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WO2024251826A1 - Glyco-engineered antibody-drug-conjugates comprising an oxime linker - Google Patents

Glyco-engineered antibody-drug-conjugates comprising an oxime linker Download PDF

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Publication number
WO2024251826A1
WO2024251826A1 PCT/EP2024/065484 EP2024065484W WO2024251826A1 WO 2024251826 A1 WO2024251826 A1 WO 2024251826A1 EP 2024065484 W EP2024065484 W EP 2024065484W WO 2024251826 A1 WO2024251826 A1 WO 2024251826A1
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Prior art keywords
antibody
glycan
linker
adc
drug
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French (fr)
Inventor
Nico Callewaert
Berre VAN MOER
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Universiteit Gent
Vlaams Instituut voor Biotechnologie VIB
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Universiteit Gent
Vlaams Instituut voor Biotechnologie VIB
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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
    • A61K47/68033—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a maytansine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1075—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of amino acids or peptide residues

Definitions

  • the present invention relates to the field of antibody-drug-conjugates (ADCs) and the application of glyco-engineering strategies in a novel site-specific conjugation technology.
  • ADCs antibody-drug-conjugates
  • the invention particularly relates to ADCs wherein a drug entity is conjugated an aminooxy-functionalized linker at di- or trisaccharide N-glycans in the hinge region, and methods for producing the same. More particularly, said conjugate is obtained through oxidized galactose- or sialyl-groups obtained from glyco-engineered eukaryotic cells which produce antibodies with an N-glycan structure that is limited to a shortened Asn- GlcNac-Gal(-Sia) branch in the hinge region of the antibody.
  • ADC Antibody-drug conjugates
  • ADCs are generated from three essential components: the antibody, the toxin and the linker in between, all contributing to the overall activity.
  • a first important feature is the targeted antigen, which is dictated by the antigen-binding domain of the antibody part of the ADC.
  • the antigen should be highly, and preferably uniquely, expressed on cells, such as tumor cells with little to no expression on healthy or undesired cells or tissue, to limit (off-target) toxicity. Moreover, efficacy improves when the antigen is uniformly expressed in the tumor tissue, rendering more cells susceptible to toxin - mediated killing. Additionally, the antigen should be accessible for binding with circulating antibodies, with the resulting interaction triggering internalization efficiently.
  • the toxin Upon internalization, the toxin is released from the ADC, either by complete degradation of the antibody or by specific cleavage of the linker moiety, to subsequently perform its cytotoxic function.
  • TME tumor micro-environment
  • alternatives that lack intracellular entry in their conjugated composition could also be functional if the toxin is released in the tumor micro-environment (TME) in a membrane - penetrable configuration, for instance by incorporating cleavage sites for metalloproteases enriched in the tumor microenvironment.
  • TEE tumor micro-environment
  • a last, debatable, requirement for antigens is the affinity of the interaction with the conjugated antibody.
  • trastuzumab is used as anti-tumor therapy both as such and in ADCs (Kadcyla (trastuzumab emtansine) and Enhertu (trastuzumab deruxtecan)), while brentuximab is only employed as a conjugate with MMAE in Adcetris (brentuximab vedotin). Possibly, this difference may be due to differential susceptibility of the specific tumors to antibody - mediated clearance.
  • the second crucial part of ADCs is the linker, which connects the antibody and the toxin and controls the stability of the conjugate.
  • the ADC is sufficiently stable in serum and does not prematurely release the toxin, which would result in systemic toxicity. Nevertheless, upon internalization in the targeted cells, the toxin should be readily releasable to perform its toxic function.
  • the final component of ADCs is the toxin (also designated as the payload), which triggers cell death in targeted cells.
  • a common feature is their potency, with IC 5 o values in or below the nM range, which is required due to the limited amount of antibody that reaches the tumor site, estimated at 0.001 - 0.01 % (Epenetos et al. Cancer Res. 1986;46(6):3183-3191).
  • Other requirements for toxins to be used in ADCs are a suitable handle for conjugation, or flexibility to allow incorporation of one; solubility in aqueous buffers; and limited hydrophobicity to reduce aggregation propensity, which would lead to shorter shelf life, faster clearance, immunogenicity and increased efflux pumping.
  • ADCs typically apply coupling to lysine- and coupling to interchain cysteine-based technologies which lack site-specificity and/or introduce structural instability.
  • Next generation ADCs typically favor more site-specific conjugation, yet so far, these require expensive nutrients during expression (noncanonical amino acids and glycan remodeling), incorporate potentially immunogenic unnatural tags (enzymatic approaches), and/or reduce structural stability. Accordingly, there exists a need for improved ADC coupling technology.
  • N-glycosylation of antibodies as a handle for site-specific conjugation to a linker-toxin moiety. More specifically, application of enzymatic Galactose oxidase activity leads to oxidation of terminal galactose residues to aldehydes, which aldehydes can be used in bio-orthogonal chemistry coupling. The resulting galactose aldehydes provide for a position for oxime ligation coupling with aminooxy-linker-toxin moieties. Furthermore, the position of N-glycosylation of the antibody was altered by introducing a neo-N-glycan site in the hinge region of antibodies to allow efficient Galactose oxidation and conjugation into novel ADCs.
  • an antibody-drug-conjugate which comprises or consists of an antibody which has an N-glycan acceptor site in the antibody hinge region and an aminooxy-linker-drug-entity, wherein said aminooxy-linker-drug-entity is coupled to the asparagine side chain of said N-glycan acceptor site through oxime ligation of the aldehyde present on the oxidized galactose residue of the disaccharidic Galactose-N-acetylglucosamine (Gal-GIcNac) or alternatively via the sialic acid residue of the trisaccharidic Sialyl-Galactose-N-acetylglucosamine (Sia-Gal-GIcNAc) glycan chain.
  • ADC antibody-drug-conjugate
  • the ADC is provided with an antibody format comprising an Fc-tail, preferably a monoclonal antibody, more preferably an IgG antibody, more preferably a human IgGl, lgG2, or lgG4 antibody.
  • an Fc-tail preferably a monoclonal antibody, more preferably an IgG antibody, more preferably a human IgGl, lgG2, or lgG4 antibody.
  • the ADC comprises an acceptor N-glycan site in the hinge region which is located at a position corresponding to amino acid 221 from the human IgGl hinge region sequence (according to EU numbering), wherein said position is an asparagine which is introduced by substitution of the native amino acid (D in human IgGl). More specifically, said substitution as present at position 6 of the hinge region of SEQ. ID NO:1.
  • the linker of the aminooxy-linker-drug-entity (or the oxime-linker-drug-entity as used interchangeably herein) in the ADC comprises or consists of a non-cleavable and/or stable linker, such as for instance a Polyethyleneglycol-based linker.
  • the drug-entity in the ADC comprises or consists of an auristatin-based drug, such as monomethyl auristatin F (MMAF) or Monomethyl auristatin E (MMAE), or an anti - mitotic drug, such as mertansine (DM1), or a DNA topoisomerase I inhibitor, such as Exatecan derivative (Dxd) .
  • an auristatin-based drug such as monomethyl auristatin F (MMAF) or Monomethyl auristatin E (MMAE)
  • an anti - mitotic drug such as mertansine (DM1)
  • Dxd DNA topoisomerase I inhibitor
  • the ADC comprises of consists of an N-glycan- aminooxy-linker-drug-entity selected from the group of Asn-GIcNAc-Gal-oxime-PEG-MMAF (for which the chemical formula is shown in Figure 18 as a non-limiting example), Asn-GIcNAc-Gal-Sia-oxime-PEG-MMAF, Asn-GIcNAc-Gal-oxime- linker-MMAE, Asn-GIcNAc-Gal-Sia-oxime-linker-MMAE, Asn-GIcNAc-Gal-oxime-linker-DMl, or Asn- GIcNAc-Gal-Sia-oxime-linker-DMl.
  • Asn-GIcNAc-Gal-Sia-oxime-linker-DMl for which the chemical formula is shown in Figure 18 as a non-limiting example
  • Asn-GIcNAc-Gal-Sia-oxime-PEG-MMAF for which the chemical
  • an antibody-drug-conjugate comprising the steps of: a. expressing an antibody comprising an antigen-binding domain specifically binding a surface exposed antigen on an eukaryotic cell, preferably a higher eukaryotic cell such as a mammalian cell, wherein said antibody has an N-glycan acceptor site in the hinge region, b.
  • eukaryotic cell has a deficiency in the N-acetyl glucosaminyl-transferase I (GNT I) enzyme and said eukaryotic cell comprises a gene encoding for an endoglucosaminidase enzyme, thereby providing for a 'Glycodelete (GD)' eukaryotic cell.
  • GNT I N-acetyl glucosaminyl-transferase I
  • the eukaryotic cell further comprises a deficiency in bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) activity, thereby providing for a 'Gycodelete LacNac (GDLN)' eukaryotic cell.
  • the linker is an aminooxy-containing molecule for ligation to the aldehyde present after oxidation of Gal or Sia on the N-glycan to form the oxime coupling, preferably a non-cleavable linker, such as a linker comprising Polyethyleneglycol.
  • the drug-entity comprises or consists of an auristatin-based drug, such as monomethyl auristatin F (MMAF) or Monomethyl auristatin E (MMAE), or an anti - mitotic drug, such as mertansine (DM1), or a DNA topoisomerase I inhibitor, such as Exatecan derivative (Dxd).
  • auristatin-based drug such as monomethyl auristatin F (MMAF) or Monomethyl auristatin E (MMAE)
  • an anti - mitotic drug such as mertansine (DM1)
  • Dxd DNA topoisomerase I inhibitor
  • the oxidation is performed using enzymatic or chemical oxidation, preferably using Galactose oxidase or sodium periodate, respectively.
  • an antibody-drug conjugate is obtained by the methods as herein before described.
  • N-glycan moieties are indicated as generally known by: a square corresponding to a N-acetylglucosamine (GIcNAc) moiety; diamond to sialic acid (Sia); light circle to galactose (Gal) ; dark circle: mannose (Man); triangle: fucose (Fuc).
  • GIcNAc N-acetylglucosamine
  • T-DM1 (commercial Kadcyla, trastuzumab DM1 conjugated)
  • trastuzumab containing the neo-glycan site N221 was expressed to a similar extent as trastuzumab wild-type (WT), both in HEK293S WT cells and in ExpiCHO cells, with efficient N- glycosylation of the N221 variant as indicated by the height of the band visible in the N221 sample lane.
  • WT trastuzumab wild-type
  • HEK293S WT and ExpiCHO cells were transfected with pcDNA3.3 vectors encoding the light and heavy chains for trastuzumab WT and N221 expression.
  • the cells were pelleted, and the resulting supernatant was analyzed for antibody by SDS PAGE and western blotting with anti-hlgG detection (heavy chain (HC) only detected) (Left).
  • the ExpiCHO expressed trastuzumab WT and N221 were treated with Endo F2 and analyzed by SDS PAGE and CBB staining (Right).
  • the molecular mass of the light chain ( LC) corresponds to 25 kDa, and of the HC to 50 kDa.
  • the molecular weight ladder used is Precision Plus ProteinTM All Blue (BioRad).
  • Trastuzumab N221 was efficiently trimmed in HEK293S GlycoDelete LacNAc (GDLN) and carried high levels of Gal-GIcNAc disaccharide at both sites (N297 and N221). Trastuzumab N221 was expressed, without hGalTl or precursors, in HEK293 GlycoDelete LacNAc (GDLN) cells. Following purification, antibody was digested with FabRICATOR, which cleaves in the hinge region, conveniently separating the N221 N-glycan from the N297 N-glycan. The detected masses corresponded to the light chain (VL-CL), N-terminal fragment of the heavy chain, carrying the N221 site (VH-CH1) and the C- terminal fragment, carrying the N297 site (CH2-CH3).
  • FIG. 5 HER2 affinity of trastuzumab was only slightly reduced upon introducing the neo-N- glycosylation site N221.
  • Trastuzumab WT and N221 were compared in an ELISA setup to assess their binding to coated HER2 (left panel). The right panel incorporates the calculated EC 5 o values.
  • Figure 6. The binding of CD16a for trastuzumab is located at the CH2 domain near the hinge, in the vicinity of the neo-N-glycosylation site N221. Possibly, some affinity loss may be caused by N- glycosylation at this site.
  • the Left structural view shows a side view, while the Right structural view shows the frontal view of the interaction between the Fc domain and CD16a.
  • the introduced neo-N- glycosylation site N221 is indicated in black.
  • Figure 9 Increasing the GaOx units during oxidation resulted in aldehydes generated at both N- glycosylation sites (N221 and N297).
  • Trastuzumab WT and N221 were incubated overnight with variable amounts of GaOx (ranging from 0.66U/nmol (N221 selective condition) to 660U/nmol (conditions described in Angelastro et al.), in the presence of 100 equivalents of aminooxy-PEG-biotin, for subsequent SDS PAGE for western blotting for streptavidin-based detection.
  • the Left panel shows the oxidation for trastuzumab expressed in HEK293S GDLN, while the Right panel shows trastuzumab expressed in HEK293S WT cells.
  • the 0.66 U/nmol condition (boxed) is the condition used in house, while the 660 u/nmol ( boxed) indicates the condition used by Angelastro et al.
