WO2017025179A1 - A transglutamine tag for efficient site-specific bioconjugation - Google Patents
A transglutamine tag for efficient site-specific bioconjugation Download PDFInfo
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- WO2017025179A1 WO2017025179A1 PCT/EP2016/001344 EP2016001344W WO2017025179A1 WO 2017025179 A1 WO2017025179 A1 WO 2017025179A1 EP 2016001344 W EP2016001344 W EP 2016001344W WO 2017025179 A1 WO2017025179 A1 WO 2017025179A1
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- YBBRCQOCSYXUOC-UHFFFAOYSA-N sulfuryl dichloride Chemical class ClS(Cl)(=O)=O YBBRCQOCSYXUOC-UHFFFAOYSA-N 0.000 description 1
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- FQZYTYWMLGAPFJ-OQKDUQJOSA-N tamoxifen citrate Chemical compound [H+].[H+].[H+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.C=1C=CC=CC=1C(/CC)=C(C=1C=CC(OCCN(C)C)=CC=1)/C1=CC=CC=C1 FQZYTYWMLGAPFJ-OQKDUQJOSA-N 0.000 description 1
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- JGVWCANSWKRBCS-UHFFFAOYSA-N tetramethylrhodamine thiocyanate Chemical compound [Cl-].C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=C(SC#N)C=C1C(O)=O JGVWCANSWKRBCS-UHFFFAOYSA-N 0.000 description 1
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- UCFGDBYHRUNTLO-QHCPKHFHSA-N topotecan Chemical compound C1=C(O)C(CN(C)C)=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 UCFGDBYHRUNTLO-QHCPKHFHSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/54—F(ab')2
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
Definitions
- the present invention pertains to the field of bioconjugation, in particular to the field of microbial transglutaminase-mediated bioconjugation and its use for the manufacture of bioconjugates, such as e.g. antibody-drug conjugates.
- Protein modification with synthetic or naturally occuring molecules is a valuable technique to study protein function or to add additional properties to a protein.
- Protein modification or bioconjugation is typically done by either chemical linkage of a further molecule to the protein of interest or enzymatically.
- Cysteine residues can be alkylated by reacting them with a-haloketones or Michael acceptors, such as maleimide derivates.
- Michael acceptors such as maleimide derivates.
- the modification of lysine residues is the oldest and and most straightforward method for labeling proteins via the primary lysine amino groups.
- the ⁇ -amino group of lysine within the protein of interest can be readily reacted with activated esters, sulfonyl chlorides, isocyanates and isothiocyanates to result in the corresponding amides, sulfonamides, ureas and thioureas (see e.g. Takaoka et al., Angew. Chem. Int. Ed. 2013, 52, 4088 - 4106). Although very efficient for bioconjugation these chemical approaches inevitably result in heterogenous mixtures. This effect becomes more pronounced in smaller molecules with fewer lysines, e.g. in antibody fragments lacking an Fc region compared to IgG type molecules.
- bioconjugation examples include the conjugation of fluorescent proteins, dyes, or the tethering with functional molecules, e.g. PEGs, porphyrins, peptides, peptide nucleic acids, and drugs (Takaoka et al., Angew. Chem. Int. Ed. 2013, 52, 4088 - 4106).
- functional molecules e.g. PEGs, porphyrins, peptides, peptide nucleic acids, and drugs (Takaoka et al., Angew. Chem. Int. Ed. 2013, 52, 4088 - 4106).
- bioconjugates may involve varying levels of difficulty depending on the desired outcome.
- bioconjugation can range from the simple and non-specific attachment of polyethylene glycol (PEG) to serum albumin (Abuchowski et al. J. of Biological Chemistry (1977) Vol. 252 (11 ):3578-3581 ) to the very demanding preparation of homogenous antibody drug bioconjugates.
- PEG polyethylene glycol
- WO 2014/001325 A1 discloses the use of sortase A for site- specific bioconjugation to Fc regions of an antibody.
- This thioacyl intermediate subsequently then can reacts with an amino-terminal glycine acting as a nucleophile.
- SrtA accepts N-terminal (oligo)glycine as a nucleophiles, creating a new peptide bond between two molecules.
- SrtA functions at physiological conditions and has been used for bioconjugation reactions to label proteins with e.g. biotin, or to functionalize a HER2-specific recombinant Fab with the plant cytotoxin gelonin (see e.g. Popp et al. (2011) Angew Chemie Int. Ed. 50: 5024-5032; Kornberger et al (2014) mAbs 6 (2): 354-366).
- target proteins are labeled carboxyterminally with the LPXTG motif followd by a purificaton tag such that the SrtA-mediated transpeptidation removes the purification tag and generates the labeled protein.
- TGases transglutaminases
- mTGases microbial transglutaminases
- TGases are a large family of enzymes detected in several organisms including mammals, invertebrates, plants and microorganisms.
- Transglutaminases are protein-glutamine ⁇ - glutamyltransferases (E.C. 2.3.2.13), which typically catalyze pH-dependent transamidation of glutamine residues to lysine residues.
- Most TGases appear to be promiscuous with respect to the lysine substrate and may even accept 5-amino-pentyl groups as lysine surrogates to serve as the amine donor.
- Transglutaminases are widely distributed in most tissues and body fluids and are thought to be involved in a variety of physiological functions.
- One example for a transglutaminase- mediated reaction is the termination of bleeding during blood coagulation by factor Xllla, which is an activated form of plasma transglutaminase which catalyzes the crosslinking between fibrin molecules (see e.g. Griffin et al. Biochem. J. (2002) 368, 377-396).
- the biological function of bacterial transglutaminases however, remains largely unknown. It has been speculated that the mTGase of Streptomyces mobaraensis cross-links inhibitory proteins during development of aerial hyphae and spores.
- the transglutaminase from S. mobaraensis exhibits no significant homology to mammalian TGases and has a unique structure compared to human TG2 being the best characterized member of the human TGase family.
- mTG2 of S. mobaraensis is calcium-independent and has only a molecular weight of 37.9 kDa, which is only about half of the molecular weight of factor Xlll-like TGases.
- mTG2 is also characterized by a higher reaction rate than its mammalian orthologue.
- mTG2 Another aspect that has contributed to the widespread use of mTG2 is the fact that the enzyme can be easily produced in larger quantities as a recombinant protein which in conjunction with its biochemical properties has led to a wide use of mTG2 also in food industry (see e.g. Sommer at al. (2011) Protein Expression and Purification 77:9-19; Marx et al (2008) Enzyme and Microbial technology 42:568-575). Random mutagenesis of mTG2 has also been employed to increase the thermodynamic stability of the enzyme (see e.g. Journal of Biotechnology 136 (2008) 156- 162), Noteably, all mutations that have resulted in an increased stability of mTG2 at elevated temperatures are located within the amino-terminal region of the enzyme.
- Microbial Transglutaminase mTG2 has also been used for site-specific and stoichiometric PEGylation of proteins such as a-lactalbumin, human growth hormone and interleukin-2 or for site-specific and stoichiometric modification of antibodies (see e.g. Jeger et al. Angew. Chem Int. Ed. 2010, 49, 9995-9997; Fontana et al. Adv Drug Deliv Rev. 2008 Jan 3;60(1): 13-28). Overall, the number of reactive glutamines was found to be low in comparision to the total number of surface-exposed glutamines.
- mTG2 when used to catalyze transglutamination on native, fully glycosylated monoclonal antibodies, was found to not recognize any glutamine residues in the respective monoclonal antibody as substrate. Only de- or aglycosylated antibodies were found to be recognized as substrates by mTG2. Interestingly, only one glutamine residue was found to serve as a acyl- glutamine substrate in the de- or aglycosylated antibodies (Q295). This strongly limits the use of mTG2 for site-specific bioconjugation of antibodies for the generation of antibody-drug conjugates, in particular for use in humans.
- hGH human growth hormone
- two glutamine residues were identified as major conjugation sites, namely Q40 and Q141. Mutagenesis of mTG resulted in an mTG variant with increased selectivity toward Q141.
- a change in the solvent which presumably results in a change in the secondary structure surrounding the reactive glutamine can be used to limit the conjugation to a single glutamine residue, and therefore increase selectivity.
- the preferred substrate sequence of mTG was also assessed using a phage-displayed peptide library utilizing a much larger library of about 10 11 linear peptides.
- Most identified clones contained an aromatic amino acid (W, F, or Y) at the -5 to -3 positions, an arginine or hydrophobic residue at the +1 or +2 positions, and hydrophobic residues at the -2 and -1 positions (see e.g. Sugimura et al., Archives of Biochemistry and Biophysics 477 (2008) 379- 383).
- ADCs antibody-drug conjugates
- the antibody specifically directs the uptake and release of a highly potent toxin to a cancer cell, a technology which has attracted much attention in recent years as a novel and effective new treatment option for cancer patients.
- ADCs which have received market approval include Kadcyla ® (trastuzumab Emtansin) and Adcetris ® (brentuximab vedotin) for the treatment of metastatic breast cancer and Hodkin lymphoma.
- ADCs can negatively impact drug efficacy, in particular if significant fractions of the material are insufficiently conjugated: Insufficiently conjugated antibodies will compete with ADCs for target binding resulting in a reduced efficacy of the properly conjugated ADCs. Increased drug loading to antibodies can also negatively impact the efficacy of the ADC. Data have demonstrated that ADCs with high drug:antibody ratios are cleared more rapidly from the blood stream resulting in reduced efficacy (see e.g. Sanderson et al. Clin. Cancer Res. 2005; 11 :843-52).
