a polypeptide or a protein containing one or more Gin residue(s) that acts as a transglutaminase amine acceptor.
  • amine donor agent refers to an agent containing one or more reactive amines (e.g., primary amines).
  • the amine donor agent can comprise an amine donor unit (e.g., primary amine H 2 ), a linker, and an agent moiety (e.g., a small molecule).
  • the amine donor agent can also be a polypeptide (e.g., an antibody) or a biocompatible polymer containing a reactive Lys (e.g., an endogenous Lys).
  • biocompatible polymer refers to a polymer (e.g., repeating monomelic or structural units) that is suitable for therapeutic or medical treatment in a recipient (e.g., human) without eliciting any undesirable local or systemic effects in the recipient.
  • a biocompatible polymer synthetic, recombinant, or native
  • a biocompatible polymer can also be a linear or a branched polymer.
  • site specificity refers to the specific conjugation or crosslinking of the amine donor agent to the polypeptide engineered with an acyl donor glutamine-containing tag at a specific site (e.g., carboxyl terminus or amino terminus of the antibody or toxin polypeptide, accessible site in the antibody (e.g., antibody light chain and/or heavy chain loops) or toxin polypeptide (e.g., polypeptide loops)).
  • a specific site e.g., carboxyl terminus or amino terminus of the antibody or toxin polypeptide, accessible site in the antibody (e.g., antibody light chain and/or heavy chain loops) or toxin polypeptide (e.g., polypeptide loops)
  • the polypeptide engineered with an acyl donor glutamine-containing tag can be an Fc-containing polypeptide, Fab-containing polypeptide, or a toxin polypeptide (e.g., a protein bound to an ion channel).
  • site specificity can also refer to the specific conjugation or crosslinking of the polypeptide (e.g., toxin polypeptide) to the biocompatible polymer engineered with an acyl donor glutamine-containing tag at a specific site (e.g., an accessible site in the biocompatible polymer).
  • Site specificity can be measured by various techniques, including, but not limited to, mass spectrometry (e.g., matrix-assisted laser-desorption ionization mass spectrometry (MALDI-MS), electrospray ionization mass spectrometry (ESI-MS), tandem mass spectrometry (MS), and time- of-flight mass spectrometry (TOF-MS)), hydrophobic interaction chromatography, ion exchange chromatography, site-directed mutagenesis, fluorescence-labeling, size exclusion chromatography, and X-ray crystallography.
  • mass spectrometry e.g., matrix-assisted laser-desorption ionization mass spectrometry (MALDI-MS), electrospray ionization mass spectrometry (ESI-MS), tandem mass spectrometry (MS), and time- of-flight mass spectrometry (TOF-MS)
  • hydrophobic interaction chromatography e.g., matrix-assisted laser-
  • antibody is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule.
  • the term is intended to encompass not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab' )2, Fv), single chain (ScFv) and domain antibodies, including shark and camelid antibodies), and fusion proteins comprising an antibody portion, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity) and antibody fragments as described herein, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site.
  • An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class.
  • immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2.
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • the subunit structures and three- dimensional configurations of different classes of immunoglobulins are well known. On one aspect, the immunoglobulin is a human, murine, or rabbit immunoglobulin.
  • Fab containing polypeptide refers to a polypeptide comprising a Fab fragment, Fab' fragment, or "(Fab')2 fragment.
  • a Fab-containing polypeptide may comprise part or all of a wild-type hinge sequence (generally at the carboxyl terminus of the Fab portion of the polypeptide).
  • a Fab-containing polypeptide may be obtained or derived from any suitable immunoglobulin, such as from at least one of the various IgGl, IgG2, IgG3, or IgG4 subtypes, or from IgA, IgE, IgD or IgM.
  • a Fab-containing polypeptide may be a Fab-containing fusion polypeptide, wherein one or more polypeptides are operably linked to a Fab-containing polypeptide.
  • a Fab fusion combines the Fab polypeptide of an immunoglobulin with a fusion partner, which in general may be any protein, polypeptide, or small molecule. Virtually any protein or small molecule may be linked to the Fab polypeptide to generate a Fab-containing fusion polypeptide.
  • Fab-containing fusion partners may include, but are not limited to, the target-binding region of a receptor, an adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine, or some other protein or protein domain.
  • a "Fab fragment” is comprised of one light chain and the CHI and variable regions of one heavy chain.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • a "Fab' fragment” contains one light chain and a portion of one heavy chain that contains the VH domain and the CHI domain and also the region between the CHI and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule.
  • a “F(ab')2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the CHI and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains.
  • a F(ab')2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.
  • Antibody fragments as used herein comprise only a portion of an intact antibody, wherein the portion preferably retains at least one, preferably most or all, of the functions normally associated with that portion when present in an intact antibody.
  • a “multispecific antibody” is one that targets more than one antigen or epitope.
  • a “bispecific,” “dual-specific” or “bifunctional” antibody is a hybrid antibody having two different antigen binding sites.
  • Bispecific antibodies are a species of multispecific antibody and may be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann (1990), Clin. Exp. Immunol. 79:315-321; and Kostelny et al. (1992), J. Immunol. 148: 1547-1553.
  • the two binding sites of a bispecific antibody will bind to two different epitopes, which may reside on the same or different protein targets.
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Further, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • the monoclonal antibodies herein may, in certain embodiments, specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81 :6851-6855 (1984)).
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may, moreover, comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • a "human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • immunoadhesin designates antibody-like or immunoglobulin- like molecules which combine the "binding domain" of a heterologous protein (an “adhesin", e.g. a receptor, ligand or enzyme) with the effector component of immunoglobulin constant domains (i.e., Fc domain).
  • adhesin e.g. a receptor, ligand or enzyme
  • the immunoadhesins comprise a fusion of the adhesin amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site (antigen combining site) of an antibody (i.e. is "heterologous") and an immunoglobulin constant domain sequence.
  • the immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgGl, IgG2, IgG3, or IgG4 subtypes, IgA, IgE, IgD or IgM.
  • the "hinge region,” “hinge sequence,” and variation thereof, as used herein, includes the meaning known in the art, which is illustrated, for example, Janeway et al., ImmunoBiology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4 th ed., 1999); Bloom et al., Protein Science (1997), 6:407-415; Humphreys et al., J. Immunol. Methods (1997), 209: 193-202.
  • wild-type amino acid refers to a sequence of amino acids or nucleic acids that occurs naturally within a certain population (e.g., human, mice, rats, cells, etc.).
  • conjugation efficiency or “crosslinking efficiency” is the ratio between the experimentally measured amount of engineered polypeptide conjugate divided by the maximum expected engineered polypeptide conjugate amount. Conjugation efficiency or crosslinking efficiency can be measured by various techniques well known to persons skilled in the art, such as hydrophobic interaction chromatography. Conjugation efficiency can also be measured at different temperature, such as room temperature or 37°C.
  • effector function refers to the biological activities attributable to the Fc region of an antibody.
  • antibody effector functions include, but are not limited to, antibody- dependent cell-mediated cytotoxicity (ADCC), Fc receptor binding, complement dependent cytotoxicity (CDC), phagocytosis, Clq binding, and down regulation of cell surface receptors (e.g., B cell receptor; BCR). See, e.g., U.S. Pat No. 6,737,056.
  • Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays known in the art for evaluating such antibody effector functions.
  • An exemplary measurement of effector function is through Fcy3 and/or Clq binding.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcRs Fc receptors
  • NK natural killer cells
  • macrophages e.g. natural killer cells, neutrophils, and macrophages
  • ADCC activity of a molecule of interest can be assessed using an in vitro ADCC assay, such as that described in U.S. Patent No. 5,500,362 or 5,821,337.
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and NK cells.
  • PBMC peripheral blood mononuclear cells
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., 1998, PNAS (USA), 95:652-656.
  • “Complement dependent cytotoxicity” or “CDC” refers to the lysing of a target in the presence of complement.
  • the complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule (e.g. an antibody) complexed with a cognate antigen.
  • a CDC assay e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods, 202: 163 (1996), may be performed.
  • Fc receptor and “FcR” describe a receptor that binds to the Fc region of an antibody.
  • the preferred FcR is a native sequence human FcR.
  • a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, FcyRIII, and FcyRIV subclasses, including allelic variants and alternatively spliced forms of these receptors.
  • FcyRll receptors include FcyRIIA (an "activating receptor") and FcyRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
  • FcRs are reviewed in Ravetch and Kinet, 1991, Ann. Rev. Immunol, 9:457-92; Capel et al., 1994, Immunomethods, 4:25-34; de Haas et al., 1995, J. Lab. Clin. Med, 126:330-41; Nimmerjahn et al., 2005, Immunity 23 :2-4.
  • FcR also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., 1976, J. Immunol., 117:587; and Kim et al., 1994, J. Immunol., 24:249).
  • beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing the proliferation of (or destroying) neoplastic or cancerous cells, inhibiting metastasis of neoplastic cells, shrinking or decreasing the size of tumor, cancer remission, decreasing cancer symptoms, increasing the quality of life of those suffering from cancer, decreasing the dose of other medications required to treat cancer, delaying the progression of cancer, curing cancer, and/or prolong survival of a cancer patients.
  • an "effective dosage” or “effective amount” of drug, compound, or pharmaceutical composition is an amount sufficient to effect any one or more beneficial or desired results.
  • beneficial or desired results include eliminating or reducing the risk, lessening the severity, or delaying the outset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease.
  • beneficial or desired results include clinical results such as reducing incidence or amelioration of one or more symptoms of various cancer-related diseases or conditions (such as gastric, head and neck, lung, ovarian, and pancreatic cancers), decreasing the dose of other medications required to treat the disease, enhancing the effect of another medication, and/or delaying the progression of the cancer in patients.
  • An effective dosage can be administered in one or more administrations.
  • an effective dosage of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly.
  • an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition.
  • an "effective dosage" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.
  • purify and grammatical variations thereof, is used to mean the removal, whether completely or partially, of at least one impurity from a mixture containing the polypeptide and one or more impurities, which thereby improves the level of purity of the polypeptide in the composition (i.e., by decreasing the amount (ppm) of impurity(ies) in the composition).
  • references to "about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to "about X” includes description of "X.” Numeric ranges are inclusive of the numbers defining the range.
  • mammals are a mammal, more preferably, a human. Mammals also include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice, and rats.
  • the present invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group, but also the main group absent one or more of the group members.
  • the present invention also envisages the explicit exclusion of one or more of any of the group members in the claimed invention.
  • the engineered polypeptide conjugates herein comprise a polypeptide (e.g., Fc-containing polypeptide, Fab-containing polypeptide, or antibody) engineered to a specific acyl donor glutamine-containing tag, wherein the polypeptide is site-specifically conjugated to an amine donor agent (e.g., a small molecule coupled to a linker) via the acyl donor glutamine-containing tag.
  • the engineered polypeptide e.g., Fc-containing polypeptide or antibody
  • an engineered polypeptide conjugate comprising the formula: polypeptide-T-A, wherein T is an acyl donor glutamine-containing tag engineered at a specific site, wherein A is an amine donor agent, wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine-containing tag at a carboxyl terminus, an amino terminus, or at an another site in the polypeptide, and wherein the acyl donor glutamine-containing tag comprises an amino acid sequence LLQGPX, wherein X is A or P (SEQ ID NO: 14), or GGLLQGPP (SEQ ID NO: 13).
  • the polypeptide is a Fc-containing polypeptide or a Fab-containing polypeptide.
  • an engineered Fc-containing polypeptide conjugate comprising the formula: (Fc-containing polypeptide)-T-A, wherein T is an acyl donor glutamine- containing tag engineered at a specific site, wherein A is an amine donor agent, wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine-containing tag at a carboxyl terminus, an amino terminus, or at an another site in the Fc-containing polypeptide, wherein the acyl donor glutamine-containing tag comprises an amino acid sequence XXQX (SEQ ID NO:35), wherein X is any amino acid, and wherein the engineered Fc-containing polypeptide conjugate comprises an amino acid substitution from glutamine to asparagine at position 295 (e.g., Q295N; EU numbering scheme).
  • Both the acyl donor glutamine-containing tag and the amine donor agent described herein are substrates for transglutaminase, and the linkage between the acyl donor glutamine-containing tag and the amine donor agent is of the formula CH 2 -CH 2 -CO- H-, wherein H- is linked to a linker and an agent moiety.
  • the transglutaminase used in the invention described herein can be obtained or made from a variety of sources.
  • the transglutaminase is a calcium dependent transglutaminase which requires calcium to induce enzyme conformational changes and allow enzyme activity.
  • transglutaminase can be derived from guinea pig liver and obtained through commercial sources (e.g., Sigma-Aldrich (St Louis, MO) and MP Biomedicals (Irvine, CA)).
  • the transglutaminase is a calcium independent transglutaminase which does not require calcium to induce enzyme conformational changes and allow enzyme activity.
  • the transglutaminase is a microbial transglutaminase derived from a microbial genome, such as transglutaminase from Streptoverticillium or Streptomyces (e.g., Streptomyces mobarensis or Streptoverticillium mobarensis).
  • the transglutaminase is a mammalian protein (e.g., human transglutaminase), a bacterial protein, a plant protein, a fungi protein (e.g., Oomycetes and Actinomicetes transglutaminases), or a prokaryotic protein.
  • the transglutaminase is from Micrococcus, Clostridium, Turolpsis, Rhizopus, Monascus, or Bacillus.
  • the transglutaminase used in the invention described herein is an engineered transglutaminase, which catalyzes transamidation of 1) one or more exogenous glutamine residues on the acyl donor glutamine-containing tag and additionally/optionally 2) endogenous glutamine residues in the antibody, with one or more lysine residues or reactive amines in the amine donor agent.
  • an engineered transglutaminase which catalyzes transamidation of 1) one or more exogenous glutamine residues on the acyl donor glutamine-containing tag and additionally/optionally 2) endogenous glutamine residues in the antibody, with one or more lysine residues or reactive amines in the amine donor agent.
  • one or more wild-type amino acid residues in the naturally occurring transglutaminase are deleted, replaced or substituted with another amino acid residue(s) to make the engineered transglutaminase.
  • the transglutaminase used in the invention described herein can also be a recombinant protein produced using recombinant techniques known to persons skilled in the art.
  • the transglutaminase used in the invention described herein can be a purified protein.
  • the purified transglutaminase is least about 50% pure.
  • pure or “purified” protein refers to a protein (e.g., transglutaminase) free from other protein contaminants.
  • the purified transglutaminase is at least about any of 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-98%, or 99% pure. In some embodiments, the purified transglutaminase is about any of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% pure.
  • the acyl donor glutamine-containing tag of the engineered polypeptide (e.g., Fc-containing, Fab-containing polypeptide, or antibody) conjugate as described herein is not spatially adjacent to a reactive Lys in the polypeptide.
  • the acyl donor glutamine-containing tag is not spatially adjacent to a reactive Lys in the carboxyl terminus, the amino terminus, or both the carboxyl and the amino termini of the polypeptide.
  • the acyl donor glutamine-containing tag comprises an amino acid sequence XXQX (SEQ ID NO: 35), wherein X can be a conventional or nonconventional amino acid, as described herein, and can be the same or a different amino acid.
  • X is L (Leu), A (Ala), G (Gly), S (Ser), V (Val), F (Phe), Y (Tyr), H (His), R (Arg), N (Asn), E (Glu), D (Asp), C (Cys), Q (Gin), I (lie), M (Met), P (Pro), T (Thr), K (Lys), or W (Trp).
  • the acyl donor glutamine-containing tag comprises an amino acid sequence selected from the group consisting of LLQGG (SEQ ID NO: 16), LLQG (SEQ ID NO: 17), LSLSQG (SEQ ID NO: 18), GGGLLQGG (SEQ ID NO: 19), GLLQG (SEQ ID NO:20), LLQ, GSPLAQSHGG (SEQ ID NO:21), GLLQGGG (SEQ ID NO:22), GLLQGG (SEQ ID NO:23), GLLQ (SEQ ID NO:24), LLQLLQGA (SEQ ID NO:25), LLQGA (SEQ ID NO:26), LLQYQGA (SEQ ID NO:27), LLQGSG (SEQ ID NO:28), LLQYQG (SEQ ID NO:29), LLQLLQG (SEQ ID NO:30), SLLQG (SEQ ID NO:31), LLQLQ (SEQ ID NO:32), LLQLLQ (SEQ ID NO
  • the acyl donor glutamine-containing tag comprises an amino acid sequence LLQGPA (SEQ ID N0:4), LLQGP (SEQ ID N0:5), LLQGPP (SEQ ID NO: 11), or GGLLQGPP (SEQ ID NO: 13).
  • the acyl donor glutamine-containing tag does not comprise an amino acid sequence selected from the group consisting of LGGQGGG (SEQ ID NO:41), GGGQGGL (SEQ ID NO:42), GXGQGGG (SEQ ID NO:43), GGXQGGG (SEQ ID NO:44), GGGQXGG (SEQ ID NO:45), and GGGQGXG (SEQ ID NO:46), wherein X is G, A, S, L, V, F, Y, R, N, or E).
  • the polypeptide (e.g., Fc-containing polypeptide, Fab-containing polypeptide, or antibody) of the engineered polypeptide conjugate comprises an amino acid modification at the last amino acid position in the carboxyl terminus relative to a wild-type polypeptide at the same position.
  • the modification is an amino acid deletion, insertion, substitution, mutation, or any combination thereof.
  • the substitution comprises replacing a wild type amino acid with another (e.g., a non-wild type amino acid).
  • the insertion comprises inserting one or more amino acid(s) (e.g., inserting one, two, three or more amino acids).
  • the other (e.g., non- wild type) or inserted amino acid is Arg.
  • the other (e.g., non-wild type) amino acid is Ala, Asn, Asp, Cys, Glu, Gin, Gly, His, He, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the last amino acid in the carboxyl terminus of the polypeptide e.g., the heavy chain of an antibody
  • the acyl donor glutamine- containing tag engineered to the C-terminus of the polypeptide comprises the amino acid sequence LLQGPA (SEQ ID NO:4) or LLQGPP (SEQ ID NO: 11).
  • the polypeptide (e.g., Fc-containing polypeptide or antibody) of the engineered polypeptide conjugate comprises an amino acid modification at position 222, 340, or 370 (EU numbering) relative to the wild-type polypeptide at the same position.
  • the modification is an amino acid deletion, insertion, substitution, mutation, or any combination thereof.
  • the substitution comprises replacing a wild type amino acid with another (e.g., a non-wild type amino acid).
  • the other (e.g., non-wild type) or inserted amino acid is Arg (e.g., K222R, K340R, or K370R (EU numbering)).
  • the insertion comprises inserting one or more amino acid(s) (e.g., inserting one, two, three or more amino acids).
  • the other (e.g., non- wild type) amino acid is Ala, Asn, Asp, Cys, Glu, Gin, Gly, His, He, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the polypeptide (e.g., an antibody) comprising the K222R substitution is engineered to the acyl donor glutamine-containing tag at the C-terminus of the light chain of the antibody, wherein the glutamine-containing tag comprises an amino acid sequence of GGLLQGPP (SEQ ID NO: 13).
  • the polypeptide (e.g., Fc-containing polypeptide, Fab-containing polypeptide, or antibody) comprises an amino acid modification at the first amino acid position in the amino terminus relative to a wild-type polypeptide at the same position.
  • the modification is an amino acid deletion, insertion, substitution, mutation, or any combination thereof.
  • the substitution comprises replacing a wild type amino acid with another (e.g., non-wild type) amino acid.
  • the insertion comprises inserting an amino acid.
  • the non-wild type or inserted amino acid is Arg.
  • the other (non-wild type or inserted) amino acid is Ala, Asn, Asp, Cys, Glu, Gin, Gly, His, He, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the polypeptide conjugate described herein comprises a full length antibody heavy chain and an antibody light chain.
  • the acyl donor glutamine-containing tag is linked to/located at the polypeptide at the carboxyl terminus of a heavy chain, a light chain, or both the heavy chain and the light chain.
  • the acyl donor glutamine-containing tag GGLLQGPP (SEQ ID NO: 13) is linked to the polypeptide at the carboxyl terminus of a light chain.
  • the acyl donor glutamine-containing tag GGLLQGPP is linked to the polypeptide comprising the Q295N mutation (EU numbering scheme) at the carboxyl terminus of a light chain.
  • the acyl donor glutamine-containing tag LLQGPA (SEQ ID NO:4), LLQGP (SEQ ID NO:5), or LLQGPP (SEQ ID NO: 11) is linked to the polypeptide comprising the Q295N mutation at the carboxyl terminus of a heavy chain.
  • the polypeptide described herein is a monoclonal antibody, a polyclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, a minibody, a diabody, or an antibody fragment.
  • the antibody is an IgG.
  • the IgG is selected from the group consisting of IgGl, IgG2, IgG3, and IgG4.
  • the antibody is an IgA, IgE, IgD, or IgM.
  • the acyl donor glutamine-containing tag is located in the polypeptide by insertion or by replacement of one or more wild-type amino acid(s) at another site on the polypeptide described herein (e.g., Fc-containing polypeptide, Fab-containing polypeptide, or antibody), wherein the other site is not an amino or a carboxyl terminus.
  • the acyl donor glutamine-containing tag is part of an antibody loop.
  • the acyl donor glutamine- containing tag can be linked to one or more heavy chain loop(s).
  • the acyl donor glutamine- containing tag can also be linked to one or more light chain loop(s) of the antibody.
  • the acyl donor glutamine-containing tag is located at both a heavy chain and a light chain loops.
  • the another site is amino acid position(s) 108, 135, 160, 168, 189-192, 190-192, 200-202, 222-223, 251-254, 252-253, 222-223, 293-297, 294-297, 295, 297, or 385 (EU numbering scheme) of the human IgGl antibody.
  • the effector function (e.g., as measured by Fcy3 and/or Clq binding) of the engineered polypeptide (e.g., Fc-containing polypeptide or antibody) conjugate described herein decreases no greater than about any of 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold relative to a wild type polypeptide (e.g., Fc-containing polypeptide or antibody).
  • the engineered polypeptide conjugate is an IgG, wherein the effector function of the IgG decreases no greater than about 2-fold relative to a wild type IgG. In other embodiments, the effector function of the IgG decreases about 2-fold relative to a wild type IgG.
  • the effector function of the IgG decreases more than about 2-fold relative to a wild type IgG.
  • the engineered Fc-containing polypeptide conjugate is an IgG, wherein the effector function of the IgG decreases no greater than about 1-fold relative to a wild type IgG.
  • the effector function of the IgG decreases about 1-fold relative to a wild type IgG.
  • the effector function of the IgG decreases more than about any of 1-fold, 3-fold, 4-fold, or 5-fold relative to a wild type IgG.
  • the effector function (e.g., as measured by Fcy3 and/or Clq binding) of the engineered polypeptide (Fc-containing polypeptide or antibody) conjugate described herein increases at least about 1-fold to 3000-fold relative to a wild type polypeptide (e.g., Fc-containing polypeptide or antibody).
  • the effector function of the engineered polypeptide (e.g., Fc-containing polypeptide or antibody) conjugate increases at least about any of 1- to 5-fold, 6- to 10-fold, 11- to 15-fold, 16- to 20-fold, 21- to 25-fold, 26- to 30- fold, 31- to 35-fold, 36- to 40-fold, 41- to 45-fold, 46- to 50-fold, 51- to 55-fold, 56- to 60-fold, 61- to 65-fold, 66- to 70-fold, 71- to 75-fold, 76- to 80-fold, 81- to 85-fold, 86- to 90-fold, 91- to 95-fold, 96- to 100-fold, 101- to 200-fold, 201- to 300-fold, 301- to 500-fold, 501- to 1000-fold, 1001- to 1500-fold, 1501- to 2000-fold, 2001- to 2500-fold, 2501- to 3000-fold relative to a wild type polypeptide (e)
  • the engineered polypeptide conjugate is an IgG, wherein the effector function of the IgG increases about 1-fold to 300-fold relative to a wild type IgG.