  • the molecular weight ladder used is Precision Plus ProteinTM All Blue (BioRad).
  • FIG. 10 Oxidation by GaOx resulted in loss of FabRICATOR cleavage of trastuzumab N221.
  • Trastuzumab N221 was oxidized overnight with GaOx in the presence of aminooxy-PEG-biotin (abbreviated as AmOx-Biotin).
  • AmOx-Biotin aminooxy-PEG-biotin
  • GaOx and/or biotin were omitted.
  • the antibody was incubated overnight with varying units of FabRICATOR, and subsequently analyzed by non-reducing SDS PAGE for CBB staining.
  • the upper panel shows the resulting CBB-stained gel, while the lower panel indicates the cleavage site of FabRICATOR, adapted from Buecheler et al.
  • FIG. 12 More MMAF conjugated to trastuzumab N221, expressed in HEK293S WT, after the second conjugation.
  • Trastuzumab N221 was oxidized overnight with 0.66U/nmol GaOx and 42.9 equivalents aminooxy - MMAF (WT #1 and LN) or with 250 equivalents (WT #2).
  • the antibodies were purified by protein A chromatography and desalted to PBS, and analyzed by SDS PAGE for western blotting using an anti - MMAF detection antibody.
  • the molecular weight ladder used is Precision Plus ProteinTM All Blue (BioRad).
  • Trastuzumab N221 was conjugated partially with MMAF. Trastuzumab N221 was oxidized overnight with 0.66U/nmol GaOx and 42.9 equivalents aminooxy - MMAF (WT #1 and GDLN), or 250 equivalents - MMAF (WT #2). After PPD catalysis of the conjugation, the antibodies were purified by protein A chromatography and desalted to PBS. The purified proteins were then incubated overnight with aminooxy-PEG-biotin, to check for unreacted aldehydes. As control, non-conjugated trastuzumab N221 (HEK wt and GDLN expressed) ("Stock”) was included with and without GaOx ("+GO").
  • Stock non-conjugated trastuzumab N221 (HEK wt and GDLN expressed)
  • MMAF conjugation of trastuzumab expressed in HEK293S WT was repeated once more, when a new batch of AmOxMMAF arrived, and was indicated as #2.
  • the molecular weight ladder used is Precision Plus ProteinTM All Blue (BioRad).
  • HER2-affinity of trastuzumab was not drastically reduced upon conjugating to the neo-N- glycosylation site N221.
  • Trastuzumab WT and N221 variants, concerning expression system and MMAF conjugation, were compared in an ELISA setup to assess their binding to coated HER2 (Left).
  • the right panel incorporates the calculated EC 5 o values.
  • Trastuzumab - MMAF was only cytotoxic to SK-BR-3 cells when linked through GlycoDelete LacNAc (GDLN) N-glycans. Antibody and antibody-drug conjugate treatments were tested for toxicity at HER2 expressing breast cancer cells (SK-BR-3). Viability was measured after a three day incubation by ATP measurement (CellTiter-Glo) and was plotted in relation to untreated cells. Error bars were calculated for biological repeats. Trastuzumab N221 (WT) was conjugated twice with MMAF (#1 versus #2), with the second repeat more efficiently conjugated which resulted in a higher DAR. Figure 16.
  • ADC treatments were tested for toxicity at HER2 expressing breast cancer cells (SK-BR-3). Viability was measured after a three day incubation by ATP measurement (CellTiter-Glo) and was plotted in relation to untreated cells (upper graph), while the calculated EC 5o was plotted (lower graph). Error bars were calculated for biological repeats.
  • FIG. 17 Bystander effect observed only for Enhertu in cocultures of SK-BR-3 and MDA-MB-231 cells.
  • ADC treatments were tested for toxicity at breast cancer cells. Viability was measured after a three day incubation by ATP measurement (CellTiter-Glo) and was plotted in relation to untreated cells. The upper panel shows the lack of toxicity for the HER2 negative MDA-MB-231 cells. The lower panel shows the toxicity in the coculture of SK-BR-3 and MDA-MB-231 cells.
  • Figure 18 Chemical formula of the Asn-GIcNAc-Gal-oxime-PEG-MMAF portion of the trastuzumab ADC produced in HEK GDLN.
  • Chemical or enzymatic oxidation approaches can be used to generate aldehydes in glycoproteins.
  • Chemical oxidation with periodate generates aldehydes from vicinol diols such as sialic acid and the backbone hydroxyl groups in, for instance, fucose.
  • vicinol diols such as sialic acid and the backbone hydroxyl groups in, for instance, fucose.
  • galactose oxidase can be used to selectively oxidize galactose at its C6 position.
  • the Left part shows the specific oxidation for each monosaccharide, while the Right part shows the impact on antibody glycosylation, with the substrate indicated with a gray background.
  • FIG. 20 Schematic representation of glyco-engineering required to provide for Glycodelete-LacNac (GDLN) production in a mammalian host cells.
  • GnT-l N acetylglucosaminyltransferase I
  • GNE bifunctional UDP - N - acetylglucosamine 2 - epimerase/N - acetylmannosamine kinase.
  • FIG. 21 Mechanism of action of antibody-drug conjugates generated with GlycoDelete LacNAc.
  • ADCs Antibody-drug conjugates
  • FIG. 22 GlycoDelete processing is required for efficient cytotoxicity of MMAF conjugated antibodies in the SK-BR-3 cell line.
  • A Schematic representation of the GDLN-ADC.
  • B Chemical representation of the drug-linker compound.
  • C Viability of SK-BR-3 cells in function of ADC concentration. ADC treatments were tested for toxicity at HER2 expressing breast cancer cells (SK-BR-3). Viability was measured after a five day incubation by ATP measurement (CellTiter-Glo) and was plotted in relation to live cells and dead cells. Error bars were calculated for biological repeats. The proposed active metabolite of the oxime- ligated ADCs is shown on the right. T-DM1 (or Kadcyla) and Enhertu are included as clinical comparators. Treatments E and F are included as unconjugated antibodies.
  • FIG. 23 GlycoDelete processing is required for efficient cytotoxicity of MMAE conjugated antibodies in the SK-BR-3 cell line.
  • A Schematic representation of the GDLN-ADC.
  • B Chemical representation of the drug-linker compound.
  • C Viability of SK-BR-3 cells in function of ADC concentration. ADC treatments were tested for toxicity at HER2 expressing breast cancer cells (SK-BR-3). Viability was measured after a five day incubation by ATP measurement (CellTiter-Glo) and was plotted in relation to live cells and dead cells. Error bars were calculated for biological repeats. The proposed active metabolite of the oxime- ligated ADCs is shown on the right. T-DM1 (or Kadcyla) and Enhertu are included as clinical comparators. Treatments E and F are included as unconjugated antibodies.
  • FIG. 24 GlycoDelete processing is required for efficient cytotoxicity of DM1 conjugated antibodies in the SK-BR-3 cell line.
  • A Schematic representation of the GDLN-ADC.
  • B Chemical representation of the drug-linker compound.
  • C Chemical representation of the conjugation in Kadcyla (T-DM1).
  • D Viability of SK-BR-3 cells in function of ADC concentration. ADC treatments were tested for toxicity at HER2 expressing breast cancer cells (SK-BR-3). Viability was measured after a five day incubation by ATP measurement (CellTiter-Glo) and was plotted in relation to live cells and dead cells. Error bars were calculated for biological repeats. The proposed active metabolite of the oxime-ligated ADCs is shown on the right. T-DM1 (or Kadcyla) is included as clinical comparator, next to the unconjugated antibodies.
  • FIG. 25 GDLN-ADC with auristatins are as potent in the SK-BR-3 cell line, if not more potent, than conventional ADC conjugated via interchain disulphide cysteines, despite lower DAR.
  • A-B Schematic representation of maleimide-based conjugation of auristatins to antibodies
  • C-D Chemical representation of the maleimide-functionalized auristatin-linker compounds.
  • E Viability of SK-BR-3 cells in function of ADC concentration. ADC treatments were tested for toxicity at HER2 expressing breast cancer cells (SK-BR-3). Viability was measured after a five day incubation by ATP measurement (CellTiter- Glo) and was plotted in relation to live cells and dead cells.
  • T-DM1 or Kadcyla
  • FIG. 26 GlycoDelete processing is required for efficient cytotoxicity of MMAF conjugated antibodies in the BT-474 cell line.
  • A Schematic representation of the GDLN-ADC.
  • B Chemical representation of the drug-linker compound.
  • C Viability of BT-474 cells in function of ADC concentration. ADC treatments were tested for toxicity at HER2 expressing breast cancer cells (BT-474). Viability was measured after a six day incubation by ATP measurement (CellTiter-Glo) and was plotted in relation to live cells and dead cells. Error bars were calculated for biological repeats. The proposed active metabolite of the oxime-ligated ADCs is shown on the right. T-DM1 (or Kadcyla) and Enhertu are included as clinical comparators. Treatments E and F are included as unconjugated antibodies.
  • FIG. 27 GlycoDelete processing is required for efficient cytotoxicity of MMAE conjugated antibodies in the BT-474 cell line.
  • A Schematic representation of the GDLN-ADC.
  • B Chemical representation of the drug-linker compound.
  • C Viability of BT-474 cells in function of ADC concentration. ADC treatments were tested for toxicity at HER2 expressing breast cancer cells (BT-474). Viability was measured after a six day incubation by ATP measurement (CellTiter-Glo) and was plotted in relation to live cells and dead cells. Error bars were calculated for biological repeats. The proposed active metabolite of the oxime-ligated ADCs is shown on the right. T-DM1 (or Kadcyla) and Enhertu are included as clinical comparators. Treatments E and F are included as unconjugated antibodies.
  • FIG. 28 GDLN-ADC with auristatins are more potent in the BT-474 cell line than conventional ADC conjugated via interchain disulphide cysteines, despite lower DAR.
  • A-B Schematic representation of maleimide-based conjugation of auristatins to antibodies
  • C-D Chemical representation of the maleimide-functionalized auristatin-linker compounds.
  • E Viability of BT-474 cells in function of ADC concentration. ADC treatments were tested for toxicity at HER2 expressing breast cancer cells (BT-474). Viability was measured after a six day incubation by ATP measurement (CellTiter-Glo) and was plotted in relation to live cells and dead cells. Error bars were calculated for biological repeats. The proposed active metabolite of the oxime-ligated ADCs is shown on the right. T-DM1 (or Kadcyla) is included as clinical comparator, next to the unconjugated antibodies.
  • FIG. 29 Western blot validation of toxin conjugation. Toxin was successfully conjugated to the antibody of interest as indicated by the anti-toxin specific western blots. Fc-detection was used to normalize the amount of antibody, while different anti-toxin detections were used specifically for each toxin: DM1, MMAF and MMAE. The molecular weight ladder used is the Precision Plus ProteinTM All Blue Prestained Protein Standards (Biorad, 1610393). Abbreviations: Mai: maleimide, AmOx: aminooxy, LN: GlycoDelete LacNAc, WT: wild type HEK expressed (oligosaccharide).
  • protein and “polypeptide” are interchangeably used further herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same.
  • peptide refers to a polymer of amino acid residues and variants and synthetic analogues of the same, wherein the peptide is of shorter length as compared to a protein or polypeptide, potentially as a result of protein digestion or proteolytic cleavage, preferably with a length of maximally 40-100 amino acids, most preferably not more than 50 amino acids.
  • recombinant (poly)peptide is meant a (poly)peptide made using recombinant techniques, i.e., through the expression of a recombinant or synthetic polynucleotide, which may be obtained in vitro and/or in a cellular context.
  • the chimeric (poly)peptide or biologically active (i.e. functional) portion thereof is recombinantly produced, it is also preferably purified, or isolated, as used interchangeably herein, or substantially free of culture medium, i.e., the impurities represent less than about 20 %, more preferably less than about 10 %, and most preferably less than about 5 % of the volume of the protein preparation.
  • isolated or “purified” is meant material that is substantially or essentially free from components that normally accompany it in its native state.
  • enriched or “enrichment” is meant herein that material or specific components are present at a substantially higher amount as compared to the non-enriched material, so typically involving a purification or isolation step.
  • expression vector includes any vector known to the skilled person, including plasmid vectors, cosmid vectors, phage vectors, such as lambda phage, viral vectors, such as adenoviral, AAV or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or Pl artificial chromosomes (PAC).
  • Expression vectors generally contain a desired coding sequence and appropriate promoter sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g. higher eukaryotes, lower eukaryotes).
  • a vector comprises a nucleotide sequence in which an expressible promoter or regulatory nucleotide sequence is operatively linked to, or associated with, a nucleotide sequence or DNA region that codes for an mRNA, such that the regulatory nucleotide sequence is able to regulate transcription or expression of the associated nucleotide sequence.
  • a regulatory nucleotide sequence or promoter of the vector is not operatively linked to the associated nucleotide sequence as found in nature, hence is heterologous to the coding sequence of the DNA region operably linked to.
  • operatively or “operably” “linked” as used herein refers to a functional linkage between the expressible promoter sequence and the DNA region or gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest, and refers to a functional linkage between the gene of interest and the transcription terminating sequence to assure adequate termination of transcription in eukaryotic cells.
  • linkage or fusion between a targeting sequence and the open reading frame of an enzyme.