- WO 2013/092983 discloses methods of functionalization of immunoglobulins with drugs employing TGase. This approach, describes the method to obtain new site by directed mutagenesis to generate a Q-donor inside the heavy chain of the antibody. This empiric m ethod needs to test high number of mutants as the rules which govern selection by TGases of glutamine residues for modification are still largely unknown. Consequently, using this method to develop new antibodies bearing Q-donor is time consuming and cost intensive.
- WO 2012/059882 further discloses specific engineered polypeptide conjugates and methods of making such conjugates using transglutaminases.
- WO 2015/097267 discloses peptide sequences which selectively react as glutamine donors with transglutaminase, such as mTG2 of S. mobaraensis which may be utilized for mTG2- mediated conjugation reactions.
- acyl glutamine-containing donor sequencederived from the dispase autolysis inducing protein of Streptomyces mobaraensis strain 40847 comprising at least the amino acid sequence TYFQAYG can be efficiently used in mTG2-mediated transglutamination reactions.
- the present invention provides in a first embodiment a protein comprising at least one acyl glutamine-containing amino donor sequence, which is covalently linked via a ⁇ - glutamyl-amide bond to an amino donor-comprising substrate, wherein the at least one acyl glutamine-containing amino acid donor sequence comprises the amino acid sequence according to SEQ ID NO: 1 (SEQ ID NO: 1 (TYFQAYG)).
- the amino donor-comprising substrate according to the present invention comprises at least an ⁇ -amino group, or at least one tripeptide having the sequence of GGG with a primary aminoterminal amino group.
- the amino donor-comprising substrate according to the invention is a lysine residue, a lysine derivative, lysine drug-conjugate, a polypeptide comprising at least one lysine residue.
- amino donor-comprising substrate according to any one of the above embodiments of the invention is covalently bound to a further molecule.
- the tripeptide GGG of the amino donor-comprising substrate is covalently bound via its carboxy terminus to a further molecule.
- the further moelcule according to the invention is one of a dye, radioisotope, drug, ribozyme, nanobody, enzyme, or linker.
- the futher molecule according to the invention is a linker, which is cleavable or non-cleavable.
- the linker according to the invention is coupled or covalently bound to a dye, radioisotope, or cytotoxin.
- the protein of the invention comprises an acyl glutamine-containing amino acid donor sequence, which [7] 2016/001344 comprises an amino acid sequence according to SEQ ID NO: 2 (X1X2 X3 TYFQAYG X4X5X 6 ), wherein
- X1 is a hydrophobic amino acid
- X2 is a negatively charged amino acid
- X3 is C or N
- X4 is C or N
- X5 is one of an amino acid with a polar, uncharged side chains
- X6 is a negatively charged amino acid.
- acyl glutamine-containing amino acid donor sequence of the invention according to any one of the above embodiments comprises an amino acid sequence according to SEQ ID NO: 2, wherein
- X1 is any one of A, V, I, L, M or G,
- X2 is one of D or E
- X4 is C or N
- X5 is S, T, or N
- X6 is one of D, or E.
- acyl glutamine-containing amino acid donor sequence of the invention comprises an amino acid sequence according to SEQ ID NO:2, wherein
- X1 is any one of A, V, I, L, M or G,
- X2 is one of D or E
- X5 is S, T, or N
- X6 is one of D, or E.
- acyl glutamine-containing amino acid donor sequence of the invention comprises the amino acid sequence according to SEQ ID NO: 2, wherein
- X1 is any one of A, V, I, L, M or G,
- X5 is T
- acyl glutamine-containing amino acid donor sequence of the invention comprises an amino acid sequence according to SEQ ID NO: 90.
- the protein according to the invention is one of an antibody, antigen-binding antibody fragment, Fc domain, enzyme, a non-immunoglobulin scaffold.
- the protein according to the invention is a monoclonal antibody, bivalent antibody, VHH, Fab, F(ab) 2> or scFv.
- the protein according to the invention is a human or humanized monoclonal antibody or fragment thereof.
- the present invention provides for a method of covalently coupling an amino donor comprising substrate of the invention to an acyl glutamine-containing amino acid donor comprising a polypeptide sequence according to SEQ ID NO:2, whereby the method comprises the step of:
- the acyl glutamine-containing amino acid donor of the inventive method is an antibody, antigen-binding antibody fragment, enzyme, or a non-immunoglobulin scaffold.
- the amino donor-comprising substrate of the inventive method is coupled or covalently bound to a dye, drug, ribozyme, nanobody, enzyme, or linker.
- the linker is cleavable or non-cleavable and is coupled to a dye, radioisotope, or cytotoxin.
- the present invention provides for a protein obtainable by the inventive method as disclosed herein.
- the present invention provides for the use of a polypeptide sequence according to any one of SEQ ID NO: 2 in the inventive method according to any one of the embodiments as disclosed herein.
- the present invention provides for a protein comprising the polypeptide comprising the amino acid sequence according to SEQ ID NO:2.
- the present invention provides for a polypeptide according to SEQ ID NO:2. In one embodiment the present invention provides for a polynucleotide encoding a polypeptide sequence according to SEQ ID NO: 2 of the invention.
- the present invention provides for a vector comprising the polynucleotide encoding the polypeptide sequence according to SEQ ID NO:2.
- the present invention provides a host cell which comprises the polynucleotide of the invention or a vector according to the invention as disclosed herein.
- the present invention provides an antibody or antigen-binding fragment thereof, bivalent antibody, or VHH antibody according to the invention which comprises at least one amino acid sequence according to SEQ ID NO:2.
- the antibody or antigen-binding fragment thereof, bivalent antibody, or VHH antibody of the invention is humanized or human.
- the antibody or antigen-binding fragment thereof according to to the invention as disclosed above is a humanized or human monoclonal antibody.
- the antibody of the invention as disclosed above is an lgG1 , lgG2, lgG3, lgG4, or IgM type antibody.
- inventive antibody as disclosed above is covalently coupled to at least one linker as disclosed above according to the inventive method as disclosed above;
- the at least one linker of the monoclonal antibody of the invention is further coupled to a dye, radioisotope, or cytotoxin.
- the inventive monoclonal antibody as disclosed herein specifically binds to cancer cell surface antigens.
- the monoclonal antibody of the invention as disclosed above is the monoclonal antibody c225 (cetuximab).
- the antibody of the invention as disclosed herein is for use in the treatment of cancer
- the inventive antibody as disclosed above is for use in the treatment of breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, preferably colorectal cancer, metastatic (mCRC), non-resctable liver metastases, Squamous Cell Carcinoma of the Head and Neck, Non-Small Cell Lung Cancer (NSCLC), Head and Neck Squamous Cell Carcinoma (HNSCC).
- mCRC metastatic
- NSCLC Non-Small Cell Lung Cancer
- HNSCC Head and Neck Squamous Cell Carcinoma
- the present invention provides for a composition comprising the inventive antibody and at least one further pharmaceutically active ingredient.
- the present invention provides a pharmaceutical comprising the inventive antibody as disclosed herein or which comprises the composition according to the invention and at least one further ingredient.
- the present invention pertains to a method of treating a subject in need thereof inflicted with cancer, wherein the treatment comprises administering to said patient a therapeutically effective amount of the pharmaceutical composition according to to the invention.
- Figure 1 a) Transglutaminase-mediated conjugation of glutamine (Q) with an amine- donor substrate, b) Amino acid sequence of a natural transglutaminase recognition sequence taken from the substrate protein DAIP (1) and engineered sequences (2) and (3) used in this study for conjugation, c) Schematic depiction of the design of an engineered transglutaminase recognition sequence (MTGtag) mimicking the functional Q298 loop in DAIP.
- TMGtag engineered transglutaminase recognition sequence
- Figure 2 MTG-mediated conjugation of engineered model peptides and fulllength monoclonal antibodies, a) Engineered model peptides 2 and 3 containing a transglutaminase recognition motif were biotinylated in MTG-catalyzed reaction with monobiotinylcadaverine (4, MBC). b) Biotinylation kinetics comparing conformational ⁇ constrained peptide 2 (o) (e.g. with an inventive peptide according to SEQ ID NO:4) with linear peptide 3 ( ⁇ ) (e.g. with inventive peptides according to SEQ ID NO:3 or SEQ ID NO:5). Error bars representing standard deviation were calculated from triplicates.
- Figure 3 Binding of biotinylated cetuximab studied in cell assays and by flow cytometry.
- Figure 4 RP-HPLC analysis of transglutaminase-promoted biotinylation of oxidized peptide 2 (gradient 10 to 80%). Peak areas of 2 and 5 were analyzed and background subtracted.
- FIG. 5 Coomassie stained 15% SDS-PAGE gel analysis of purified cetuximab wildtype
- cetuximab variants 7 and 8 e.g. variant 7 corresponds to cetuximab according to the invention, which has been carboxyterminally fused to SEQ ID NO:90 and locked or constrained by a disulfide bridge, and variant corresponds to cetuximab carboxyterminally fused to SEQ ID NO:246 without disulfide bridge formation and not conformationally constrained.
- Antibodies were reduced in the presence of 2-mercaptoethanol prior to loading (2 g/lane). Prestained protein ladder (New England Biolabs) is indicating protein sizes in kDa.
- Figure 7 Schematic representation of a heavy chain of 10 used for tryptic-digestion prior to MALDI-MS-analysis.
- Figure 8 MALDI-TOF-MS spectra of trypsin digested heavy chain of 10.
- Figure 9 MALDI-TOF-MS spectra of trypsin digested heavy chain of 10 in detail.
- FIG. 10 (A) Peptides derived from trypsin digestion were analyzed by using the bioinformatics tool GPMAW. Peptides modified by either carbamidomethyl (CAM) at cysteine residues or monobiotinyl-cadaverine (MBC) at glutamine residues are underlined in cyan, peptides identified without modifications are underlined in magenta. (B) Statistical analysis of residue coverage indicating that a 56% coverage of the residues was achieved.