  • the effector function of the IgG increases about any of 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 40-fold, 60-fold, 80-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 1500-fold, 2000-fold, 2500-fold, or 3000-fold relative to a wild type IgG.
  • the amine donor agent has the formula: X-Y-Z, wherein X is an amine donor unit; Y is a linker; and Z is an agent moiety.
  • the number of the amine donor agents which may be conjugated via the acyl donor glutamine-containing tag to the polypeptide is dependent on the number of acyl donor glutamine-containing tags which are linked/inserted to the polypeptide(s) as well as the number of Gin on the acyl donor glutamine-containing tag.
  • two amine donor agents may be site-specifically conjugated to an antibody at the carboxyl termini of the two heavy chains and/or two amine donor agents may be site-specifically conjugated to the same antibody at the carboxyl termini of the two light chains.
  • the amine donor unit of the present invention is a primary amine ( H 2 ) that provides a substrate for transglutaminase to allow conjugation of the agent moiety to the polypeptide via the acyl donor glutamine-containing tag.
  • H 2 primary amine
  • the linkage between the acyl donor glutamine-containing tag and the amine donor unit is of the formula CH 2 -CH 2 -CO- H-, wherein H- is linked to a linker and an agent moiety.
  • the linker of the present invention can be a cleavable or a non-cleavable linker.
  • the linker (with amine donor unit) or the amine donor agent can be released from the polypeptide.
  • the linker can be a peptide linker (e.g., conventional or nonconventional amino acid(s)) and/or a non-peptide linker.
  • a non-peptide linker include an alkyl linker and a PEG linker.
  • the amine donor unit-linker (e.g., X-Y) is a linear unit comprising an agent moiety.
  • the amine donor unit-linker is a branched unit (e.g., at least 2 units) comprising at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more agent moieties.
  • Exemplary amine donor unit-linkers include, but are not limited to, Ac-Lys-Gly, aminocaproic acid, Ac-Lys-P-Ala, amino-PEG2-C2, amino-PEG3-C2, amino-PEG6-C2, Ac-Lys- Val-Cit (citrulline)-PABC (p-aminobenzyloxycarbonyl), aminocaproyl-Val-Cit-PABC, amino- PEG6-C2-Val-Cit-PABC, aminocaproyl-Val-Cit-PABC, [(3R,5R)-l- ⁇ 3-[2-(2- aminoethoxy)ethoxy]propanoyl ⁇ piperidine-3,5-diyl]bis-Val-Cit-PABC, [(3S,5S)-l- ⁇ 3-[2-(2- aminoethoxy)ethoxy]propanoyl ⁇ piperidine-3,5-diyl]bis-Val-Cit-PABC-, Ac-
  • a small molecule is a cytotoxic agent, an immunosuppressive agent, or an imaging agent (e.g., a fluorophore).
  • the cytotoxic agent is a chemotherapeutic agent.
  • cytotoxic agent examples include, but are not limited to, an anthracycline, an auristatin, a camptothecin, a combretastatin, a dolastatin, a duocarmycin, an enediyne, a geldanamycin, an indolino-benzodiazepine dimer, a maytansine, a puromycin, a taxane, a vinca alkaloid, SN-38, a tubulysin, a hemiasterlin, a spliceostatin, a pladienolide, and stereoisomers, isosteres, analogs, or derivatives thereof.
  • the anthracyclines are derived from bacteria Strepomyces and have been used to treat a wide range of cancers, such as leukemias, lymphomas, breast, uterine, ovarian, and lung cancers.
  • exemplary anthracyclines include, but are not limited to, daunorubicin, doxorubicin (i.e., adriamycin), epirubicin, idarubicin, valrubicin, and mitoxantrone.
  • Dolastatins and their peptidic analogs and derivatives, auristatins are highly potent antimitotic agents that have been shown to have anticancer and antifungal activity. See, e.g., U.S. Pat. No. 5,663,149 and Pettit et al., Antimicrob. Agents Chemother. 42:2961-2965, 1998.
  • Exemplary dolastatins and auristatins include, but are not limited to, dolastatin 10, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), MMAD (Monomethyl Auristatin D or monomethyl dolastatin 10), MMAF (Monomethyl Auristatin F or N-methylvaline-valine-dolaisoleuine- dolaproine-phenylalanine), MMAE (Monomethyl Auristatin E or N-methylvaline-valine- dolaisoleuine-dolaproine-norephedrine), 5 -benzoyl valeric acid-AE ester (AEVB), and other novel auristatins (such as the ones described in U.S.
  • the auristatin is 0101 (2-methylalanyl-iV-[(3R,4S, 5S)-3-methoxy-l- ⁇ (2S)-2- [(1R.2R)- 1 -methoxy-2-methyl-3 -oxo-3- ⁇ [(15)-2-phenyl- 1 -(1 ,3-thiazol-2- yl)ethyl]amino ⁇ propyl]pyrrolidin-l-yl ⁇ -5-methyl-l-oxoheptan-4-yl]-N-methyl-L-valinamide) having the following structure:
  • the auristatin is 3377 (N,2-dimethylalanyl-N- ⁇ (l S,2R)-4- ⁇ (2S)-2- [(lR,2R)-3- ⁇ [(l S)-l-carboxyl-2-phenylethyl]amino ⁇ -l-methoxy-2-methyl-3- oxopropyl]pyrrolidin- 1 -yl ⁇ -2-methoxy- 1 -[(1 S)-l -methylpropyl]-4-oxobutyl ⁇ -N-m ethyl -L- valinamide) having the following structure:
  • the auristatin is 0131 (2-methyl-L-proly-N-[(3R,4S,5S)-l- ⁇ (2S)-2- [(lR,2R)-3- ⁇ [(l S)-l-carboxy-2-phenylethyl]amino ⁇ -l-methoxy-2-methyl-3- oxopropyl]pyrrolidin-l-yl ⁇ -3-methoxy-5-methyl-l-oxoheptan-4-yl]-N-methyl-L-valinamide) having the following structure:
  • the auristatin is 0121(2-methyl-L-proly-N-[(3R,4S,5S)-l- ⁇ (2S)-2- [(lR,2R)-3- ⁇ [(2S)-l-methoxy-l-oxo-3-phenylpropan-2-yl]amino ⁇ -l-methoxy-2 -methyl -3- oxopropyl]pyrrolidin-l-yl ⁇ -3-methoxy-5-methyl-l-oxoheptan-4-yl]-N-methyl-L-valinamide) having the following structure:
  • Camptothecin is a cytotoxic quinoline alkaloid which inhibits the enzyme topoisomerase I.
  • Examples of camptothecin and its derivatives include, but are not limited to, topotecan and irinotecan, and their metabolites, such as SN-38.
  • Combretastatins are natural phenols with vascular disruption properties in tumors.
  • Exemplary combretastatins and their derivatives include, but are not limited to, combretastatin A- 4 (CA-4) and ombrabulin.
  • Duocarmycins are DNA alkylating agents with cytotoxic potency. See Boger and Johnson, PNAS 92:3642-3649 (1995). Exemplary duocarmycins include, but are not limited to, duocarmycin A, duocarmycin Bl, duocarmycin B2, duocarmycin CI, duocarmycin C2, duocarmycin D, duocarmycin SA, and CC-1065.
  • Enediynes are a class of anti-tumor bacterial products characterized by either nine- and ten-membered rings or the presence of a cyclic system of conjugated triple-double-triple bonds.
  • Exemplary enediynes include, but are not limited to, calicheamicin, esperamicin, and dynemicin.
  • Geldanamycins are benzoquinone ansamycin antibiotic that bind to Hsp90 (Heat Shock Protein 90) and have been used antitumor drugs.
  • exemplary geldanamycins include, but are not limited to, 17-AAG (17-N-Allylamino-17-Demethoxygeldanamycin) and 17-DMAG (17- Dimethylaminoethylamino- 17-demethoxygeldanamycin).
  • Hemiasterlin and its analogues bind to the tubulin, disrupt normal microtubule dynamics, and, at stoichiometric amounts, depolymerize microtubules.
  • Maytansines or their derivatives maytansinoids inhibit cell proliferation by inhibiting the mcirotubules formation during mitosis through inhibition of polymerization of tubulin. See Remillard et al., Science 189: 1002-1005 (1975).
  • Exemplary maytansines and maytansinoids include, but are not limited to, mertansine (DM1) and its derivatives as well as ansamitocin.
  • PBDs Pyrrolobenzodiazepine dimers
  • IGNs indolino-benzodiazepine dimers
  • PBD and IGN molecules are based on the natural product athramycin, and interact with DNA in a sequence-selective manner, with a preference for purine-guanine-purine sequences.
  • Exemplary PBDs and their analogs include, but are not limited to, SJG-136.
  • Spliceostatins and pladienolides are anti-tumor compounds which inhibit splicing and interact with spliceosome, SF3b.
  • spliceostatins include, but are not limited to, spliceostatin A, and FR901464.
  • pladienolides include, but are not limited to, Pladienolide B, Pladienolide D, and E7107.
  • Taxanes are diterpenes that act as anti-tubulin agents or mitotic inhibitors.
  • Exemplary taxanes include, but are not limited to, paclitaxel (e.g., TAXOL ® ) and docetaxel (TAXOTERE ® ).
  • Tubulysins are natural products isolated from a strain of mycobacteria that has been shown to depolymerize microtubules and induce mitotic arrest.
  • Exemplary tubulysins include, but are not limited to, tubulysin A, tubulysin B, and tubulysin D.
  • Vinca alkyloids are also anti-tubulin agents.
  • Exemplary vinca alkyloids include, but are not limited to, vincristine, vinblastine, vindesine, and vinorelbine.
  • the agent moiety is an immunosuppressive agent.
  • immunosuppressive agent include, but are not limited to, gancyclovier, etanercept, tacrolimus, sirolimus, voclosporin, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolgate mofetil, methotrextrate, and glucocorticoid and its analogs.
  • the agent moiety is an imaging agent (e.g., a fluorophore or a chelator), such as fluorescein, rhodamine, lanthanide phosphors, and their derivatives thereof.
  • fluorophores include, but are not limited to, fluorescein isothiocyanate (FITC) (e.g., 5-FITC), fluorescein amidite (FAM) (e.g., 5-FAM), eosin, carboxyfluorescein, erythrosine, Alexa Fluor ® (e.g., Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700, or 750), carboxytetramethylrhodamine (TAMRA) (e.g., 5,-TAMRA), tetramethylrhodamine (TMR), and sulforhod
  • chelators include, but are not limited to, l,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA), l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA), l,4,7-triazacyclononane,l- glutaric acid-4,7-acetic acid (NODAGA), diethylenetrianiixiepentaacetic acid (DTPA), and 1,2- bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) (BAPTA).
  • DOTA l,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid
  • NOTA l,4,7-triazacyclononane-l,4,7-triacetic acid
  • NODAGA diethylenetrianiixiepentaacetic acid
  • BAPTA
  • the agent moiety is a polypeptide.
  • the polypeptide is an antibody, such as a humanized, human, chimeric, or murine monoclonal antibody.
  • the agent moiety is a toxin polypeptide (or a toxin protein).
  • a toxin polypeptide include, but are not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, tricothecenes, inhibitor cystine knot (ICK) peptides (e.g., ceratotoxins), and conotoxin (e.g., KIIIA or SmIIIa).
  • ICK inhibitor cystine knot
  • therapeutic radioisotopes or other labels can be incorporated in the agent moiety (e.g., by binding to a chelator) for conjugation of a polypeptide (e.g., a Fc- containing or Fab-containing polypeptide) to an amine donor agent that bears a chelator.
  • a polypeptide e.g., a Fc- containing or Fab-containing polypeptide
  • Examples of a radioisotope or other labels include, but are not limited to, 3 H, 14 C, 15 N, 35 S, 18 F,
  • the agent moiety is a biocompatible polymer.
  • the polypeptide can be conjugated to the biocompatible polymer via the acyl donor glutamine-containing tag to improve the biological characteristics of the polypeptide, e.g., to increase serum half-life and bioactivity, and/or to extend in vivo half-lives.
  • biocompatible polymers include water-soluble polymer, such as polyethylene glycol (PEG) or its derivatives thereof and zwitterion-containing biocompatible polymers (e.g., a phosphorylcholine containing polymer).
  • X is H 2 (i.e., thus forming a covalent bond with glutamine as CH 2 -CH 2 -CO- H-), m is a 0 to 20, n is 1 to 8, p is 0 to 3, q is 0 or 1, amino acid is any conventional or nonconventional amino acid and Z is a cytotoxic agent or an imaging agent.
  • Conventional or naturally occurring amino acids are divided into groups based on common side-chain properties: (1) non-polar: Norleucine, Met, Ala, Val, Leu, He; (2) polar without charge: Cys, Ser, Thr, Asn, Gin; (3) acidic (negatively charged): Asp, Glu; (4) basic (positively charged): Lys, Arg; and (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe, His.
  • Conventional amino acids include L or D stereochemistry. Unconventional amino acids are non-naturally occurring amino acids.
  • an uncoventioanl amino acid examples include, but are not limited to, aminoadipic acid, beta-alanine, beta- aminopropionic acid, aminobutyric acid, piperidinic acid, aminocaprioic acid, aminoheptanoic acid, aminoisobutyric acid, aminopimelic acid, citmlline, diaminobutyric acid, desmosine, diaminopimelic acid, diaminopropionic acid, N-ethylglycine, N-ethylaspargine, hyroxylysine, allo-hydroxylysine, hydroxyproline, isodesmosine, allo-isoleucine, N-methylglycine, sarcosine, N-methylisoleucine, N-methylvaline, norvaline, norleucine, orithine, 4-hydroxyproline, ⁇ - carboxyglutamate, ⁇ - ⁇ , ⁇ , ⁇ -trimethyllysine, ⁇ - ⁇ -acetyllysine,
  • the amine donor agent is selected from the group consisting of Alexa 488 cadaverine, 5-FITC cadaverine, Alexa 647 cadaverine, Alexa 350 cadaverine, 5- TAMRA cadaverine, 5-FAM cadaverine, SRI 01 cadaverine, 5,6-TAMRA cadaverine, 5-FAM lysine, Ac-Lys-Gly-MMAD, amino-PEG3 -C2-MM AD, amino-PEG6-C2-MMAD, amino-PEG3- C2-amino-nonanoyl-MMAD, aminocaproyl-Val-Cit-PABC-MMAD, Ac-Lys-P-Ala-MMAD, Aminocaproyl-MMAD, amino-PEG6-C2-Val-Cit-PABC-MMAD, Ac-Lys-Val-Cit-PABC- MMAD, Ac-Lys-Val-Cit-PABC- MMAD, Ac-Lys-Val-Cit-
  • the acyl donor glutamine-containing tag comprises the amino acid sequence LLQGPA (SEQ ID NO:4), LLQGP (SEQ ID NO:5), LLQGPP (SEQ ID NO: 11), or GGLLQGPP (SEQ ID NO: 13) and the amine donor agent is Ac-Lys-Val-Cit-P ABC-MMAD, Ac-Lys-Val-Cit- PABC-0101, or amino-PEG6-C2-MMAD.
  • Exemplary structures of the amine donor agent are listed in Table 1. Table 1
  • the invention provides an engineered toxin polypeptide conjugate comprising the formula: (toxin polypeptide)-T-A, wherein T is an acyl donor glutamine- containing tag engineered at a specific site, wherein A is an amine donor agent, wherein the amine donor agent is a biocompatible polymer comprising a reactive amine, wherein the biocompatible polymer is site-specifically conjugated to the acyl donor glutamine-containing tag at a carboxyl terminus, an amino terminus, or elsewhere at an another site in the toxin polypeptide, and wherein the acyl donor glutamine-containing tag comprises an amino acid sequence LLQGPX, wherein X is A or P (SEQ ID NO: 14), or GGLLQGPP (SEQ ID NO: 13).
  • the toxin polypeptide can be site-specifically conjugated to the biocompatible polymer via the acyl donor glutamine-containing tag as described herein to improve the biological characteristics of the toxin polypeptide, e.g., to increase the serum half-life and bioactivity, and/or to extend its in vivo half-lives.
  • toxin polypeptide is a ceratotoxin or a conotoxin (e.g., KIIIA or SmIIIa).
  • the biocompatible polymer is a water soluble polymer such as PEG derivative or a zwitterion-containing biocompatible polymer.
  • the invention provides a method for preparing an engineered polypeptide conjugate comprising the formula: polypeptide-T-A, wherein T is an acyl donor glutamine-containing tag engineered at a specific site, wherein A is an amine donor agent, and wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine-containing tag at a carboxyl terminus, an amino terminus, or elsewhere at an another site in the polypeptide, and wherein the acyl donor glutamine-containing tag comprises an amino acid sequence LLQGPX, wherein X is A or P (SEQ ID NO: 14), or GGLLQGPP (SEQ ID NO: 13), comprising the steps of: a) providing an engineered polypeptide-T molecule comprising the polypeptide and the acyl donor glutamine-containing tag; b) contacting the amine donor agent with the engineered polypeptide-T molecule
  • the invention provides a method for preparing an engineered Fc- containing polypeptide conjugate comprising the formula: (Fc-containing polypeptide)-T-A, wherein T is an acyl donor glutamine-containing tag engineered at a specific site; wherein A is an amine donor agent; and wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine-containing tag at a carboxyl terminus, an amino terminus, or elsewhere at an another site in the Fc-containing polypeptide, wherein the acyl donor glutamine-containing tag comprises an amino acid sequence XXQX (SEQ ID NO: 35), wherein X is any amino acid (e.g., X can be the same or a different amino acid), and wherein the engineered Fc-containing polypeptide conjugate comprises an amino acid substitution from glutamine to asparagine at position 295 (e.g., Q295N; EU numbering scheme), comprising the steps of: a) providing an engineered (Fc-containing polypeptid
  • the engineered (Fc-containing polypeptide)-! is expressed in CHO cells.
  • the acyl donor glutamine-containing tag comprises an amino acid sequence selected from the group consisting of LLQGG (SEQ ID NO: 16), LLQG (SEQ ID NO: 17), LSLSQG (SEQ ID NO: 18), GGGLLQGG (SEQ ID NO: 19), GLLQG (SEQ ID NO:20), LLQ, GSPLAQSHGG (SEQ ID NO:21), GLLQGGG (SEQ ID NO:22), GLLQGG (SEQ ID NO:23), GLLQ (SEQ ID NO:24), LLQLLQGA (SEQ ID NO:25), LLQGA (SEQ ID NO:26), LLQYQGA (SEQ ID NO:27), LLQGSG (SEQ ID NO:28), LLQYQG (SEQ ID NO:29), LLQLLQG (SEQ ID NO:30), SLLQG (SEQ ID NO: 16),
  • the acyl donor glutamine-containing tag comprises an amino acid sequence LLQGPA (SEQ ID NO:4), LLQGP (SEQ ID NO:5), LLQGPP (SEQ ID NO: 1 1), or GGLLQGPP (SEQ ID NO: 13).
  • the engineered polypeptide conjugate prepared using the methods described herein has conjugation efficiency of at least about 51%. In some embodiments, the engineered polypeptide conjugate has conjugation efficiency of at least about any of 51%-60%, 61%-70%, 71%-80%, 81%-90%, or 91%- 100%. In some embodiments, the engineered polypeptide conjugate has conjugation efficiency of about any of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%), 99.9%, or 100%. For example, the engineered polypeptide (e.g., Fc-containing polypeptide) comprising the Q295N mutation (EU numbering scheme) has conjugation efficiency of at least about 99.8%.
  • the concentration ratio between the amine donor agent contacted and the engineered polypeptide-T molecule contacted is from about 2: 1 to about 800: 1.
  • the concentration ratio between the amine donor agent (e.g., a cytotoxic drug) and the engineered polypeptide attached to an acyl donor glutamine-containing tag loaded or used for the transglutaminase-catalyzed conjugation reaction can be about 20: 1.
  • the concentration ratio between the amine donor agent contacted and the engineered polypeptide-T molecule contacted is about any of 2: 1, 3 : 1, 4: 1, 5 : 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 15 : 1, 20: 1, 25 : 1, 30: 1, 35: 1, 40: 1, 45: 1, 50: 1, 60: 1, 70: 1, 80: 1, 90: 1, 100: 1, 200: 1, 300: 1, 400: 1, 500: 1, 600: 1, 700: 1, or 800: 1.
  • the antibody-drug-conjugate when a polypeptide (e.g., antibody) is conjugated with an amine donor agent via an acyl donor glutamine-containing tag at a specific site (e.g., C-terminus), the antibody-drug-conjugate is more stable (e.g., longer in vivo half-life). Accordingly, in some embodiments, the engineered polypeptide conjugate as described herein is present in a subject (e.g., a mammal) at at least about 50% after at least about 1 day in vivo.
  • a subject e.g., a mammal
  • the engineered polypeptide conjugate is present in a subject at at least about any of 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% after at least about any of 2 hours, 2-6 hours, 6-12 hours, 12-18 hours, 18-24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, or 2 weeks in vivo.
  • the methods provided herein further comprise a purification step.
  • the engineered Fc-containing polypeptide conjugates described herein can be purified using various purification methods, such as, e.g., hydroxylapatite chromatography; dialysis; affinity chromatography; hydrophobic interaction chromatography (HIC) (e.g, fractionation on a HIC); ammonium sulphate precipitation; polyethylene glycol or polyethylene glycol derivative precipitation, anion or cation exchange chromatography; reverse phase HPLC; chromatography on silica; chromatofocusing; SDS-PAGE, gel filtration, size exclusion chromatography, and weak partitioning chromatography.
  • HIC hydrophobic interaction chromatography
  • At least one purification step comprises a step of affinity chromatography method.
  • Protein A ligand synthetic, recombinant, or native
  • Synthetic or recombinant Protein A ligand may be purchased commercially from GE Healthcare (Piscataway, NJ), Pierce (Rockford, IL), Sigma-Aldrich (St.
  • Applied Biosystems Fester City, CA
  • native Protein A ligand e.g., MABSELECTTM, PROSEPTM Va, and PROSEPTM Ultra Plus
  • MABSELECTTM, PROSEPTM Va, and PROSEPTM Ultra Plus may be purchased commercially from GE Healthcare (Piscataway, NJ) or Millipore (Billerica, MA).
  • the purified engineered Fc-containing polypeptide conjugate, the purified engineered Fab-containing polypeptide conjugate, or the purified toxin polypeptide conjugates resulting from the purification step is highly pure, i.e., at least about any of 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-98%, or 99% pure.
  • the purified engineered polypeptide conjugate is about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure.
  • the engineered polypeptide conjugates of the present invention are useful in various applications including, but are not limited to, therapeutic treatment methods and diagnostic treatment methods.
  • the invention provides a method for treating a cancer in a subject. Accordingly, in some embodiments, provided is a method of treating a cancer in a subject in need thereof comprising administering to the subject an effective amount of a composition (e.g., pharmaceutical composition) comprising the engineered polypeptide conjugates as described herein.