  • inducible promoter refers to a promoter that can be switched 'on' or 'off' (thereby regulating gene transcription) in response to external stimuli such as, but not limited to, temperature, pH, certain nutrients, specific cellular signals, et cetera. It is used to distinguish between a "constitutive promoter", by which a promoter is meant that is continuously switched 'on', i.e. from which gene transcription is constitutively active.
  • Binding means any interaction, be it direct or indirect.
  • a direct interaction implies a contact between the binding partners.
  • An indirect interaction means any interaction whereby the interaction partners interact in a complex of more than two molecules. The interaction can be completely indirect, with the help of one or more bridging molecules, or partly indirect, where there is still a direct contact between the partners, which is stabilized by the additional interaction of one or more molecules.
  • the binding or association maybe non-covalent - wherein the juxtaposition is energetically favoured by for instance hydrogen bonding or van der Waals or electrostatic interactions - or it may be covalent, for instance by peptide or disulphide bonds.
  • affinity generally refers to the degree to which a ligand, chemical, protein or peptide binds to another (target) protein or peptide so as to shift the equilibrium of single protein monomers toward the presence of a complex formed by their binding.
  • antibody refers to an immunoglobulin (Ig) molecule or a molecule comprising an immunoglobulin (Ig) domain, which specifically binds with an antigen.
  • Antibodies can further be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins.
  • antigen-binding domain refers to a portion of any antibody or antibody-like structure that by itself has high affinity for an antigenic determinant, or epitope, and contains one or more complementarity determining regions (CDRs) accounting for such specificity, typically at least 3 CDRs, or in conventional antibodies, such as monoclonal antibodies, defined by 6 CDRs.
  • CDRs complementarity determining regions
  • Non-limiting examples of antigen-binding domains or active antibody fragments include immunoglobulin domains, Fab, F(ab)'2, scFv, heavy-light chain dimers, immunoglobulin single variable domains (ISVDs), Nanobodies (or VHH antibodies), domain antibodies, and single chain structures, such as a complete light chain or complete heavy chain.
  • the term 'antibody' or 'Fc-fusion' as used herein further refers to the genetic linking or fusion of antigenbinding fragments or antigen-binding domains with an Fc constant domain as to obtain dimers forming an antibody structure when expressed in a recombinant host.
  • antibody fragments, or single domain antibodies such as ISVDs may be C-terminally fused to the N-terminus of an Fc domain, preferably via a linker or hinge region, as known in the art and/or as described herein.
  • IgG immunoglobulins
  • IgA immunoglobulin A
  • IgM immunoglobulin M
  • IgD immunoglobulin D
  • IgE immunoglobulins
  • IgG comprises the majority, representing 60 % of total serum Igs in humans.
  • human IgG class of antibodies the IgGl antibody subclass is most abundant, binds the FcR receptor and shows distinct properties compared with the lgG2, lgG3, and lgG4 subclasses and is the most exploited subclass in therapeutic antibodies.
  • the human IgG molecule is composed of two identical fragment antigen binding (Fab) domains and one fragment crystallizable (Fc) domain that make it multivalent and multifunctional.
  • the two Fab fragments each consist of a heterodimer of a light chain and the N-terminal part of the heavy chain, whereas the C -terminal half of the two heavy chains dimerizes to form the Fc fragment of the IgG antibody.
  • the N-terminal domains of the Fab fragment are the variable domains (V L and V H ) that are responsible for antigen recognition, whereas the C-terminal part of the heavy chains compose the Fc fragment that is responsible for humoral and cellular effector functions.
  • the two Fabs and the Fc-tail are connected by the hinge region, which facilitates the spatial alignment of the three moieties for binding to antigens and effector ligands.
  • the "hinge region” of an antibody is defined herein as the amino acid sequence for the antibody region that connects the antigen-binding region (or Fab region) and Fc regions together and contributes flexibility between these regions.
  • the hinge length is linked with IgG functionality, and is generally considered as a three-part structure, an upper, core, and lower section.
  • Fc domains or “Fc-regions” or “Fc-tails”, as interchangeably used herein, refer to the single Fc chain and/or the dimeric Fc domain of an Fc-containing proteins. Specifically in antibodies, said Fc domain is thus responsible for antibody function, and Antibody Fc engineering stands for engineering functions of antibodies, which are effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), and controlling serum half-life. Engineered Fc domains may therefore be present in the form of mutants or variants containing amino acid substitutions, insertions or deletions as to allow different modifications of the Fc in post-translational modifications, dimerization behavior, effector function, serum half-life, among others.
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • Fc region sequences often include the hinge region, as to provide for an Fc fragment that can be used for fusion to an antigen-binding domain, such as a Fab, or an ISVD.
  • an antigen-binding domain such as a Fab, or an ISVD.
  • conventional antibody numbering annotations are known in the art, such as for instance IMGT numbering (LeFranc, 2014; Frontiers in Immunology. 5 (22): 1-22), Kabat numbering (Kabat, E.A. et al., Sequences of proteins of immunological interest.
  • Antibodies can be obtained by immunization of animals or humans, or made recombinantly by introducing a particular exogenous sequence in a host cell for production of an antibody. Most common is the manufacturing of monoclonal antibodies (mAbs) which are human-like antibodies produced from a cell lineage made by cloning a unique white blood cell, and derived from mouse proteins (murine), human proteins, or chimeric (mouse/human parts) or humanized proteins.
  • mAbs monoclonal antibodies
  • glycoslation acceptor site refers to a position within a polypeptide which can be N- or O-glycosylated.
  • N-linked glycans are typically attached to the side chain of an Asparagine (Asn), while O-linked glycans are commonly linked to the hydroxyl oxygen side chain residue of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline sidechains.
  • Antibody Fc tails conventionally have an Asn at position 297 (EU numbering) functioning as N- glycan acceptor site, though alternative N-glycosylation sites may be present and/or introduced by substitution or mutation of single or multiple amino acids in the original antibody Fc part.
  • N-glycosylation acceptor site refers to a position within a polypeptide , such as an antibody, which can be N-glycosylated.
  • N-linked glycans are typically attached to Asparagine (Asn) which resides in a consensus site.
  • An "NXT”, “NXS”, “NXC” or “NXV” motif refers to the consensus sequences Asn-Xaa-Thr/Ser or Asn-Xaa-Cys/Val, wherein Xaa can be any amino acid except proline (Shrimal, S. and Gilmore, R., J Cell Sci. 126(23), 2013, Sun, S.
  • N-glycosylation acceptor sites are specific to the consensus sequence Asn-Xaa-Thr/Ser or Asn-Xaa-Cys/Val. It has been shown in the art that the presence of proline between Asn and Thr/Ser leads to inefficient N- glycosylation.
  • glycoproteins Glycosylation sites and glycan structures present on proteins, referred to herein as glycoproteins, can be determined in part by enrichment of glycopeptides, or by enzymatically or chemically releasing glycans.
  • the structure of released glycans and remaining peptides can be determined by mass spectrometry and liquid chromatography/mass spectrometry. Fragmentation techniques can be used to obtain glycan structures and amino acid sequences of the peptide backbone of glycopeptides with mass spectrometry, though often a low fragmentation efficiency is obtained with glycopeptides, and customized approaches are often desired to a certain analytical setting.
  • glycocan generally refers in the art to glycosidically linked monosaccharides, oligosaccharides and polysaccharides. Hence, carbohydrate portions of a glycoconjugate, such as a glycoprotein, glycolipid, or a proteoglycan are referred to herein as a "glycan”. Glycans can be homo- or heteropolymers of monosaccharide residues, and can be linear or branched.
  • N-linked glycans may be composed of N-acetylgalactosamine (GalNAc), Galactose (Gal), neuraminic acid (such as sialic acid (Sia), most common being N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Glc), and N-acetyl-9- O-acetylneuraminic acid), N-acetylglucosamine (GIcNAc), Fucose (Fuc), Mannose (Man), and other monosaccharides, as also exemplified further herein.
  • GalNAc N-acetylgalactosamine
  • Gal Gal
  • neuraminic acid such as sialic acid (Sia)
  • Sia most common being N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Glc), and N-acetyl-9
  • O-linked glycans are assembled one sugar at a time on a serine or threonine residue of a peptide chain in the Golgi apparatus.
  • N-linked glycans there are no known consensus sequences but the position of a proline residue at either -1 or +3 relative to the serine or threonine is favourable for O-linked glycosylation.
  • Complex N-glycans in the art refers to structures with typically one, two or more (e.g. up to six) outer branches, most often linked to an inner core structure Man3GlcNAc2.
  • the term “complex N-glycans” is well known to the skilled person and defined in literature. For instance, a complex N-glycan may have at least one branch, or at least two, of alternating GIcNAc and optionally also Galactose (Gal) residues that may terminate in a variety of oligosaccharides but typically will not terminate with a Mannose residue.
  • 'Glycoproteins' as used in the application refers to proteins that, in their normal physiological context and/or their functional form, contain oligosaccharide chains (N-glycans) covalently attached to their polypeptide side-chains.
  • a glycoprotein comprises also proteins with an artificially introduced glycosylation site, particularly an artificially introduced N-glycosylation site.
  • a glycoprotein typically a recombinant glycoprotein, for example a heterologous recombinant glycoprotein (which does not occur normally in the eukaryotic organism) is produced as several glycoforms when it is made in a eukaryotic organism such as a N-glycosylation-engineered eukaryotic organism.
  • '(glyco)protein' and 'enzyme' e.g. endoglucosaminidase, glycosyltransferase, mannosidase, mannosyltransferase
  • 'enzyme' e.g. endoglucosaminidase, glycosyltransferase, mannosidase, mannosyltransferase
  • part of the protein may be sufficient to achieve an (e.g. therapeutic, enzymatic) effect.
  • variants i.e. proteins in which one or more amino acids have been substituted with other amino acids, but which retain functionality or even show improved functionality
  • variants of the enzymes optimized for enzymatic activity i.e. proteins in which one or more amino acids have been substituted with other amino acids, but which retain functionality or even show improved functionality
  • a glycoprotein refers to the protein itself; a glycoprotein may be either in its glycosylated or non-glycosylated form.
  • a 'glycosylated' protein is a (glyco)protein that carries at least one oligosaccharide chain.
  • An N-glycosylated protein, particularly an N-glycosylated recombinant glycoprotein, is a glycoprotein which carries at least one oligosaccharide chain on an N-glycan.
  • An 'endoglucosaminidase' as used herein refers to enzymes that hydrolyse the bond between the anomeric carbon of a non-terminal beta-linked N-acetylglucosamine residue in an oligosaccharide of a glycoprotein or a glycolipid, and its aglycon, thereby releasing mono- or oligosaccharides from glycoproteins or glycolipids or sugar polymers.
  • Endoglucosaminidases are a subset of the glycosidases, and may or may not have other enzymatic activities (such as e.g. glycosyltransferase activity).
  • a particular class of endoglucosaminidases is formed by the endo-p-N-acetylglucosaminidases or mannosyl- glycoprotein endo-p-/V-acetylglucosaminidases, indicated as EC 3.2.1.96 in the International Union of Biochemistry and Molecular Biology (IUBMB) nomenclature.
  • This particular class of enzymes are capable of catalyzing the endohydrolysis of the N, N '-diacetylchitobiosyl unit in high-mannose glycopeptides and glycoproteins containing the -[Man(GlcNAc)j]Asn- structure.
  • GIcNAc /V-acetyl-D-glucosamine residue
  • These enzymes can remove sugar chains (hybrid N- glycans, high mannose N-glycans and neoglycoforms of N-glycans as shown herein) while leaving one GIcNAc residue on the protein.
  • examples of these include, but are not limited to Endo A, Endo BH, Endo CE, Endo D, Endo Fl, Endo H, Endo M, Endo T (see also W02006/050584), and ENGase.
  • Other examples are known to the skilled person and can for instance be found on www.cazy.org, in particular under the Glycoside Hydrolase Family 85 and 18.
  • a 'glycosyltransferase' as used in the application is any of a group of enzymes that catalyze the transfer of glycosyl groups in biochemical reactions, in particular glycosyl transfer to asparagine-linked sugar residues to give N-linked glycoproteins.
  • Glycosyltransferases fall under EC 2.4 in the IUBMB nomenclature, a particular class of glycosyltransferases are hexosyltransferases (EC 2.4.1).
  • enzymes such as, but not limited to, N-acetylglucosaminyl transferases, N-acetylgalactosaminyltransferases, sialyltransferases, fucosyltransferases, galactosyltransferases, and mannosyltransferases.
  • Mannosyltransferases are excluded for specific embodiments of N-glycosylation- engineered yeast cells described in the application.
  • Mannosyltransferases' as used in the application refers to enzymes that catalyze the transfer of a mannosyl group to an acceptor molecule, typically another carbohydrate, in the Golgi apparatus.
  • Mannosyltransferases are typically endogenous enzymes in fungi and yeast and involved in the synthesis of high-mannose type glycans.
  • a "higher eukaryotic cell” as used herein refers to eukaryotic cells that are not cells from unicellular organisms.
  • a higher eukaryotic cell is a cell from (or derived from, in case of cell cultures) a multicellular eukaryote such as a human cell line or another mammalian cell line (e.g. a CHO cell line).
  • a multicellular eukaryote such as a human cell line or another mammalian cell line (e.g. a CHO cell line).