- GPMAW bioinformatics tool
- Figure 11 SDS-PAGE gel analysis of cetuximab wild-type (wt) and variants 9 and 10 comparing antibodies with C-terminal lysine from the wild-type sequence of cetuximab (+K447) and antibodies lacking this C-terminal lysine (-K447).
- Intramolecular cross-linking catalyzed by MTG has been observed in preliminary experiments between the MTG-tags and the C-terminal Lys residue from the wild-type sequence of cetuximab (+K447). This cross-linkage could be completely abolished by omitting this lysine (-K447).
- Figure 2 Determination of antibody conjugation efficiency using TAMRA-cadaverine and fluorescence analysis.
- Figure 13 Determination of free thiols in antibodies 7 and 8 using Ellman's reagent.
- Figure 14 (A) Western Blot using streptavidin-AP, NBT/BCIP for the detection of biotinylated cetuximab. Arrows indicate the position of cetuximab (bidtinylated) and of mTG2. (B) Coomassie stained protein gel with lanes loaded as indicated (C) fluorescence readout for the detection cetuximab comprising MTG-tag 1 (SEQ ID NO:90), or MTG-tag2 (SEQ ID NO:246) conjugated to fluorescent amino-donor substrates GGG-TAMRA or TAMRA-cadavarine.
- MTG-tag 1 SEQ ID NO:90
- MTG-tag2 SEQ ID NO:246 conjugated to fluorescent amino-donor substrates GGG-TAMRA or TAMRA-cadavarine.
- FIG. 15 Cytotoxic payloads with different primary amines used in transglutaminase- mediated conjugation reactions.
- MMAE Monomethy auristatin E
- PEG polyethylene glycol
- MMAE Monomethyl auristatin E
- vc-PABC cleavable valine-citrulline-p- aminobenzylcarbamate
- vc-PABC Monomethyl auristatin E
- vc-PABC Monomethyl auristatin E
- vc-PABC Monomethyl auristatin E with cleavable valine-citrulline-p- aminobenzylcarbamate
- MMAF Monomethyl auristatin F
- vc-PABC Monomethyl auristatin F
- FIG. 16 Hydrophobic interaction chromatography (HIC) of antibody-drug-conjugates
- ADCs synthesized by catalysis of microbial transglutaminase (MTG).
- ADCs were generated by enzyme-mediated conjugation of cytotoxic payloads 1-4 as shown in Figure 15 to an lgG1 antibody (cetuximab) with a carboxy-terminal MTG-tag at each of its heavy chains.
- A Blank run
- B Unconjugated antibody control
- C ADCs with a different drug-to-antibody ratio (DAR) obtained by conjugation to payload 1
- D ADCs with a different drug-to-antibody ratio (DAR) obtained by conjugation to payload 2
- E ADCs with a different drug-to- antibody ratio (DAR) obtained by conjugation to payload 3
- F ADCs with a different drug-to-antibody ratio (DAR) obtained by conjugation to payload 4.
- Sequences according to SEQ ID NO: 1 -SEQ ID NO:294 are according to the invention and may be used in in accordance with the embodiments of the present invention:
- the present invention is solved according to a first embodiment by a protein which comprises at least one acyl glutamine-containing amino acid donor sequence covalently linked via a ⁇ -glutamyl-amid bond to an amino donor-comprising substrate, wherein the at least one acyl glutamine-containing amino acid donor sequence comprises at least the amino acid sequence according to SEQ ID NO: 1 (SEQ ID NO: 1 (TYFQAYG)).
- a protein which comprises at least one, e.g. 1 , 2, 3, 4, 5, 6, 7, or 8, preferably 1-6 (e.g.
- acyl glutamine-containing amino acid donor sequences whereby the at least one acyl glutamine-containing amino acid donor sequence according to the invention is covalently linked via a ⁇ -glutamyl-amid bond to an amino donor-comprising substrate and wherein the at least one acyl glutamine-containing amino acid donor sequence comprises at least the amino acid sequence according to SEQ ID NO: 1 (TYFQAYG).
- acyl glutamine-containing amino acid donor sequence pertains to an amino acid sequence, which is at least 7 amino acids in length, e.g.
- the acyl glutamine-containing amino acid donor sequence according to the invention may e.g. in addition the the amino acid sequence according to SEQ ID NO: 1 comprise natural or non-natural amino acids, or naturally occuring amino acid derivates.
- the inventive acyl glutamine-containing amino acid donor sequence may comprise 1, 2, 3, 4, 5, 6, 7 or 8 non-natural amino acids, or naturally occuring amino acid derivates.
- non-natural amino acids which may be comprised in the acyl glutamine- containing amino acid donor sequence of the invention may include azidonorleucine, 3-(1- naphthyl)alanine, 3-(2-naphthyl)alanine, p-ethynyl-phenylalanine, p-propargyl-oxy- phenylalanine, m-ethynyl-phenylalanine, 6-ethynyl-tryptophan, 5-ethynyl-tryptophan, (R)-2- amino ⁇ 3-(4-ethynyl-1 H-pyrrol-3-yl)propanoic acid, p-bromophenylalanine, p- idiophenylalanine, p-azidophenylalanine, 3-(6-chloroindolyl)alanine, 3-(6- bromoindoyl)alanine, 3-(5-bromoindolyl
- naturally occuring amino acid derivatives may include 4- hydroxyproline, ⁇ - ⁇ , ⁇ , ⁇ -trimethyllysine, 3-methylhistidine, 5-hydroxylysine, O-phosphoserine, ⁇ -carboxyglutamate, ⁇ - ⁇ -acetyllysine, ⁇ - ⁇ -methylarginine, N-acetylserine, ⁇ , ⁇ , ⁇ - trimethylalanine.
- ⁇ -glutamyl-amid bond refers to an isopeptide bond, e.g. an amide bond, which does not form part of the peptide-bond backbone of the respective polypeptide or protein, which is formed between the gamma-carbon of the glutamyl residue of the inventive acyl glutamine-containing amino acid donor sequence and a primary (1°) amine of the amino donor-comprising substrate accoding to the invention.
- the amino donor-comprising substrate according to the invention comprises at least an ⁇ -amino group or at least one tripeptide having the sequence of GGG with a primary aminoterminal amino group.
- the amino donor-comprising substrate according to the invention may be lysine, such as L-lysine, or a lysine derivative, or structural mimetics therof, such as e.g.
- the amino donor-comprising substrate according to the invention may e.g. also comprise lysine derivatives which have been coupled to a dye (e.g. TAMRA, or Alexa- Fluor® dyes), or other molecule such as e.g. biotin.
- the amino donor-comprising substrate according to the invention may e.g. also be a GGG tripeptide, which e.g. may further be coupled to a dye or molecule, such as TAMRA-ethylenediamine or biotin, e.g. :
- the amino donor-comprising substrate according to the invention may e.g. also include polypeptides comprising at least one lysine residue.
- the amino donor-comprising substrate of the invention is covaently bound to a further molecule.
- the amino donor-comprising substrate according to the invention as disclosed above such as the tripeptide as disclosed above, may be covalently bound to a further molecule, preferably via its carboxyterminus.
- the further molecule may be covelently bound via a peptide bond, isopeptide bond, or any other covalent bond, such as e.g. a disulfide bond.
- the further molecule according to the invention as disclosed above is one of a dye, radioisotope, drug, ribozyme, nanobody, enzyme, or linker.
- a dye which may be covalently bound to the amino donor-comprising substrate according to the invention may be a fluorophore.
- the amino donor-comprising substrate according to the invention may be covalently bound to a fluorophore.
- fluorophore refers to a chemical compound, which when excited by exposure to a particular wavelength of light, emits light at a different wavelength, e.g.
- fluorophores that may be covalently bound to the amino donor-comprising substrate according to the invention may include 1,8-ANS, 4-methylumbelliferone, 7-amino-4-methylcoumarin, 7- hydroxy-4-methylcoumarin, Acridine, Alexa Fluor 350TM, Alexa Fluor 405TM, A CA, AMCA-X, ATTO Rho6G, ATTO Rho11 , ATTO Rho12, ATTO Rho13, ATTO Rho14, ATTO Rho101, Pacific Blue, Alexa Fluor 430TM, Alexa Fluor 480TM, Alexa Fluor 488TM, BODIPY 492/515, Alexa Fluor 532TM, Alexa Fluor 546TM, Alexa Fluor 555TM, Alexa Fluor 594TM, BODIPY 505/515, Cy2, cyQUANT GR, FITC, Fluo-3, Fluo-4, GFP (EGFP), mHoneydew, Oregon GreenTM 488, Oregon GreenTM 514, EYFP, D
- Fluorophores according to the invention may e.g. also include quantum dots.
- the term quantum dot as used in the present invention refers to a single spherical nanocrystal of semiconductor material where the radius of the nanocrystal is less than or equal to the size of the exciton Bohr radius for that semiconductor material (the value for the exciton Bohr radius can be calculated from data found in handbooks containing information on semiconductor properties, such as the CRC Handbook of Chemistry and Physics, 83rd ed., Lide, David R. (Editor), CRC Press, Boca Raton, Fla. (2002)). Quantum dots are known in the art, as they are described in references, such as Weller, Angew. Chem. Int. Ed. Engl.
- Quantum dots may e.g. be from about 1 nm to about 1000 nm diameter, e.g.
- a QD is capable of emitting electromagnetic radiation upon excitation (i.e., the QD is photoluminescent) and includes a "core" of one or more first semiconductor materials, and may be surrounded by a "shell” of a second semiconductor material.
- a QD core surrounded by a semiconductor shell is referred to as a "core/shell” QD.
- the surrounding "shell" material will preferably have a bandgap energy that is larger than the bandgap energy of the core material and may be chosen to have an atomic spacing close to that of the "core” substrate.