  • a composition e.g., pharmaceutical composition
  • cancers include, but are not limited to, a solid cancer (such as bladder, breast, cervical, choriocarcinoma, colon, esophageal, gastric, glioblastoma, head and neck, kidney, liver, lung (e.g., Non Small Cell Lung Cancer (NSCLC)), oral, ovarian, pancreatic, prostate, and skin cancer); and a liquid cancer (such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T- PLL), large granular lymphocytic leukemia, and adult T-cell leukemia).
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • AML acute myelogenous leukemia
  • CML chronic myelogenous leukemia
  • provided is a method of inhibiting tumor growth or progression in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising the engineered polypeptide conjugates as described herein.
  • a method of inhibiting metastasis of cancer cells or tumors e.g., solid or liquid tumors
  • a method of inducing tumor regression in a subject in need thereof comprising administering to the subject an effective amount of a composition comprising the engineered polypeptide conjugates as described herein.
  • a method of detecting, diagnosing, and/or monitoring a condition associated with a cancer-related protein e.g., Trop-2, BRCA1, BRCA2, HER2, VEGF, CD20, CD25, EFGR, 5T4, CD22, etc.
  • a cancer-related protein e.g., Trop-2, BRCA1, BRCA2, HER2, VEGF, CD20, CD25, EFGR, 5T4, CD22, etc.
  • a method of diagnosing cancer in a subject suspected of suffering from cancer comprising a) contacting a sample of the subject with the engineered polypeptide conjugates as described herein under conditions that result in binding of the engineered polypeptide conjugates with a cancer-related protein, and b) determining binding of the engineered polypeptide conjugates to the cancer-related protein.
  • the agent moiety in the engineered polypeptide conjugates as described herein can be a detectable moiety such as an imaging agent and an enzyme-substrate label.
  • the engineered polypeptide conjugates as described herein can also be used for in vivo diagnostic assays, such as in vivo imaging (e.g., PET or SPECT), or a staining reagent.
  • the methods described herein further comprise a step of treating a subject with an additional form of therapy.
  • the additional form of therapy is an additional anti-cancer therapy including, but not limited to, chemotherapy, radiation, surgery, hormone therapy, and/or additional immunotherapy.
  • the additional form of therapy comprises administering one or more therapeutic agent in addition to the engineered polypeptide conjugates as described herein.
  • the therapeutic agents include, but are not limited to, an antibody-drug conjugate (e.g., brentuximab vedotin (ADCETRIS ® ) and ado-trastuzumab emtansine (KADCYLA ® )), an antibody (e.g., an anti-VEGF antibody, an anti-HER2 antibody, anti-CD25 antibody, and/or an anti-CD20 antibody), an angiogenesis inhibitor, a cytotoxic agent (e.g., docetaxel, cisplatin, doxorubicin, mitomycin, tamoxifen, or fluorouracil), and an anti-inflammatory agent (e.g., prednisone, and progesterone).
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the engineered polypeptide conjugates as described herein in a pharmaceutically acceptable excipient or carrier.
  • the engineered polypeptide conjugates can be administered alone or in combination with one or more other engineered polypeptide conjugates of the invention or in combination with one or more other drugs (or as any combination thereof).
  • the pharmaceutical compositions, methods and uses of the invention thus also encompass embodiments of combinations (coadministration) with other active agents, as detailed below.
  • the term "co-administration,” “co-administered,” or “in combination with” is intended to mean and does refer to the following: (i) simultaneous administration of a combination of an engineered polypeptide conjugate disclosed herein and therapeutic agent(s) to a patient in need of treatment, when such components are formulated together into a single dosage form which releases said components at substantially the same time to said patient; (ii) substantially simultaneous administration of such combination of an engineered polypeptide conjugate disclosed herein and therapeutic agent(s) to a patient in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at substantially the same time by said patient, whereupon said components are released at substantially the same time to said patient; (iii) sequential administration of such combination of an engineered polypeptide conjugate disclosed herein and therapeutic agent(s) to a patient in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at consecutive times by said patient with a significant time interval between each administration, whereupon said components are
  • the engineered polypeptide conjugates disclosed herein are suitable to be administered as a formulation in association with one or more pharmaceutically acceptable excipient(s).
  • excipient is used herein to describe any ingredient other than the compound(s) of the invention.
  • excipient(s) to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.
  • pharmaceutically acceptable excipient includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
  • isotonic agents including, but not limited to, sugars, polyalcohols (e.g., mannitol, sorbitol) or sodium chloride are included in the pharmaceutical composition.
  • additional examples of pharmaceutically acceptable substances include, but are not limited to, wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody.
  • the engineered polypeptide conjugates described herein can be deimmunized to reduce immunogenicity upon administration to a subject using known techniques such as those described, e.g., in PCT Publication W098/52976 and WO00/34317.
  • Pharmaceutical compositions of the present invention and methods for their preparation are readily apparent to those skilled in the art. Such compositions and methods for their preparation may be found, for example, in Remington's Pharmaceutical Sciences, 22nd Edition (Mack Publishing Company, 2012). Pharmaceutical compositions are preferably manufactured under GMP conditions.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses.
  • a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. Any method for administering peptides, proteins or antibodies accepted in the art may suitably be employed for the engineered polypeptide conjugates disclosed herein.
  • parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue, thus generally resulting in the direct administration into the blood stream, into muscle, or into an internal organ.
  • parenteral administration includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like.
  • parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intrasynovial injection or infusions; and kidney dialytic infusion techniques.
  • parenteral administration is the intravenous or the subcutaneous route.
  • Formulations of a pharmaceutical composition suitable for parenteral administration typically generally comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
  • a pharmaceutically acceptable carrier such as sterile water or sterile isotonic saline.
  • Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration.
  • injectable formulations may be prepared, packaged, or sold in unit dosage
  • the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen free water) prior to parenteral administration of the reconstituted composition.
  • a suitable vehicle e.g. sterile pyrogen free water
  • Parenteral formulations also include aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.
  • parenteral administration forms include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired.
  • Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, or in a liposomal preparation.
  • Formulations for parenteral administration may be formulated to be immediate and/or modified release. Modified release formulations include controlled, delayed, sustained, pulsed, targeted and programmed release formulations.
  • sterile injectable solutions can be prepared by incorporating the engineered Fc-containing polypeptide, e.g., antibody-drug conjugate or bispecific antibody, in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile filtered solution thereof.
  • the proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • Dosage regimens may be adjusted to provide the optimum desired response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the patients/subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are generally dictated by and directly dependent on (a) the unique characteristics of the agent moiety (e.g., small molecules such as cytotoxic agent) and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • agent moiety e.g., small molecules such as cytotoxic agent
  • the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals are generally dictated by and directly dependent on (a) the unique characteristics of the agent moiety (e.g., small molecules such as cytotoxic agent) and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • the dose and dosing regimen is adjusted in accordance with methods well-known in the therapeutic arts. That is, the maximum tolerable dose can be readily established, and the effective amount providing a detectable therapeutic benefit to a patient may also be determined, as can the temporal requirements for administering each agent to provide a detectable therapeutic benefit to the patient. Accordingly, while certain dose and administration regimens are exemplified herein, these examples in no way limit the dose and administration regimen that may be provided to a patient in practicing the present invention.
  • dosage values may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. Further, the dosage regimen with the compositions of this invention may be based on a variety of factors, including the type of disease, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular antibody employed. Thus, the dosage regimen can vary widely, but can be determined routinely using standard methods.
  • doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and/or laboratory values.
  • the present invention encompasses intra-patient dose-escalation as determined by the skilled artisan. Determining appropriate dosages and regimens are well-known in the relevant art and would be understood to be encompassed by the skilled artisan once provided the teachings disclosed herein.
  • the total monthly dose of an engineered polypeptide conjugate disclosed herein is typically in the range of about 0.01 mg to about 1200 mg per patient, depending, of course, on the mode of administration.
  • an intravenous monthly dose may require about 1 to about 1000 mg/patient.
  • the total monthly dose may be administered in single or divided doses and may, at the physician's discretion, fall outside of the typical range given herein.
  • an exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an engineered polypeptide conjugate e.g., an Fc-containing polypeptide conjugate, Fab-containing polypeptide conjugate, antibody conjugate, or toxin polypeptide conjugate, disclosed herein is about 0.01 to about 1000 mg/patient/month.
  • the engineered Fc-containing polypeptide conjugate may be administered at about 1 to about 200 or about 1 to about 150 mg/patient/month.
  • the patient is human.
  • kits of the invention include one or more containers comprising a purified engineered polypeptide conjugate and instructions for using the conjugate for treating a disease.
  • the instructions comprise a description of administration of the engineered polypeptide conjugate to treat a disease, such as cancer (e.g., colon, esophageal, gastric, head and neck, lung, ovarian, or pancreatic cancer).
  • the kit may further comprise a description of selecting an individual suitable for treatment based on identifying whether that individual has the disease and the stage of the disease.
  • the instructions relating to the use of the engineered polypeptide conjugate generally include information as to dosage, dosing schedule, and route of administration for the intended treatment.
  • the containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub- unit doses.
  • Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine- readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
  • kits of this invention are in suitable packaging.
  • suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like.
  • packages for use in combination with a specific device such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump.
  • a kit may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the container may also have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is an engineered polypeptide as described herein.
  • the container may further comprise a second pharmaceutically active agent.
  • Kits may optionally provide additional components such as buffers and interpretive information.
  • the kit comprises a container and a label or package insert(s) on or associated with the container.
  • Example 1 Proteolysis of the C-terminal heavy chain TG6 tag during CHO expression
  • TG transglutaminase tags TG7-TG17 (SEQ ID NOS: 2-11; Table 1) were designed to contain proline to minimize the proteolysis.
  • HIC hydrophobic interaction chromatography
  • mass spectrometry as described by Strop et al. Chem Biol, 20(2): 161-7 (2013), hereby incorporated by reference herein in its entirety.
  • TG tags e.g., TG11 and TG12
  • the desired payload e.g., AcLys-VC-PABC-0101
  • AcLys-VC-PABC-0101 AcLys-VC-PABC is acetyl-lysine-valine- citrulline-p-aminobenzyloxycarbonyl, and 0101 is 2-methylalanyl-N-[(3R,4,S', 5 ) S)-3-methoxy-l- ⁇ (25)-2-[(lR,2R)-l-methoxy-2-methyl-3-oxo-3- ⁇ [(l ⁇ )-2-phenyl-l-(l,3-thiazol-2- yl)ethyl]amino ⁇ propyl]pyrrolidin-l -yl ⁇ -5-methyl- 1 -oxoheptan-4-yl]-N-methyl-L-val
  • Figures 1A-1D Accordingly, these results demonstrate that TG9 and TGI 7 tags prevent proteolysis at the C- terminus of an antibody's heavy chain when expressed in CHO cells.
  • Example 2 Proteolysis of the C-terminal light chain LCQ04 tag during CHO expression
  • the LCQ05 tag (GGLLQGPP (SEQ ID NO: 13)) was created having two prolines similar to the TGI 7 tag described above.
  • the LCQ05 TG tag appeared homogeneous when expressed in both CHO and HEK293 cells and could be conjugated to the desired linker-payload (e.g., AcLys- VC-PABC-0101) at comparable levels as LCQ04.
  • Figures 2A-2B Accordingly, these results demonstrate that LCQ05 tag prevents proteolysis at the C-terminus of an antibody's light chain when expressed in CHO cells.
  • the new acyl donor glutamine-containing tags were validated by comparing the biophysical characteristics of the unconjugated and conjugated TG6 tag and TGI 7 tag. More specifically, analytical size exclusion chromatography (SEC) showed similar low level of aggregates, and the conjugation efficiency was comparable under the same conditions.
  • SEC analytical size exclusion chromatography
  • the run was performed at 0.8 mL/min with a 35 minutes linear gradient 0-100% B.
  • MS Mass spectrometry
  • LC/MS Liquid Chromatography-Mass Spectrometry
  • antibody-drug conjugates were deglycosylated with PNGase F (NEB, cat#P0704L) under non-denaturing conditions at 37°C overnight.
  • ADCs 500 ng
  • Both antibody conjugates were also tested for in vitro efficacy on BxPC3 and OVCAR3 cell lines for cytotoxicity, and the IC50 values obtained were comparable. See Table 2 and Figures 6A-6B. More specifically, BxPC3 and OVCAR3 cells at 2000 and 3000 cells/well, respectively, were diluted in 100 uL growth media (serum free DMEM, Cellgro Mediatech, Manassas, VA). The next day, 25 uL of five times of ADCs (in serum free DMEM) were added.
  • CelltiterGlo assays (Promega, Madison, WI) were performed 4 days after treatment. Cells were added with 1 : 1 diluted reagent after media removal. The plates were read on MSpectraMax M5 plate reader (Molecular Devices, Downingtown, PA).
  • the PK (pharmacokinetics) was further tested by injecting female rats with a single dose of AB-TG6-AcLys-VC-PABC-0101 or AB-TG17-AcLys-VC-PABC-0101 (174-2) at 5 mg/kg.
  • the animals were 12 adult Sprague Dawley female rats at approximately 3 months of age. (Harlan, Livermore, CA).
  • compounds were dosed intravenously through the lateral tail vein, and samples were withdrawn from the contralateral tail vein at appropriate time points, except for a terminal bleed which was obtained via cardiac puncture on anesthetized adult rats. Samples from one group of rats were processed for serum while samples from a separate group of rats were processed for plasma. Both sample types were stored frozen at -80°C until analysis.
  • Example 4 Q295N mutant in the heavy chain of the mAb eliminates unwanted conjugation due to trace amounts of aglycosylated antibodies expressed during mAb expression
  • transglutaminase can recognize Q295 in aglycosylated IgGs, and conjugation can be achieved at this site. See MS/MS confirmation of conjugation of AmPEG6- MMAD to tryptic peptide EEQ*YNSTYR (SEQ ID NO: 15) in Figure 9A.
  • EEQ*YNSTYR SEQ ID NO: 15
  • FIG. 9A When antibodies are expressed in the CHO or HEK293 expression system, most of the produced proteins are gycoyslated at position N297. The presence of glycans at position N297 prevents conjugation to Q295. However, there is typically a small fraction of aglycosylated antibodies that are also produced. It is this contaminating aglycosylated antibody fraction, which is suitable for conjugation at position Q295.
  • conjugated EEQ*YNSTYR SEQ ID NO: 15
  • standard peptide was spiked into a fixed amount (2.5 ⁇ g) of unconjugated tryptic digests of mAbl HC (heavy chain), and a standard curve for quantification was generated.
  • AmPEG6-MMAD amino-polyethylene glycol-6 propionyl monomethyl auristatin D
  • Figure 9B A single point mutant Q295N was created to abolish the undesired conjugation.
  • the peptide mass fingerprint of the Q295N mutant revealed only conjugation at the designed glutamine tag site for both the mAbl light chain and heavy chain molecules with no additional identified sites.

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Abstract

The present invention provides engineered polypeptide conjugates (e.g., antibody-drug- conjugates) comprising specific acyl donor glutamine-containing tags and amine donor agents. The invention also provides methods of making such engineered polypeptide conjugates using transglutaminase and methods of using thereof.

Description

ENGINEERED POLYPEPTIDE CONJUGATES
Related Applications
This application claims the benefits of U.S. Provisional Application No. 61/860,757, filed July 31, 2013, and U.S. Provisional Application No. 62/028,236, filed July 23, 2014, which are hereby incorporated by reference in their entireties.
Field
The present invention relates generally to engineered polypeptide conjugates (e.g., antibody-drug conjugates) comprising specific acyl donor glutamine-containing tags and amine donor agents. The invention also relates to methods for making such engineered polypeptide conjugates using transglutaminase and methods of using thereof.
Background
Antibody therapy provides targeted therapeutic treatment in patients with various disorders, such as cancers and immunological diseases, and therefore has played an important role in biological research. Different approaches of targeted antibody therapy, including antibody-drug conjugates (ADC), have been explored. See, e.g., Doronian et al., Bioconj. Chem. 19: 1960-1963 (2008); and Junutula et al., Nature Biotechnology 26: 925-932 (2008).
In the case of antibody-drug conjugates (i.e., immunoconjugates), cytotoxic small molecules (drugs) are generally linked or conjugated to antibodies for targeted local delivery of the drug moieties to tumors. Chemical modification has been widely used for conjugating drugs to antibodies either through lysine side chain amines or through cysteine sulfhydryl groups activated by reducing interchain disulfide bonds. However, these types of "residue-specific" conjugation lead to a heterogeneous mixture of conjugates having different molar ratios of drug to antibody, different and non-specific conjugation sites, different efficiency, safety, and pharmacokinetics, and different clearance of antibody drug conjugates. See, e.g., Tanaka et al, FEBS Letters 579:2092-2096 (2005); and Wang et al., Protein Sci. 14: 2436-2446 (2005). Further, inclusion bodies or incorrect disulfide bridges may also be formed in cysteine- introduced antibodies. See, e.g., Gentle et al., Bioconj. Chem. 15:658-663 (2004). Reactive cysteine residues engineered at specific sites of antibodies (e.g., THIOMAB) for specific drug conjugation with defined stoichiometry has also been explored. See Junutula et al., Nature Biotechnology, 26: 925-932 (2008). However, expression and conjugation of such cysteine engineered antibodies and antibody-drug conjugates are complicated processes which require lengthy reaction procedures (e.g., reductions and oxidations). See, e.g., Gomez et al., Biotechnology and Bioengineering, 105(4): 748-760 (2009). Antibody aggregates may also be generated during the process of making the cysteine engineered antibodies and the antibody-drug conjugates.
Enzymatic approaches using a transglutaminase for protein conjugation have been explored recently as an alternative to "residue-specific" conjugation of antibodies/proteins and drugs. Transglutaminases (EC2.3.2.13; protein-glutamine:gamma-glutamyl transferee; protein- glutamine: amine γ-glutamyltransferase; CAS 80146-85-6) belong to a family of enzymes that catalyze the acyl addition to a primary amine wherein the gamma-carboxamide group of peptide- bound γ-glutanyl residue is the acyl donor and the primary amine is the acyl acceptor and the amine donor. Transglutaminases have been used, for example, for the attachment of proteins to proteins. See, e.g., Tanaka et al, FEBS Letters 579:2092-2096 (2005). Enzymatic modification of antibodies using transglutaminases has also been reported. See Josten et al. J. of Immunological Methods 240:47-54 (2000); Takazawa et al., Biotechnology and Bioengineering 86(4): 399-404 (2004); Mindt et al., Bioconj. Chem 19:271-27 (2008), and Jeger et al., Angewandte Chemie, 49(51): 9995-9997 (2010). Protein conjugation or modification using transglutaminase provides the advantages of high selectivity, simplified reaction procedures, and mild reaction conditions. WO2012059882 describes site-specific conjugation of antibodies and small molecules mediated by a transglutaminase.
All publications, patents, and patent applications cited herein are hereby incorporated by reference herein in their entirety for all purposes to the same extent as if each individual publication, patent, and patent application were specifically and individually indicated to be so incorporated by reference. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls.
Summary
The present invention provides transglutaminase-mediated antibody drug conjugates comprising a specific acyl donor glutamine-containing tag engineered at a specific site of the antibody and an amine donor agent (e.g., linker-payload). The invention also provides a homogenous site-specific transglutaminase-mediated antibody drug conjugate (e.g., at least about 99.8% site-specific) comprising the amino acid substitution from glutamine (Q) to asparagine (N) at position 295 of the antibody (Q295N; EU numbering scheme).
In one aspect, this invention provides an engineered polypeptide conjugate comprising the formula: polypeptide-T-A, wherein T is an acyl donor glutamine-containing tag engineered at a specific site, wherein A is an amine donor agent, wherein the amine donor agent is site- specifically conjugated to the acyl donor glutamine-containing tag at a carboxyl terminus, an amino terminus, or at an another site in the polypeptide, and wherein the acyl donor glutamine- containing tag comprises an amino acid sequence GGLLQGPP (SEQ ID NO: 13), LLQGPA (SEQ ID NO:4), or LLQGPP (SEQ ID NO: 11).
In another aspect, the invention provides an engineered Fc-containing polypeptide conjugate comprising the formula: (Fc-containing polypeptide-T-A), wherein T is an acyl donor glutamine-containing tag engineered at a specific site, wherein A is an amine donor agent, wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine- containing tag at a carboxyl terminus, an amino terminus, or at an another site in the Fc- containing polypeptide, wherein the acyl donor glutamine-containing tag comprises an amino acid sequence XXQX (SEQ ID NO:35), wherein X is any amino acid (e.g., X can be the same or different amino acid), and wherein the engineered Fc-containing polypeptide conjugate comprises an amino acid substitution from glutamine to asparagine at position 295 (Q295N; EU numbering scheme). In some embodiments, the acyl donor glutamine-containing tag is LLQGPA (SEQ ID NO:4), LLQGP (SEQ ID NO:5), LLQGPP (SEQ ID NO: 11) or GGLLQGPP (SEQ ID NO: 13).
In some embodiments, the acyl donor glutamine-containing tag is not spatially adjacent to a reactive Lys (i.e., the ability to form a covalent bond as an amine donor in the presence of an acyl donor and a transglutaminase) in the polypeptide or the Fc-containing polypeptide.
In some embodiments, the polypeptide or the Fc-containing polypeptide comprises an amino acid modification at the last amino acid position in the carboxyl terminus relative to a wild-type polypeptide at the same position. The amino acid modification can be an amino acid deletion, insertion, substitution, mutation, or any combination thereof.
In some embodiments, the polypeptide conjugate comprises a full length antibody heavy chain and an antibody light chain, wherein the acyl donor glutamine-containing tag is located at the carboxyl terminus of a heavy chain, a light chain, or both the heavy chain and the light chain.
In some embodiments, the polypeptide conjugate comprises an antibody, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, a minibody, a diabody, or an antibody fragment. In some embodiments, the antibody is an IgG.
In some embodiments, the amine donor agent comprises the formula: X-Y-Z, wherein X is an amine donor unit; Y is a linker; and Z is an agent moiety. In some embodiments, the amine donor unit-linker (X-Y) is a branched unit (e.g., at least 2 units) and the agent moiety comprises at least about 2 agent moieties.
In some embodiments, the amine donor unit-linker (X-Y) is selected from the group consisting of Ac-Lys-Gly, aminocaproic acid, Ac-Lys-P-Ala, amino-PEG2-C2, amino-PEG3-C2, amino-PEG6-C2, Ac-Lys-Val-Cit-PABC, amino-PEG6-C2-Val-Cit-PABC, aminocaproyl-Val- Cit-PABC, [(3R,5R)-l-{3-[2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit- PABC, [(3S,5S)-l-{3-[2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit- PABC, putrescine, and Ac-Lys-putrescine.
In some embodiments, the agent moiety is a cytotoxic agent, including, but not limited to, an anthracycline, an auristatin, a camptothecin, a combretastatin, a dolastatin, a duocarmycin, an enediyne, a geldanamycin, an indolino-benzodiazepine dimer, a maytansine, a puromycin, a pyrrolobenzodiazepine dimer, a taxane, a vinca alkaloid, a tubulysin, a hemiasterlin, a spliceostatin, a pladienolide, and stereoisomers, isosteres, analogs, or derivatives thereof. For example, the cytotoxic agent is MMAD (Monomethyl Auristatin D) or 0101 (2-methylalanyl-N- [(3R, 4S, 5S)-3 -methoxy- 1 - { (2S)-2- [( \R, 2R)- 1 -methoxy-2-methyl-3 -oxo-3 - { [( 15)-2-phenyl- 1 -(1,3- thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-l -yl}-5-methyl-l -oxoheptan-4-yl]-N-m ethyl -L- valinamide).