  • the term generally refers to mammalian cells, human cell lines and insect cell lines. More particularly, the term refers to vertebrate cells, even more particularly to mammalian cells or human cells.
  • the higher eukaryotic cells as described herein will typically be part of a cell culture (e.g. a cell line, such as a HEK or CHO cell line).
  • Yeast cells can be from the species Saccharomyces (e.g. Saccharomyces cerevisiae), Hansenula (e.g. Hansenula polymorpha), Arxula (e.g. Arxula adeninivorans), Yarrowia (e.g. Yarrowia lipolytica), Kluyveromyces (e.g. Kluyveromyces lactis), or Komagataella phaffii (Kurtzman, C.P. (2009) J Ind Microbiol Biotechnol.
  • Saccharomyces e.g. Saccharomyces cerevisiae
  • Hansenula e.g. Hansenula polymorpha
  • Arxula e.g. Arxula adeninivorans
  • Yarrowia e.g. Yarrowia lipolytica
  • Kluyveromyces e.g. Kluyveromyces lactis
  • the lower eukaryotic cells are Pichia cells, and in a most particular embodiment Pichia pastoris cells.
  • the filamentous fungus cell is Myceliopthora thermophila (also known as Cl by the company Dyadic), Aspergillus species (e.g. Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus japonicus), Fusarium species (e.g. Fusarium venenatum), Hypocrea and Trichoderma species (e.g. Trichoderma reesei).
  • the "lower or higher eukaryotic cell” or “eukaryotic cell” of the present invention is a glyco-engineered cell.
  • a “glyco-engineered cell” refers to a cell that has been genetically modified so that it expresses proteins with an altered N-glycan structure and/or O-glycan structure as compared to in a wild type background.
  • the naturally occurring modifications on glycoproteins have been altered by genetic engineering of enzymes involved in the glycosylation pathway.
  • sugar chains in N-linked glycosylation may be divided in three types: high-mannose (typically yeast), complex (typically mammalian) and hybrid type glycosylation.
  • O-glycan patterns exist, for example with yeast oligomannosylglycans differing from mucin-type O- glycosylation in mammalian cells.
  • the different types of N- and O-glycosylation are all well known to the skilled person and defined in the literature. Considerable effort has been directed towards the identification and optimization of strategies for the engineering of eukaryotic cells that produce glycoproteins having a desired N-and/or O-glycosylation pattern and are known in the art (e.g. De Pourcq, K. et al., Appl Microbiol Biotechnol. 87(5), 2010).
  • glyco-engineered cells or the glyco-engineered expression system which are used are described in patent applications W02010015722 and WO2015032899 (further designated herein as GlycoDelete cells, or cells having a GlycoDelete background) and in Meuris L. et al (2014) Nat. Biotechn.
  • beta-1, 4-galactosyltransferase' refers to an enzyme that has exclusive specificity for the donor substrate UDP-galactose; all transfer galactose in a betal,4 linkage to similar acceptor sugars: GIcNAc, Glc, and Xyl.
  • the beta-1, 4-galactosyltransferase adds galactose to N- acetylglucosamine residues that are either monosaccharides or the nonreducing ends of glycoprotein carbohydrate chains.
  • Particularly preferred beta-1, 4-galactosyltransferases are of the mammalian type, even more particularly are of the human type.
  • 'UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase' or abbreviated GNE enzyme in the eukaryotic cell of the present invention refers to a bifunctional enzyme that initiates and regulates the biosynthesis of N-acetylneuraminic acid (NeuAc), a precursor of sialic acids. It is a ratelimiting enzyme in the sialic acid biosynthetic pathway. The enzyme is allosterically regulated and hence is subject to feedback inhibition by cytidine monophosphate-N-acetylneuraminic acid (CMP-Neu5Ac), the end product of neuraminic acid biosynthesis.
  • CMP-Neu5Ac cytidine monophosphate-N-acetylneuraminic acid
  • a eukaryotic cell lacking 'UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase' or GNE in the present invention can refer to a lower eukaryotic cell which - by nature - does not possess this enzyme. Alternatively, it refers to a higher eukaryotic cell which has a gene disruption (or a gene deletion or a mutation which generates a non-functional enzyme) in this enzyme (also see Figure 20).
  • the ADC is produced in a Glycodelete background of a higher eukaryotic cell, i.e.
  • the above N-glycan structures may additionally comprise a fucose branched on the Asn-GIcNAc (e.g. as shown for the fucose (triangle) present in the structure of the right panel of Figure 19).
  • a therapeutically active agent or “therapeutically active composition” means any molecule or composition of molecules that has or may have a therapeutic effect (i.e., curative or prophylactic effect) in the context of treatment of a disease (as described further herein).
  • a therapeutically active agent is a disease-modifying agent, which can be a cytotoxic agent, such as a toxin, or a cytotoxic drug, or an enzyme capable of converting a prodrug into a cytotoxic drug, or a radionuclide, or a cytotoxic cell, or which can be a non-cytotoxic agent.
  • a therapeutically active agent has a curative effect on the disease.
  • the ADC, or pharmaceutical composition comprising the ADC of the invention may act as a therapeutically active agent, when beneficial in treating patients with a disease related to the target of the antibody part of the ADC its antigen, or at least with a disease that may benefit from a treatment of targeting the cells expressing the antigen of the antibody part of the ADC as described herein, or patients suffering from another disease.
  • subject relates to any organism such as a vertebrate, particularly any mammal, including both a human and another mammal, for whom diagnosis, therapy or prophylaxis is desired, e.g., an animal such as a rodent, a rabbit, a cow, a sheep, a horse, a dog, a cat, a lama, a pig, or a non-human primate (e.g., a monkey).
  • the rodent may be a mouse, rat, hamster, guinea pig, or chinchilla.
  • the subject is a human, a rat or a non-human primate.
  • the subject is a human.
  • a subject is a subject with or suspected of having a disease or disorder, in particular a disease or disorder as disclosed herein, also designated “patient” herein.
  • patient a disease or disorder as disclosed herein.
  • the aforementioned terms do not imply that symptoms are present.
  • treatment refers to a substance/composition used in therapy, i.e., in the prevention or treatment of a disease or disorder.
  • disease or disorder refer to any pathological state, in particular to the diseases or disorders as defined herein.
  • treatment or “treating” or “treat” can be used interchangeably and are defined by a therapeutic intervention that slows, interrupts, arrests, controls, stops, reduces, or reverts the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve a total elimination of all disease-related signs, symptoms, conditions, or disorders.
  • Therapeutic treatment is thus designed to treat an illness or to improve a person's health, rather than to prevent an illness.
  • Treatment may also refer to a prophylactic treatment which relates to a medication, or a treatment designed and used to prevent a disease from occurring.
  • the present invention provides for provide a novel innovative Antibody-drug conjugate (ADC) design and manufacturing method for producing the same, and is based on the surprising finding that site-specific coupling of payload via engineered N-glycan chains present in the hinge region. More specifically, by making use of N-glycan structures defined by a 'shortened branch' (GIcNac-Gal or GIcNac-Gal-Sia) homogenous structure obtained using Glycodelete or Glycodelete LacNac production hosts for the antibody of said ADC, the potency in cytotoxicity was positively impacted as compared to the use of wildtype bi-antennary N-glycan structures on the same antibody as conjugation site.
  • ADC Antibody-drug conjugate
  • the present application discloses for the first time that ADCs can be efficiently produced with GlycoDelete oxime-ligation-based coupling technology. Terminal galactose residues of the disaccharide N-glycan at the neo-glycan D221N acceptor site in the hinge region are oxidized by galactose oxidase into aldehydes. The resulting aldehydes subsequently react with aminooxy-functionalized toxins in so- called oxime ligation reactions.
  • This GlycoDelete-ADC technology yields ADCs (also referred to herein as GDLN-ADCs) that are more potent than ADCs that are generated via oxidation and oxime ligation with antibodies that have not been produced in GlycoDelete LacNAc host cells, comprising larger, bulkier and more heterogenous N-glycan chains (WT-ADCs).
  • WT-ADCs heterogenous N-glycan chains
  • GDLN-ADCs outperformed WT-ADCs when the toxin is conjugated via stable, non-cleavable linkers (exemplified herein with MMAF and DM1), as well as with the MMAE toxin that is conjugated via the valine-citrulline protease cleavable linker.
  • stable, non-cleavable linkers exemplified herein with MMAF and DM1
  • the impact of GlycoDelete processing is most evident for ADCs with non-cleavable linkers, which may stem from a difference in hydrophilicity of the active metabolite.
  • the active metabolite for DM1- and MMAF-conjugated GDLN-ADCs is the toxin-oxime linker-disaccharide- asparagine.
  • the WT-ADCs are assumed to be degraded to an active metabolite carrying an oligosaccharide instead of a disaccharide, with a larger size and an increased hydrophilicity which could delay or prevent its lysosomal escape into the cytosol (see also Figure 21).
  • the superiority of the in vitro cytotoxicity is less pronounced compared to WT-ADCs, which may be a consequence of the cathepsin cleavage of the dipeptide in the linker which eliminates at least partially the impact of the N-glycan hydrophilicity.
  • GDLN-ADCs still outperform WT-ADCs with cleavable linkers, leading to the mechanistic assumption that the protease degradation of the linker could be a rate-limiting step in lysosomal ADC maturation, which is less impactful for GDLN-ADCs capable of lysosomal escape, yielding a more potent ADC.
  • the DAR of GDLN-ADCs is limited by the number of hinge N-glycans and the galactosylation efficiency at DAR 1.6-2.0, while the maleimide-generated ADCs can be conjugated with up to a DAR of 8, or at least, as exemplified herein to a conjugation reaction optimized for DAR of 4.
  • a first aspect of the invention relates to an antibody-drug-conjugate (ADC) comprising an antibody which has an N-glycan acceptor site in the hinge region and an aminooxy- or oxime-linker-drug-entity coupled to the N-glycan of said N-glycan acceptor site, wherein said N-glycan consists of a Galactose- N- acetylglucosamine (Gal-GIcNAc) or Sialyl-Galactose- N-Acetylglucosamine (Sia-Gal-GIcNAc) glycan chain.
  • ADC antibody-drug-conjugate
  • the antibody-drug-conjugate comprises an antibody format which preferably comprises a monoclonal antibody, which has a GIcNAc-Gal-oxime-linker-drug entity or GIcNAc-Gal-Sia- oxime-linker-drug-entity on an N-glycan acceptor site present in the hinge region.
  • trisaccharidic glycan entity is providing a slightly higher hydrophilicity as compared to the disaccharidic glycan entity exemplified in the application because of the sialic acid presence, though still much lower in hydrophilicity as compared to the WT native N-glycan branches, making these also potent alternatives for the GDLN-ADC coupling technology disclosed herein.
  • an antibody-drug-conjugate comprising an antibody which has an N-glycan acceptor site in the hinge region and an oxime-linker-drug-entity, wherein said oxime- linker-drug-entity is coupled to the asparagine side chain of said N-glycan acceptor site via an oxidized form (which is presented herein as an aldehyde) of the galactose residue of Galactose-N- Acetylglucosamine (Gal-GIcNAc) or via an oxidized form of the sialic acid residue of Sialyl-Galactose-N- Acetylglucosamine (Sia-Gal-GIcNAc) glycan chain.
  • ADC antibody-drug-conjugate
  • the present invention relates to the provision of newly generated antibody-drug conjugates by oxidation of galactose or sialyl moieties present in glycan chains obtained from glycol-engineered antibody production hosts, and conjugation to the resulting aldehyde group on said sugar.
  • SEQ ID NO:1 hinge region of human IgGl
  • SEQ ID NO:2 D221N-mutant hinge region of human IgGl
  • SEQ ID NO:3 human IgGl Fc region (including the hinge region)
  • the middle hinge regions contain two conserved cysteine residues (Cys226 and Cys229) to form interchain disulfide bonds between the two heavy chains to join these together and the lower hinge sequence is relatively conserved and is responsible for the flexibility and positioning of the Fc region relative to the Fab arms and affects the binding of Fc to Fc_R.
  • an N-glycan acceptor site was introduced in the hinge region of human IgGl by substitution of the D221 to an N (Blundell et al. J Immunol (2019) 202 (5): 1595-1611). Said amino acid site is however not conserved in the hinge region of lgG2, 3, or 4 (as described for instance in Rayner et al. (2015; JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 13, pp. 8420-8438; see figure 2)).
  • the N-glycosylation acceptor site in the hinge region of an ADC with an antibody derived from an lgG3 may for instance be obtained by substituting the D in the upper region (position 8 of the upper hinge) to an N; or alternatively, for antibodies based on the lgG2 or lgG4 hinge region by introducing an Asn in the upper hinge.
  • the latter may be of particular interest when an ADC is desired lacking effector functions.
  • Antibodies without effector function can also be generated by removing the Asn at position N297 (and/or by enzymatically removing the Fc N-glycan).
  • VHH-Fc fusions as known in the art, known as potent antibodies, are generally composed of the VHH sequence (in monovalent or multivalent/multispecific format), linked (directly or via a linker, such as a GS-linker) to an Fc region, wherein said Fc region sequence may be based on the human IgGl, wherein the EPKSC N- terminal sequence is removed or replaced with EPKSS, thus both allowing to replace the D following to an N, as to obtain a hinge region with an N-glycan acceptor site.