- the core and/or the shell can be a semiconductor material including, but not limited to, those of the groups ll-VI (ZnS, ZnSe, ZnTe, US, CdSe, CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, and the like) and lll-V (GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, and the like) and IV (Ge, Si, and the like) materials, PbS, PbSe, and an alloy or a mixture thereof.
- ll-VI ZnS, ZnSe, ZnTe, US, CdSe, CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe
- Preferred shell materials include ZnS.
- Quantum dots may be coupled to the inventive linker, enzyme, or protein by any method known in the art such as e.g. the methods disclosed in Nanotechnology. 2011 Dec 9;22(49):494006; Colloids and Surfaces B: Biointerfaces 84 (2011) 360-368.
- the amino donor-comprising substrate of the invention may be covalently bound to a radioisotope such as e.g. 47 Ca, 14 C, 137 Cs, S7 Cr, ⁇ Co, 60 Co, 67 Cu, 67 Ga, 123
- Typical chelators that may e.g.
- DPTA DPTA
- EDTA Ethylenediamine-tetraacetic acid
- EGTA Ethyleneglycol-O, 0'-bis(2- aminoethyl)-N, N, N ⁇ N'-tetraacetic acid
- NTA Nitrilotriacetic acid
- HEDTA N-(2- Hydroxyethyl)-ethylenediamine-N,N',N'-triacetic acid
- DTPA 2-[Bis[2-
- the amino donor-comprising substrate according to the invention may be covalently bound to a drug.
- drug refers to any chemical compound or molecule which interferes with the physiological function of a cell, e.g. a cancer or tumor cell.
- Drugs which may be linked to the amino donor-comprising substrate according to the invention may include cytostatic agents, or cytotoxic agents.
- cytostatic agents that may be used for covalent coupling to the amino donor- comprising substrate according to the invention include alkylating agents, antimetabolites, antibiotics, mitotic inhibitors, hormones, or hormone antagonists. Alkylating agents may e.g.
- Busulfan (Myleran), Carboplatin (Paraplatin), Chlorambucil, Cisplatin, Cyclophosphamide (Cytoxan), dacarbazine (DTIC-Dome), Estramustine Phosphate, Ifosphamide, Mechlorethamine (Nitrogen Mustard), Melphalan (Phenylalanine Mustard), Procarbazine, Thiotepa, Uracil Mustard, antimetabolites may e.g.
- mitotic inhibitors may e.g. include Etoposide (VP-16, VePesid), Teniposide (VM-26, Vumon), Vinblastine, Vincristine, Vindesine, hormones, or hormone antagonists which may e.g.
- Buserelin Conjugate Equine Estrogen (Premarin), Cortisone, Chlorotriansene (Tace), Dexamethasone (Decadron), Diethylstilbestrol (DES), Ethinyl Estradiol (Estinyl), Fluoxymesterone (Halotestin), Flutamide, Goserelin Acetate (Zoladex), Hydroxyprogesterone Caproate (Delalutin), Leuprolide, Medroxyprogesterone Acetate (Provera), Megestrol Acetate (Megace), Prednisone, Tamoxifen (Nolvadex), Testolactone (Teslac), Testosterone.
- amino donor-comprising substrate according to the invention may be covalently bound to an enzyme, such as L-Asparaginase.
- the amino donor-comprising substrate of the invention may e.g. be covalently bound to a ribozyme or nanobody.
- ribozyme refers to an enzymatically active RNA molecule having trans-splicing activity and self-splicing activity.
- the ribozyme can serve to inhibit the activity of the cancer-specific gene by a trans-splicing reaction, thereby exhibiting a selective anticancer effect. Any ribozyme may be used in the present invention, as long as it can inactivate a cancer-specific gene and activate the cancer therapeutic gene.
- the amino donor-comprising substrate of the invention may e.g. be covalently bound to a nanobody.
- a nanobody refers to an antibody comprising a small single variable domain such as e.g. VHH of antibodies obtained from camelids and dromedaries, e.g. Camelus baclrianus and Calelus dromaderius including new world members such as llama species (Lama paccos, Lama glama and Lama vicugna).
- Antibodies from the species have been characterized with respect to size, structural complexity and antigenicity for human subjects.
- IgG antibodies from this family of mammals as found in nature lack light chains, and are thus structurally distinct from the typical four chain quarternary structure having two heavy and two light chains, for antibodies from other animals (see e.g. WO94/04678, Hamers-Casterman C, Nature 363: 446-448; Harmsen (2007) Appl Microb Biotechnol 77: 13-22).
- the amino donor-comprising substrate of the invention may e.g. be covalently bound to a linker.
- linker or “linker peptide” refers to a synthetic or artifical amino acid sequence that connects or links two molecules, such as e.g. two polypeptide sequences that link two polypeptide domains, or e.g. a protein and a cytostatic drug, or toxin.
- synthetic or “artifical” as used in the present invention refers to amino acid sequences that are not naturally occurring.
- the linker which is covalently bound to the amino donor- comprising substrate of the invention is cleavable or non-cleavable.
- cleavable refers to linkers which may be cleaved by proteases, acids, or by reduction of a disulfide body (e.g. glutathion-mediated or glutathion sensitive).
- cleavable linkers may comprise valine-citrulline linkers, hydrazone linkers, or disulfide linkers.
- Non-cleavable linkers which may e.g.
- amino donor-comprising substrate of the invention comprise maleimidocaproyl linker to MMAF (mc-MMAF), N- maleimidomethylcyclohexane-1-carboxylate (MCC), or mercapto-acetamidocaproyl linkers.
- the linker according to the invention is coupled or covalently bound to a dye, radioisotope, or cytotoxin.
- the linker according to the invention which is covalently bound to the amino donor-comprising substrate of the invention may be e.g. coupled or covalently bound to a dye, radioisotope, or cytotoxin.
- the term "coupled” as used for the linker according to the invention refers to the fact that the dye, radioisotope or cytoxin is non-covalently attached to the linker molecule according to the invention, e.g. via ionic, or hydrophobic interctions.
- the linker may comprise streptavidin and the dye, radioisotope or cytotoxin may be covalently bound to biotion.
- the radioisotope may be chelated to one of the chelators as disclosed above, which may be covalently bound to biotion.
- the radioisotope may e.g. be incorporated into the biotin, e.g. [8,9-3H]biotin (see e.g. Robinson et al., J Biol Chem. 1983 May 25,258(10):6660-4.).
- the linker may e.g. be covalently bound to a cytotoxin, which may e.g.
- Cytotoxins which are suited for covalent attachment to linker molecules include those e.g. disclosed above and may be grouped into two main classes: The first class includes cytotoxins which disrupt microtubule assembly and the second class cytotoxins which target DNA structure. Accordingly, cytotoxins which may e.g. be covalently bound to the linker according to the invention include doxorubicin, calicheamicin, auristatin, maytansine duoarmycin and analogs thereof, a-amaitin, tubulysin and analogs thereof.
- the amino donor- comprising substrate of the invention which is coupled to a cleavable or non-cleavable linker as disclosed above and which are further coupled to a cytotoxin as disclosed above, may e.g. be used in transglutaminase-mediated bioconjugation reations.
- the bioconjugation reaction is catalyzed by mTG2.
- the bioconjugation reaction may e.g.
- acyl glutamine-containing amino acid donor sequence substrate according to the invention as disclosed above comprising at least the amino acid sequence according to SEQ ID NO:1 (TYFQAYG) and the amino donor-comprising substrate of the invention as disclosed above in the presence of mTG2 under conditions which allow the mTG2 catalyzed reaction.
- the acyl glutamine-containing amino acid donor sequence of the invention comprises an amino acid sequence according to SEQ ID NO: 2 (X 1 X 2 X3 TYFQAYG X4X5X6), wherein
- Xi is a hydrophobic amino acid
- X 2 is a negatively charged amino acid
- Xa is C or N
- X5 is one of an amino acid with with a polar, uncharged side chains, and Xe is a negatively charged amino acid.
- Xi may comprise any one of the amino acids A, I, L, M, F, W, Y, V, X2 may comrise the amino acids D, E, 3, A may be one of C or N, X5 may be one of S, T, N or Q and Xe may be one of D, or E. Accordingly, the acyl glutamine-containing amino acid donor sequence of the invention may comprise an amino acid sequence according to any one of SEQ ID NO:1 - SEQ ID NO:294, e.g.
- the acyl glutamine-containing amino acid donor sequence of the invention may comprise any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID
- Xi may comprise any one of the amino acids A, V, I, L, M or G
- X 2 may comprise the amino acids D
- E may comprise the amino acids
- X3 may be C
- X4 may be C or N
- X 5 may be one of S, T, or N
- Xe may be one of D, or E.
- the acyl glutamine-containing amino acid donor sequence of the invention may comprise an amino acid sequence according to any one of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 , SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO.-21 , SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID ⁇ .
- SEQ ID NO:140 SEQ ID NO:141 , SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO-.59, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:l5, SEQ ID N0.16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111 , SEQ ID NO:112,
- the acyl glutamine-containing amino acid donor sequence of the invention comprises an amino acid sequence according to SEQ ID NO:4 (XiX 2 C TYFQAYG CXsXe) in which both X 3 and X4 are C such that a disulfide bond may be formed between the two cysteine residues X3 and X4 which will form a loop-like structure, with amino residues TYFQAG forming the loop-like structure.
- acyl glutamine-containing amino acid donor sequence Xi may be any one of the amino acids A, V, I, L, M or G, X 2 may be D, or E, X3 and t are both C, X 5 may be one of S, T or N and X 6 may be one of the amino acids D, or E.