In some embodiments, the amine donor agent (X-Y-Z) includes, but is not limited to, Alexa 488 cadaverine, 5-FITC cadaverine, Alexa 647 cadaverine, Alexa 350 cadaverine, 5- TAMRA cadaverine, 5-FAM cadaverine, SRI 01 cadaverine, 5,6-TAMRA cadaverine, 5-FAM lysine, Ac-Lys-Gly (acetyl -lysine-glycine)-MM AD, amino-PEG3 -C2-MM AD, amino-PEG6-C2- MMAD, amino-PEG3-C2-amino-nonanoyl-MMAD, aminocaproyl-Val-Cit-PABC-MMAD, amino-PEG-C2-Val-Cit-PABC-MMAD, Ac-Lys-Val-Cit-PABC (acetyl-lysine-valine-citrulline- /?-aminobenzyloxycarbonyl)-MMAD, aminocaproyl-MMAD, Ac-Lys-P-Ala-MMAD, amino- PEG2-C2-MMAE, aminocaproyl-MMAE, amino-PEG3 -C2-MM AE, aminocaproyl-MMAF, aminocaproyl-Val-Cit-PABC-MMAE, amino-PEG-6-C2-Val-Cit-PABC-MMAE, Ac-Lys-Val- Cit-PABC -MM AE, aminocaproyl-Val-Cit-PABC-MMAF, amino-PEG-6-C2-Val-Cit-PABC- MMAF, Ac-Lys-Val-Cit-PABC-MMAF, Ac-Lys-Val-Cit-PABC-0101, putrescinyl- geldanamycin, Ac-Lys-putrescinyl-geldanamycin, aminocaproyl-3377, amino-PEG6-C2-3377, aminocaproyl-0131, amino-PEG6-C2-0131, [(3R,5R)-l-{3-[2-(2- aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit-PABC-MMAD, [(3R,5R)-l-{3- [2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit-PABC-MMAE, and 2- aminoethoxy-PEG6-NODAGA.
In another aspect, the invention provides a method for preparing an engineered Fc- containing polypeptide conjugate comprising the formula: (Fc-containing polypeptide-T-A), wherein T is an acyl donor glutamine-containing tag engineered at a specific site, wherein A is an amine donor agent, wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine-containing tag at a carboxyl terminus, an amino terminus, or at an another site in the Fc-containing polypeptide, wherein the acyl donor glutamine-containing tag comprises an amino acid sequence XXQX (SEQ ID NO:35), wherein X is any amino acid (e.g., X can be the same or a different amino acid), and wherein the engineered Fc-containing polypeptide conjugate comprises an amino acid substitution from glutamine to asparagine at position 295 (Q295N; EU numbering scheme), comprising the steps of: a) providing an engineered (Fc-containing polypeptide)-! molecule comprising the Fc-containing polypeptide and the acyl donor glutamine-containing tag; b) contacting the amine donor agent with the engineered (Fc- containing polypeptide)-! molecule in the presence of a transglutaminase; and c) allowing the engineered (Fc-containing polypeptide)-! to covalently link to the amine donor agent to form the engineered Fc-containing polypeptide conjugate. In some embodiments, the engineered Fc- containing polypeptide-! is expressed in CHO cells
In another aspect, the invention provides a method for preparing an engineered polypeptide conjugate comprising the formula: polypeptide-!-A, wherein ! is an acyl donor glutamine-containing tag engineered at a specific site, wherein A is an amine donor agent, wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine- containing tag at a carboxyl terminus, an amino terminus, or at an another site in the polypeptide, and wherein the acyl donor glutamine-containing tag comprises an amino acid sequence LLQGPX, wherein X is A or P (SEQ ID NO: 14), or GGLLQGPP (SEQ ID NO: 13), comprising the steps of: a) providing an engineered polypeptide-! molecule comprising the polypeptide and the acyl donor glutamine-containing tag; b) contacting the amine donor agent with the engineered polypeptide-! molecule in the presence of a transglutaminase; and c) allowing the engineered polypeptide-! to covalently link to the amine donor agent to form the engineered Fc-containing polypeptide conjugate. In some embodiments, the engineered polypeptide-! molecule is expressed in CHO cells. In some embodiments, the engineered polypeptide conjugate (e.g., the engineered Fc- containing polypeptide conjugate, the engineered Fab-containing polypeptide conjugate, or the engineered antibody conjugate) as described herein has conjugation efficiency of at least about 51%.
In another aspect, the invention provides a pharmaceutical composition comprising the engineered polypeptide conjugate as described herein (e.g., the engineered Fc-containing polypeptide conjugate, the engineered Fab-containing polypeptide conjugate, or the engineered antibody conjugate) and a pharmaceutically acceptable excipient.
In another aspect, the invention provides a method of treating a cancer in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition comprising the engineered polypeptide conjugate (e.g., the engineered Fc-containing polypeptide conjugate, the engineered Fab-containing polypeptide conjugate, or the engineered antibody conjugate) as described herein.
In another aspect, the invention provides a method of inhibiting tumor growth or progression in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition comprising the engineered polypeptide conjugate (e.g., the engineered Fc-containing polypeptide conjugate, the engineered Fab-containing polypeptide conjugate, or the engineered antibody conjugate) as described herein.
In another aspect, the invention provides a method of diagnosing cancer in a subject suspected of suffering from cancer, comprising a) contacting a sample of the subject with the engineered polypeptide conjugate (e.g., the engineered Fc-containing polypeptide conjugate, the engineered Fab-containing polypeptide conjugate, or the engineered antibody conjugate) as described herein under conditions that result in binding of the engineered polypeptide conjugate with a cancer-related protein, and b) determining binding of the engineered polypeptide conjugate to the cancer-related protein.
In another aspect, the invention provides an engineered polypeptide conjugate (e.g., the engineered Fc-containing polypeptide conjugate, the engineered Fab-containing polypeptide conjugate, or the engineered antibody conjugate) purified by the methods described herein.
Brief Description of the Drawings
Figures 1A-1D depict graphs showing different percentages of the clipping of four different acyl donor glutamine-containing tags (the "glutamine tags") expressed in CHO cells. Figure 1A depicts the results of the glutamine tag TG6 (SEQ ID NO: 1) in Ab-TG6. Figure IB depicts the results of the glutamine tag TG9 (SEQ ID NO:4) in Ab-TG9. Figure 1C depicts the results of the glutamine tag TG10 (SEQ ID NO: 5) in Ab-TGIO. Figure ID depicts the results for the glutamine tag TG17 (SEQ ID NO: 11) in Ab-TG17.
Figures 2A-2B show that the glutamine tag LCQ04 (SEQ ID NO: 12) expressed in CHO cells was clipped from the C-terminus of an antibody light chain (2 A), whereas no clipping was observed in the glutamine tag LCQ05 (SEQ ID NO: 13) in Ab-LCQ05 (2B).
Figures 3A-3B show that similar low levels of aggregates were observed for the glutamine tags TG6 (SEQ ID NO: l) and TG17 (SEQ ID NO: 11) in Ab-TG6-AcLys-VC-PABC- 0101 (Figure 3 A) and Ab-TG17-AcLys-VC-PABC-0101 (Figure 3B) using analytical size exclusion chromatography (SEC).
Figures 4A-4B show that similar Drug-Antibody-Ratio (DAR) was observed for the glutamine tags TG6 (SEQ ID NO: l) and TG17 (SEQ ID NO: 11) in Ab-TG6 and Ab-TG6- AcLys-VC-PABC-0101 (Figure 4A) and Ab-TG17 and Ab-TG17-AcLys-VC-PABC-0101 (Figure 4B) using Hydrophobic Interaction Chromatography (HIC).
Figures 5A-5B show that similar Drug-Antibody-Ratio (DAR) was observed for the glutamine tags TG6 (SEQ ID NO: l) and TG17 (SEQ ID NO: 11) in Ab-TG6 and Ab-TG17 (Figure 5 A) and Ab-TG6-AcLys-VC-PABC-0101 and Ab-TG17-AcLys-VC-PABC-0101 (Figure 5B) using Mass Spectrometry (MS).
Figures 6A-6B show that antibody conjugates Ab-TG6-AcLys-VC-PABC-0101 and Antibody-TG17-AcLys-VC-PABC-0101 had the similar in vitro efficacy for cytotoxicity in BxPC3 cell line (Figure 6A) and OVCAR3 cell line (Figure 6B).
Figures 7A-7B show that antibody conjugate (Ab-TG17-AcLys-PABC-0101) having the glutamine tag TG17 (SEQ ID NO: 11) had similar activity in in vivo tumor models (% body weight change in Figure 7A; and tumor volume in Figure 7B) in comparison to the antibody conjugate (Ab-TG6-AcLys-PABC-0101) having the glutamine tag TG6 (SEQ ID NO: 1).
Figure 8 shows the pharmacokinetics for antibody conjugates Ab-TG6-AcLys-VC- PABC-0101, Antibody-TG17-AcLys-VC-PABC-0101, Ab-TG6, and Ab-TG17.
Figure 9A shows that transglutaminase can recognize Q295 in aglycosylated IgGs, and conjugation can be achieved at this site (conjugation of Amino-PEG6-MMAD to a tryptic peptide (SEQ ID NO: 15)).
Figure 9B shows that 1.3% of the total injected Q295 peptide was conjugated with amino- PEG6-MMAD in the heavy chain of the antibody, assuming 100% digestion efficiency. Detailed Description
The present invention provides transglutaminase-mediated antibody-drug conjugates comprising a specific acyl donor glutamine-containing tag engineered at a specific site of the antibody and an amine donor agent (e.g., linker-payload). The invention also provides a homogenous site-specific transglutaminase-mediated antibody-drug conjugate comprising the amino acid substitution from glutamine (Q) to asparagine (N) at position 295 (Q295N; EU numbering scheme). The inventors have discovered that proteolysis of an acyl donor glutamine- containing tag engineered at C-terminus of an antibody's heavy chain or light chain, when expressed in CHO cells, can be prevented by using several newly designed glutamine-containing tags having unique sequences (e.g., LLQGPA (SEQ ID NO:4), LLQGPP (SEQ ID NO: 11), and GGLLQGPP (SEQ ID NO: 13)). The inventors have also discovered that mutation at position 295 (i.e., Q295N; EU numbering scheme) of the antibody drug conjugate eliminates the off- target conjugation of a small percentage of aglycosylated antibody at position 295 and yields highly homogenous conjugates that are better than 99.8% site-specific.
Accordingly, in one aspect, the invention provides an engineered polypeptide conjugate comprising the formula: polypeptide-T-A, wherein T is an acyl donor glutamine-containing tag engineered at a specific site; wherein A is an amine donor agent; wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine-containing tag at a carboxyl terminus, an amino terminus, or at an another site in the polypeptide, and wherein the acyl donor glutamine-containing tag comprises an amino acid sequence LLQGPX, wherein X is A or P (SEQ ID NO: 14), or GGLLQGPP (SEQ ID NO: 13). In some embodiments, the polypeptide is an Fc-containing polypeptide or a Fab-containing polypeptide.
In another aspect, the invention provides an engineered Fc-containing polypeptide conjugate comprising the formula: (Fc-containing polypeptide)-T-A, wherein T is an acyl donor glutamine-containing tag engineered at a specific site; wherein A is an amine donor agent; wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine- containing tag at a carboxyl terminus, an amino terminus, or at an another site in the Fc- containing polypeptide, wherein the acyl donor glutamine-containing tag comprises an amino acid sequence XXQX (SEQ ID NO:35), wherein X is any amino acid, and wherein the engineered Fc-containing polypeptide conjugate comprises an amino acid substitution from glutamine to asparagine at position 295 (Q295N; EU numbering scheme).
In another aspect, the invention provides a method for preparing an engineered polypeptide conjugate comprising the formula: polypeptide-T-A, wherein T is an acyl donor glutamine-containing tag engineered at a specific site, wherein A is an amine donor agent; wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine- containing tag at a carboxyl terminus, an amino terminus, or at an another site in the polypeptide, and wherein the acyl donor glutamine-containing tag comprises an amino acid sequence LLQGPX, wherein X is A or P (SEQ ID NO: 14), or GGLLQGPP (SEQ ID NO: 13), comprising the steps of: a) providing an engineered polypeptide-T molecule comprising the polypeptide and the acyl donor glutamine-containing tag; b) contacting the amine donor agent with the engineered polypeptide-T molecule in the presence of a transglutaminase; and c) allowing the engineered polypeptide-T to covalently link to the amine donor agent to form the engineered polypeptide conjugate. In some embodiments, the engineered polypeptide is an Fc-containing polypeptide or a Fab-containing polypeptide. In some embodiments, the engineered polypeptide-T molecule is expressed in CHO cells.
General Techniques and Definitions
Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art.
The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual^ Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, molecular biology, biochemistry, immunology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Throughout this specification and claims, the word "comprise," or variations such as "comprises" or "comprising," will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably herein to refer to chains of amino acids of any length, preferably, relatively short (e.g., 10-100 amino acids). The chain may be linear or branched, it may comprise modified amino acids, and/or may be interrupted by non-amino acids. The terms also encompass an amino acid chain that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that the polypeptides can occur as single chains or associated chains.
The term "Fc-containing polypeptide" as used herein refers to a polypeptide (e.g., an antibody or an immunoadhesin) comprising the carboxyl terminal polypeptide sequences of an immunoglobulin heavy chain. The Fc-containing polypeptide may comprise native or variant Fc regions (i.e., sequences). The Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. An Fc- containing polypeptide may comprise part or all of a wild-type hinge sequence (generally at amino terminus of the Fc-containing polypeptide). An Fc-containing polypeptide may also be a dimer. An Fc-containing polypeptide may be obtained or derived from any suitable immunoglobulin, such as from at least one of the various IgGl, IgG2, IgG3, or IgG4 subtypes, or from IgA, IgE, IgD or IgM. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, for example, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Glu216, or from Ala231, to the carboxyl -terminus thereof.
An Fc-containing polypeptide may be an Fc-containing fusion polypeptide, wherein one or more polypeptides are operably linked to an Fc-containing polypeptide. An Fc fusion combines the Fc polypeptide of an immunoglobulin with a fusion partner, which in general may be any protein, polypeptide, or small molecule. Virtually any protein or small molecule may be linked to the Fc region to generate an Fc-containing fusion polypeptide. Fc-containing fusion partners may include, but are not limited, the target-binding region of a receptor, an adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine, or some other protein or protein domain.
The term "acyl donor glutamine-containing tag", "glutamine tag," "Q-containing tag", or "Q-tag" as used herein refers to a polypeptide or a protein containing one or more Gin residue(s) that acts as a transglutaminase amine acceptor.
The term "amine donor agent" or "acyl acceptor" as used herein refers to an agent containing one or more reactive amines (e.g., primary amines). For example, the amine donor agent can comprise an amine donor unit (e.g., primary amine H2), a linker, and an agent moiety (e.g., a small molecule). The amine donor agent can also be a polypeptide (e.g., an antibody) or a biocompatible polymer containing a reactive Lys (e.g., an endogenous Lys).
As used herein, the term "biocompatible polymer" refers to a polymer (e.g., repeating monomelic or structural units) that is suitable for therapeutic or medical treatment in a recipient (e.g., human) without eliciting any undesirable local or systemic effects in the recipient. A biocompatible polymer (synthetic, recombinant, or native) can be a water soluble or water insoluble polymer. A biocompatible polymer can also be a linear or a branched polymer.
As used herein, the term "site specificity," "site-specifically conjugated," or "site- specifically crosslinked" refers to the specific conjugation or crosslinking of the amine donor agent to the polypeptide engineered with an acyl donor glutamine-containing tag at a specific site (e.g., carboxyl terminus or amino terminus of the antibody or toxin polypeptide, accessible site in the antibody (e.g., antibody light chain and/or heavy chain loops) or toxin polypeptide (e.g., polypeptide loops)). The polypeptide engineered with an acyl donor glutamine-containing tag can be an Fc-containing polypeptide, Fab-containing polypeptide, or a toxin polypeptide (e.g., a protein bound to an ion channel). The term "site specificity," "site-specifically conjugated," or "site-specifically crosslinked" can also refer to the specific conjugation or crosslinking of the polypeptide (e.g., toxin polypeptide) to the biocompatible polymer engineered with an acyl donor glutamine-containing tag at a specific site (e.g., an accessible site in the biocompatible polymer). Site specificity can be measured by various techniques, including, but not limited to, mass spectrometry (e.g., matrix-assisted laser-desorption ionization mass spectrometry (MALDI-MS), electrospray ionization mass spectrometry (ESI-MS), tandem mass spectrometry (MS), and time- of-flight mass spectrometry (TOF-MS)), hydrophobic interaction chromatography, ion exchange chromatography, site-directed mutagenesis, fluorescence-labeling, size exclusion chromatography, and X-ray crystallography. As used herein, the term "spatially adjacent to" refers to interference with the desired transglutaminase reaction (e.g., lysine residue that is located such that it can interfere with the conjugation reaction by serving as an amine donor agent).
As used herein, the term "antibody" is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, unless otherwise indicated by context, the term is intended to encompass not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab' )2, Fv), single chain (ScFv) and domain antibodies, including shark and camelid antibodies), and fusion proteins comprising an antibody portion, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity) and antibody fragments as described herein, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site. An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three- dimensional configurations of different classes of immunoglobulins are well known. On one aspect, the immunoglobulin is a human, murine, or rabbit immunoglobulin.
The term "Fab containing polypeptide" as used herein refers to a polypeptide comprising a Fab fragment, Fab' fragment, or "(Fab')2 fragment. A Fab-containing polypeptide may comprise part or all of a wild-type hinge sequence (generally at the carboxyl terminus of the Fab portion of the polypeptide). A Fab-containing polypeptide may be obtained or derived from any suitable immunoglobulin, such as from at least one of the various IgGl, IgG2, IgG3, or IgG4 subtypes, or from IgA, IgE, IgD or IgM. A Fab-containing polypeptide may be a Fab-containing fusion polypeptide, wherein one or more polypeptides are operably linked to a Fab-containing polypeptide. A Fab fusion combines the Fab polypeptide of an immunoglobulin with a fusion partner, which in general may be any protein, polypeptide, or small molecule. Virtually any protein or small molecule may be linked to the Fab polypeptide to generate a Fab-containing fusion polypeptide. Fab-containing fusion partners may include, but are not limited to, the target-binding region of a receptor, an adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine, or some other protein or protein domain.
A "Fab fragment" is comprised of one light chain and the CHI and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
A "Fab' fragment" contains one light chain and a portion of one heavy chain that contains the VH domain and the CHI domain and also the region between the CHI and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule.
A "F(ab')2 fragment" contains two light chains and two heavy chains containing a portion of the constant region between the CHI and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F(ab')2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.
"Antibody fragments" as used herein comprise only a portion of an intact antibody, wherein the portion preferably retains at least one, preferably most or all, of the functions normally associated with that portion when present in an intact antibody.
A "multispecific antibody" is one that targets more than one antigen or epitope. A "bispecific," "dual-specific" or "bifunctional" antibody is a hybrid antibody having two different antigen binding sites. Bispecific antibodies are a species of multispecific antibody and may be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann (1990), Clin. Exp. Immunol. 79:315-321; and Kostelny et al. (1992), J. Immunol. 148: 1547-1553. The two binding sites of a bispecific antibody will bind to two different epitopes, which may reside on the same or different protein targets.
The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Further, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
The monoclonal antibodies herein may, in certain embodiments, specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81 :6851-6855 (1984)).
"Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may, moreover, comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1 : 105-115 (1998); Harris, Biochem. Soc. Transactions 23 : 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).
A "human antibody" is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
As used herein, the term "immunoadhesin" designates antibody-like or immunoglobulin- like molecules which combine the "binding domain" of a heterologous protein (an "adhesin", e.g. a receptor, ligand or enzyme) with the effector component of immunoglobulin constant domains (i.e., Fc domain). Structurally, the immunoadhesins comprise a fusion of the adhesin amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site (antigen combining site) of an antibody (i.e. is "heterologous") and an immunoglobulin constant domain sequence. The immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgGl, IgG2, IgG3, or IgG4 subtypes, IgA, IgE, IgD or IgM.
The "hinge region," "hinge sequence," and variation thereof, as used herein, includes the meaning known in the art, which is illustrated, for example, Janeway et al., ImmunoBiology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4th ed., 1999); Bloom et al., Protein Science (1997), 6:407-415; Humphreys et al., J. Immunol. Methods (1997), 209: 193-202.
As used herein, the term "wild-type amino acid," "wild-type IgG," "wild-type bispecific antibody," or "wild-type mAb" refers to a sequence of amino acids or nucleic acids that occurs naturally within a certain population (e.g., human, mice, rats, cells, etc.).
As used herein, the term "conjugation efficiency" or "crosslinking efficiency" is the ratio between the experimentally measured amount of engineered polypeptide conjugate divided by the maximum expected engineered polypeptide conjugate amount. Conjugation efficiency or crosslinking efficiency can be measured by various techniques well known to persons skilled in the art, such as hydrophobic interaction chromatography. Conjugation efficiency can also be measured at different temperature, such as room temperature or 37°C.
The term "effector function" refers to the biological activities attributable to the Fc region of an antibody. Examples of antibody effector functions include, but are not limited to, antibody- dependent cell-mediated cytotoxicity (ADCC), Fc receptor binding, complement dependent cytotoxicity (CDC), phagocytosis, Clq binding, and down regulation of cell surface receptors (e.g., B cell receptor; BCR). See, e.g., U.S. Pat No. 6,737,056. Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays known in the art for evaluating such antibody effector functions. An exemplary measurement of effector function is through Fcy3 and/or Clq binding.
As used herein "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g. natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. ADCC activity of a molecule of interest can be assessed using an in vitro ADCC assay, such as that described in U.S. Patent No. 5,500,362 or 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and NK cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., 1998, PNAS (USA), 95:652-656.
"Complement dependent cytotoxicity" or "CDC" refers to the lysing of a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule (e.g. an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods, 202: 163 (1996), may be performed.
As used herein, "Fc receptor" and "FcR" describe a receptor that binds to the Fc region of an antibody. The preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, FcyRIII, and FcyRIV subclasses, including allelic variants and alternatively spliced forms of these receptors. FcyRll receptors include FcyRIIA (an "activating receptor") and FcyRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. FcRs are reviewed in Ravetch and Kinet, 1991, Ann. Rev. Immunol, 9:457-92; Capel et al., 1994, Immunomethods, 4:25-34; de Haas et al., 1995, J. Lab. Clin. Med, 126:330-41; Nimmerjahn et al., 2005, Immunity 23 :2-4. "FcR" also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., 1976, J. Immunol., 117:587; and Kim et al., 1994, J. Immunol., 24:249).
As used herein, "treatment" is an approach for obtaining beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing the proliferation of (or destroying) neoplastic or cancerous cells, inhibiting metastasis of neoplastic cells, shrinking or decreasing the size of tumor, cancer remission, decreasing cancer symptoms, increasing the quality of life of those suffering from cancer, decreasing the dose of other medications required to treat cancer, delaying the progression of cancer, curing cancer, and/or prolong survival of a cancer patients.
As used herein, an "effective dosage" or "effective amount" of drug, compound, or pharmaceutical composition is an amount sufficient to effect any one or more beneficial or desired results. For prophylactic use, beneficial or desired results include eliminating or reducing the risk, lessening the severity, or delaying the outset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. For therapeutic use, beneficial or desired results include clinical results such as reducing incidence or amelioration of one or more symptoms of various cancer-related diseases or conditions (such as gastric, head and neck, lung, ovarian, and pancreatic cancers), decreasing the dose of other medications required to treat the disease, enhancing the effect of another medication, and/or delaying the progression of the cancer in patients. An effective dosage can be administered in one or more administrations. For purposes of this invention, an effective dosage of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective dosage" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.