  • HEK higher eukaryotic cell
  • Galactosylation in HEK cells was observed to be higher as compared to ExpiCHO cells initially, and could be further optimized to near completeness by hGalTl and precursors uridine, galactose and MnCL.
  • NaO 4 sodium periodate
  • alO 4 sodium periodate
  • several other comparable reagents known in the art can be used (also see figure 19).
  • the resulting aldehydes can subsequently be used in conjugations with aminooxy- and hydrazide-based linkers and reductive amination, among others.
  • This fungal enzyme of 68 kDa originally isolated from Fusarium graminearum, generates hydrogen peroxide next to oxidized galactose (Parikka et al. Oxidation with galactose oxidase: Multifunctional enzymatic catalysis. J Mol Catal B Enzym. 2015;120:47-59; Whittaker JW. Free Radical Catalysis by Galactose Oxidase. Chem Rev. 2003;103(6):2347-2364).
  • GaOx is a naturally secreted protein, both products are generated at the extracellular site. It is therefore hypothesized that the main function of GaOx is the production of peroxide, with which it can combat bacteria in its proximity.
  • GaOx is a multidomain protein, with an N - terminal carbohydrate binding domain (CBD), hypothesized to tether the enzyme to the fungal cell wall, and two C - terminal domains which both contribute to the active site.
  • CBD carbohydrate binding domain
  • GaOx requires Cu 2+ as cofactor and a tyrosine radical in its active site. In its catalytic cycle, galactose is first bound and deprotonated at its C6 hydroxyl group.
  • the oxime-linker-drug-entity is coupled to the asparagine side chain of said N-glycan acceptor site on the antibody hinge region.
  • the term “linked to”, “coupled to”, or “fused to”, as used herein, and interchangeably used herein as “connected to”, “conjugated to”, “ligated to” refers, in particular, to “chemical and/or enzymatic conjugation” resulting in a stable covalent link.
  • the glycoprotein-conjugates comprise a linker between the LacNAc or Sia- LacNAc N-glycan and the toxin or drug moiety.
  • Certain linkers are more useful than others and the use of a specific linker will depend on the intended application. For example oximes (aminooxy) and hydrazones, in particular derived from aliphatic aldehydes, show less stability over time in water or at lower pH. Aromatically stabilized structures can be more useful to stably link a glycan to a conjugated moiety.
  • Such stabilized linkers are also within the scope of the present application, as they can limit adverse effects due to premature release of the conjugated moiety, particularly when the conjugated moiety is a toxic substance intended for killing of a tumor cell.
  • BICYCLO[6.1.0]NON-4-YNE REAGENTS as well as aromatically stabilized triazole linkers and sulfamide linkers. It is within common technical knowledge that increased stability of a conjugate can also result from reduced aggregation tendency of any of the moieties comprised within said conjugate.
  • Non-cleavable linkers are divided into two groups, namely thioether or maleimidocaproyl (MC). They consist of stable bonds that prevent proteolytic cleavage and ensure greater plasma stability than their cleavable counterparts.
  • MC maleimidocaproyl
  • the most stable linkers currently used are the non-degradable linkers, which incorporate no cleavable functionality, also referred to herein as non-cleavable linkers.
  • linker and conjugation chemistry must be uniquely tailored to correlate each unique facet: the antibody, the conjugated moiety, and the profile of the disease to be treated.
  • antibody-drug conjugates and linkers used herein see for example Jessica R. McCombs and Shawn C. Owen, AAPS J. 17(2), 2015 and Lu, J. et al., Int J Mol Sci. 17(4), 2016 as well as a recent review by Pillow, T.H., Pharm Pat Anal. 6(1), 2017 describing a novel quaternary ammonium salt linker useful in conjugates for the treatment of cancer and infectious diseases.
  • Still other suitable spacers or linkers will be clear to the skilled person, and may generally be any linker or spacer used in the art.
  • linkers or spacers are suitable for use in applications which are intended for pharmaceutical use.
  • a linker between the glycan and the drug moiety in the ADC may in certain aspects also be a suitable amino acid sequence, and in particular amino acid sequences of between 1 and 50, or more specifically, between 1 and 30 amino acid residues.
  • GS linkers such as for example (GS)n or (GGGSS)n or (GSS)n, as described in WO 99/42077 and the (G4S)3, GS30, GS15, GSg and GS 7 linkers described in the applications by Ablynx mentioned herein (see for example WO 06/040153 and WO 06/122825), as well as hinge-like regions, such as the hinge regions of naturally occurring heavy chain antibodies or similar sequences (such as described in WO 94/04678).
  • Still other suitable linkers generally comprise organic compounds or polymers, in particular those suitable for use in polypeptides for pharmaceutical use.
  • poly(ethyleneglycol) (PEG) moieties have been used to link antibody domains, see for example WO 04/081026. It is encompassed within the scope of the invention that the length, the degree of flexibility and/or other properties of the linker may have some influence on the properties of the final glycoprotein-conjugate of the invention, including but not limited to the affinity, specificity or avidity for a specific target. Based on the disclosure herein, the skilled person will be able to determine the optimal linker for use in a specific glycoprotein of the invention, optionally after some limited routine experiments.
  • the length and flexibility of the linker is preferably such that it allows each building block to bind to its cognate target.
  • the skilled person will be able to determine the optimal linker for use in a specific glycoprotein of the invention, optionally after some limited routine experiments.
  • these linkers may be the same or different.
  • the skilled person will be able to determine the optimal linkers for use in a specific polypeptide of the invention, optionally after some limited routine experiments.
  • ADCs with longer linkers including for example carbohydrates, which can provide the ADCs with higher hydrophilicity and accordingly improved water-solubility.
  • ADCs comprising linkers with more carbohydrates are thus also within the scope of the present application.
  • linkers modified with PEG or consisting of PEG can be useful to increase the hydrophilic properties of a glycoproteinconjugate. Conjugation methods to link drug/toxin moieties to the oxidized N-glycans of the antibody
  • extended chemical adaptations as known in the art may be applied on to provide for optimal customized oxime ligations, such as for example but not limited to : reduction of the oxime bond or application of reverse Pictet-Spengler ligations, both leading to increased stabilization; or alternatively hydrazone formation to reduce the stabilization and inducing lability of the bond in acidic condition (applicable for instance when required in the lysosome).
  • vedotin vedotin
  • Adcetris Adcetris
  • Padcev Enfortumab vedotin
  • Polivy Polivy
  • Tivdak tisotumab vedotin
  • mafodotin monomethyl auristatin F MMAF
  • Blenrep monomethyl auristatin E
  • Both auristatins are synthetic mimetics of dolastatin , a marine tetrapeptide natural product isolated from the sea hare
  • Dolabella auricularia selected from hundreds of candidates in structure activity relationship (SAR) studies for their high potency, water solubility, stability and possibility for conjugation.
  • Cell death is triggered, with in vitro potencies of 0.1 to 10 nM, respectively for MMAE and MMAF, by inhibiting tubulin binding to GTP and thereby disturbing microtubule dynamics, which results in G2/M cell cycle arrest and apoptosis.
  • the difference in the toxicity between MMAE and MMAF is caused by differential cell penetration, with the non-charged MMAE more efficiently entering cells via diffusion. Nevertheless, upon cell entry, either by methyl- esterification or by conjugation to antibodies, MMAF is lOOx more potent, because of its higher affinity for tubulin due to the additional negative charge.
  • a further aspect relates to methods to produce the ADCs of the invention.
  • such methods start by introducing an expression vector comprising a nucleotide sequence encoding the antibody part(s) of the ADC according to the invention in a suitable (higher) eukaryotic cell of choice, followed by expressing the antibody for some time, purifying the N-glycosylated antibody, oxidizing the gal or sia into aldehydes on the N-glycan site of said antibody and linking of a specific aminooxy-conjugation moiety to the purified oxidized N-glycan of the antibody through oxime ligation.
  • the oxidation reaction and the coupling method itself is generally carried out in vitro.
  • the antibody expressed in the cells comprises an N-glycan acceptor site in the hinge region, and the eukaryotic cell has a deficiency in the N-acetyl glucosaminyl-transferase I (GNT I) enzyme and comprises a gene encoding for an endoglucosaminidase enzyme, thereby providing for a 'Glycodelete (GD)' eukaryotic cell (and optionally deficient in GNE activity).
  • GNT I N-acetyl glucosaminyl-transferase I
  • the method to produce a ADC comprises the steps of :
  • the diol to be oxidized originates from a LacNAc disaccharide present on the glycoprotein of the invention.
  • Oximes are formed by subsequent reaction of the resulting free aldehyde groups with aminooxy-containing molecules, commonly described as LacNAc oxidation-oxime ligation chemistry.
  • catalysts like para-phenylenediamine, 2-aminophenols or 2-(aminomethyl)benzimidazoles.
  • Oxime and hydrazine conjugation are a promising alternative to clickchemistry, a more complex biorthogonal modification strategy. Under reductive amination conditions, the dialdehyde can be reacted with amines, which results in stable oxazepine derivatives.
  • said method to produce an ADC optionally comprises the steps of
  • Galactose Oxidase (GAO or GaOx or GalOx)
  • GEO or GaOx or GalOx the enzyme Galactose Oxidase
  • oxime bonds are formed, optionally in the presence of a catalase, all of this is well-known in the art.
  • linkers As described before is particularly envisaged herein.
  • linkers to modulate the stability of the glycoprotein-conjugates as described before is particularly envisaged herein.
  • Example 1 Engineering the antibody for improved oxidation by Galactose oxidase.
  • ADCs antibody - drug conjugates
  • N-glycans For the creation of antibody - drug conjugates (ADCs) wherein the conjugation is made at the N-glycans, we introduced an additional N-glycosylation site besides the conventional N297, said neo-N- glycosylation site being located in the antibody hinge region which is more accessible for enzymatic treatment, especially for galactose oxidation of the N-glycan.
  • Figure 1 shows the expression of trastuzumab N221 to a similar extent as trastuzumab WT in both HEK293S cells and ExpiCHO cells, which was confirmed by the similar expression yields for both antibody variants after purification.
  • a high efficiency of the neo-N-glycosylation site was assumed in both expression systems, given that the N221 antibody variant of HEK293S expression ran completely at a higher molecular mass, for trastuzumab N221 expressed in ExpiCHO and HEK293S cells.
  • the trastuzumab (WT and N221-engineered variant) antibodies were recombinantly produced in HEK293S cells. Additionally, by applying the HEK293S cell expression platform, another layer of variation in recombinant production of glyco-engineered antibodies was possible by introducing the 'GlycoDelete' concept ( Figure 20). As such, the N-glycan would be reduced in size and homogenized.
  • GlycoDelete cells were further manipulated by knocking out GNE (GDLN), in order to abolish the formation of CMP-sialic acid, the precursor for sialylation.
  • trastuzumab variants processed by EndoT in the HEK293S 'GlycoDelete LacNAc' (GDLN) cells.
  • Trastuzumab WT and N221 were expressed without hGalTl or precursors and analyzed, after purification, by mass spectrometry after FabRICATOR digest (this protease specifically cleaves in the hinge region, conveniently in between the two N-glycosylation sites, yielding the N-terminal fragment VH-CH1 with the N221 glycan (SEQ.
  • Example 4 Conjugating antibody with aminooxy - functionalized linkers: testing the specific oxidation of trastuzumab N221.
  • trastuzumab N221 expressed in ExpiCHO yielded some biotinylation.
  • the lack of biotin signal for trastuzumab WT is a combination of limited galactose content and reduced activity of GaOx on the Fc N-glycan.
  • galactosylation was increased with hGalTl and precursors with trastuzumab N221 in ExpiCHO cells, only a small increase in biotinylation could be observed.
  • a higher biotin signal was expected, since DSA FACE data indicated a larger fraction of trastuzumab N221 that carried galactose residues.
  • the lack of efficient oxidation or conjugation of CHO expressed trastuzumab N221 proved another rationale to switch to HEK293S expression for trastuzumab (next to expression yield loss and the GDLN option).
  • the reduced GaOx activity at N297 N-glycans of antibodies could be used to our advantage, by combining ADCC with ADC.
  • ADC By selectively oxidizing and conjugating toxin to the neo-N-glycosylation site N221, the Fc N-glycan remained unaltered, which we assume may result in potent ADCC activity.
  • Angelastro et al. by increasing the oxidase units, the Fc N-glycan became oxidizable (ChemSusChem. 2022;15(9)).
  • Conjugating the galactose residues of the Fc N-glycan next to the N221 N- glycan would increase the drug-to-antibody-ratio (DAR), which could be useful in certain applications.
  • DAR drug-to-antibody-ratio
  • trastuzumab WT (lacking the accessible N221 site) was found to be more efficiently oxidized in conditions with more GaOx units. Yet, for the other antibody variants, the best conditions were found with intermediate amounts of GaOx and differed depending on the number of galactose residues present ( Figure 9).
  • the optimal condition resulting in the higher streptavidin signal was 660U/nmol, 330U/nmol, 165U/nmol and 16.5U/nmol.
  • Example 5 Generating trastuzumab - drug conjugates with GaOx.
  • MMAF monomethyl auristatin F
  • Figure 11 After PPD-based catalysis of the conjugation, the reactions were analyzed by SDS PAGE and western blotting.