- the acyl glutamine-containing amino acid donor sequence of the invention may comprise an amino acid sequence according to any one of SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11 , SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO: 106, SEQ ID NO:107, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131 , SEQ ID
- the peptides of the invention may be synthesized according to any known method in the art, e.g. according to the methods disclosed in Atherton, E.; Sheppard, R.C. (1989). Solid Phase peptide synthesis: a practical approach. Oxford, England: IRL Press. ISBN 0-19-963067-4, or Stewart, J.M.; Young, J.D. (1984). Solid phase peptide synthesis (2nd ed.), ISBN 0-935940- 03-0). Peptides may e.g.
- Peptides may then e.g. cleaved from the resin by a standard cleavage cocktail of 94% TFA, 2% triethylsilane, 2% anisole, 2% H2O.
- a standard cleavage cocktail 94% TFA, 2% triethylsilane, 2% anisole, 2% H2O.
- DTT dithiotreitol
- Dislufide bridge formation between residues X3 and X* may be achieved under oxidizing conditions, e.g. 1 mg ml "1 in 100 mM (NH 4 )2C03 aq. pH 8.4, whereby the oxidatoin reation may be monitored by RP-HPLC.
- Xi may be any one of amino acids A, V, I, L, M or G
- X 2 is E
- X 3 * are both C
- Xs is T
- X 6 is E.
- the acyl glutamine-containing amino acid donor sequence of the invention may comprise any one of SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90.
- acyl glutamine-containing amino acid donor sequence of the invention comprises the amino acid sequence according to SEQ ID NO:90 (GECTYFQAYGCTE).
- acyl glutamine-containing amino acid donor sequence according to the invention as disclosed above may e.g. be covalently bound to a protein of interest, such as e.g. enzymes or antibodies.
- a protein of interest such as e.g. enzymes or antibodies.
- polynucleotides encoding the acyl glutamine-containing amino acid donor sequence of the invention may be added to cDNA constructs encoding light or heavy chains of antibodies by way of polymerase chain reaction (PCR).
- PCR polymerase chain reaction
- SOE- PCR may be used according to the principle disclosed in PCR Methods Appl. 1993 May;2(4):301-4, or e.g. in Critical Reviews in Oral Biology and Medicine, 4(3/4):581-590 (1993).
- the protein comprising the acyl glutamine-containing amino acid donor sequence according to the invention as disclosed above may be an antibody, antigen-binding antibody fragment, Fc domain, enzyme, a non-immunoglobulin scaffold.
- the acyl glutamine-containing amino acid donor sequence of the invention may be covalently bound to an antibody, antigen-binding fragment, Fc domain, enzyme, a non-immunoglobulin scaffold.
- the protein according to the invention comprising the inventive acyl glutamine- containing amino acid donor sequence may be an antibody, whereby the term "antibody” refers to 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.
- a target such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.
- the term “antibody” encompasses not only intact polyclonal or monoclonal antibodies, but also, unless otherwise specified, any antigen binding fragment thereof that competes with the intact antibody for specific binding, fusion proteins comprising an antigen binding portion, any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site, antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies).
- monoclonal antibody refers to antibodies displaying a single binding specificity and indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
- the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma (e.g. murine or human) method first described by Kohler et at., Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat.No.4,816,567).
- a "monoclonal antibody” may also be isolated from phage antibody libraries using the techniques described in Clackson et at., Nature, 352:624-628 (1991) and Marks et at., J. Mol. Biol., 222:581-597 (1991).
- human monoclonal antibodies refers to monoclonal antibodies, which have variable and constant regions derived from human germline immunoglobulin sequences.
- humanized as used in the context of the present invention preferably refers to a monoclonal antibody in which the amino acid sequence is essentially identical to that of a human variant, despite the non-human origin of some of its complementarity determining region (CD ) segments responsible for the ability of the antibody to bind to its target antigen.
- CD complementarity determining region
- chimeric monoclonal antibody refers to a monoclonal antibody in which murine Fab fragments are spliced to human Fc.
- the protein according to the invention as disclosed above is an antigen- binding fragment.
- antigen binding fragement refers to, for example, Fab, Fab', F(ab') 2 , Fd, Fv, domain antibodies, also referred to as “nanobodies” (dAbs, or VHH, e.g., shark and camelid antibodies, see e.g.
- a "bispecific antibody” as used according to the invention is an antibody that can bind two different target molecules
- “bivalent antibody” as according to the invention is an antibody that can bind two different sites of one target molecule.
- the inventive protein may be a nanobody which comprise the acyl glutamine-containing amino acid donor sequence according to the invention, e.g. fused to the carboxyterminus of the nanobody.
- the nanobody according to the invention may then e.g. be used in a mTG2-mediated bioconjugation, whereby the amino donor-comprising substrate may e.g. be further coupled to a linker-cytotoxin as disclosed above, or may e.g.
- nanobodies of the invention may e.g. be used for cancer treatment, or imaging purposes.
- nanobodies of the invention coupled to 68 Ga which specifically bind EGFR, or HER2 may be used for Pet imaging to detect or image EGFR or HER2 expressing tumors.
- the term "specific binding” or any grammatical variation thereof refers to the ability of the inventive antibody, antigen-binding fragment (e.g. Fab, Fab", F(ab Fd, Fv), monoclonal antibody, single-chain antibody (scFv) or nanobody to bind to bind to its respective target (e.g.
- EGFR EGFR, or HER2
- K D K D of at least about 1 x 10 "6 , e.g. 1 x 10 "6 , 1 x 10 7 M, 1 x 10 8 M, 1 x 10- 9 M, 1 x 10- 10 M, 1 x 10 "11 M, 1 x 10 "12 M, 1 x 10 13 M, 1 x 10 "14 M, 1 x 10 15 M, preferably between 1 x 10 0 M and 1 x 10 ⁇ 15 M, more preferably between about 1x10 12 M to about 1x10 " 15 .
- immunoglobulin as used in the present invention may be used interchangeably with the term "antibody”.
- the basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
- An IgM antibody consists of 5 of the basic heterotetramer units along with an additional polypeptide called a J chain, and contains 10 antigen binding sites, while IgA antibodies comprise from 2-5 of the basic 4-chain units which can polymerize to form polyvalent assemblages in combination with the J chain.
- the 4-chain unit is generally about 150,000 daltons.
- Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype.
- Each H and L chain also has regularly spaced intrachain disulfide bridges.
- Each H chain has at the Niter-minus, a variable domain (VH) followed by three constant domains (CH) for each of the a and Y chains and four CH domains for ⁇ and ⁇ isotypes.
- Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain at its other end.
- VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (Cm)- Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
- Cm heavy chain
- immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated ⁇ , ⁇ , ⁇ , ⁇ and ⁇ , respectively.
- the ⁇ and a classes are further divided into subclasses on the basis of relatively minor differences in the CH sequence and function, e.g., humans express the following subclasses: lgG1, lgG2A, lgG2B, lgG3, lgG4, lgA1 and Igic1.
- IgG comprises the major class as it normally exists as the second most abundant protein found in plasma.
- Fc domains of the lgG1 subclass are often used as the immunoglobulin moiety, because lgG1 has the langest serurn half-life of any of the serum proteins.
- the Fc moiety may, e.g., also comprise Fc variants such as those disclosed in WO 02/094852 (e.g. Fc amino acid sequence with variants Fc-488, Fc4, Fc5, Fe6, Fc7, and Fc8 as disclosed therein) to reduce effctor function of the Fc moeity.
- Fc variants such as those disclosed in WO 02/094852 (e.g. Fc amino acid sequence with variants Fc-488, Fc4, Fc5, Fe6, Fc7, and Fc8 as disclosed therein) to reduce effctor function of the Fc moeity.
- the protein according to the invention may be a fusion protein comprising the the acyl glutamine-containing amino acid donor sequence according to the invention and a Fc domain to extend its serum half-life, whereby the the acyl glutamine-containing amino acid donor sequence according to the invention is fused to the carboxyterminus of the Fc moiety.
- the terminal lysine (K) residue may e.g. be removed in Fc moieties e.g. in IgG-type antibodies, or in Fc-fusion proteins, by PCR-based site-directed mutagenesis to avoid intermolecular crosslinking. PCR-based site-directed mutagenesis may e.g. be done according to the method disclosed in Nucleic Acids Res.
- primer pairs which may be used for the site-directed mutagenesis may e.g. also be selected according to the the web- based automated tool available at: http://bioinformatics.org/primerx.
- the corresponding cDNAs which encode the protein according to the invention lacking a terminal lysine residue may be obtained by custom gene synthesis (see e.g. Nature 432, 1050-1054 (23 December 2004); Nucleic Acids Res. 2007 Apr; 35(8): e61.; ACS Synth. Biol. 3, 97-106 (2014); Nat. Methods 6, 343-345 (2009); Nucleic Acids Res. 40, e55 (2012); Proc. Natl. Acad. Sci. USA 105, 20404-20409 (2008)).
- the protein according to the invention is a non-immunoglobulin scaffold comprising the inventive acyl glutamine-containing amino acid donor sequence.
- non- immunoglobulin scaffold as used for the protein according to the invention refers to affinity proteins that are not based on Ig molecules.
- Non-immunoglobulin scaffold proteins include e.g. those disclosed in Nature Biotechnology 23, 1257-1268, 2005, Trends Biotechnol. 2015 Jul;33(7):408-18.
- the protein according to the invention may be an albumin binding domain, phytocystaton protein (Adhiron), adnectin, Z-domain of S.
- the protein according to the invention as disclosed herein may comprise the inventive acyl glutamine-containing amino acid donor sequence, whereby the inventive acyl glutamine-containing amino acid donor sequence may be present at the aminoterminus or at the carboxyterminus of the protein according to the invention.