The term "purify," and grammatical variations thereof, is used to mean the removal, whether completely or partially, of at least one impurity from a mixture containing the polypeptide and one or more impurities, which thereby improves the level of purity of the polypeptide in the composition (i.e., by decreasing the amount (ppm) of impurity(ies) in the composition).
Reference to "about" a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X." Numeric ranges are inclusive of the numbers defining the range.
An "individual" or a "subject" is a mammal, more preferably, a human. Mammals also include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice, and rats.
It is understood that wherever embodiments are described herein with the language "comprising," otherwise analogous embodiments described in terms of "consisting of and/or "consisting essentially of are also provided.
Where aspects or embodiments of the invention are described in terms of a Markush group or other grouping of alternatives, the present invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group, but also the main group absent one or more of the group members. The present invention also envisages the explicit exclusion of one or more of any of the group members in the claimed invention.
The residue designations in this application are based on the EU numbering scheme of the constant domain (Edelman et al., Proc. Natl. Acad. Sci. USA, 63(l):78-85 (1969). Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. Throughout this specification and claims, the word "comprise," or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The materials, methods, and examples are illustrative only and not intended to be limiting
Engineered Polypeptide Conjugates
The engineered polypeptide conjugates herein comprise a polypeptide (e.g., Fc-containing polypeptide, Fab-containing polypeptide, or antibody) engineered to a specific acyl donor glutamine-containing tag, wherein the polypeptide is site-specifically conjugated to an amine donor agent (e.g., a small molecule coupled to a linker) via the acyl donor glutamine-containing tag. The engineered polypeptide (e.g., Fc-containing polypeptide or antibody) can also be modified to eliminate the off-target conjugation between the acyl donor glutamine-containing tag and the amine donor agent to achieve highly homogenous polypeptide conjugates that are at least about 99% site-specific.
In one aspect, provided is an engineered polypeptide conjugate comprising the formula: polypeptide-T-A, wherein T is an acyl donor glutamine-containing tag engineered at a specific site, wherein A is an amine donor agent, wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine-containing tag at a carboxyl terminus, an amino terminus, or at an another site in the polypeptide, and wherein the acyl donor glutamine-containing tag comprises an amino acid sequence LLQGPX, wherein X is A or P (SEQ ID NO: 14), or GGLLQGPP (SEQ ID NO: 13). In some embodiments, the polypeptide is a Fc-containing polypeptide or a Fab-containing polypeptide. In another aspect, provided is an engineered Fc-containing polypeptide conjugate comprising the formula: (Fc-containing polypeptide)-T-A, wherein T is an acyl donor glutamine- containing tag engineered at a specific site, wherein A is an amine donor agent, wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine-containing tag at a carboxyl terminus, an amino terminus, or at an another site in the Fc-containing polypeptide, wherein the acyl donor glutamine-containing tag comprises an amino acid sequence XXQX (SEQ ID NO:35), wherein X is any amino acid, and wherein the engineered Fc-containing polypeptide conjugate comprises an amino acid substitution from glutamine to asparagine at position 295 (e.g., Q295N; EU numbering scheme).
Both the acyl donor glutamine-containing tag and the amine donor agent described herein are substrates for transglutaminase, and the linkage between the acyl donor glutamine-containing tag and the amine donor agent is of the formula CH2-CH2-CO- H-, wherein H- is linked to a linker and an agent moiety. The transglutaminase used in the invention described herein can be obtained or made from a variety of sources. In some embodiments, the transglutaminase is a calcium dependent transglutaminase which requires calcium to induce enzyme conformational changes and allow enzyme activity. For example, transglutaminase can be derived from guinea pig liver and obtained through commercial sources (e.g., Sigma-Aldrich (St Louis, MO) and MP Biomedicals (Irvine, CA)). In some embodiments, the transglutaminase is a calcium independent transglutaminase which does not require calcium to induce enzyme conformational changes and allow enzyme activity. In some embodiments, the transglutaminase is a microbial transglutaminase derived from a microbial genome, such as transglutaminase from Streptoverticillium or Streptomyces (e.g., Streptomyces mobarensis or Streptoverticillium mobarensis). Commercially available calcium independent transglutaminase such as ACTIVA™ (Ajinomoto, Japan) is suitable for the present invention. In some embodiments, the transglutaminase is a mammalian protein (e.g., human transglutaminase), a bacterial protein, a plant protein, a fungi protein (e.g., Oomycetes and Actinomicetes transglutaminases), or a prokaryotic protein. In some embodiments, the transglutaminase is from Micrococcus, Clostridium, Turolpsis, Rhizopus, Monascus, or Bacillus.
In some embodiments, the transglutaminase used in the invention described herein is an engineered transglutaminase, which catalyzes transamidation of 1) one or more exogenous glutamine residues on the acyl donor glutamine-containing tag and additionally/optionally 2) endogenous glutamine residues in the antibody, with one or more lysine residues or reactive amines in the amine donor agent. For example, one or more wild-type amino acid residues in the naturally occurring transglutaminase are deleted, replaced or substituted with another amino acid residue(s) to make the engineered transglutaminase.
In some embodiments, the transglutaminase used in the invention described herein can also be a recombinant protein produced using recombinant techniques known to persons skilled in the art. In some embodiments, the transglutaminase used in the invention described herein can be a purified protein. For example, the purified transglutaminase is least about 50% pure. As used herein, "pure" or "purified" protein refers to a protein (e.g., transglutaminase) free from other protein contaminants. In some embodiments, the purified transglutaminase is at least about any of 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-98%, or 99% pure. In some embodiments, the purified transglutaminase is about any of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% pure.
In some embodiments, the acyl donor glutamine-containing tag of the engineered polypeptide (e.g., Fc-containing, Fab-containing polypeptide, or antibody) conjugate as described herein is not spatially adjacent to a reactive Lys in the polypeptide. For example, the acyl donor glutamine-containing tag is not spatially adjacent to a reactive Lys in the carboxyl terminus, the amino terminus, or both the carboxyl and the amino termini of the polypeptide.
In some embodiments, the acyl donor glutamine-containing tag comprises an amino acid sequence XXQX (SEQ ID NO: 35), wherein X can be a conventional or nonconventional amino acid, as described herein, and can be the same or a different amino acid. In some embodiments, X is L (Leu), A (Ala), G (Gly), S (Ser), V (Val), F (Phe), Y (Tyr), H (His), R (Arg), N (Asn), E (Glu), D (Asp), C (Cys), Q (Gin), I (lie), M (Met), P (Pro), T (Thr), K (Lys), or W (Trp). In some embodiments, the acyl donor glutamine-containing tag comprises an amino acid sequence selected from the group consisting of LLQGG (SEQ ID NO: 16), LLQG (SEQ ID NO: 17), LSLSQG (SEQ ID NO: 18), GGGLLQGG (SEQ ID NO: 19), GLLQG (SEQ ID NO:20), LLQ, GSPLAQSHGG (SEQ ID NO:21), GLLQGGG (SEQ ID NO:22), GLLQGG (SEQ ID NO:23), GLLQ (SEQ ID NO:24), LLQLLQGA (SEQ ID NO:25), LLQGA (SEQ ID NO:26), LLQYQGA (SEQ ID NO:27), LLQGSG (SEQ ID NO:28), LLQYQG (SEQ ID NO:29), LLQLLQG (SEQ ID NO:30), SLLQG (SEQ ID NO:31), LLQLQ (SEQ ID NO:32), LLQLLQ (SEQ ID NO:33), LLQGR (SEQ ID NO:34), LLQGPP (SEQ ID NO: 11), LLQGP A (SEQ ID NO:4), GGLLQGPP (SEQ ID NO: 13), GGLLQGA (SEQ ID NO: 12), LLQGA (SEQ ID NO: l), LLQGPGK (SEQ ID NO:2), LLQGPG (SEQ ID NO:3), LLQGP (SEQ ID NO:5), LLQP (SEQ ID NO:6), LLQPGK (SEQ ID NO: 7), LLQAPGK (SEQ ID NO: 8), LLQGAPG (SEQ ID NO: 9), and LLQGAP (SEQ ID NO: 10). In some embodiments, the acyl donor glutamine-containing tag comprises an amino acid sequence LLQGPA (SEQ ID N0:4), LLQGP (SEQ ID N0:5), LLQGPP (SEQ ID NO: 11), or GGLLQGPP (SEQ ID NO: 13). In some embodiments, the acyl donor glutamine-containing tag does not comprise an amino acid sequence selected from the group consisting of LGGQGGG (SEQ ID NO:41), GGGQGGL (SEQ ID NO:42), GXGQGGG (SEQ ID NO:43), GGXQGGG (SEQ ID NO:44), GGGQXGG (SEQ ID NO:45), and GGGQGXG (SEQ ID NO:46), wherein X is G, A, S, L, V, F, Y, R, N, or E).
In some embodiments, the polypeptide (e.g., Fc-containing polypeptide, Fab-containing polypeptide, or antibody) of the engineered polypeptide conjugate comprises an amino acid modification at the last amino acid position in the carboxyl terminus relative to a wild-type polypeptide at the same position. In some embodiments, the modification is an amino acid deletion, insertion, substitution, mutation, or any combination thereof. In some embodiments, the substitution comprises replacing a wild type amino acid with another (e.g., a non-wild type amino acid). In some embodiments, the insertion comprises inserting one or more amino acid(s) (e.g., inserting one, two, three or more amino acids). In some embodiments, the other (e.g., non- wild type) or inserted amino acid is Arg. In some embodiments, the other (e.g., non-wild type) amino acid is Ala, Asn, Asp, Cys, Glu, Gin, Gly, His, He, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. For example, in some embodiments, the last amino acid in the carboxyl terminus of the polypeptide (e.g., the heavy chain of an antibody) can be deleted, and the acyl donor glutamine- containing tag engineered to the C-terminus of the polypeptide comprises the amino acid sequence LLQGPA (SEQ ID NO:4) or LLQGPP (SEQ ID NO: 11).
In some embodiments, the polypeptide (e.g., Fc-containing polypeptide or antibody) of the engineered polypeptide conjugate comprises an amino acid modification at position 222, 340, or 370 (EU numbering) relative to the wild-type polypeptide at the same position. In some embodiments, the modification is an amino acid deletion, insertion, substitution, mutation, or any combination thereof. In some embodiments, the substitution comprises replacing a wild type amino acid with another (e.g., a non-wild type amino acid). In some embodiments, the other (e.g., non-wild type) or inserted amino acid is Arg (e.g., K222R, K340R, or K370R (EU numbering)). In some embodiments, the insertion comprises inserting one or more amino acid(s) (e.g., inserting one, two, three or more amino acids). In some embodiments, the other (e.g., non- wild type) amino acid is Ala, Asn, Asp, Cys, Glu, Gin, Gly, His, He, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. For example, in some embodiments, the polypeptide (e.g., an antibody) comprising the K222R substitution is engineered to the acyl donor glutamine-containing tag at the C-terminus of the light chain of the antibody, wherein the glutamine-containing tag comprises an amino acid sequence of GGLLQGPP (SEQ ID NO: 13).
In some embodiments, the polypeptide (e.g., Fc-containing polypeptide, Fab-containing polypeptide, or antibody) comprises an amino acid modification at the first amino acid position in the amino terminus relative to a wild-type polypeptide at the same position. In some embodiments, the modification is an amino acid deletion, insertion, substitution, mutation, or any combination thereof. In some embodiments, the substitution comprises replacing a wild type amino acid with another (e.g., non-wild type) amino acid. In some embodiments, the insertion comprises inserting an amino acid. In some embodiments, the non-wild type or inserted amino acid is Arg. In some embodiments, the other (non-wild type or inserted) amino acid is Ala, Asn, Asp, Cys, Glu, Gin, Gly, His, He, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
In some embodiments, the polypeptide conjugate described herein comprises a full length antibody heavy chain and an antibody light chain. In some embodiments, the acyl donor glutamine-containing tag is linked to/located at the polypeptide at the carboxyl terminus of a heavy chain, a light chain, or both the heavy chain and the light chain. For example, the acyl donor glutamine-containing tag GGLLQGPP (SEQ ID NO: 13) is linked to the polypeptide at the carboxyl terminus of a light chain. In one variation, the acyl donor glutamine-containing tag GGLLQGPP (SEQ ID NO: 13) is linked to the polypeptide comprising the Q295N mutation (EU numbering scheme) at the carboxyl terminus of a light chain. In another variation, the acyl donor glutamine-containing tag LLQGPA (SEQ ID NO:4), LLQGP (SEQ ID NO:5), or LLQGPP (SEQ ID NO: 11) is linked to the polypeptide comprising the Q295N mutation at the carboxyl terminus of a heavy chain.
In some embodiments, the polypeptide described herein is a monoclonal antibody, a polyclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, a minibody, a diabody, or an antibody fragment.
In some embodiments, the antibody is an IgG. In some embodiments, the IgG is selected from the group consisting of IgGl, IgG2, IgG3, and IgG4.
In some embodiments, the antibody is an IgA, IgE, IgD, or IgM.
In some embodiments, the acyl donor glutamine-containing tag is located in the polypeptide by insertion or by replacement of one or more wild-type amino acid(s) at another site on the polypeptide described herein (e.g., Fc-containing polypeptide, Fab-containing polypeptide, or antibody), wherein the other site is not an amino or a carboxyl terminus. For example, the acyl donor glutamine-containing tag is part of an antibody loop. The acyl donor glutamine- containing tag can be linked to one or more heavy chain loop(s). The acyl donor glutamine- containing tag can also be linked to one or more light chain loop(s) of the antibody. In some embodiments, the acyl donor glutamine-containing tag is located at both a heavy chain and a light chain loops. In some embodiments, the another site is amino acid position(s) 108, 135, 160, 168, 189-192, 190-192, 200-202, 222-223, 251-254, 252-253, 222-223, 293-297, 294-297, 295, 297, or 385 (EU numbering scheme) of the human IgGl antibody.
In some embodiments, the effector function (e.g., as measured by Fcy3 and/or Clq binding) of the engineered polypeptide (e.g., Fc-containing polypeptide or antibody) conjugate described herein decreases no greater than about any of 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold relative to a wild type polypeptide (e.g., Fc-containing polypeptide or antibody). In some embodiments, the engineered polypeptide conjugate is an IgG, wherein the effector function of the IgG decreases no greater than about 2-fold relative to a wild type IgG. In other embodiments, the effector function of the IgG decreases about 2-fold relative to a wild type IgG. In other embodiments, the effector function of the IgG decreases more than about 2-fold relative to a wild type IgG. In some embodiments, the engineered Fc-containing polypeptide conjugate is an IgG, wherein the effector function of the IgG decreases no greater than about 1-fold relative to a wild type IgG. In other embodiments, the effector function of the IgG decreases about 1-fold relative to a wild type IgG. In some embodiments, the effector function of the IgG decreases more than about any of 1-fold, 3-fold, 4-fold, or 5-fold relative to a wild type IgG.
In some embodiments, the effector function (e.g., as measured by Fcy3 and/or Clq binding) of the engineered polypeptide (Fc-containing polypeptide or antibody) conjugate described herein increases at least about 1-fold to 3000-fold relative to a wild type polypeptide (e.g., Fc-containing polypeptide or antibody). In some embodiments, the effector function of the engineered polypeptide (e.g., Fc-containing polypeptide or antibody) conjugate increases at least about any of 1- to 5-fold, 6- to 10-fold, 11- to 15-fold, 16- to 20-fold, 21- to 25-fold, 26- to 30- fold, 31- to 35-fold, 36- to 40-fold, 41- to 45-fold, 46- to 50-fold, 51- to 55-fold, 56- to 60-fold, 61- to 65-fold, 66- to 70-fold, 71- to 75-fold, 76- to 80-fold, 81- to 85-fold, 86- to 90-fold, 91- to 95-fold, 96- to 100-fold, 101- to 200-fold, 201- to 300-fold, 301- to 500-fold, 501- to 1000-fold, 1001- to 1500-fold, 1501- to 2000-fold, 2001- to 2500-fold, 2501- to 3000-fold relative to a wild type polypeptide (e.g., Fc-containing polypeptide or antibody). In some embodiments, the engineered polypeptide conjugate is an IgG, wherein the effector function of the IgG increases about 1-fold to 300-fold relative to a wild type IgG. In some embodiments, the effector function of the IgG increases about any of 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 40-fold, 60-fold, 80-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 1500-fold, 2000-fold, 2500-fold, or 3000-fold relative to a wild type IgG.
In some embodiments, the amine donor agent has the formula: X-Y-Z, wherein X is an amine donor unit; Y is a linker; and Z is an agent moiety.
The number of the amine donor agents which may be conjugated via the acyl donor glutamine-containing tag to the polypeptide (e.g., Fc-containing polypeptide, Fab-containing polypeptide, or antibody) is dependent on the number of acyl donor glutamine-containing tags which are linked/inserted to the polypeptide(s) as well as the number of Gin on the acyl donor glutamine-containing tag. For example, two amine donor agents may be site-specifically conjugated to an antibody at the carboxyl termini of the two heavy chains and/or two amine donor agents may be site-specifically conjugated to the same antibody at the carboxyl termini of the two light chains.
The amine donor unit of the present invention is a primary amine ( H2) that provides a substrate for transglutaminase to allow conjugation of the agent moiety to the polypeptide via the acyl donor glutamine-containing tag. Accordingly, the linkage between the acyl donor glutamine-containing tag and the amine donor unit is of the formula CH2-CH2-CO- H-, wherein H- is linked to a linker and an agent moiety.
The linker of the present invention can be a cleavable or a non-cleavable linker. For example, the linker (with amine donor unit) or the amine donor agent can be released from the polypeptide. In some embodiments, the linker can be a peptide linker (e.g., conventional or nonconventional amino acid(s)) and/or a non-peptide linker. Examples of a non-peptide linker include an alkyl linker and a PEG linker.
In some embodiments, the amine donor unit-linker (e.g., X-Y) is a linear unit comprising an agent moiety. In other embodiments, the amine donor unit-linker is a branched unit (e.g., at least 2 units) comprising at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more agent moieties.
Exemplary amine donor unit-linkers include, but are not limited to, Ac-Lys-Gly, aminocaproic acid, Ac-Lys-P-Ala, amino-PEG2-C2, amino-PEG3-C2, amino-PEG6-C2, Ac-Lys- Val-Cit (citrulline)-PABC (p-aminobenzyloxycarbonyl), aminocaproyl-Val-Cit-PABC, amino- PEG6-C2-Val-Cit-PABC, aminocaproyl-Val-Cit-PABC, [(3R,5R)-l-{3-[2-(2- aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, [(3S,5S)-l-{3-[2-(2- aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit-PABC-, Ac-Lys-putrescine, or 2-aminoethoxy. The agent moiety of the engineered polypeptide of the present invention includes a small molecule, a protein or polypeptide, and a biocompatible polymer.
In some embodiments, a small molecule is a cytotoxic agent, an immunosuppressive agent, or an imaging agent (e.g., a fluorophore). In some embodiments, the cytotoxic agent is a chemotherapeutic agent.
Examples of a cytotoxic agent include, but are not limited to, an anthracycline, an auristatin, a camptothecin, a combretastatin, a dolastatin, a duocarmycin, an enediyne, a geldanamycin, an indolino-benzodiazepine dimer, a maytansine, a puromycin, a taxane, a vinca alkaloid, SN-38, a tubulysin, a hemiasterlin, a spliceostatin, a pladienolide, and stereoisomers, isosteres, analogs, or derivatives thereof.
The anthracyclines are derived from bacteria Strepomyces and have been used to treat a wide range of cancers, such as leukemias, lymphomas, breast, uterine, ovarian, and lung cancers. Exemplary anthracyclines include, but are not limited to, daunorubicin, doxorubicin (i.e., adriamycin), epirubicin, idarubicin, valrubicin, and mitoxantrone.
Dolastatins and their peptidic analogs and derivatives, auristatins, are highly potent antimitotic agents that have been shown to have anticancer and antifungal activity. See, e.g., U.S. Pat. No. 5,663,149 and Pettit et al., Antimicrob. Agents Chemother. 42:2961-2965, 1998. Exemplary dolastatins and auristatins include, but are not limited to, dolastatin 10, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), MMAD (Monomethyl Auristatin D or monomethyl dolastatin 10), MMAF (Monomethyl Auristatin F or N-methylvaline-valine-dolaisoleuine- dolaproine-phenylalanine), MMAE (Monomethyl Auristatin E or N-methylvaline-valine- dolaisoleuine-dolaproine-norephedrine), 5 -benzoyl valeric acid-AE ester (AEVB), and other novel auristatins (such as the ones described in U.S. Publication No. 2013/0129753). In some embodiments, the auristatin is 0101 (2-methylalanyl-iV-[(3R,4S, 5S)-3-methoxy-l-{(2S)-2- [(1R.2R)- 1 -methoxy-2-methyl-3 -oxo-3-{ [(15)-2-phenyl- 1 -(1 ,3-thiazol-2- yl)ethyl]amino}propyl]pyrrolidin-l-yl}-5-methyl-l-oxoheptan-4-yl]-N-methyl-L-valinamide) having the following structure:
Figure imgf000026_0001
In some embodiments, the auristatin is 3377 (N,2-dimethylalanyl-N-{(l S,2R)-4-{(2S)-2- [(lR,2R)-3-{[(l S)-l-carboxyl-2-phenylethyl]amino}-l-methoxy-2-methyl-3- oxopropyl]pyrrolidin- 1 -yl }-2-methoxy- 1 -[(1 S)-l -methylpropyl]-4-oxobutyl }-N-m ethyl -L- valinamide) having the following structure:
Figure imgf000027_0001
In other embodiments, the auristatin is 0131 (2-methyl-L-proly-N-[(3R,4S,5S)-l-{(2S)-2- [(lR,2R)-3-{[(l S)-l-carboxy-2-phenylethyl]amino}-l-methoxy-2-methyl-3- oxopropyl]pyrrolidin-l-yl}-3-methoxy-5-methyl-l-oxoheptan-4-yl]-N-methyl-L-valinamide) having the following structure:
Figure imgf000027_0002
In other embodiments, the auristatin is 0121(2-methyl-L-proly-N-[(3R,4S,5S)-l-{(2S)-2- [(lR,2R)-3-{[(2S)-l-methoxy-l-oxo-3-phenylpropan-2-yl]amino}-l-methoxy-2 -methyl -3- oxopropyl]pyrrolidin-l-yl}-3-methoxy-5-methyl-l-oxoheptan-4-yl]-N-methyl-L-valinamide) having the following structure:
Figure imgf000027_0003
Camptothecin is a cytotoxic quinoline alkaloid which inhibits the enzyme topoisomerase I. Examples of camptothecin and its derivatives include, but are not limited to, topotecan and irinotecan, and their metabolites, such as SN-38.
Combretastatins are natural phenols with vascular disruption properties in tumors.
Exemplary combretastatins and their derivatives include, but are not limited to, combretastatin A- 4 (CA-4) and ombrabulin.
Duocarmycins are DNA alkylating agents with cytotoxic potency. See Boger and Johnson, PNAS 92:3642-3649 (1995). Exemplary duocarmycins include, but are not limited to, duocarmycin A, duocarmycin Bl, duocarmycin B2, duocarmycin CI, duocarmycin C2, duocarmycin D, duocarmycin SA, and CC-1065.