  • trastuzumab N221 expressed in HEK293S WT and GDLN cells without hGalTl or precursors, in order to produce sufficient material to test cytotoxicity in vitro with HER2 overexpressing cancer cells.
  • the conjugatable MMAF was lowered to an excess of the toxin, in respect to the antibodies, to 42.9 x. After overnight oxidation and conjugation, the reactions were catalyzed with I mM PPD and purified by protein A chromatography and desalted to PBS.
  • trastuzumab N221, expressed in HEK293S WT cells was repeated the oxidation and conjugation of trastuzumab N221, expressed in HEK293S WT cells, to aminooxy-MMAF.
  • the GDLN variant was omitted for this repetition, since this conjugate had been shown to be toxic on HER2 overexpressing cells, as disclosed below.
  • a new batch of toxin was used to conjugation trastuzumab N221 with 250 equivalents during its overnight oxidation.
  • trastuzumab from a new antibody expression batch which was expressed in HEK293S WT cells with 1 % hGalTl and precursors, and as such carried more galactose residues.
  • the profile for conjugated trastuzumab N221 GDLN depicts a peak in the DAR2 section, which could be explained by oxidation of and conjugation at the Fc N-glycan, given that the analysis was run in reduced phase. Additional evidence for Fc conjugation can be found in the DARI section, which shows two peaks, with identical molecular mass after deconvolution, which may be the conjugates with different conjugation sites. If we assume that the largest peak is conjugated at N221, given its more accessible location, the little peak would be Fc N- glycan conjugated.
  • the ratio between the two peaks indicates that about 15 - 20 % would be conjugated at N297, which is more than anticipated based on the aminooxy-biotin conjugation experiment (3.33 %).
  • DAR determination we used native SEC MS.
  • the profile after deconvolution was only manageable for annotation for the GDLN variant since the WT variants proved too diverse for peak calling.
  • the drug load varied from DARO to DAR4 with on average 1.61 toxins conjugated to trastuzumab N221. This number is slightly higher than obtained with reduced HPLC MS, which could be due to technical differences between the methods.
  • the profiles show DAR3 and DAR4 species which further indicates the oxidation and conjugation of Fc N-glycans.
  • HER2 positive breast cancer cell line SK-BR-3 is often used in ADC research and was adopted here as well, for straightforward comparison with efficacies disclosed in literature.
  • the HER2 negative cell line MDA-MB-231 is often used as negative control, to test for non-targeted toxicity.
  • FACS fluorescence - assisted cell sorting
  • trastuzumabMMAF-conjugated derivatives were seeded in 96 well plates for 24h to subsequently exchange their growth medium with DMEM medium supplemented with ADCs. The cells were incubated over weekend with the treatments. Next, the cells were washed in PBS and analyzed for cell viability by ATP measurements with CellTiter-Glo.
  • trastuzumab-LacNAc (LN) - MMAF for SK-BR-3 cells, compared to the clinically relevant trastuzumab-drug conjugates Kadcyla and Enhertu, which showed the superiority of the conjugate generated with our GaOx/Oxime ligation platform ( Figure 16).
  • Enhertu was unable to induce cell death to single digit viability percentages.
  • novel antibody - drug conjugates are superior in clinical settings to first generation ADCs which can be (partially) attributed to bystander effect.
  • the latter characteristic results in improved solid tumor killing due to the membrane permeability of the active metabolite of the ADC, leading to cell death in cells with a negative or reduced antigen status.
  • the notorious heterogeneity of solid tumors which can result in incomplete responses in patients, highlights the need for improved ADCs, such as bystander effect-competent variants.
  • Example 7 Confirmation of the in vitro cytotoxicity of trastuzumab-GaOx/oxime coupling on disaccharide N-glycans in hinge region.
  • trastuzumab with its hinge sequence mutated to include the D221N N-glycosylation site, was oxidized at terminal galactose residues with galactose oxidase (GaOx) and conjugated to aminooxy-functionalized drug-linkers. After purification from the oxidation/conjugation reaction via protein A chromatography and desalting to phosphate buffered saline (pH 7.4), the ADCs were compared with approved trastuzumab-based ADCs (Kadcyla or T-DMl and Enhertu) for in vitro cytotoxicity in the HER2+ cancer cell lines SK-BR-3.
  • a first ADC was generated with the GaOx/Oxime technology employed MMAF as cytotoxin, with the non- cleavable PEG4 as linker.
  • the linker does not harbour a cleavage site and is classified as a stable linker.
  • the cytotoxicity of Trastuzumab D221N MMAF (HEK GDLN) and Trastuzumab D221N MMAF (HEK WT) was compared in the SK-BR-3 cell line to T-DMl (Kadcyla) and Enhertu (Figure 22).
  • the ADCs of which the antibody was produced in HEK GDLN host cells was cytotoxic to these HER2+ breast cancer cells (EC 5 o of 11 pM), whereas the corresponding ADC construct with WT N-glycan structure did not show cytotoxicity.
  • the active metabolite generated in the SK-BR-3 cell line is believed to be Asn+ N-glycan + Oxime linker + MMAF, when there is no cleavable linker present (such as is the case for MMAF).
  • the metabolites differ in hydrophilicity: the GDLN-ADC metabolite carries a disaccharide, while the WT- ADC metabolite carries an oligosaccharide (see right panel Figure 22). Possibly, the increased hydrophilicity of the WT-ADC metabolite is too large to cross the lysosomal membrane. Without escaping the lysosome, the metabolite cannot perform its cytotoxic function (in the cytosol, where the microtubules are).
  • Example 8 In vitro cytotoxicity for ADC coupling technology with cleavable linkers.
  • Monomethyl auristatin E (MMAE)-based ADCs are typically constructed using cleavable linkers to obtain the required cytotoxicity, so as a second set of ADCs we conjugated trastuzumab with our GaOx/Oxime technology and MMAE as cytotoxin via the cleavable valine-citrulline-PABC linker.
  • This linker can be digested by cathepsin protease upon lysosomal delivery.
  • the conjugated GDLN-ADC molecule is lower in size than the conjugated WT-N-Glycan-ADC, and therefore may escape from the lysosome more easily. Further investigation remains to check whether protease processing of the linker is suboptimal or delayed in SK- BR-3, which would generate a bottleneck, making the WT-ADC metabolite less active, or active on a different time scale.
  • Example 9 Biosimilar ADC production with GaOx/ oxime hinge coupling technology.
  • the GlycoDelete-ADC molecule wherein the N-glycan is limited to a disaccharide does show cytotoxicity, though it is less active than T-DM1 (EC 5 o for Kadcyla: 51.3 pM versus 259 pM for the GDLN-ADC-DM1 compound).
  • the five-fold difference may reflect the anticipated lower DAR (drug to antibody ratio) of GDLN-ADC (1.6) versus T-DM1 (3.5), in combination with the suboptimal linker chemistry.
  • the novel ADC coupling technology applied in the hinge region with in addition short N-glycan structures allows for an improved effect as compared to applying native N-glycans as site-specific coupling technology.
  • Example 10 ADC GaOx/oxime-based hinge coupling technology versus maleimide coupling.
  • the NHS conjugation chemistry used in Kadcyla targets primary amines such as the N-termini and lysine amino acids. Yet, this conjugation strategy is less preferred than targeting cysteines residues with maleimide-functionalized toxins, after partial reduction of the antibody interchain disulfide bonds.
  • MMAF and MMAE auristatin-based GlycoDelete-ADCs
  • GDLN-ADC is 5x more potent than its maleimide alternative (EC 5 o of 11.1 pM versus 50.7 pM)
  • GDLN-ADC (MMAE) conjugate is similarly potent as its maleimide alternative (EC 5 o of 90.6 pM versus 143 pM), despite its lower anticipated DAR (GDLN-ADC at most 1.6-1.8, Mal-MMAE at most 4.4).
  • Example 11 Confirmation of in vitro cytotoxicity effect in additional cancer cell line.
  • GDLN-ADC conjugation chemistry
  • the GDLN-ADC (MMAE) conjugate is 3.5x more potent as its maleimide alternative (EC 5 o of 72.5 pM versus 259 pM), despite its lower anticipated DAR (GDLN-ADC at most 1.6-1.8, Mal-MMAE at most 4.4).
  • the data presented here supports the superiority of GDLN-ADCs over WT-ADCs and the relevance of the GDLN-ADC technology as an alternative to generate clinically relevant ADCs.
  • cDNA codon optimized for mammalian expression
  • IDT gBIocks The cDNA contained the full length LC or HC sequences of trastuzumab, with the latter sequence additionally modified with the novel N221 N-glycosylation site (D221N).
  • D221N novel N221 N-glycosylation site
  • Trastuzumab was expressed in ExpiCHO cells (ThermoFisher Scientific), according to the manufacturer's protocol. Briefly, 6x 10 6 cells/ml, grown at 37 °C and 8% CO2 were transfected with 0.8 pg DNA/ml using the ExpiFectamine CHO reagent. For efficient antibody expression, LC and HC were transfected at a 2:1 ratio respectively. One day after transfection, ExpiCHO enhancer and ExpiCHO feed were added according to the manufacturer's instructions. Subsequently, the cultures were further incubated at 32 °C and 5% CO2. Cells were fed a second time 5 days post-transfection. Cultures were harvested as soon as cell viability dropped below 70 - 75%.
  • trastuzumab was also expressed in HEK293S cells (WT and 'GlycoDelete LacNAc' (abbreviated herein as GDLN or LN) modified). Prior to transfection, cells (ThermoFisher Scientific) were cultured in FreeStyle293 expression media (Life Technologies) supplemented with equal volumes of Ex- Cell-293 (Sigma), at 37 °C with 8% CO2 while shaking at 130 rpm. Mammalian expression plasmids encoding the LC and HCs of trastuzumab were transfected, at a 2:1 ratio respectively, into FreeStyle293 cells using polyethylenimine (PEI).
  • PEI polyethylenimine
  • HEK293S cells were seeded at 3x 10 6 cells/mL in Freestyle-293 medium (ThermoFisher Scientific).
  • 2.25 pg DNA/ml cells was added to the cells and incubated on a shaking platform at 37 °C and 8% CO2, for 5 min.
  • 4.5 pg PEI/ml cells was added to the cultures, and cells were further incubated for 5 h, after which an equal culture volume of Ex-Cell-293 was added to the cells.
  • Transfections were incubated until cell viability dropped below 70 - 75%, after which cells were pelleted (10', 300 g) and supernatants were filtered before purification.
  • APTS-labeled N-glycans were treated overnight in 5 mM ammonium acetate pH 5 with exoglycosidases to verify the N-glycan composition.
  • the following enzymes were used: a-fucosidase O (P0749, NEB), a- mannosidase (P0768, NEB), p-hexosaminidase (GKX-5003, Agilent), -galactosidase (GKX-5014, Agilent), a-sialidase (In house produced, Arthrobacter ureafaciens).
  • Non-PNGase F treated samples were taken along as controls.
  • Endo F2 digest was performed on purified trastuzumab variants for SDS PAGE analysis of treated samples, to verify the high occupancy of the N221 site.
  • Antibody and Endo F2 (P0772, NEB) were incubated at 1U/10 ug trastuzumab overnight at 37 °C.
  • trastuzumab N22 In order to determine the N-glycosylation for the specific sites in trastuzumab N221, we digested 750 pg trastuzumab N221 overnight at 37 °C in PBS with 70 units FabRICATOR (A0-FR1-020, Genovis). This protease cleaves antibodies specifically below the hinge region, successfully separating the N221 glycan from the N297 N-glycan, respectively located at the F(ab')j and Fc/2 fragments. We next separated the fragments based on their size with SEC (HiLoad 16/600 200 pg column) and subjected the pooled elution fractions to DSA FACE.
  • SEC HiLoad 16/600 200 pg column
  • LC-MS N-glycosylation analysis of GlycoDelete truncated trastuzumab by intact protein mass spectrometry (MS) was included to analyze the recombinant proteins more accurately.
  • LC-MS was performed on an Ultimate 3000 HPLC (Thermo Fisher Scientific, Bremen, Germany) equipped with a Poroshell 300SB-C8 column (Thermo Scientific 1.0 mm of LD. x 150 mm), in-line connected with an ESI source to an LTQ XL mass spectrometer (Thermo Fischer Scientific).
  • ESI parameters were used: a surface-induced dissociation of 30 V, a spray voltage of 5.0 kV, capillary temperature of 325 °C, capillary voltage of 35 V and a sheath gas (N2) flow rate setting of 7.
  • Binding of trastuzumab variants to antigen HER2 was validated by ELISA. Briefly, 75 ng HER2 (10004- H08H, Sino Biological) was coated overnight at 4 °C. After one hour blocking in 4% milk (in PBS, 0.5% Tween20), a serial dilution of the trastuzumab variants was incubated for one hour at room temperature.
  • trastuzumab variants to CD16a 158V was validated by ELISA.
  • 200 ng trastuzumab was coated overnight at 4 °C. After one hour blocking in 4% milk (in PBS, 0.5% Tween20), a serial dilution of the CD16a 158V (#1O389-H27H1, Sino Biological) was incubated for two hours at room temperature. Next, antibody was detected with anti-HisTag antibody coupled to HRP (R931-25, Invitrogen). In between each step, the ELISA plate was washed three times in PBS-Tween20.