- a determining factor in positioning the acyl glutamine-containing amino acid donor sequence of the invention to the amino- or carboxyterminus of the inventive protein as diclosed above is whether or not it will interfere with the intended use of the protein.
- the protein according to the invention is an antibody, e.g. a monoclonal antibody
- carboxyterminal fusion proteins with the inventive acyl glutamine-containing amino acid donor sequence are preferred, whereby the acyl glutamine-containing amino acid donor sequence according to SEQ ID NO:2 may be fused to the light chain, or heavy chain, or both heavy and light chain, thereby providing an monoclonal antibody comprising 2 (e.g. on both light chains (LCs), or on both heavy chains (HCs)) or 4 acyl glutamine-containing amino acid donor sequences according to the invention.
- the present invention provides for a method of covalently coupling an amino donor-comprising substrate according to the invention as disclosed above to a acyl glutamine-containing amino acid donor of the invention which comprises a polypeptide sequence according to SEQ ID NO:2, whereby the method comprises the step of:
- the amino donor-comprising substrate according to the invention and the acyl glutamine-containing amino acid donor according to the invention in the presence of transglutaminase, preferably mTG2, to obtain the inventive protein as disclosed above.
- the amino donor-comprising substrate according to the invention as disclosed above and the acyl glutamine-containing amino acid donor according to the invention are contacted in the presence of mTG2 to obtain the inventive protein as disclosed above.
- the term "contacted" as used in the inventive method refers to any situation in which two or more molecules, such as the amino donor-comprising substrate and the acyl glutamine-containing amino acid donor in accordance with the invention, are brought into intimate physical contact with one another, e.g. form part of the same reaction mixture or aqueous solution.
- the mTG2-mediated reaction according to the inventive method may comprise reacting the acyl glutamine- containing amino acid donor according to the invention with an 1-60 molar equivalent, or 1-50 molar equivalent of the amino donor-comprising substrate according to the invention, e.g.
- the concentration of the acyl glutamine-containing amino acid donor according to the invention may be from about 0.1 mg/ml to about 100 mg/ml, e.g.
- mTG2 may be e.g. present in an amount from about 0.01 mol equivalents to about 2 mol equivalents to the substrate, e.g. to the protein comprising the the acyl glutamine-containing amino acid donor sequence, from about 0.05 mol equivalents to about 1.5 mol equivalents, from about 0.1 mol equivalents to about 1.125 mol equivalents, from about 0.125 mol equivalents to about 1.75 mol equivalents, from about 0.25 mol equivalents, 0.3 mol equivalents, 0.4 mol equivalents, 0.5 mol equivalents, 0.6 mol equivalents, 0.7 mol equivalents, 0.8 mol equivalents to about 1 mol equivalents, or from about 1 mol equivalents to about 2.5 mol equivalents, preferably mTG2 is used in the inventive method in an amout of about 1 mol equivalents.
- the reaction may e.g. be allowed to proceed 25°C-37°C for about 1-5 hours, or for about 2-4 hours, or for about 2.5 hours to about 3.5h, or for about 1 hour, 2 hours, 3 hours, 4 hours.
- the inventive method may e.g. be carried out in any suitable buffer such as HEPES (4-2-hydroxyethyl-1- piperazineethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), or PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid) at a pH of about pH7.0.
- the acyl glutamine-containing amino acid donor according to the invention which may be used in the inventive method may be as disclosed above, e.g. the inventive acyl glutamine-containing amino acid donor may be an antibody, antigen-binding antibody fragment, enzyme, or a non-immunoglobulin scaffold as disclosed above.
- Microbial transglutaminease such as e.g. mTG2 which may be used according to the embodiments of the present invention, may be obtained from Streptoverticillium mobaraense, or any of its substrains, such as e.g. 40847, according to the method disclosed in Biochem. J. (1994) 299:825-829.
- Streptoverticillium mobaraense may e.g. cultivated as described by Ando et al. (1989) Agric. Biol. Chem.
- 0.1 ml of the spore suspension may be grown in 500 ml of a medium containing: polypeptone, 2.0%; yeast extract, 0.2%; K2HPO4, 0.2%; MgS0 4 ,7H 2 0, 0.1 % ; potato starch, 2.0 % ; glucose, 0.5 % ; pH 7.0.
- Cultivation may be continued at 30°C, and the culture may be aerated and shaken at 90 rev. /min for 9-11 days until the maximum of enzyme activity was reached.
- the culture fluid may then be separated from the mycel by centrifugation at 10000 g for 10 min and subsequent filtration over a folded filter paper.
- the supernatant may directly be utilized in chromatography or stored at - 20 °C for later use.
- 1.0 ml of the cell suspension may be transferred to 3.5 L of a medium containing 2.5 % potato starch instead of 2.0 %, and 1.0 % glucose instead of 0.5 %.
- the culture may be grown with aeration (2 litres/min) and stirring at 140-180 rev./min for the first day and subsequently at 300- 350 revJmin. Maximum of enzyme activity is typically achieved after 7 days.
- Streptoverticillium mobaraense may e.g. be obtained from ATCC® (ATCC 29032), or e.g. DSM, Braunschweig, Germany.
- the inventive amino donor-comprising substrate which is used in the inventive method is coupled or covalently bound to a dye, drug, ribozyme, nanobody, enzyme, or linker as disclosed above.
- the acyl glutamine-containing amino acid donor may be an antibody or antigen binding fragment as defined above which comprises at its carboxyterminus the inventive acyl glutamine-containing amino acid sequence according to SEQ ID NO:2, or e.g. according to any one of SEQ ID NO:7-294 as disclosed herein.
- the amino donor-comprising substrate may be as defined above, e.g. may be a peptide comprising at least one lysine residue, or the tripeptide GGG as disclosed above which may e.g. be coupled to a linker, dye, drug, ribozyme, nanobody, or enzyme as disclosed above.
- the linker used in the inventive method may e.g. be further linked to a cytotoxic drug (payload), dye, or radioisotope as defined above.
- the acyl glutamine-containing amino acid donor and the amino donor-comprising substrate may e.g. be present in a reaction mixture in a ratio of about 1 :40 molar equivalents (antibody: amino donor-comprising substrate) or in any molar equivalent disclosed above, with microbial transglutaminase 2 present in an amount as disclosed above, e.g. in a 1:1 molar equivalent to the antibody comprising the inventive acyl glutamine-containing amino acid donor sequence as disclosed above.
- the reaction may e.g.
- the present invention provides for a protein according to the invention which is obtainable by the inventive method as disclosed above.
- the protein obtainable according to the present invention may be any protein as disclosed herein, e.g. an antibody, antigen binding fragment, enzyme, or a non-immunoglobulin scaffold as disclosed herein, which comprises the amino acid sequence according to SEQ ID NO:2.
- the protein obtainable by the inventive method may comprise an antibody drug conjugate comprising the at least one acyl glutamine-containing amino acid donor sequence according to SEQ ID NO:2 as disclosed herein, preferably 2, more preferably 4 acyl glutamine-containing amino acid donor sequences as disclosed above which are covalently bound via an isopeptide bond formed between the acyl glutamine-containing amino acid donor of the invention (e.g. X1X2 X3 TYFQAYG X4X5X6), whereby the isopeptide bond is formed between the central glutamine residue (underlined) and the amino group of the amino donor-comprising substrate.
- an antibody drug conjugate comprising the at least one acyl glutamine-containing amino acid donor sequence according to SEQ ID NO:2 as disclosed herein, preferably 2, more preferably 4 acyl glutamine-containing amino acid donor sequences as disclosed above which are covalently bound via an isopeptide bond formed between the acyl glutamine-containing amino acid donor of the invention (e.g. X
- the present invention pertains to the use of a polypeptide sequence according to SEQ ID NO:2 in the inventive method as disclosed above. Accordingly, the present invention pertains to the use of one or more, e.g. 1 , 2, 3, 4, 5, or more of the inventive polypeptide sequences disclosed herein in the inventive method.
- the present invention pertains to the use of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID
- SEQ ID NO: 244 SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261 , SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267, SEQ ID NO: 268, SEQ ID NO: 269, SEQ ID NO: 270, SEQ ID NO: 271, SEQ ID NO: 272, SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO: 277
- polypeptide according to SEQ ID NO:2 may be carboxyterminally fused the HC or LC of an antibody as disclosed above.
- sequences in which both X3, X» are C as disclosed above are preferred, accordingly, the use of polypeptide sequences SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO NO:7,
- the present invention also provides polynucleotides encoding an inventive polypeptide according to SEQ ID NO:2.
- the present invention provides for polynucleotides encoding the inventive polypeptide according to SQE ID NO:2 of the following structure:
- N denotes any nucleotide (A, C, T, G)
- Y denotes a pyrimidine base (C or T)
- R denotes a purine base (A or G).
- Base nomenclature is according to the Nomenclature Committee of the International Union of Biochemistry (NC-IUB), see e.g. J. Biol. Chem., 1986, 261 , 13-17.
- NIMN 1 - NNN 6 indicate codons 1-6, whereby codon NNN1 encodes amino acid Xi of SEQ ID NO-.2, NNN 2 amino acid X 2 of SEQ ID NO:2, NNN 3 amino acid X3 of SEQ ID NO:2, NNN 4 amino acid X4 of SEQ ID NO:2, NNN 5 amino acid X5 of SEQ ID NO:2 and NNN 6 amino acid Xe of SEQ ID NO:2.
- codon usage should be optimized for the specific host, e.g.
- the different codon usage by the respective host should be considered.
- the inventive polynucleotide is used for expression in a rodent or human cell lines, such as CHO or HEK 293 cells, for the manufacture of a therapeutic, the corresponding codon usage should be used to increase e.g. protein production (Trends in Molecular Medicine, November 2014, Vol. 20, No. 11).