Enediynes are a class of anti-tumor bacterial products characterized by either nine- and ten-membered rings or the presence of a cyclic system of conjugated triple-double-triple bonds. Exemplary enediynes include, but are not limited to, calicheamicin, esperamicin, and dynemicin.
Geldanamycins are benzoquinone ansamycin antibiotic that bind to Hsp90 (Heat Shock Protein 90) and have been used antitumor drugs. Exemplary geldanamycins include, but are not limited to, 17-AAG (17-N-Allylamino-17-Demethoxygeldanamycin) and 17-DMAG (17- Dimethylaminoethylamino- 17-demethoxygeldanamycin).
Hemiasterlin and its analogues (e.g., HTI-286) bind to the tubulin, disrupt normal microtubule dynamics, and, at stoichiometric amounts, depolymerize microtubules.
Maytansines or their derivatives maytansinoids inhibit cell proliferation by inhibiting the mcirotubules formation during mitosis through inhibition of polymerization of tubulin. See Remillard et al., Science 189: 1002-1005 (1975). Exemplary maytansines and maytansinoids include, but are not limited to, mertansine (DM1) and its derivatives as well as ansamitocin.
Pyrrolobenzodiazepine dimers (PBDs) and indolino-benzodiazepine dimers (IGNs) are anti-tumor agents that contain one or more immine functional groups, or their equivalents, that bind to duplex DNA. PBD and IGN molecules are based on the natural product athramycin, and interact with DNA in a sequence-selective manner, with a preference for purine-guanine-purine sequences. Exemplary PBDs and their analogs include, but are not limited to, SJG-136.
Spliceostatins and pladienolides are anti-tumor compounds which inhibit splicing and interact with spliceosome, SF3b. Examples of spliceostatins include, but are not limited to, spliceostatin A, and FR901464. Examples of pladienolides include, but are not limited to, Pladienolide B, Pladienolide D, and E7107.
Taxanes are diterpenes that act as anti-tubulin agents or mitotic inhibitors. Exemplary taxanes include, but are not limited to, paclitaxel (e.g., TAXOL®) and docetaxel (TAXOTERE®).
Tubulysins are natural products isolated from a strain of mycobacteria that has been shown to depolymerize microtubules and induce mitotic arrest. Exemplary tubulysins include, but are not limited to, tubulysin A, tubulysin B, and tubulysin D.
Vinca alkyloids are also anti-tubulin agents. Exemplary vinca alkyloids include, but are not limited to, vincristine, vinblastine, vindesine, and vinorelbine.
In some embodiments, the agent moiety is an immunosuppressive agent. Examples of an immunosuppressive agent include, but are not limited to, gancyclovier, etanercept, tacrolimus, sirolimus, voclosporin, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolgate mofetil, methotrextrate, and glucocorticoid and its analogs.
In some embodiments, the agent moiety is an imaging agent (e.g., a fluorophore or a chelator), such as fluorescein, rhodamine, lanthanide phosphors, and their derivatives thereof. Examples of fluorophores include, but are not limited to, fluorescein isothiocyanate (FITC) (e.g., 5-FITC), fluorescein amidite (FAM) (e.g., 5-FAM), eosin, carboxyfluorescein, erythrosine, Alexa Fluor® (e.g., Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700, or 750), carboxytetramethylrhodamine (TAMRA) (e.g., 5,-TAMRA), tetramethylrhodamine (TMR), and sulforhodamine (SR) (e.g., SR101). Examples of chelators include, but are not limited to, l,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA), l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA), l,4,7-triazacyclononane,l- glutaric acid-4,7-acetic acid (NODAGA), diethylenetrianiixiepentaacetic acid (DTPA), and 1,2- bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) (BAPTA).
In some embodiments, the agent moiety is a polypeptide. In some embodiments, the polypeptide is an antibody, such as a humanized, human, chimeric, or murine monoclonal antibody.
In some embodiments, the agent moiety is a toxin polypeptide (or a toxin protein). Examples of a toxin polypeptide include, but are not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, tricothecenes, inhibitor cystine knot (ICK) peptides (e.g., ceratotoxins), and conotoxin (e.g., KIIIA or SmIIIa).
In some embodiments, therapeutic radioisotopes or other labels can be incorporated in the agent moiety (e.g., by binding to a chelator) for conjugation of a polypeptide (e.g., a Fc- containing or Fab-containing polypeptide) to an amine donor agent that bears a chelator. Examples of a radioisotope or other labels include, but are not limited to, 3H, 14C, 15N, 35S, 18F,
32 , 33t> 64r 68^ 89^ 90v 99T 123T 124T 125T 131T 111T 131T 153 C m 186τ> 188τ> 211 » . 212D -
P, P, Cu, Ga, Zr, Y, Tc, I, I, I, I, In, In, Sm, Re, Re, At, Bi, and 153Pb.
In some embodiments, the agent moiety is a biocompatible polymer. The polypeptide can be conjugated to the biocompatible polymer via the acyl donor glutamine-containing tag to improve the biological characteristics of the polypeptide, e.g., to increase serum half-life and bioactivity, and/or to extend in vivo half-lives. Examples of biocompatible polymers include water-soluble polymer, such as polyethylene glycol (PEG) or its derivatives thereof and zwitterion-containing biocompatible polymers (e.g., a phosphorylcholine containing polymer).
In some embodiments, the amine donor agent (X-Y-Z) is
Figure imgf000030_0001
wherein X is H2 (i.e., thus forming a covalent bond with glutamine as CH2-CH2-CO- H-), m is a 0 to 20, n is 1 to 8, p is 0 to 3, q is 0 or 1, amino acid is any conventional or nonconventional amino acid and Z is a cytotoxic agent or an imaging agent.
Conventional or naturally occurring amino acids are divided into groups based on common side-chain properties: (1) non-polar: Norleucine, Met, Ala, Val, Leu, He; (2) polar without charge: Cys, Ser, Thr, Asn, Gin; (3) acidic (negatively charged): Asp, Glu; (4) basic (positively charged): Lys, Arg; and (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe, His. Conventional amino acids include L or D stereochemistry. Unconventional amino acids are non-naturally occurring amino acids. Examples of an uncoventioanl amino acid include, but are not limited to, aminoadipic acid, beta-alanine, beta- aminopropionic acid, aminobutyric acid, piperidinic acid, aminocaprioic acid, aminoheptanoic acid, aminoisobutyric acid, aminopimelic acid, citmlline, diaminobutyric acid, desmosine, diaminopimelic acid, diaminopropionic acid, N-ethylglycine, N-ethylaspargine, hyroxylysine, allo-hydroxylysine, hydroxyproline, isodesmosine, allo-isoleucine, N-methylglycine, sarcosine, N-methylisoleucine, N-methylvaline, norvaline, norleucine, orithine, 4-hydroxyproline, γ- carboxyglutamate, ε-Ν,Ν,Ν-trimethyllysine, ε-Ν-acetyllysine, O-phosphoserine, N-acetyl serine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-Ν-methylarginine, and other similar amino acids and amino acids (e.g., 4-hydroxyproline).
In some embodiments, the amine donor agent is selected from the group consisting of Alexa 488 cadaverine, 5-FITC cadaverine, Alexa 647 cadaverine, Alexa 350 cadaverine, 5- TAMRA cadaverine, 5-FAM cadaverine, SRI 01 cadaverine, 5,6-TAMRA cadaverine, 5-FAM lysine, Ac-Lys-Gly-MMAD, amino-PEG3 -C2-MM AD, amino-PEG6-C2-MMAD, amino-PEG3- C2-amino-nonanoyl-MMAD, aminocaproyl-Val-Cit-PABC-MMAD, Ac-Lys-P-Ala-MMAD, Aminocaproyl-MMAD, amino-PEG6-C2-Val-Cit-PABC-MMAD, Ac-Lys-Val-Cit-PABC- MMAD, Ac-Lys-Val-Cit-PABC-0101, amino-PEG3-C2-Val-Cit-PABC-MMAD, amino-PEG3- C2- Val-Cit-P ABC-MMAD, amino-PEG6-C2- Val-Cit-P ABC-0101, aminocaproyl-MMAE, amino-PEG3 -C2-MM AE, amino-PEG2-C2-MMAE, aminocaproyl-MMAF, aminocaproyl- Val- Cit-P ABC -MMAE, amino-PEG6-C2-Val-Cit-PABC-MMAF, aminocaproyl-Val-Cit-PABC- MMAF, amino-PEG2-C2-MMAF, amino-PEG3-C2-MMAF, putrescinyl-geldanamycin, Ac-Lys- putrescinyl-geldanamycin, aminocaproyl-3377, aminocaproyl-0131, amino-PEG6-C2-0131, amino-PEG6-C2-3377, aminocaproyl -0121, amino-PEG6-C2-0121, [(3R,5R)-l-{3-[2-(2- aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit-P ABC-MMAD, [(3R,5R)-l-{3- [2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis- Val-Cit-P ABC-MMAE, and 2- aminoethoxy-PEG6-NODAGA (or 2,2'-(7-(l-amino-28-carboxy-25-oxo-3,6,9, 12, 15,18,21- heptaoxa-24-azaoctacosan-28-yl)-l,4,7-triazonane-l,4-diyl)diacetic acid). In some embodiments, the acyl donor glutamine-containing tag comprises the amino acid sequence LLQGPA (SEQ ID NO:4), LLQGP (SEQ ID NO:5), LLQGPP (SEQ ID NO: 11), or GGLLQGPP (SEQ ID NO: 13) and the amine donor agent is Ac-Lys-Val-Cit-P ABC-MMAD, Ac-Lys-Val-Cit- PABC-0101, or amino-PEG6-C2-MMAD. Exemplary structures of the amine donor agent are listed in Table 1. Table 1
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
In another aspect, the invention provides an engineered toxin polypeptide conjugate comprising the formula: (toxin polypeptide)-T-A, wherein T is an acyl donor glutamine- containing tag engineered at a specific site, wherein A is an amine donor agent, wherein the amine donor agent is a biocompatible polymer comprising a reactive amine, wherein the biocompatible polymer is site-specifically conjugated to the acyl donor glutamine-containing tag at a carboxyl terminus, an amino terminus, or elsewhere at an another site in the toxin polypeptide, and wherein the acyl donor glutamine-containing tag comprises an amino acid sequence LLQGPX, wherein X is A or P (SEQ ID NO: 14), or GGLLQGPP (SEQ ID NO: 13). For example, the toxin polypeptide can be site-specifically conjugated to the biocompatible polymer via the acyl donor glutamine-containing tag as described herein to improve the biological characteristics of the toxin polypeptide, e.g., to increase the serum half-life and bioactivity, and/or to extend its in vivo half-lives. In some embodiments, toxin polypeptide is a ceratotoxin or a conotoxin (e.g., KIIIA or SmIIIa). In some embodiments, the biocompatible polymer is a water soluble polymer such as PEG derivative or a zwitterion-containing biocompatible polymer.
Methods for Making the Engineered Polypeptide Conjugates
The methods for making the engineered polypeptide conjugates described herein are also provided in the present invention. In one aspect, the invention provides a method for preparing an engineered polypeptide conjugate comprising the formula: polypeptide-T-A, wherein T is an acyl donor glutamine-containing tag engineered at a specific site, wherein A is an amine donor agent, and wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine-containing tag at a carboxyl terminus, an amino terminus, or elsewhere at an another site in the polypeptide, and wherein the acyl donor glutamine-containing tag comprises an amino acid sequence LLQGPX, wherein X is A or P (SEQ ID NO: 14), or GGLLQGPP (SEQ ID NO: 13), comprising the steps of: a) providing an engineered polypeptide-T molecule comprising the polypeptide and the acyl donor glutamine-containing tag; b) contacting the amine donor agent with the engineered polypeptide-T molecule in the presence of a transglutaminase; and c) allowing the engineered polypeptide-T to covalently link to the amine donor agent to form the engineered polypeptide conjugate. In some embodiments, the polypeptide is a Fc-containing or Fab-containing polypeptide, or an antibody. In some embodiments, the engineered polypeptide- T molecule is expressed in CHO cells.
In another aspect, the invention provides a method for preparing an engineered Fc- containing polypeptide conjugate comprising the formula: (Fc-containing polypeptide)-T-A, wherein T is an acyl donor glutamine-containing tag engineered at a specific site; wherein A is an amine donor agent; and wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine-containing tag at a carboxyl terminus, an amino terminus, or elsewhere at an another site in the Fc-containing polypeptide, wherein the acyl donor glutamine-containing tag comprises an amino acid sequence XXQX (SEQ ID NO: 35), wherein X is any amino acid (e.g., X can be the same or a different amino acid), and wherein the engineered Fc-containing polypeptide conjugate comprises an amino acid substitution from glutamine to asparagine at position 295 (e.g., Q295N; EU numbering scheme), comprising the steps of: a) providing an engineered (Fc-containing polypeptide)-T molecule comprising the Fc-containing polypeptide and the acyl donor glutamine-containing tag; b) contacting the amine donor agent with the engineered (Fc-containing polypeptide)-T molecule in the presence of a transglutaminase; and c) allowing the engineered (Fc-containing polypeptide)-T to covalently link to the amine donor agent to form the engineered Fc-containing polypeptide conjugate. In some embodiments, the engineered (Fc-containing polypeptide)-! is expressed in CHO cells. In some embodiments, the acyl donor glutamine-containing tag comprises an amino acid sequence selected from the group consisting of LLQGG (SEQ ID NO: 16), LLQG (SEQ ID NO: 17), LSLSQG (SEQ ID NO: 18), GGGLLQGG (SEQ ID NO: 19), GLLQG (SEQ ID NO:20), LLQ, GSPLAQSHGG (SEQ ID NO:21), GLLQGGG (SEQ ID NO:22), GLLQGG (SEQ ID NO:23), GLLQ (SEQ ID NO:24), LLQLLQGA (SEQ ID NO:25), LLQGA (SEQ ID NO:26), LLQYQGA (SEQ ID NO:27), LLQGSG (SEQ ID NO:28), LLQYQG (SEQ ID NO:29), LLQLLQG (SEQ ID NO:30), SLLQG (SEQ ID NO:31), LLQLQ (SEQ ID NO:32), LLQLLQ (SEQ ID NO:33), LLQGR (SEQ ID NO:34), LLQGPP (SEQ ID NO: 1 1), LLQGPA (SEQ ID NO:4), GGLLQGPP (SEQ ID NO: 13), GGLLQGA (SEQ ID NO: 12), LLQGA (SEQ ID NO: l), LLQGPGK (SEQ ID NO:2), LLQGPG (SEQ ID NO:3), LLQGP (SEQ ID NO:5), LLQP (SEQ ID NO:6), LLQPGK (SEQ ID NO:7), LLQAPGK (SEQ ID NO:8), LLQGAPG (SEQ ID NO:9), and LLQGAP (SEQ ID NO: 10). In some embodiments, the acyl donor glutamine-containing tag comprises an amino acid sequence LLQGPA (SEQ ID NO:4), LLQGP (SEQ ID NO:5), LLQGPP (SEQ ID NO: 1 1), or GGLLQGPP (SEQ ID NO: 13).
In some embodiments, the engineered polypeptide conjugate prepared using the methods described herein has conjugation efficiency of at least about 51%. In some embodiments, the engineered polypeptide conjugate has conjugation efficiency of at least about any of 51%-60%, 61%-70%, 71%-80%, 81%-90%, or 91%- 100%. In some embodiments, the engineered polypeptide conjugate has conjugation efficiency of about any of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%), 99.9%, or 100%. For example, the engineered polypeptide (e.g., Fc-containing polypeptide) comprising the Q295N mutation (EU numbering scheme) has conjugation efficiency of at least about 99.8%.
In some embodiments, the concentration ratio between the amine donor agent contacted and the engineered polypeptide-T molecule contacted is from about 2: 1 to about 800: 1. For example, the concentration ratio between the amine donor agent (e.g., a cytotoxic drug) and the engineered polypeptide attached to an acyl donor glutamine-containing tag loaded or used for the transglutaminase-catalyzed conjugation reaction can be about 20: 1. In some embodiments, the concentration ratio between the amine donor agent contacted and the engineered polypeptide-T molecule contacted is about any of 2: 1, 3 : 1, 4: 1, 5 : 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 15 : 1, 20: 1, 25 : 1, 30: 1, 35: 1, 40: 1, 45: 1, 50: 1, 60: 1, 70: 1, 80: 1, 90: 1, 100: 1, 200: 1, 300: 1, 400: 1, 500: 1, 600: 1, 700: 1, or 800: 1.
In some embodiments, when a polypeptide (e.g., antibody) is conjugated with an amine donor agent via an acyl donor glutamine-containing tag at a specific site (e.g., C-terminus), the antibody-drug-conjugate is more stable (e.g., longer in vivo half-life). Accordingly, in some embodiments, the engineered polypeptide conjugate as described herein is present in a subject (e.g., a mammal) at at least about 50% after at least about 1 day in vivo. For example, the engineered polypeptide conjugate is present in a subject at at least about any of 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% after at least about any of 2 hours, 2-6 hours, 6-12 hours, 12-18 hours, 18-24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, or 2 weeks in vivo.
In some embodiments, the methods provided herein further comprise a purification step. The engineered Fc-containing polypeptide conjugates described herein can be purified using various purification methods, such as, e.g., hydroxylapatite chromatography; dialysis; affinity chromatography; hydrophobic interaction chromatography (HIC) (e.g, fractionation on a HIC); ammonium sulphate precipitation; polyethylene glycol or polyethylene glycol derivative precipitation, anion or cation exchange chromatography; reverse phase HPLC; chromatography on silica; chromatofocusing; SDS-PAGE, gel filtration, size exclusion chromatography, and weak partitioning chromatography.
In some embodiments, at least one purification step comprises a step of affinity chromatography method. Protein A ligand (synthetic, recombinant, or native) may be used to affinity purify the engineered Fc-containing polypeptide conjugates described herein. Synthetic or recombinant Protein A ligand may be purchased commercially from GE Healthcare (Piscataway, NJ), Pierce (Rockford, IL), Sigma-Aldrich (St. Louis, MO), or Applied Biosystems (Foster City, CA), and native Protein A ligand (e.g., MABSELECT™, PROSEP™ Va, and PROSEP™ Ultra Plus ) may be purchased commercially from GE Healthcare (Piscataway, NJ) or Millipore (Billerica, MA).
In some embodiments, the purified engineered Fc-containing polypeptide conjugate, the purified engineered Fab-containing polypeptide conjugate, or the purified toxin polypeptide conjugates resulting from the purification step is highly pure, i.e., at least about any of 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-98%, or 99% pure. For example, the purified engineered polypeptide conjugate is about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure.
Methods of Using the Engineered Polypeptide Conjugates
The engineered polypeptide conjugates of the present invention are useful in various applications including, but are not limited to, therapeutic treatment methods and diagnostic treatment methods.
In one aspect, the invention provides a method for treating a cancer in a subject. Accordingly, in some embodiments, provided is a method of treating a cancer in a subject in need thereof comprising administering to the subject an effective amount of a composition (e.g., pharmaceutical composition) comprising the engineered polypeptide conjugates as described herein. As used herein, cancers include, but are not limited to, a solid cancer (such as bladder, breast, cervical, choriocarcinoma, colon, esophageal, gastric, glioblastoma, head and neck, kidney, liver, lung (e.g., Non Small Cell Lung Cancer (NSCLC)), oral, ovarian, pancreatic, prostate, and skin cancer); and a liquid cancer (such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T- PLL), large granular lymphocytic leukemia, and adult T-cell leukemia).
In some embodiments, provided is a method of inhibiting tumor growth or progression in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising the engineered polypeptide conjugates as described herein. In other embodiments, provided is a method of inhibiting metastasis of cancer cells or tumors (e.g., solid or liquid tumors) in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising the engineered polypeptide conjugates as described herein. In other embodiments, provided is a method of inducing tumor regression in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising the engineered polypeptide conjugates as described herein.
In another aspect, provided is a method of detecting, diagnosing, and/or monitoring a condition associated with a cancer-related protein (e.g., Trop-2, BRCA1, BRCA2, HER2, VEGF, CD20, CD25, EFGR, 5T4, CD22, etc.) in vivo or in vitro. Accordingly, in some embodiments, provided is a method of diagnosing cancer in a subject suspected of suffering from cancer, comprising a) contacting a sample of the subject with the engineered polypeptide conjugates as described herein under conditions that result in binding of the engineered polypeptide conjugates with a cancer-related protein, and b) determining binding of the engineered polypeptide conjugates to the cancer-related protein.
The agent moiety in the engineered polypeptide conjugates as described herein can be a detectable moiety such as an imaging agent and an enzyme-substrate label. The engineered polypeptide conjugates as described herein can also be used for in vivo diagnostic assays, such as in vivo imaging (e.g., PET or SPECT), or a staining reagent.
In some embodiments, the methods described herein further comprise a step of treating a subject with an additional form of therapy. In some embodiments, the additional form of therapy is an additional anti-cancer therapy including, but not limited to, chemotherapy, radiation, surgery, hormone therapy, and/or additional immunotherapy.
In some embodiments, the additional form of therapy comprises administering one or more therapeutic agent in addition to the engineered polypeptide conjugates as described herein. The therapeutic agents include, but are not limited to, an antibody-drug conjugate (e.g., brentuximab vedotin (ADCETRIS®) and ado-trastuzumab emtansine (KADCYLA®)), an antibody (e.g., an anti-VEGF antibody, an anti-HER2 antibody, anti-CD25 antibody, and/or an anti-CD20 antibody), an angiogenesis inhibitor, a cytotoxic agent (e.g., docetaxel, cisplatin, doxorubicin, mitomycin, tamoxifen, or fluorouracil), and an anti-inflammatory agent (e.g., prednisone, and progesterone).
Pharmaceutical Compositions
The present invention also provides a pharmaceutical composition comprising the engineered polypeptide conjugates as described herein in a pharmaceutically acceptable excipient or carrier. The engineered polypeptide conjugates can be administered alone or in combination with one or more other engineered polypeptide conjugates of the invention or in combination with one or more other drugs (or as any combination thereof). The pharmaceutical compositions, methods and uses of the invention thus also encompass embodiments of combinations (coadministration) with other active agents, as detailed below.
As used herein, the term "co-administration," "co-administered," or "in combination with" is intended to mean and does refer to the following: (i) simultaneous administration of a combination of an engineered polypeptide conjugate disclosed herein and therapeutic agent(s) to a patient in need of treatment, when such components are formulated together into a single dosage form which releases said components at substantially the same time to said patient; (ii) substantially simultaneous administration of such combination of an engineered polypeptide conjugate disclosed herein and therapeutic agent(s) to a patient in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at substantially the same time by said patient, whereupon said components are released at substantially the same time to said patient; (iii) sequential administration of such combination of an engineered polypeptide conjugate disclosed herein and therapeutic agent(s) to a patient in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at consecutive times by said patient with a significant time interval between each administration, whereupon said components are released at substantially different times to said patient; and (iv) sequential administration of such combination of an engineered polypeptide conjugate disclosed herein and therapeutic agent(s) to a patient in need of treatment, when such components are formulated together into a single dosage form which releases said components in a controlled manner whereupon they are concurrently, consecutively, and/or overlappingly released at the same and/or different times to said patient, where each part may be administered by either the same or a different route.