  • HRP activity was analyzed by incubating TMB substrate (BD OptEIA) in the wells for 10 minutes, followed by acidification with 2N H2SO4. Absorbance was measured at 450 nm and 655 nm, and plotted from duplicated experiments.
  • Trastuzumab was oxidized with 0.66U GaOx/nmol antibody overnight in 100 mM sodium phosphate at pH 7 at 28 °C, with 100 equivalents aminooxy-PEG-biotin. After oxidation, conjugation was catalyzed for one hour with para-phenylenediamine (PPD). Successful conjugation was verified with SDS PAGE and western blotting for streptavidin-based detection (#21851, Invitrogen). No GaOx unit adjustments for the number of galactose residues were implemented.
  • trastuzumab N221 (HEK293S WT and HEK293S GDLN expressed with hGalTl or precursors) was oxidized with 0.66U GaOx/nmol overnight in 100 mM sodium phosphate at pH 7 at 28 °C. During oxidation, the remainder of available aminooxy-functionalized MMAF toxin was divided over the two reactions, for 42.9 molar equivalents of toxin compared to 4 mg antibody.
  • the conjugation was catalyzed by 1 mM PPD for lh.
  • Antibodies were then purified by protein A chromatography as above and desalted over HiPrep 26/10 Desalting columns (Cytiva) to PBS.
  • trastuzumab N221 expressed in HEK293S WT cells, with 1% GalT and precursors (1 ISM MnCI2, 5 mM galactose and 1 mM uridine), was oxidized with 0.66U GaOx/nmol overnight in 100 mM sodium phosphate at pH 7 at 28 °C.
  • the molar equivalents of MMAF toxin were increased to 250 equivalents.
  • the conjugation reaction was treated with PPD and purified, identically to the first oxidation and conjugation.
  • the purified trastuzumab - drug conjugates were analyzed SDS PAGE and western blotting for specific detection with anti-MMAF antibody (MA542538, Fisher Scientific) and anti-mouse antibody conjugated to DyLight800 (SA5-35521, ThermoFisher Scientific).
  • peptide MS was performed after digest with 0.5 pg GluC (P8100, NEB), or with the combination of 0.5 pg Trypsin (V5111, Promega) and 0.5 pg LysC (P8109, NEB) in triethylammonium bicarbonate (TEAB). After heat denaturation in 8M urea at 95°C for 10 minutes, reduction in 5 mM DTT at 55°C for 30 minutes and alkylation in 10 mM IAA at RT for 15 minutes, proteases GluC or trypsin and LysC were incubated with antibodies and MMAF conjugates for 4h to overnight. Next, the resulting peptides were analyzed by MS.
  • TEAB triethylammonium bicarbonate
  • DAR determination of MMAF conjugates was performed in collaboration with RIC (Kortrijk, Belgium) by implementing reduced RPLC MS and native SEC MS.
  • the former method was run with a Q-TOF (6540) setup using 0.1% TFA in H2O (buffer A) and 0.1% TFA (buffer B) in acetonitrile as mobile phases in a gradient mode.
  • the native SEC MS technique was run with 100 mM ammonium acetate in isocratic mode on an Orbitrap Q - Exactive Plus.
  • Trastuzumab N221 was oxidized and conjugated with the MMAE drug-linker compound (MedChemExpress, HY-153263), as described above for the MMAF drug-linker.
  • Antibody was oxidized overnight with 0.66U GaOx per nmol antibody, in the presence of 50 molar equivalents of aminooxytoxin linker (in 50-100 mM sodium phosphate buffer pH 7). After overnight incubation, the oxime ligation was catalyzed by addition of PPD. After an incubation of two hours, the reaction mixture was diluted in protein A binding buffer and subjected to protein A affinity chromatography. The eluted antibody-drug conjugate was subsequently desalted to phosphate buffered saline pH 7.4.
  • Trastuzumab N221 was oxidized and conjugated to aminooxy-PEG4-azide (BroadPharm, BP-23595), as described above for the MMAF and MMAE conjugations.
  • aminooxy-PEG4-azide BroadPharm, BP-23595
  • the azide-modified antibody was then conjugated with 15 molar equivalents DBCO-PEG4-DM1 (MedChemExpress, HY-136261).
  • the conjugation was terminated after 72h and purified by protein A chromatography.
  • the final antibody-drug conjugate was subsequently desalted to phosphate buffered saline pH 7.4. Conjugation of antibody with maleimide-MMAF and maleimide-MMAE
  • Trastuzumab was reduced with 2.2 molar equivalents TCEP for 90 minutes at 37 °C in 50 mM sodium phosphate pH 7. Subsequently, the reduced cysteines were conjugated with 10 molar equivalents maleimide-functionalized toxins for lh at 25 °C.
  • Maleimide-functionalized toxins were bought from MedChem Express: Maleimidocaproyl MMAF (HY-15578) and MC-Val-Cit-PAB-MMAE (HY-15575). Next, the reactions were diluted in protein A binding buffer and purified with protein A chromatography. The final antibody-drug conjugate was subsequently desalted to phosphate buffered saline pH 7.4.
  • HER2 status was verified by FACS analysis.
  • Cells were stained with live/dead stain LDeFI506 (65-0866-18, eBiosciences), fixed in 4 % PFA and stained with 5 g/ml trastuzumab WT (HEK293S WT or ExpiCHO produced) in FACS buffer (PBS, 1 % BSA, 0.5 mM EDTA) for 30 minutes.
  • FACS buffer PBS, 1 % BSA, 0.5 mM EDTA
  • the AF633 conjugated anti-hlgG antibody was incubated at 4 g/ml on the cells for 30 minutes.
  • the cells were analyzed in the LSRII flow cytometer (BD).
  • SK-BR-3 or BT-474cells were seeded at 5.000 or 10.000 cells/well at day 0.
  • the medium was exchanged for DMEM medium, supplemented with the treatment (a serial dilution of antibody or the toxin conjugates).
  • the cells were then incubated for 5-6 days (for 5.000 cells/well) or 2.5 - 3 days (for the 10.000 cells/well).
  • dead cell debris and free ATP was removed by washing the cells three or two times, resp. with PBS.
  • Cells were then analyzed for viability with the Cel ITiter Gio assay (G7572, Promega). Briefly, equal amounts of CellTiter-Glo reagent were mixed with the cell culture. Cell lysis was induced for two minutes on an orbital shaker, after which the luminescence signal was read for Is using a plate reader (GloMax, ProMega).
  • the in vitro toxicity assay was adapted at day 0 with the seeding of 5000 SK-BR-3 cells with 5000 MDA-MB-231 cells per well, premixed for all wells.
  • non-treated cells were included. Additionally, for BT-474 cells, as control for dead cells, non-treated cells were treated with 30 % ethanol after the PBS wash. The viability of treated cells was analyzed relative to the live and dead cells. Finally, in BT-474 cells, a longer incubation period was required when alternative (weaker, less potent) toxins were used as compared to MMAF.

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Abstract

The present invention relates to the field of antibody-drug-conjugates (ADCs) and the application of glyco-engineering strategies in a novel site-specific conjugation technology. The invention particularly relates to ADCs wherein a drug entity is conjugated an aminooxy-functionalized linker at di- or trisaccharide N-glycans in the hinge region, and methods for producing the same. More particularly, said conjugate is obtained through oxidized galactose- or sialyl-groups obtained from glyco-engineered eukaryotic cells which produce antibodies with an N-glycan structure that is limited to a shortened Asn- GlcNac-Gal(-Sia) branch in the hinge region of the antibody.

Description

GLYCO-ENGINEERED ANTIBODY-DRUG-CONJUGATES COMPRISING AN OXIME LINKER
FIELD OF THE INVENTION
The present invention relates to the field of antibody-drug-conjugates (ADCs) and the application of glyco-engineering strategies in a novel site-specific conjugation technology. The invention particularly relates to ADCs wherein a drug entity is conjugated an aminooxy-functionalized linker at di- or trisaccharide N-glycans in the hinge region, and methods for producing the same. More particularly, said conjugate is obtained through oxidized galactose- or sialyl-groups obtained from glyco-engineered eukaryotic cells which produce antibodies with an N-glycan structure that is limited to a shortened Asn- GlcNac-Gal(-Sia) branch in the hinge region of the antibody.
BACKGROUND
Antibody-drug conjugates (ADC) have been developed to deliver potent cytotoxins to specific cells, based on their antigen expression, and are typically applied as anti-tumor therapy for patients with resistant or refractory (solid) tumors. Currently, several ADCs are marketed after approval by the FDA.
ADCs are generated from three essential components: the antibody, the toxin and the linker in between, all contributing to the overall activity. A first important feature is the targeted antigen, which is dictated by the antigen-binding domain of the antibody part of the ADC. The antigen should be highly, and preferably uniquely, expressed on cells, such as tumor cells with little to no expression on healthy or undesired cells or tissue, to limit (off-target) toxicity. Moreover, efficacy improves when the antigen is uniformly expressed in the tumor tissue, rendering more cells susceptible to toxin - mediated killing. Additionally, the antigen should be accessible for binding with circulating antibodies, with the resulting interaction triggering internalization efficiently. Upon internalization, the toxin is released from the ADC, either by complete degradation of the antibody or by specific cleavage of the linker moiety, to subsequently perform its cytotoxic function. Despite internalization being required for all approved ADC formats, alternatives that lack intracellular entry in their conjugated composition could also be functional if the toxin is released in the tumor micro-environment (TME) in a membrane - penetrable configuration, for instance by incorporating cleavage sites for metalloproteases enriched in the tumor microenvironment. A last, debatable, requirement for antigens is the affinity of the interaction with the conjugated antibody. Higher affinity results in improved tumor selectivity, yet halts effective tumor penetration, especially when the maximal tolerated dose (MTD) is limited, which is the case for most approved ADCs. Correlated with the antigen selection, the antibody itself also influences the final conjugate. Firstly, given the differences between ADCs and antibodies in the tumor killing mechanism, the antibody of interest by itself can be but does not need to be efficacious. For example, trastuzumab is used as anti-tumor therapy both as such and in ADCs (Kadcyla (trastuzumab emtansine) and Enhertu (trastuzumab deruxtecan)), while brentuximab is only employed as a conjugate with MMAE in Adcetris (brentuximab vedotin). Possibly, this difference may be due to differential susceptibility of the specific tumors to antibody - mediated clearance.
The second crucial part of ADCs is the linker, which connects the antibody and the toxin and controls the stability of the conjugate. Ideally, the ADC is sufficiently stable in serum and does not prematurely release the toxin, which would result in systemic toxicity. Nevertheless, upon internalization in the targeted cells, the toxin should be readily releasable to perform its toxic function.
The final component of ADCs is the toxin (also designated as the payload), which triggers cell death in targeted cells. All toxins in approved conjugates either target DNA (directly or indirectly through topoisomerase I inhibition) and are thus toxic to both proliferating and resting cells, or they target microtubules for toxicity uniquely in dividing cells. More recently investigated toxins employ different mechanisms such as the inhibition of RNA polymerase, of the RNA spliceosome and of anti - apoptotic processes using Bcl-xL inhibitors. A common feature is their potency, with IC5o values in or below the nM range, which is required due to the limited amount of antibody that reaches the tumor site, estimated at 0.001 - 0.01 % (Epenetos et al. Cancer Res. 1986;46(6):3183-3191). Other requirements for toxins to be used in ADCs are a suitable handle for conjugation, or flexibility to allow incorporation of one; solubility in aqueous buffers; and limited hydrophobicity to reduce aggregation propensity, which would lead to shorter shelf life, faster clearance, immunogenicity and increased efflux pumping.
The current generation of approved ADCs typically apply coupling to lysine- and coupling to interchain cysteine-based technologies which lack site-specificity and/or introduce structural instability. Next generation ADCs typically favor more site-specific conjugation, yet so far, these require expensive nutrients during expression (noncanonical amino acids and glycan remodeling), incorporate potentially immunogenic unnatural tags (enzymatic approaches), and/or reduce structural stability. Accordingly, there exists a need for improved ADC coupling technology.
SUMMARY OF THE INVENTION
In the present invention we used the N-glycosylation of antibodies as a handle for site-specific conjugation to a linker-toxin moiety. More specifically, application of enzymatic Galactose oxidase activity leads to oxidation of terminal galactose residues to aldehydes, which aldehydes can be used in bio-orthogonal chemistry coupling. The resulting galactose aldehydes provide for a position for oxime ligation coupling with aminooxy-linker-toxin moieties. Furthermore, the position of N-glycosylation of the antibody was altered by introducing a neo-N-glycan site in the hinge region of antibodies to allow efficient Galactose oxidation and conjugation into novel ADCs.
Finally, our glyco-engineering technology as further discussed and referred to herein, allowed to compare two different N-glycosylation trees: a native biantennary N-glycosylation structure and a 'shortbranch' N-glycosylation structure as produced in eukaryotic hosts engineered to produce so-called 'GlycoDelete' N-glycans (see Callewaert et al., W02010/015722 and WO2015032899, and below). The present application discloses ADCs generated from antibodies with both types of N-glycan chains, further conjugated upon galactose oxidation of the N-glycans, to provide aminooxy-based coupling technology for the toxin moiety.