- Codons NNN1 - NNN 6 according to the invention may not, however, code for a stop codon, e.g. TAG (amfce/), TAA (ochre), or TGA (opa/).
- NNN1 may be any one of codons GCT, GCC, GCA, GCG, GTT, GTC, GTA.GTG, ATT, ATC, ATA CTT, CTC, CTA, CTG, TTA, TTG, ATG, GGT, GGC, GGA, GGG, NNN 2 may be any one of codons GAT, GAC, GAA, GAG, NNN 3)
- the present invention also provides for a vector which comprises the inventive polynucleotides as disclosed above.
- the vector according to the invention may e.g. be an expression vector.
- expression vector as used for the vector according to the invention to a nucleic acid vector that comprises a gene expression controlling region, such as a promoter or promoter component, operably linked to a nucleotide sequence encoding at least one polypeptid, e.g. at least the inventive polypeptide sequence according to any of of SEQ ID NO:2- SEQ ID NO:294.
- Expression vectors which may e.g.
- the polynucleotides may e.g. be comprised 5' or 3' to an multiple cloning site (MCS), which will allow to generate in-frame fusion proteins when a corresponding cDNA encoding e.g. an antibody light or heavy chain, is cloned into the MCS.
- MCS multiple cloning site
- the present invention also provides a host cell which comprises the inventive polynucleotide sequence as disclosed above, e.g. according to SEQ ID NO:2, or e.g. any of SEQ ID NO:7-294, or which comprises a vector according to the invention as disclosed above.
- a host cell according to the invention comprising the inventive polynucleotide or a vector according to the invention as disclosed above refers to any type of cell that can contain the vector according to the invention.
- the host cell can be a eukaryotic cell, e.g., plant, animal, fungi, or algae (e.g.
- the host cell can be a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human.
- the host cell can be an adherent cell or a suspended cell, i.e., a cell that grows in suspension.
- a host cell according to the invention may e.g.
- the present invention also provides for an antibody or antigen-binding fragment thereof, bivalent antibody, or VHH antibody comprising at least one amino acid sequence according to SEQ ID NO:2.
- Antibodies, antigen-binding fragments thereof, bivalent antibodies, or VHH antibodies according to the invention are as disclosed above.
- an antibody according to the invention may comprise at least one amino acid sequence according to SEQ ID NO:2 (e.g. the acyl glutamine-containing amino acid donor according to the invention), e.g. as a carboxyterminal fusion to a light or heavy chain.
- the antibody according to the invention preferably comprises 2, or 4 inventive amino acid sequences according to SEQ ID NO:2, e.g. the antibody of the invention may comprise two light chains, or two heavy chains which comprise at their carboxy terminus the inventive amino acid sequence according to SEQ ID NO:2.
- both heavy and light chains comprise as carboxyterminal fusions the inventive amino acid sequence according to SEQ ID NO:2.
- the inventive antibodies may e.g. also be modified by PCR-based site directed mutagenesis as disclosed above to remove terminal lysine residues in the Fc domain.
- the antibody according to the invention as disclosed above is a human or humanized monoclonal antibody as disclosed above, preferably, an lgG1 , lgG2, lgG3, lgG4, or IgM type antibody.
- the inventive antibody comprises at least one linker covalently coupled to it according to the inventive method disclosed above.
- the inventive antibody may comprise one, two, three, or four linker (e.g. cleavable or non-cleavable as disclosed above), which have been covalently coupled to the antibody according to the inventive method disclosed herein.
- the inventive linker is further coupled to a dye, radioisotope, or cytotoxin as disclosed above.
- cancer cell surface antigens include, but are not limited to EGFR (epidermal grwoth factor receptor), HER2, HER4, AFP, ⁇ ⁇ ⁇ 3 integrin, MUC16, CD4, CD20, CD22, CD30, CD33, CD52, CD56, CD66e, CD140b, CD227, EpCam, GD3, PSMA, or VEGF.
- Cancer cell surface antigens according to the invention also include cancer stem cell such as e.g.
- SLAMs signal lymphocyte activation molecule family receptors; see Yilmaz et al., "SLAM family markers are conserved among hematopoietic stem cells from old and reconstituted mice and markedly increase their purity," Hematopoiesis 107: 924-930 (2006)), such as CD150, CD244, and CD48, and those markers disclosed in U.S. Patent No. 6,004,528.
- the antibody according to the invention is cetuximab (c225), or e.g. a biosimilar thereof which binds to EGFR.
- cetuximab c225
- biosimilar as used for the inventive cetuximab is used in a manner that is consistent with the working definition promulgated by the US FDA which defines a biosimilar product to be one that is "highly similar” to a reference product (despite minor differences in clinically inactive components). In practice there can be no clinically meaningful differences between the reference product and the biosimilar product in terms of safety, purity, and potency (Public Health Service (PHS) Act ⁇ 262).
- cetuximab (or e.g. its biosimilar) according to the invention comprises at least one, preferably at least two, more preferably four acyl glutamine-containing amino acid donor sequences according to the invention.
- cetuximab according to the invention comprises two, or preferably four linkers as defined above further coupled to a cytotoxin as above.
- cetuximab (or e.g. its biosimilar) according to the invention may be used in the treatment of cancer.
- cancer refers to a cellular disorder characterized by uncontrolled or disregulated cell proliferation, decreased cellular differentiation, inappropriate ability to invade surrounding tissue, and/or ability to establish new growth at ectopic sites.
- cancer further encompasses primary and metastatic cancers.
- inventive antibody cetuximab may e.g.
- mCRC metastatic
- NSCLC Non-Small Cell Lung Cancer
- HNSCC Head and Neck Squamous Cell Carcinoma
- Cetuximab c225, or its biosimilar
- EGFR epidermal growth factor receptor
- RAS wild-type metastatic colorectal cancer
- Cetuximab according to the invention may also be used in combination with irinotecan- based chemotherapy, or e.g. in first-line treatment in combination with FOLFOX, or e.g. as a single agent in patients who have failed oxaliplatin- and irinotecan-based therapy and who are intolerant to irinotecan.
- the present invention provides for a composition which comprises the inventive antibody as disclosed above, which comprises at least one further pharmaceutically active ingredient.
- the composition according to the invention may comprise cetuximab (or e.g. its biosimilar) in aqueous or lyophilized form and at least one further chemotherapeutic agent, wherein the agent is selected from the group comprising capecitabine, 5-fluoro-2'-deoxyuiridine, irinotecan, 6-mercaptopurine (6- MP), cladribine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine, pemetrexed, pentostatin, thioguanine; leucovorin, cisplatin, carboplatin
- the present invention provides a pharmaceutical composition which comprises the inventive antibody as disclosed above, or the composition according to the invention and at least one further ingredient. Accordingly, the present invention provides for pharmaceutical compositions which comprise the inventive antibody and which may comprise excipients and/or stabilizers and/or surfactants and/or preservatives.
- excipients as used herein means a component of a pharmaceutical product that is not an active ingredient such as, for example, diluents.
- the excipients that are useful in preparing the inventive pharmaceutical composition are generally safe and non-toxic.
- Surfactants that may be used with the inventive pharmaceutical composition include e.g. anionic surfactants such as e.g.
- cationic surfactants such as e.g. quaternary ammonium and pyridinium cationic surfactants, or non-ionic surfactants, such as e.g. Sorbitan esters, polysorbates, e.g.
- Polysorbat 20 Polyoxyethylen-(20)-sorbitanmonolaurat
- Polysorbat 21 Polyoxyethylen-(4)-sorbitanmonolaurat
- Polysorbat 40 Polyoxyethylen-(20)- sorbitanmonopalmitat
- Polysorbat 60 Polyoxyethylen-(20)-sorbitan-monostearat
- Polysorbat 61 Polyoxyethylen-(4)-sorbitanmonostearat
- Polysorbat 65 Polyoxyethylen-(20)- sorbitantristearat
- Polysorbat 80 Polyoxyethylen-(20)-sorbitanmonooleat
- Polysorbat 81 Polyoxyethylen-(5)-sorbitanmonooleat
- Polysorbat 85 Polyoxyethylen-(20)-sorbitantrioleat
- Polysorbat 120 Polyoxyethylen-(20)-sorbitantrioleat
- Polysorbat 120
- the present invention provides for a method of treating a subject in need thereof inflicted with cancer, wherein the treatment comprises administering to said subject (e.g. a patient, wherein the patient is preferably a human) a therapeutically effective amount of the pharmaceutical composition as disclosed herein. Accordingly, the present invention provides for a method of treating treating a subject in need thereof inflicted with cancer, e.g.
- cetuximab inventice c225 (cetuximab) antibody
- cetuximab e.g. cetuximab according to the invention
- Peptides were cleaved from the resin by a standard cleavage cocktail of 94% TFA, 2% triethylsilane, 2% anisole, 2% H20. Cysteine containing peptides were cleaved in the presence of dithiotreitol (DTT) to suppress unwanted oxidation of cysteins. After 2 h of cleavage, peptides were precipitated in cold diethylether and washed twice with diethylether. The disulfide-bridged crude peptide was oxidized at 1 mg ml-1 in 100 mM (NH- 2CO3 aq. pH 8.4 and oxidation monitored by RP-HPLC. Afterwards, peptides were isolated by preparative RP-HPLC.
- DTT dithiotreitol
- ESI mass spectra were collected on a Shimadzu LCMS-2020 equipped with a Phenomenex Jupiter 5u C4 LC column (50 x 1 mm, 5 pm, 300 A) by using an eluent system of 0.1% aq. formic acid (eluent A) and acetonitrile containing 0.1% formic acid (eluent B).