Generally, the engineered polypeptide conjugates disclosed herein are suitable to be administered as a formulation in association with one or more pharmaceutically acceptable excipient(s). The term 'excipient' is used herein to describe any ingredient other than the compound(s) of the invention. The choice of excipient(s) to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In some embodiments, isotonic agents, including, but not limited to, sugars, polyalcohols (e.g., mannitol, sorbitol) or sodium chloride are included in the pharmaceutical composition. Additional examples of pharmaceutically acceptable substances include, but are not limited to, wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody.
In some embodiments, the engineered polypeptide conjugates described herein can be deimmunized to reduce immunogenicity upon administration to a subject using known techniques such as those described, e.g., in PCT Publication W098/52976 and WO00/34317. Pharmaceutical compositions of the present invention and methods for their preparation are readily apparent to those skilled in the art. Such compositions and methods for their preparation may be found, for example, in Remington's Pharmaceutical Sciences, 22nd Edition (Mack Publishing Company, 2012). Pharmaceutical compositions are preferably manufactured under GMP conditions.
A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. Any method for administering peptides, proteins or antibodies accepted in the art may suitably be employed for the engineered polypeptide conjugates disclosed herein.
The pharmaceutical compositions of the invention are typically suitable for parenteral administration. Parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue, thus generally resulting in the direct administration into the blood stream, into muscle, or into an internal organ. For example, parenteral administration includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intrasynovial injection or infusions; and kidney dialytic infusion techniques. In some embodiments, parenteral administration is the intravenous or the subcutaneous route.
Formulations of a pharmaceutical composition suitable for parenteral administration typically generally comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water. Exemplary parenteral administration forms include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, or in a liposomal preparation. Formulations for parenteral administration may be formulated to be immediate and/or modified release. Modified release formulations include controlled, delayed, sustained, pulsed, targeted and programmed release formulations. For example, in one aspect, sterile injectable solutions can be prepared by incorporating the engineered Fc-containing polypeptide, e.g., antibody-drug conjugate or bispecific antibody, in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
Dosage regimens may be adjusted to provide the optimum desired response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for the patients/subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are generally dictated by and directly dependent on (a) the unique characteristics of the agent moiety (e.g., small molecules such as cytotoxic agent) and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
Thus, the skilled artisan would appreciate, based upon the disclosure provided herein, that the dose and dosing regimen is adjusted in accordance with methods well-known in the therapeutic arts. That is, the maximum tolerable dose can be readily established, and the effective amount providing a detectable therapeutic benefit to a patient may also be determined, as can the temporal requirements for administering each agent to provide a detectable therapeutic benefit to the patient. Accordingly, while certain dose and administration regimens are exemplified herein, these examples in no way limit the dose and administration regimen that may be provided to a patient in practicing the present invention.
It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. Further, the dosage regimen with the compositions of this invention may be based on a variety of factors, including the type of disease, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular antibody employed. Thus, the dosage regimen can vary widely, but can be determined routinely using standard methods. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and/or laboratory values. Thus, the present invention encompasses intra-patient dose-escalation as determined by the skilled artisan. Determining appropriate dosages and regimens are well-known in the relevant art and would be understood to be encompassed by the skilled artisan once provided the teachings disclosed herein. For administration to human subjects, the total monthly dose of an engineered polypeptide conjugate disclosed herein is typically in the range of about 0.01 mg to about 1200 mg per patient, depending, of course, on the mode of administration. For example, an intravenous monthly dose may require about 1 to about 1000 mg/patient. The total monthly dose may be administered in single or divided doses and may, at the physician's discretion, fall outside of the typical range given herein.
An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an engineered polypeptide conjugate, e.g., an Fc-containing polypeptide conjugate, Fab-containing polypeptide conjugate, antibody conjugate, or toxin polypeptide conjugate, disclosed herein is about 0.01 to about 1000 mg/patient/month. In certain embodiments, the engineered Fc-containing polypeptide conjugate may be administered at about 1 to about 200 or about 1 to about 150 mg/patient/month. In some embodiments, the patient is human.
Kits
The invention also provides kits (or articles of manufacture) for use in the treatment of the disorders described above. Kits of the invention include one or more containers comprising a purified engineered polypeptide conjugate and instructions for using the conjugate for treating a disease. For example, the instructions comprise a description of administration of the engineered polypeptide conjugate to treat a disease, such as cancer (e.g., colon, esophageal, gastric, head and neck, lung, ovarian, or pancreatic cancer). The kit may further comprise a description of selecting an individual suitable for treatment based on identifying whether that individual has the disease and the stage of the disease.
The instructions relating to the use of the engineered polypeptide conjugate generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub- unit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine- readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
The kits of this invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. A kit may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an engineered polypeptide as described herein. The container may further comprise a second pharmaceutically active agent.
Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container.
EXAMPLES
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.
Example 1: Proteolysis of the C-terminal heavy chain TG6 tag during CHO expression
Clipping of the last two amino acids (-GA) from an acyl donor glutamine-containing tag (e.g., TG6 tag (LLQGA (SEQ ID NO: l)) was observed when the TG6 tag was positioned at the C-terminus of an antibody's heavy chain (e.g., mAbl, which is an anti-Trop2 antibody as used in all of the examples described herein). Under tested expression conditions, 10-90% of the TG6 tag was found to be proteolyzed. This C-terminal clipping appears specific to CHO cells, as it was not observed during expression of Ab-TG6 in HEK293 cells. When the -GA was lost from the tag, no conjugation of a desired payload (e.g., drug or agent moiety) to the antibody was observed at the clipped TG6 tag (i.e., LLQ) at the C-terminus of the antibody.
A new set of the acyl donor glutamine-containing tags at the end of the C-terminus of an antibody (e.g., mAbl) was created to prevent the observed clipping. TG (transglutaminase) tags TG7-TG17 (SEQ ID NOS: 2-11; Table 1) were designed to contain proline to minimize the proteolysis. The newly designed tags were first expressed in HEK293 cells and tested for homogeneity and conjugatability by HIC (hydrophobic interaction chromatography) and mass spectrometry as described by Strop et al. Chem Biol, 20(2): 161-7 (2013), hereby incorporated by reference herein in its entirety. In contrast to TG6, TG tags (e.g., TG11 and TG12) that contained proline at the C-terminus to the reactive glutamine showed no conjugation to the linker and the desired payload (e.g., AcLys-VC-PABC-0101 (AcLys-VC-PABC is acetyl-lysine-valine- citrulline-p-aminobenzyloxycarbonyl, and 0101 is 2-methylalanyl-N-[(3R,4,S', 5)S)-3-methoxy-l- {(25)-2-[(lR,2R)-l-methoxy-2-methyl-3-oxo-3-{[(l^)-2-phenyl-l-(l,3-thiazol-2- yl)ethyl]amino}propyl]pyrrolidin-l -yl } -5-methyl- 1 -oxoheptan-4-yl]-N-methyl-L-valinamide)). The remaining TG tags were conjugated to high efficiency. Some C-terminal heterogeneity was observed for TG7, TG8, and TG12-15.
TG tags TG9, TG10, and TGI 7, which appeared homogeneous in HEK293, were expressed in CHO cells to test whether C-terminal clipping would occur. The expression of TG10 (finished in -LLQGP (SEQ ID NO:5)) in CHO cells showed an 11% clipping of -GP. Remaining two tags (TG9 and TGI 7) did not show any C-terminal clipping under the same condition in which around -33.6 % clipping was observed in the TG6 tag. Figures 1A-1D. Accordingly, these results demonstrate that TG9 and TGI 7 tags prevent proteolysis at the C- terminus of an antibody's heavy chain when expressed in CHO cells.
Table 1
Figure imgf000048_0001
Example 2: Proteolysis of the C-terminal light chain LCQ04 tag during CHO expression
Clipping of the last two amino acids (-GA) from the LCQ04 tag (GGLLQGA (SEQ ID NO: 12)) was also observed when the LCQ04 tag was positioned at the C-terminus of an antibody's light chain having K222R mutation (EU numbering scheme), although at much lesser degree than in the antibody's heavy chain. This C-terminal clipping appears specific to CHO cells, as it was not observed during HEK293 expression. Under tested expression conditions, about 5% of the LCQ04 tag was found to be proteolyzed.
The LCQ05 tag (GGLLQGPP (SEQ ID NO: 13)) was created having two prolines similar to the TGI 7 tag described above. The LCQ05 TG tag appeared homogeneous when expressed in both CHO and HEK293 cells and could be conjugated to the desired linker-payload (e.g., AcLys- VC-PABC-0101) at comparable levels as LCQ04. Figures 2A-2B. Accordingly, these results demonstrate that LCQ05 tag prevents proteolysis at the C-terminus of an antibody's light chain when expressed in CHO cells.
Example 3: Biophysical and efficacy comparison of TG6 and TG17 tags
The new acyl donor glutamine-containing tags were validated by comparing the biophysical characteristics of the unconjugated and conjugated TG6 tag and TGI 7 tag. More specifically, analytical size exclusion chromatography (SEC) showed similar low level of aggregates, and the conjugation efficiency was comparable under the same conditions. Figures 3A-3B. In SEC, about 15 ug of samples (antibody-TG6 and antibody-TG17, or antibody-TG6- AcLys-VC-PABC-0101 and antibody-TG6-AcLys-VC-PABC-0101) were injected in a TSKGEL® G3000SW (Tosoh Bioscience LLC, King of Prussia, PA) size exclusion chromatography column on an Agilent HP 1100 HPLC (Santa Clara, CA) and run at 0.5mL/min with the mobile phase (170mM KPi; 210mM KC1; 15% Isopropanol). The samples were also applied to the HIC (Hydrophobic Interaction Chromatography) column and showed similar DAR (drug antibody ratio) for the TG6 and TGI 7 tags as well as in mass spectrometry. Figures 4A-4B and 5A-5B. For HIC, 20 ug of antibody drug conjugate (e.g., antibody carrying TG6 or TG17 tag and conjugated to AcLys-VC-PABC-0101) in 0.75 M ammonium sulfate was loaded onto a TSKGEL® Butyl - PR column (Tosoh Bioscience, King of Prussia, PA) on an Agilent HP 1100 HPLC (Santa Clara, CA). The mobile phase buffer A was 1.5 M ammonium sulfate and 50 mM potassium phosphate at pH=7.0; buffer B was 50 mM potassium phosphate and 20% Isopropanol at pH=7.0. The run was performed at 0.8 mL/min with a 35 minutes linear gradient 0-100% B. For Mass spectrometry (MS), prior to Liquid Chromatography-Mass Spectrometry (LC/MS) analysis, antibody-drug conjugates were deglycosylated with PNGase F (NEB, cat#P0704L) under non-denaturing conditions at 37°C overnight. ADCs (500 ng) were loaded into a reverse phase column (Michrom-Bruker, Auburn, CA). LS/MS analysis was performed using Agilent 1100 series HPLC system coupled to an Orbitrap Velos Pro (Thermo Fisher Scientific, Waltham, MA) mass spectrometer. The resulting mass spectra were deconvoluted using ProMass software (Thermo Fisher Scientific). These results demonstrate that the new glutamine tags (e.g., TGI 7) have similar biophysical characteristics as the TG6 tag.
In vitro efficacy in BxPC3 and OVCAR3 cell lines
Both antibody conjugates (Antibody-TG6-AcLys-VC-PABC-0101 or Antibody-TG17- AcLys-VC-PABC-0101) were also tested for in vitro efficacy on BxPC3 and OVCAR3 cell lines for cytotoxicity, and the IC50 values obtained were comparable. See Table 2 and Figures 6A-6B. More specifically, BxPC3 and OVCAR3 cells at 2000 and 3000 cells/well, respectively, were diluted in 100 uL growth media (serum free DMEM, Cellgro Mediatech, Manassas, VA). The next day, 25 uL of five times of ADCs (in serum free DMEM) were added. CelltiterGlo assays (Promega, Madison, WI) were performed 4 days after treatment. Cells were added with 1 : 1 diluted reagent after media removal. The plates were read on MSpectraMax M5 plate reader (Molecular Devices, Downingtown, PA).
Table 2
Figure imgf000050_0001
These results demonstrate that Antibody-TG6-AcLys-VC-PABC-0101 and Antibody- TG17-AcLys-VC-PABC-0101 have similar in vitro efficacy in BxPC3 and OVCAR3 cell lines for cytotoxicity.
In vivo efficacy in Pan0123 xenograft model
In vivo efficacy studies of the antibody conjugates were performed with antibody- expressing Pancreatic Pan0123 PDX xenograft tumors. 2x2x2 mm3 tumor fragments were implanted subcutaneously into 5-8 weeks old SCID (Severe Combined Immunodeficient) mice until the tumor sizes reached at least 300 mm3. Control antibody conjugate and Trop-2 targeting antibody conjugates (Ab-TG6-AcLys-VC-PABC-0101 and Ab-TG17-AcLys-VC- PABC-0101) were given to the SCID mice at 1.5 mg/kg as single dose bolus injection through tail vein. All experimental animals were monitored for body weight changes weekly. Tumor volume was measured once a week by a Caliper device and calculated with the following formula: Tumor volume = (length x width2) / 2. The tumor volume and body weight were monitored up to 80 days after tumor implantation. These results demonstrate that antibody conjugate having the TGI 7 tag had similar activity in comparison to the antibody conjugate having the TG6 tag. Figures 7A-7B.
Pharmacokinetics
The PK (pharmacokinetics) was further tested by injecting female rats with a single dose of AB-TG6-AcLys-VC-PABC-0101 or AB-TG17-AcLys-VC-PABC-0101 (174-2) at 5 mg/kg.
The animals were 12 adult Sprague Dawley female rats at approximately 3 months of age. (Harlan, Livermore, CA). For PK and plasma stability experiments, compounds were dosed intravenously through the lateral tail vein, and samples were withdrawn from the contralateral tail vein at appropriate time points, except for a terminal bleed which was obtained via cardiac puncture on anesthetized adult rats. Samples from one group of rats were processed for serum while samples from a separate group of rats were processed for plasma. Both sample types were stored frozen at -80°C until analysis.
Serum was collected at 8 time points up to 14 days with 3 rats per group. ELISA (Enzyme-Linked Immunosorbent Assay) was performed to measure total antibody and antibody conjugate in plasma as described by Strop et al. Chem Biol. 20(2): 161-167 (2013). The results were similar for TG6 and TG17. Figure 8.
Example 4: Q295N mutant in the heavy chain of the mAb eliminates unwanted conjugation due to trace amounts of aglycosylated antibodies expressed during mAb expression
In addition to conjugation of the antibody and the desired payload at an engineered site, a small amount of aglycosylated antibody present in starting material led to antibody-drug conjugation at position Q295, resulting in approximately 1.3% of off-target conjugation. Such off-target conjugation can be eliminated by the Q295N mutant (EU numbering scheme), which in turn results in highly homogenous antibody-drug conjugates that are better than 99.8% site- specific.
More specifically, transglutaminase can recognize Q295 in aglycosylated IgGs, and conjugation can be achieved at this site. See MS/MS confirmation of conjugation of AmPEG6- MMAD to tryptic peptide EEQ*YNSTYR (SEQ ID NO: 15) in Figure 9A. When antibodies are expressed in the CHO or HEK293 expression system, most of the produced proteins are gycoyslated at position N297. The presence of glycans at position N297 prevents conjugation to Q295. However, there is typically a small fraction of aglycosylated antibodies that are also produced. It is this contaminating aglycosylated antibody fraction, which is suitable for conjugation at position Q295.
To estimate the amount of this off-target conjugation to the Q295 site, conjugated EEQ*YNSTYR (SEQ ID NO: 15) standard peptide was spiked into a fixed amount (2.5 μg) of unconjugated tryptic digests of mAbl HC (heavy chain), and a standard curve for quantification was generated. Using the precursor ion extracted chromatogram, it was estimated that 1.3% of the total injected Q295 peptide was conjugated with AmPEG6-MMAD (amino-polyethylene glycol-6 propionyl monomethyl auristatin D) in the mAbl HC molecule assuming 100% digestion efficiency. Figure 9B. A single point mutant Q295N was created to abolish the undesired conjugation. The peptide mass fingerprint of the Q295N mutant revealed only conjugation at the designed glutamine tag site for both the mAbl light chain and heavy chain molecules with no additional identified sites.
Although the disclosed teachings have been described with reference to various applications, methods, and compositions, it will be appreciated that various changes and modifications can be made without departing from the teachings herein and the claimed invention below. The foregoing examples are provided to better illustrate the disclosed teachings and are not intended to limit the scope of the teachings presented herein. While the present teachings have been described in terms of these exemplary embodiments, the skilled artisan will readily understand that numerous variations and modifications of these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the current teachings.
All references cited herein, including patents, patent applications, papers, text books, and the like, and the references cited therein, to the extent that they are not already, are hereby incorporated by reference in their entirety. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls.
The foregoing description and Examples detail certain specific embodiments of the invention and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the invention may be practiced in many ways and the invention should be construed in accordance with the appended claims and any equivalents thereof.

Claims

WHAT IS CLAIMED IS:
1. An engineered polypeptide conjugate comprising the formula: polypeptide-T-A, wherein T is an acyl donor glutamine-containing tag engineered at a specific site, wherein A is an amine donor agent, wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine-containing tag at a carboxyl terminus, an amino terminus, or at an another site in the polypeptide, and wherein the acyl donor glutamine-containing tag comprises an amino acid sequence GGLLQGPP (SEQ ID NO: 13).
2. An engineered Fc-containing polypeptide conjugate comprising the formula: (Fc- containing polypeptide-T-A), wherein T is an acyl donor glutamine-containing tag engineered at a specific site, wherein A is an amine donor agent, wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine-containing tag at a carboxyl terminus, an amino terminus, or at an another site in the Fc-containing polypeptide, wherein the acyl donor glutamine-containing tag comprises an amino acid sequence XXQX (SEQ ID NO:35), wherein X is any amino acid, and wherein the engineered Fc-containing polypeptide conjugate comprises an amino acid substitution from glutamine to asparagine at position 295 (Q295N; EU numbering scheme).
3. An engineered Fc-containing polypeptide conjugate comprising the formula: (Fc- containing polypeptide-T-A), wherein T is an acyl donor glutamine-containing tag engineered at a specific site, wherein A is an amine donor agent, wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine-containing tag at a carboxyl terminus, an amino terminus, or at an another site in the Fc-containing polypeptide, wherein the engineered Fc-containing polypeptide conjugate comprises an amino acid substitution from glutamine to asparagine at position 295 (Q295N; EU numbering scheme), and wherein the acyl donor glutamine-containing tag comprises an amino acid sequence selected from the group consisting of LLQGG (SEQ ID NO: 16), LLQG (SEQ ID NO: 17), LSLSQG (SEQ ID NO: 18), GGGLLQGG (SEQ ID NO: 19), GLLQG (SEQ ID NO:20), LLQ, GSPLAQSHGG (SEQ ID NO:21), GLLQGGG (SEQ ID NO:22), GLLQGG (SEQ ID NO:23), GLLQ (SEQ ID NO:24), LLQLLQGA (SEQ ID NO:25), LLQGA (SEQ ID NO:26), LLQYQGA (SEQ ID NO:27), LLQGSG (SEQ ID NO:28), LLQYQG (SEQ ID NO:29), LLQLLQG (SEQ ID NO:30), SLLQG (SEQ ID N0:31), LLQLQ (SEQ ID NO:32), LLQLLQ (SEQ ID NO:33), LLQGR (SEQ ID NO:34), LLQGPP (SEQ ID NO: 11), LLQGPA
(SEQ ID NO:4), GGLLQGPP (SEQ ID NO: 13), GGLLQGA (SEQ ID NO: 12), LLQGA (SEQ ID NO: l), LLQGPGK (SEQ ID NO:2), LLQGPG (SEQ ID NO:3), LLQGP
(SEQ ID NO: 5), LLQP
(SEQ ID NO: 6), LLQPGK
(SEQ ID NO: 7), LLQAPGK
(SEQ ID NO: 8), LLQGAPG (SEQ ID NO: 9), and LLQGAP (SEQ ID NO: 10).
The engineered polypeptide conjugate of claim 1 or the engineered Fc-containing polypeptide conjugate of claim 2 or 3, wherein the acyl donor glutamine-containing tag is not spatially adjacent to a reactive Lys in the polypeptide or the Fc-containing polypeptide.
The engineered polypeptide conjugate or the engineered Fc-containing polypeptide conjugate of claim 4, wherein the polypeptide or the Fc-containing polypeptide comprises an amino acid modification at the last amino acid position in the carboxyl terminus relative to a wild-type polypeptide at the same position.
The engineered polypeptide conjugate or the engineered Fc-containing polypeptide conjugate of claim 5, wherein the modification is an amino acid deletion.
The engineered polypeptide conjugate or the engineered Fc-containing polypeptide conjugate of claim 6, wherein the polypeptide conjugate comprises a full length antibody heavy chain and an antibody light chain.
The engineered polypeptide conjugate or the engineered Fc-containing polypeptide conjugate of claim 7, wherein the acyl donor glutamine-containing tag is located at the carboxyl terminus of a heavy chain, a light chain, or both the heavy chain and the light chain.
9. The engineered polypeptide conjugate or the engineered Fc-containing polypeptide conjugate of any one of claims 1-8, wherein the polypeptide comprises an antibody, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, a minibody, a diabody, or an antibody fragment.
10. The engineered polypeptide conjugate or the engineered Fc-containing polypeptide conjugate of claim 9, wherein the antibody is an IgG.
11. The engineered polypeptide conjugate or the engineered Fc-containing polypeptide conjugate of any one of claims 1-10, wherein the amine donor agent comprises the formula: X-Y-Z, wherein X is an amine donor unit; Y is a linker; and Z is an agent moiety.
12. The engineered polypeptide conjugate or the engineered Fc-containing polypeptide conjugate of claim 11, wherein the amine donor unit-linker (X-Y) is selected from the group consisting of Ac-Lys-Gly, aminocaproic acid, Ac-Lys-P-Ala, amino-PEG2-C2, amino-PEG3-C2, amino-PEG6-C2, Ac-Lys-Val-Cit-PABC, aminocaproyl-Val-Cit- PABC, [(3R,5R)-l-{3-[2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis- Val-Cit-PABC, [(3S,5S)-l-{3-[2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5- diyl]bis-Val-Cit-PABC, putrescine, 2-aminoethoxy-PEG6, and Ac-Lys-putrescine.
13. The engineered polypeptide conjugate or the engineered Fc-containing polypeptide conjugate of claim 11 or 12, wherein the agent moiety is a cytotoxic agent.
14. The engineered polypeptide conjugate or the engineered Fc-containing polypeptide conjugate of claim 13, wherein the cytotoxic agent is selected from the group consisting of anthracycline, an auri statin, a camptothecin, a combretastatin, a dolastatin, a duocarmycin, an enediyne, a geldanamycin, an indolino-benzodiazepine dimer, a maytansine, a puromycin, a pyrrolobenzodiazepine dimer, a taxane, a vinca alkaloid, a tubulysin, a hemiasterlin, a spliceostatin, a pladienolide, and stereoisomers, isosteres, analogs, or derivatives thereof.
15. The engineered polypeptide conjugate or the engineered Fc-containing polypeptide conjugate of claim 13, wherein the cytotoxic agent is MMAD (Monomethyl Auristatin D) or 0101 (2-methylalanyl-N-[(3R,4^ 55)-3-methoxy-l-{(25)-2-[(lR,2R)- 1 -methoxy-2-methyl-3 -oxo-3 - { [( 15)-2-phenyl- 1 -( 1 , 3 -thiazol-2- yl)ethyl]amino}propyl]pyrrolidin-l -yl } -5-methyl- 1 -oxoheptan-4-yl]-N-methyl-L- valinamide).
16. The engineered polypeptide conjugate or the engineered Fc-containing polypeptide conjugate of any one of claims 11-15, wherein the amine donor agent is selected from the group consisting of Alexa 488 cadaverine, 5-FITC cadaverine, Alexa 647 cadaverine, Alexa 350 cadaverine, 5-TAMRA cadaverine, 5-FAM cadaverine, SR101 cadaverine, 5,6-TAMRA cadaverine, 5-FAM lysine, Ac-LysGly-MMAD, amino- PEG3 -C2-MMAD, amino-PEG6-C2-MMAD, amino-PEG3-C2-amino-nonanoyl- MMAD, aminocaproyl-Val-Cit-PABC-MMAD, amino-PEG-C2-Val-Cit-PABC- MMAD, Ac-Lys-Val-Cit-PABC-MMAD, aminocaproyl-MMAD, Ac-Lys-p-Ala- MMAD, amino-PEG2-C2-MMAE, aminocaproyl-MMAE, amino-PEG3 -C2-MMAE, aminocaproyl-MMAF, aminocaproyl-Val-Cit-PABC-MMAE, amino-PEG-6-C2-Val- Cit-PABC-MMAE, Ac-Lys-Val-Cit-PABC-MMAE, aminocaproyl-Val-Cit-PABC- MMAF, amino-PEG-6-C2-Val-Cit-PABC-MMAF, Ac-Lys-Val-Cit-PABC-MMAF, Ac-Lys-Val-Cit-P ABC-0101, putrescinyl-geldanamycin, Ac-Lys-putrescinyl- geldanamycin, aminocaproyl-3377, amino-PEG6-C2-3377, aminocaproyl-0131, amino-PEG6-C2-0131, aminocaproyl-0121, amino-PEG6-C2-0121, [(3R,5R)-l-{3- [2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit-PABC-MMAD, [(3R,5R)-l-{3-[2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit- PABC-MMAE, and 2-aminoethoxy-PEG6-NODAGA.
17. The engineered polypeptide conjugate or the engineered Fc-containing polypeptide conjugate of claim 11, wherein the amine donor unit-linker (X-Y) is a branched unit and the agent moiety comprises at least about 2 agent moieties.
18. The engineered polypeptide conjugate or the engineered Fc-containing polypeptide conjugate of any one of claims 1 and 4-17, comprising an amino acid substitution from glutamine to asparagine at position 295 (Q295N; EU numbering scheme).
19. A pharmaceutical composition comprising the engineered polypeptide conjugate or the engineered Fc-containing polypeptide conjugate of any one of claims 1-18, and a pharmaceutically acceptable excipient.
20. A method of treating a cancer in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19.
21. A method of inhibiting tumor growth or progression in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19.
22. A method for preparing the engineered Fc-containing polypeptide conjugate of claim 2 or 3, comprising the steps of:
a) providing an engineered (Fc-containing polypeptide)-! molecule comprising the Fc-containing polypeptide and the acyl donor glutamine-containing tag;
b) contacting the amine donor agent with the engineered (Fc-containing polypeptide)- T molecule in the presence of a transglutaminase; and
c) allowing the engineered (Fc-containing polypeptide)-! to covalently link to the amine donor agent to form the engineered Fc-containing polypeptide conjugate.
23. A method for preparing an engineered polypeptide conjugate comprising the formula: polypeptide-!-A, wherein ! is an acyl donor glutamine-containing tag engineered at a specific site, wherein A is an amine donor agent, wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine-containing tag at a carboxyl terminus, an amino terminus, or at an another site in the polypeptide, and wherein the acyl donor glutamine-containing tag comprises an amino acid sequence LLQGPX, wherein X is A or P (SEQ ID NO: 14), or GGLLQGPP (SEQ ID NO: 13),
comprising the steps of:
a) providing an engineered polypeptide-! molecule comprising the polypeptide and the acyl donor glutamine-containing tag;
b) contacting the amine donor agent with the engineered polypeptide-! molecule in the presence of a transglutaminase; and c) allowing the engineered polypeptide-T to covalently link to the amine donor agent to form the engineered Fc-containing polypeptide conjugate.
24. The method of claim 22 or 23, wherein the engineered polypeptide conjugate or the engineered Fc-containing polypeptide conjugate has conjugation efficiency of at least about 51%.
25. The method of any one of claims 22-24, wherein the transglutaminase is a microbial, purified, or engineered transglutaminase.
26. The method of any one of claims 22-25, further comprising a purification step, wherein the engineered polypeptide conjugate or the engineered Fc-containing polypeptide conjugate is purified by a chromatography step.
27. The method of any one of claims 22-26, wherein the engineered polypeptide-T molecule or Fc-containing polypeptide-T is expressed in CHO cells.
28. The method of claim 23, wherein the polypeptide is Fc-containing or Fab-containing polypeptide.
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Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016030791A1 (en) 2014-08-28 2016-03-03 Pfizer Inc. Stability-modulating linkers for use with antibody drug conjugates
WO2016166629A1 (en) 2015-04-13 2016-10-20 Pfizer Inc. Therapeutic antibodies and their uses
WO2016170447A1 (en) 2015-04-24 2016-10-27 Rinat Neuroscience Corp. Recombinant microbial transglutaminases
WO2017125831A1 (en) 2016-01-21 2017-07-27 Pfizer Inc. Mono and bispecific antibodies for epidermal growth factor receptor variant iii and cd3 and their uses
WO2017134547A1 (en) 2016-02-01 2017-08-10 Pfizer Inc. Tubulysin analogs and methods for their preparation
WO2018025168A1 (en) 2016-08-03 2018-02-08 Pfizer Inc. Heteroaryl sulfone-based conjugation handles, methods for their preparation, and their use in synthesizing antibody drug conjugates
CN108602878A (en) * 2015-12-18 2018-09-28 卫材R&D管理有限公司 Immunoglobulin is conjugated in C-terminal lysine
WO2018220584A1 (en) 2017-06-02 2018-12-06 Pfizer Inc. Antibodies specific for flt3 and their uses
WO2019057772A1 (en) 2017-09-19 2019-03-28 Paul Scherrer Institut Transglutaminase conjugation method and linker
WO2019082020A1 (en) 2017-10-27 2019-05-02 Pfizer Inc. Antibodies and antibody-drug conjugates specific for cd123 and uses thereof
WO2019152705A1 (en) 2018-02-01 2019-08-08 Pfizer Inc. Antibodies specific for cd70 and their uses
WO2019224715A1 (en) 2018-05-23 2019-11-28 Pfizer Inc. Antibodies specific for cd3 and uses thereof
WO2019224716A2 (en) 2018-05-23 2019-11-28 Pfizer Inc. Antibodies specific for gucy2c and uses thereof
WO2020128893A1 (en) 2018-12-21 2020-06-25 Pfizer Inc. Combination treatments of cancer comprising a tlr agonist
WO2020188061A1 (en) 2019-03-19 2020-09-24 Paul Scherrer Institut Transglutaminase conjugation method with a glycine based linker
US10973920B2 (en) 2014-06-30 2021-04-13 Glykos Finland Oy Saccharide derivative of a toxic payload and antibody conjugates thereof
RU2762939C2 (en) * 2016-06-10 2021-12-24 Эйсай Ар Энд Ди Менеджмент Ко., Лтд. Immunoglobulins conjugated with lysine
WO2022013775A1 (en) 2020-07-17 2022-01-20 Pfizer Inc. Therapeutic antibodies and their uses
WO2022058594A1 (en) 2020-09-18 2022-03-24 Araris Biotech Ag Transglutaminase conjugation method with amino acid-based linkers
WO2022084560A1 (en) 2020-10-25 2022-04-28 Araris Biotech Ag Means and methods for producing antibody-linker conjugates
US11396551B2 (en) 2018-02-01 2022-07-26 Pfizer Inc. Chimeric antigen receptors targeting CD70
WO2023072934A1 (en) 2021-10-25 2023-05-04 Araris Biotech Ag Methods for producing antibody-linker conjugates
WO2023161291A1 (en) 2022-02-22 2023-08-31 Araris Biotech Ag Peptide linkers comprising two or more payloads
WO2023166418A2 (en) 2022-03-03 2023-09-07 Pfizer Inc. Multispecific antibodies and uses thereof

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2930801C (en) * 2010-11-05 2019-05-28 Rinat Neuroscience Corporation Engineered polypeptide conjugates and methods for making thereof using transglutaminase
JP6521959B2 (en) * 2013-07-31 2019-05-29 ライナット ニューロサイエンス コーポレイション Engineered polypeptide conjugates
BR112016024370A2 (en) 2014-04-25 2017-10-10 Pfizer high drug loading antibody-drug conjugates
EP3419670A2 (en) 2016-02-26 2019-01-02 Regeneron Pharmaceuticals, Inc. Optimized transglutaminase site-specific antibody conjugation
KR20200088402A (en) * 2017-11-14 2020-07-22 데비오팜 리서치 ＆ 매뉴팩처링 에스아 Ligand-drug-conjugate as substrate for selective cleavage by cathepsin B exopeptidase activity
AU2019386945A1 (en) 2018-11-30 2021-07-22 Bristol-Myers Squibb Company Antibody comprising a glutamine-containing light chain C-terminal extension, conjugates thereof, and methods and uses
WO2020123425A2 (en) 2018-12-12 2020-06-18 Bristol-Myers Squibb Company Antibodies modified for transglutaminase conjugation, conjugates thereof, and methods and uses
WO2023173132A1 (en) 2022-03-11 2023-09-14 Regeneron Pharmaceuticals, Inc. Anti-glp1r antibody-drug conjugates comprising glp1 peptidomimetics and uses thereof

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR901464A (en) 1943-01-23 1945-07-27 Boehringer & Soehne Gmbh Process for obtaining vanillin
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US5500362A (en) 1987-01-08 1996-03-19 Xoma Corporation Chimeric antibody with specificity to human B cell surface antigen
US5663149A (en) 1994-12-13 1997-09-02 Arizona Board Of Regents Acting On Behalf Of Arizona State University Human cancer inhibitory pentapeptide heterocyclic and halophenyl amides
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
WO1998052976A1 (en) 1997-05-21 1998-11-26 Biovation Limited Method for the production of non-immunogenic proteins
WO2000034317A2 (en) 1998-12-08 2000-06-15 Biovation Limited Method for reducing immunogenicity of proteins
US6737056B1 (en) 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
WO2012059882A2 (en) 2010-11-05 2012-05-10 Rinat Neuroscience Corporation Engineered polypeptide conjugates and methods for making thereof using transglutaminase
US20130129753A1 (en) 2011-11-17 2013-05-23 Pfizer Inc. Cytotoxic peptides and antibody drug conjugates thereof

Family Cites Families (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2155762C (en) 1994-08-23 2000-04-25 Haruya Sato Protein modification method
US6919076B1 (en) 1998-01-20 2005-07-19 Pericor Science, Inc. Conjugates of agents and transglutaminase substrate linking molecules
WO1999051625A2 (en) * 1998-04-02 1999-10-14 Rigel Pharmaceuticals, Inc. Peptides causing formation of compact structures
EP1121425B1 (en) * 1998-10-13 2005-06-29 The University Of Georgia Research Foundation, Inc. Stabilized bioactive peptides and methods of identification, synthesis and use
JP2003527561A (en) 1999-01-22 2003-09-16 スミスクライン・ビーチャム・コーポレイション Methods for site-specific labeling of proteins and uses therefor
EP1562630A4 (en) 2002-10-31 2009-02-18 Univ Northwestern Injectible bioadhesive polymeric hydrogels as well as related methods of enzymatic preparation
US6995245B2 (en) 2003-05-30 2006-02-07 Centocor, Inc. Formation of novel erythropoietin conjugates using transglutaminase
WO2004106939A2 (en) 2003-06-03 2004-12-09 Paul Scherrer Institut Method for the linkage of bifunctional chelating agents and (radioactive) transition metal complexes to proteins and peptides
WO2005082023A2 (en) 2004-02-23 2005-09-09 Genentech, Inc. Heterocyclic self-immolative linkers and conjugates
US20060019258A1 (en) 2004-07-20 2006-01-26 Illumina, Inc. Methods and compositions for detection of small interfering RNA and micro-RNA
AU2005286607B2 (en) 2004-09-23 2011-01-27 Genentech, Inc. Cysteine engineered antibodies and conjugates
CA2623652C (en) 2005-09-26 2013-11-26 Medarex, Inc. Antibody-drug conjugates and methods of use
US9127130B2 (en) 2006-08-11 2015-09-08 Starpharma Pty Ltd. Polylysine dendrimer contrast agent
US20090181037A1 (en) 2007-11-02 2009-07-16 George Heavner Semi-Synthetic GLP-1 Peptide-FC Fusion Constructs, Methods and Uses
WO2010002042A1 (en) 2008-07-04 2010-01-07 国立大学法人 九州大学 Enzyme substrate for labeling of protein
JP5563572B2 (en) 2008-07-23 2014-07-30 ハンミ サイエンス カンパニー リミテッド Bioactive polypeptide drug conjugates using non-peptidic polymers with tri-terminal functional groups
EP2350013A4 (en) 2008-10-13 2012-10-17 San Diego State University Res Foundation Compositions for labeling and identifying autophagosomes and methods for making and using them
US10865233B2 (en) 2008-12-18 2020-12-15 Dana-Farber Cancer Institute, Inc. NKG2D-fc for immunotherapy
RU2540012C2 (en) * 2009-07-29 2015-01-27 Дайити Санкио Компани, Лимитед Motilin-like peptide compound having imparted transmucosal absorbing ability
SG181130A1 (en) 2009-12-06 2012-07-30 Biogen Idec Hemophilia Inc Factor viii-fc chimeric and hybrid polypeptides, and methods of use thereof
RU2519031C1 (en) 2010-01-19 2014-06-10 Ханми Сайенс Ко., Лтд. Liquid formulations for long-acting conjugate of g-csf
MX2012008453A (en) 2010-01-19 2012-11-21 Hanmi Science Co Ltd Liquid formulations for long-acting erythropoietin conjugate.
AR081755A1 (en) 2010-04-02 2012-10-17 Hanmi Holdings Co Ltd FORMULATION OF PROLONGED ACTION OF THE FOLICULES STIMULATING HORMONE WHERE AN IMMUNOGLOBULIN FRAGMENT, PREPARATION METHOD AND METHOD TO TREAT A SUBJECT SUFFERING A REPRODUCTIVE DISORDER ARE USED
MX2014005728A (en) 2011-11-11 2014-05-30 Rinat Neuroscience Corp Antibodies specific for trop-2 and their uses.
EP2793947B1 (en) * 2011-12-23 2021-02-03 Innate Pharma Enzymatic conjugation of polypeptides
JP6521959B2 (en) * 2013-07-31 2019-05-29 ライナット ニューロサイエンス コーポレイション Engineered polypeptide conjugates
CN106211774B (en) * 2013-08-02 2020-11-06 辉瑞公司 anti-CXCR 4 antibodies and antibody-drug conjugates

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR901464A (en) 1943-01-23 1945-07-27 Boehringer & Soehne Gmbh Process for obtaining vanillin
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US5500362A (en) 1987-01-08 1996-03-19 Xoma Corporation Chimeric antibody with specificity to human B cell surface antigen
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
US5663149A (en) 1994-12-13 1997-09-02 Arizona Board Of Regents Acting On Behalf Of Arizona State University Human cancer inhibitory pentapeptide heterocyclic and halophenyl amides
WO1998052976A1 (en) 1997-05-21 1998-11-26 Biovation Limited Method for the production of non-immunogenic proteins
WO2000034317A2 (en) 1998-12-08 2000-06-15 Biovation Limited Method for reducing immunogenicity of proteins
US6737056B1 (en) 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
WO2012059882A2 (en) 2010-11-05 2012-05-10 Rinat Neuroscience Corporation Engineered polypeptide conjugates and methods for making thereof using transglutaminase
US20130129753A1 (en) 2011-11-17 2013-05-23 Pfizer Inc. Cytotoxic peptides and antibody drug conjugates thereof

Non-Patent Citations (41)

* Cited by examiner, † Cited by third party
Title
"Remington's Pharmaceutical Sciences", 2012, MACK PUBLISHING COMPANY
AUSUBEL ET AL.: "Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology", 2002, WILEY, JOHN & SONS, INC.
BLOOM ET AL., PROTEIN SCIENCE, vol. 6, 1997, pages 407 - 415
BOGER; JOHNSON, PNAS, vol. 92, 1995, pages 3642 - 3649
CAPEL ET AL., IMMUNOMETHODS, vol. 4, 1994, pages 25 - 34
CLYNES ET AL., PNAS (USA), vol. 95, 1998, pages 652 - 656
COLIGAN ET AL.: "Short Protocols in Protein Science", 2003, WILEY, JOHN & SONS, INC.
DE HAAS ET AL., J LAB. CLIN. MED, vol. 126, 1995, pages 330 - 41
DORONIAN ET AL., BIOCONJ. CHEM., vol. 19, 2008, pages 1960 - 1963
EDELMAN ET AL., PROC. NATL. ACAD SCI. USA, vol. 63, no. 1, 1969, pages 78 - 85
GAZZANO-SANTORO ET AL., J IMMUNOL. METHODS, vol. 202, 1996, pages 163
GENTLE ET AL., BIOCONJ. CHEM., vol. 15, 2004, pages 658 - 663
GOMEZ ET AL., BIOTECHNOLOGY AND BIOENGINEERING, vol. 105, no. 4, 2009, pages 748 - 760
GUYER ET AL., J. IMMUNOL., vol. 117, 1976, pages 587
HARLOW; LANE: "Using Antibodies: A Laboratory Manual", 1998, COLD SPRING HARBOR LABORATORY PRESS
HARRIS, BIOCHEM. SOC. TRANSACTIONS, vol. 23, 1995, pages 1035 - 1038
HUMPHREYS ET AL., J IMMUNOL. METHODS, vol. 209, 1997, pages 193 - 202
HURLE; GROSS, CURR. OP. BIOTECH., vol. 5, 1994, pages 428 - 433
JANEWAY ET AL.: "ImmunoBiology: the immune system in health and disease", 1999, ELSEVIER SCIENCE LTD.
JEGER ET AL., ANGEWANDTE CHEMIE, vol. 49, no. 51, 2010, pages 9995 - 9997
JONES ET AL., NATURE, vol. 321, 1986, pages 522 - 525
JOSTEN ET AL., J OF IMMUNOLOGICAL METHODS, vol. 240, 2000, pages 47 - 54
JUNUTULA ET AL., NATURE BIOTECHNOLOGY, vol. 26, 2008, pages 925 - 932
KIM ET AL., J IMMUNOL., vol. 24, 1994, pages 249
KOSTELNY ET AL., J IMMUNOL., vol. 148, 1992, pages 1547 - 1553
MINDT ET AL., BIOCONJ. CHEM, vol. 19, 2008, pages 271 - 27
MORRISON ET AL., PROC. NATL. ACAD SCI. USA, vol. 81, 1984, pages 6851 - 6855
NIMMERJAHN ET AL., IMMUNITY, vol. 23, 2005, pages 2 - 4
PETTIT ET AL., ANTIMICROB. AGENTS CHEMOTHER., vol. 42, 1998, pages 2961 - 2965
PRESTA, CURR. OP. STRUCT. BIOL., vol. 2, 1992, pages 593 - 596
RAVETCH; KINET, ANN. REV. IMMUNOL., vol. 9, 1991, pages 457 - 92
REMILLARD ET AL., SCIENCE, vol. 189, 1975, pages 1002 - 1005
RIECHMANN ET AL., NATURE, vol. 332, 1988, pages 323 - 329
SAMBROOK J.; RUSSELL D: "Molecular Cloning: A Laboratory Manual", 2000, COLD SPRING HARBOR LABORATORY PRESS
SONGSIVILAI; LACHMANN, CLIN. EXP. IMMUNOL., vol. 79, 1990, pages 315 - 321
STROP ET AL., CHEM BIOL., vol. 20, no. 2, 2013, pages 161 - 167
STROP ET AL., CHEM BIOL., vol. 20, no. 2, 2013, pages 161 - 7
TAKAZAWA ET AL., BIOTECHNOLOGY AND BIOENGINEERING, vol. 86, no. 4, 2004, pages 399 - 404
TANAKA ET AL., FEBS LETTERS, vol. 579, 2005, pages 2092 - 2096
VASWANI; HAMILTON, ANN. ALLERGY, ASTHMA & IMMUNOL., vol. 1, 1998, pages 105 - 115
WANG ET AL., PROTEIN SCI., vol. 14, 2005, pages 2436 - 2446

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US10973920B2 (en) 2014-06-30 2021-04-13 Glykos Finland Oy Saccharide derivative of a toxic payload and antibody conjugates thereof
AU2015308179B2 (en) * 2014-08-28 2020-07-02 Pfizer Inc., Stability-modulating linkers for use with antibody drug conjugates
US10967068B2 (en) 2014-08-28 2021-04-06 Pfizer Inc. Stability-modulating linkers for use with antibody drug conjugates
WO2016030791A1 (en) 2014-08-28 2016-03-03 Pfizer Inc. Stability-modulating linkers for use with antibody drug conjugates
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WO2016170447A1 (en) 2015-04-24 2016-10-27 Rinat Neuroscience Corp. Recombinant microbial transglutaminases
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US20200009263A1 (en) * 2015-12-18 2020-01-09 Eisai R&D Management Co., Ltd. C-terminal lysine conjugated immunoglobulins
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WO2017125831A1 (en) 2016-01-21 2017-07-27 Pfizer Inc. Mono and bispecific antibodies for epidermal growth factor receptor variant iii and cd3 and their uses
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US11753669B2 (en) 2016-06-10 2023-09-12 Eisai R&D Management Co., Ltd. Lysine conjugated immunoglobulins
US11712480B2 (en) 2016-08-03 2023-08-01 Pfizer Inc. Heteroaryl sulfone-based conjugation handles, methods for their preparation, and their use in synthesizing antibody drug conjugates
WO2018025168A1 (en) 2016-08-03 2018-02-08 Pfizer Inc. Heteroaryl sulfone-based conjugation handles, methods for their preparation, and their use in synthesizing antibody drug conjugates
WO2018220584A1 (en) 2017-06-02 2018-12-06 Pfizer Inc. Antibodies specific for flt3 and their uses
WO2019057772A1 (en) 2017-09-19 2019-03-28 Paul Scherrer Institut Transglutaminase conjugation method and linker
WO2019082020A1 (en) 2017-10-27 2019-05-02 Pfizer Inc. Antibodies and antibody-drug conjugates specific for cd123 and uses thereof
US11613581B2 (en) 2017-10-27 2023-03-28 Pfizer, Inc. Antibodies and antibody-drug conjugates specific for CD123 and uses thereof
WO2019152705A1 (en) 2018-02-01 2019-08-08 Pfizer Inc. Antibodies specific for cd70 and their uses
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US11396551B2 (en) 2018-02-01 2022-07-26 Pfizer Inc. Chimeric antigen receptors targeting CD70
US11987634B2 (en) 2018-02-01 2024-05-21 Pfizer Inc. Antibodies specific for CD70 and their uses
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WO2019224716A2 (en) 2018-05-23 2019-11-28 Pfizer Inc. Antibodies specific for gucy2c and uses thereof
WO2020128893A1 (en) 2018-12-21 2020-06-25 Pfizer Inc. Combination treatments of cancer comprising a tlr agonist
WO2020188061A1 (en) 2019-03-19 2020-09-24 Paul Scherrer Institut Transglutaminase conjugation method with a glycine based linker
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