- eluent A 0.1% aq. formic acid
- acetonitrile containing 0.1% formic acid eluent B
- Peptides were dissolved at 50 mg/ml in DMSO and diluted to 1.25 mg/ml in 100 mM HEPES pH 7.0 for enzymatic conjugation. Monobiotinyl cadaverine was added at a 5 fold molar excess over peptide and enzymatic reactions initiated by addition of microbial transglutaminase at an enzyme/substrate ratio of 1/50 (w/w). For peptide biotinylation assays involving peptide 2 (SEQ ID NO: 90) and reference peptide GGGSLLQG (SEQ ID NO:295), the amount of microbial transglutaminase was reduced to an enzyme/substrate ratio of 1/385 and 1/182 (w/w), respectively.
- PCR was used to generate c225 (Cetuximab) constructs lacking the carboxyterminal lysine residue of the heavy chain.
- Plasmids encoding C225 including a terminal lysine residue and the carboxyterminal amino acid sequence according to SEQ ID NO:246 were amplified using primers having the sequences according to SEQ ID NO:296, SEQ ID NO:299 (both at Ipmole) for 10 cycles (PCR conditions: 3min @ 98°C (initial denaturing), 10s @ 98°C, 30s @ 55°C, 15 s @ 72°C), followed by 20 cycles using primers having the sequences according top SEQ ID NO:296, SEQ ID NO:300 (10 pmole primers) with the following cycling conditions: 10 s @ 98°C, 30s @ 55°C, 15 s @ 72°C, final extension 5min@ 72°C.
- the resulting amplification product was gel purified and subsequently subjected to a restrictoin digest using EcoRV/BamHI (3h @ 37°C).
- the restriction fragment was then purified using a PCR purification kit.
- the fragment was then cloned into an EcoRV/BamHI digested pTT5_C225_HC vector (encoding the C225 heavy chain).
- Antibody conjugation using transglutaminase Antibodies were adjusted to 0.5 mg/ml and incubated with 40 mol equiv per conjugation site of monobiotinyl cadaverine (MBC) and 1 mol equiv microbial transglutaminase (mTGase) in 100 mM HEPES buffer pH 7.0 for 3 h at 25°C.
- MBC monobiotinyl cadaverine
- mTGase microbial transglutaminase
- reactions were analyzed by 15% SDS-PAGE and biotinylated heavy chains visualized by semi-dry western blot with streptavidin-alkaline phosphatase conjugate and NBT/BCIP.
- Antibody conjugation efficiency was determined by antibody labeling with N- (Tetramethylrodaminyl) cadaverine (TAMRA-cadaverine) and measurement of fluorescence at an excitation wavelength of 547 nm and an emission wavelength of 573 nm.
- TAMRA-cadaverine N- (Tetramethylrodaminyl) cadaverine
- MMG transglutaminase
- TAMRA-cadaverine conjugated antibodies was determined by using a standard curve of TAMRA-cadaverine ranging from 9.72 to 0.19 ⁇ in PBS (40 ⁇ per well) and fluorescence readout using a plate reader (Tecan Infinite M1000).
- reaction were also analyzed by 15% SDS-PAGE and TAMRA labeled heavy chains visualized by fluorescence readout.
- EBC-1 and CHO-K1 cells were cultured in DMEM with 4 mM L-glutamine (Sigma- Aldrich) and DMEM-F12 + GlutaMaxTM (Gibco®), respectively, both supplemented with 10% fetal bovine (Sigma-Aldrich) at 37 °C and 5% C02.
- Cell binding experiments were performed by using EGFR- overexpressing EBC-1 [3] and EGFR-negative CHO-K1 cells in combination with confocal fluorescence microscopy and flow cytometry, respectively. Washing and incubation steps were performed at 4 °C using PBS with 1% BSA. For microscopy based experiments, cells were grown on glass coverslips followed by consecutive labelling with 100 nM of respective antibody-conjugates and 1 :200 diluted Streptavidin Alexa Fluor® 488 conjugate (Life Technologies).
- Cetuximab heavy chain variants 9 and 10 (9: with inventive MTG-tag according to SEQ ID NO:90, 10: with inventive MTG-tag according to SEQ ID NO:246) with terminal lysine residues (K447) removed were conjugated to biotin-cadavarine, or GGGYK-Biotin.
- Assay conditions for carrying out the mTG2-mediated conjugation reactions were as follows:
- Cetuximab (according to the invention) 0.5 mg/ml
- TAM RA/TAM RA-cadaverine 50 molar
- cetuximab comprising the inventive MTG-tag1 , or MTG-tag2 was fluorescently labled, while wild-type cetuximab without the inventive polypeptide sequences according to SEQ ID NO:90, SEQ ID NO:246 is not fluorescently labeled indicating that the inventive MTG tags were efficiently recognized my mTG2.
- ADCs Antibody drug conjugates
- MTG-promoted conjugation reactions Antibody drug conjugates (ADCs) were synthesized in MTG-catalyzed reactions containing 1 mg/ml cetuximab tagged with SEQ ID NO: 246 (e.g. cetuximab comprising at SEQ ID NO: 246 at the carboxyterminus of each of the heavy chains), 20 equivalents of cytotoxic payload 1-4, and 0.1 equivalents of microbial transglutaminase (MTG). Reactions were performed in PBS pH 7.4 at 37°C for 16h and stopped by the addition of 1 volume of HIC buffer A.
- SEQ ID NO: 246 e.g. cetuximab comprising at SEQ ID NO: 246 at the carboxyterminus of each of the heavy chains
- MTG microbial transglutaminase
- ADCs were evaluated by hydrophobic interaction chromatography (HIC) on a TSKgel Butyl- NPR column (Tosoh Bioscience, 4.6 mm x 3.5 cm, 2.5 pm) using an Agilent Infinity 1260 HPLC.
- the HIC method was applied using a mobile phase of 50 mM NaH 2 P0 4 , 1.5 M (NH 4 ) 2 S0 2 pH 7.5 (Buffer A) and 50 mM NaH 2 P0 4 pH 7.5 (Buffer B).
- ADCs (45 pg) in 0.75 M (NH 4 ) 2 S0 2 were loaded and eluted with a gradient consisting of 2.5 min 0% Buffer B followed by a linear gradient into 100% Buffer B over 25 min with a flow rate of 0.9 ml/min.
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CA2994741A CA2994741A1 (en) | 2015-08-07 | 2016-08-04 | A transglutamine tag for efficient site-specific bioconjugation |
JP2018506297A JP7008014B2 (en) | 2015-08-07 | 2016-08-04 | Transglutamine tags for effective site-specific bioconjugation |
CN201680046587.7A CN107922479A (en) | 2015-08-07 | 2016-08-04 | Transglutamin-ase 9 label for effective locus specificity Bioconluaate |
EP16753248.0A EP3331904B1 (en) | 2015-08-07 | 2016-08-04 | A transglutamine tag for efficient site-specific bioconjugation |
US15/750,725 US11130818B2 (en) | 2015-08-07 | 2016-08-04 | Transglutamine tag for efficient site-specific bioconjugation |
AU2016306907A AU2016306907A1 (en) | 2015-08-07 | 2016-08-04 | A transglutamine tag for efficient site-specific bioconjugation |
ES16753248T ES2855998T3 (en) | 2015-08-07 | 2016-08-04 | Transglutamine tag for efficient site-specific bioconjugation |
IL256810A IL256810B (en) | 2015-08-07 | 2018-01-09 | A transglutamine tag for efficient site-specific bioconjugation |
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WO2019057772A1 (en) | 2017-09-19 | 2019-03-28 | Paul Scherrer Institut | Transglutaminase conjugation method and linker |
WO2020112588A1 (en) * | 2018-11-30 | 2020-06-04 | Bristol-Myers Squibb Company | Antibody comprising a glutamine-containing light chain c-terminal extension, conjugates thereof, and methods and uses |
WO2020188061A1 (en) | 2019-03-19 | 2020-09-24 | Paul Scherrer Institut | Transglutaminase conjugation method with a glycine based linker |
EP3865155A1 (en) | 2020-02-13 | 2021-08-18 | Orano Med | Process for site-specific modification of an antibody |
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 |
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 |
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WO2017147542A2 (en) * | 2016-02-26 | 2017-08-31 | Regeneron Pharmaceuticals, Inc. | Optimized transglutaminase site-specific antibody conjugation |
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- 2016-08-04 JP JP2018506297A patent/JP7008014B2/en active Active
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Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2019057772A1 (en) | 2017-09-19 | 2019-03-28 | Paul Scherrer Institut | Transglutaminase conjugation method and linker |
WO2020112588A1 (en) * | 2018-11-30 | 2020-06-04 | Bristol-Myers Squibb Company | Antibody comprising a glutamine-containing light chain c-terminal extension, conjugates thereof, and methods and uses |
WO2020188061A1 (en) | 2019-03-19 | 2020-09-24 | Paul Scherrer Institut | Transglutaminase conjugation method with a glycine based linker |
EP3865155A1 (en) | 2020-02-13 | 2021-08-18 | Orano Med | Process for site-specific modification of an antibody |
WO2021160724A1 (en) | 2020-02-13 | 2021-08-19 | Orano Med | Process for site-specific modification of an antibody |
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 |
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 |
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ES2855998T3 (en) | 2021-09-27 |
IL256810A (en) | 2018-03-29 |
CA2994741A1 (en) | 2017-02-16 |
JP7008014B2 (en) | 2022-02-10 |
EP3331904B1 (en) | 2020-12-23 |
IL256810B (en) | 2021-10-31 |
US20190194344A1 (en) | 2019-06-27 |
AU2016306907A1 (en) | 2018-03-29 |
WO2017025179A8 (en) | 2018-04-19 |
AU2016306907A9 (en) | 2019-07-25 |
US11130818B2 (en) | 2021-09-28 |
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JP2018529321A (en) | 2018-10-11 |
CN107922479A (en) | 2018-04-17 |
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