Surprisingly, toxicity was only obtained for the ADCs produced with short-branch N-glycan ADCs produced by GlycoDelete cells , and not with non-engineered or wild type N-glycan ADCs. As demonstrated for the non-limiting novel ADCs exemplified herein, these disaccharide or trisaccharide short-branch N-glycan coupled ADCs even show a superiority in potency on HER2 positive cancer cells when compared with clinically approved ctrastuzumab-based conjugates, Kadcyla and Enhertu, with respectively a lysine specific conjugation approach and an interchain cysteine conjugation technique, at least when coupled to a highly potent MMAF toxin. Additional in vitro experiments using different biosimilar constructs, allowing a comparison of ADCs with identical toxin, though different in coupling and/or linker technology, further provide proof of concept that our novel ADCs may be considered as a valid alternative with improved properties over the status quo.
In a first aspect an antibody-drug-conjugate (ADC) is provided which comprises or consists of an antibody which has an N-glycan acceptor site in the antibody hinge region and an aminooxy-linker-drug-entity, wherein said aminooxy-linker-drug-entity is coupled to the asparagine side chain of said N-glycan acceptor site through oxime ligation of the aldehyde present on the oxidized galactose residue of the disaccharidic Galactose-N-acetylglucosamine (Gal-GIcNac) or alternatively via the sialic acid residue of the trisaccharidic Sialyl-Galactose-N-acetylglucosamine (Sia-Gal-GIcNAc) glycan chain.
In a further aspect the ADC is provided with an antibody format comprising an Fc-tail, preferably a monoclonal antibody, more preferably an IgG antibody, more preferably a human IgGl, lgG2, or lgG4 antibody.
In another aspect the ADC comprises an acceptor N-glycan site in the hinge region which is located at a position corresponding to amino acid 221 from the human IgGl hinge region sequence (according to EU numbering), wherein said position is an asparagine which is introduced by substitution of the native amino acid (D in human IgGl). More specifically, said substitution as present at position 6 of the hinge region of SEQ. ID NO:1.
In a specific embodiment, the linker of the aminooxy-linker-drug-entity (or the oxime-linker-drug-entity as used interchangeably herein) in the ADC comprises or consists of a non-cleavable and/or stable linker, such as for instance a Polyethyleneglycol-based linker.
In yet another embodiment, the drug-entity in the ADC comprises or consists of an auristatin-based drug, such as monomethyl auristatin F (MMAF) or Monomethyl auristatin E (MMAE), or an anti - mitotic drug, such as mertansine (DM1), or a DNA topoisomerase I inhibitor, such as Exatecan derivative (Dxd) .
In yet another embodiment, the ADC comprises of consists of an N-glycan- aminooxy-linker-drug-entity selected from the group of Asn-GIcNAc-Gal-oxime-PEG-MMAF (for which the chemical formula is shown in Figure 18 as a non-limiting example), Asn-GIcNAc-Gal-Sia-oxime-PEG-MMAF, Asn-GIcNAc-Gal-oxime- linker-MMAE, Asn-GIcNAc-Gal-Sia-oxime-linker-MMAE, Asn-GIcNAc-Gal-oxime-linker-DMl, or Asn- GIcNAc-Gal-Sia-oxime-linker-DMl.
In yet another aspect a method is provided to produce an antibody-drug-conjugate comprising the steps of: a. expressing an antibody comprising an antigen-binding domain specifically binding a surface exposed antigen on an eukaryotic cell, preferably a higher eukaryotic cell such as a mammalian cell, wherein said antibody has an N-glycan acceptor site in the hinge region, b. oxidizing the galactose residue of the Galactose-N-acetylglucosamine (Gal-GIcNac) N-glycan or oxidizing the sialic acid of the Sialyl-Galactose-N-acetylglucosamine (Sia-Gal-GIcNAc) N- glycan present on the asparagine of said N-glycan acceptor site in the hinge region of said antibody of step a) to obtain an aldehyde for coupling the drug entity, and c. connecting an oxime-linker-drug-entity or aminooxy-linker-drug-entity by oxime ligation to said aldehyde of the oxidized N-glycan, d. obtaining the antibody-N-glycan-oxime-linked-drug ADC molecule, wherein said eukaryotic cell has a deficiency in the N-acetyl glucosaminyl-transferase I (GNT I) enzyme and said eukaryotic cell comprises a gene encoding for an endoglucosaminidase enzyme, thereby providing for a 'Glycodelete (GD)' eukaryotic cell.
In another aspect in the method for producing an ADC the eukaryotic cell further comprises a deficiency in bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) activity, thereby providing for a 'Gycodelete LacNac (GDLN)' eukaryotic cell. In yet another aspect in the method the linker is an aminooxy-containing molecule for ligation to the aldehyde present after oxidation of Gal or Sia on the N-glycan to form the oxime coupling, preferably a non-cleavable linker, such as a linker comprising Polyethyleneglycol.
In yet another aspect in the method the drug-entity comprises or consists of an auristatin-based drug, such as monomethyl auristatin F (MMAF) or Monomethyl auristatin E (MMAE), or an anti - mitotic drug, such as mertansine (DM1), or a DNA topoisomerase I inhibitor, such as Exatecan derivative (Dxd).
In yet another aspect in the method to produce an ADC the oxidation is performed using enzymatic or chemical oxidation, preferably using Galactose oxidase or sodium periodate, respectively.
In yet another aspect an antibody-drug conjugate is obtained by the methods as herein before described.
DESCRIPTION OF THE FIGURES
The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes.
Throughout the Figures present in the application as filed, the different N-glycan moieties are indicated as generally known by: a square corresponding to a N-acetylglucosamine (GIcNAc) moiety; diamond to sialic acid (Sia); light circle to galactose (Gal) ; dark circle: mannose (Man); triangle: fucose (Fuc).
Throughout the Figures as present in the application as filed, for clarity and consistency, the different ADC molecules applied in the examples were numbered and defined as listed here below:
A. Trastuzumab D221N, Oxime-conjugated to MMAF (HEK GDLN expressed)
B. Trastuzumab D221N, Oxime-conjugated to MMAF (HEK WT expressed)
C. T-DM1 (commercial Kadcyla, trastuzumab DM1 conjugated)
D. Enhertu (commercial; trastuzumab DXd conjugated)
E. Trastuzumab D221N (HEK GDLN expressed)
F. Trastuzumab D221N (HEK WT expressed)
G. Trastuzumab D221N, Oxime-conjugated to MMAE (HEK GDLN expressed)
H. Trastuzumab D221N, Oxime-conjugated to MMAE (HEK WT expressed)
I. Trastuzumab D221N, Oxime-conjugated to DM1 (HEK GDLN expressed)
J. Trastuzumab D221N, Oxime-conjugated to DM1 (HEK WT expressed)
K. Trastuzumab, Maleimide-conjugated to MMAF L. Trastuzumab, Maleimide-conjugated to MMAE
Figure 1. Trastuzumab containing the neo-glycan site N221 was expressed to a similar extent as trastuzumab wild-type (WT), both in HEK293S WT cells and in ExpiCHO cells, with efficient N- glycosylation of the N221 variant as indicated by the height of the band visible in the N221 sample lane. HEK293S WT and ExpiCHO cells were transfected with pcDNA3.3 vectors encoding the light and heavy chains for trastuzumab WT and N221 expression. Upon 25 - 30 % cell death, the cells were pelleted, and the resulting supernatant was analyzed for antibody by SDS PAGE and western blotting with anti-hlgG detection (heavy chain (HC) only detected) (Left). After purification via protein A chromatography, the ExpiCHO expressed trastuzumab WT and N221 were treated with Endo F2 and analyzed by SDS PAGE and CBB staining (Right). The molecular mass of the light chain ( LC) corresponds to 25 kDa, and of the HC to 50 kDa. The molecular weight ladder used is Precision Plus Protein™ All Blue (BioRad).
Figure 2. No galactosylation was observed for trastuzumab transiently expressed in ExpiCHO cells, in contrast to the variable galactose levels upon its transient expression in HEK293S cells. Trastuzumab WT and N221 were expressed in ExpiCHO and HEK293S WT cells. After purification via protein A and SEC, the antibodies were subjected to DSA FACE for N-glycan analysis. In this process, their full glycosylation profile was analyzed, giving a mixture of N297 and N221 glycans. The native N-glycans were verified by exoglycosidase digest (not shown).
Figure 3. Addition of 1 % hGalTl and precursors during HEK293S expression improved antibody galactosylation tremendously, with the main glycoform being double galactosylated. Trastuzumab WT and N221 were co-transfected along with 1 % hGalTl in HEK293S cells. During expression, precursors (uridine, galactose and MnCL) were added. After protein A chromatography, the total N-glycan glycoforms, derived from N221 and/or N297 asparagines, were analyzed by DSA FACE.
Figure 4. Trastuzumab N221 was efficiently trimmed in HEK293S GlycoDelete LacNAc (GDLN) and carried high levels of Gal-GIcNAc disaccharide at both sites (N297 and N221). Trastuzumab N221 was expressed, without hGalTl or precursors, in HEK293 GlycoDelete LacNAc (GDLN) cells. Following purification, antibody was digested with FabRICATOR, which cleaves in the hinge region, conveniently separating the N221 N-glycan from the N297 N-glycan. The detected masses corresponded to the light chain (VL-CL), N-terminal fragment of the heavy chain, carrying the N221 site (VH-CH1) and the C- terminal fragment, carrying the N297 site (CH2-CH3).
Figure 5. HER2 affinity of trastuzumab was only slightly reduced upon introducing the neo-N- glycosylation site N221. Trastuzumab WT and N221 were compared in an ELISA setup to assess their binding to coated HER2 (left panel). The right panel incorporates the calculated EC5o values. Figure 6. The binding of CD16a for trastuzumab is located at the CH2 domain near the hinge, in the vicinity of the neo-N-glycosylation site N221. Possibly, some affinity loss may be caused by N- glycosylation at this site. The Left structural view shows a side view, while the Right structural view shows the frontal view of the interaction between the Fc domain and CD16a. The introduced neo-N- glycosylation site N221 is indicated in black. Figure adapted from PBD 1HZH, PVUO.
Figure 7. No loss of affinity between CD16a 158V and trastuzumab was detected upon N-glycosylation of N221 in HEK293S WT cells. Trastuzumab WT and N221 were compared in an ELISA setup to assess their binding to CD16a V158 (Left). The right panel incorporates the calculated EC5o values.
Figure 8. The Galactose N-glycans on Trastuzumab N221 were oxidized with a much higher efficiency as compared to trastuzumab WT. Trastuzumab variants were oxidized overnight with the standard Galactose oxidase (GaOx) concentration (0.66U/nmol) in the presence of aminooxy-PEG-biotin. All oxidation reactions were compared to a control in which the enzyme was omitted. The Left panel shows the streptavidin - based western blot detection of conjugation, while the right panel shows the loading distribution of the antibodies by CBB staining. The molecular weight ladder used is Precision Plus Protein™ All Blue (BioRad). Abbreviations: +Gal (condition for higher galactosylation with hGalTl and precursors), LN (GlycoDelete LacNAc).
Figure 9. Increasing the GaOx units during oxidation resulted in aldehydes generated at both N- glycosylation sites (N221 and N297). Trastuzumab WT and N221 were incubated overnight with variable amounts of GaOx (ranging from 0.66U/nmol (N221 selective condition) to 660U/nmol (conditions described in Angelastro et al.), in the presence of 100 equivalents of aminooxy-PEG-biotin, for subsequent SDS PAGE for western blotting for streptavidin-based detection. The Left panel shows the oxidation for trastuzumab expressed in HEK293S GDLN, while the Right panel shows trastuzumab expressed in HEK293S WT cells. The 0.66 U/nmol condition (boxed) is the condition used in house, while the 660 u/nmol ( boxed) indicates the condition used by Angelastro et al. The molecular weight ladder used is Precision Plus Protein™ All Blue (BioRad).
Figure 10. Oxidation by GaOx resulted in loss of FabRICATOR cleavage of trastuzumab N221. Trastuzumab N221 was oxidized overnight with GaOx in the presence of aminooxy-PEG-biotin (abbreviated as AmOx-Biotin). As controls, GaOx and/or biotin were omitted. After PPD catalysis of conjugation, the antibody was incubated overnight with varying units of FabRICATOR, and subsequently analyzed by non-reducing SDS PAGE for CBB staining. The upper panel shows the resulting CBB-stained gel, while the lower panel indicates the cleavage site of FabRICATOR, adapted from Buecheler et al. (J Pharm Pharmacol. 2018;70(5):625-635). The molecular weight ladder used is Precision Plus Protein™ All Blue (BioRad). Figure 11. Conjugation of MMAF to trastuzumab N221 was assumed to be completed with a minimum of 250 equivalents, in respect to the antibody. Trastuzumab WT and N221 were oxidized overnight with 0.66U/nmol GaOx in the presence of varying amounts of aminooxy-MMAF. Afterwards, the conjugation was catalyzed with PPD and analyzed by SDS PAGE, with western blotting for anti-hlgG detection. Two controls were included: one lacking GaOx with 1000 eq. MMAF, and one lacking MMAF with GaOx. The molecular weight ladder used is Precision Plus Protein™ All Blue (BioRad).
Figure 12. More MMAF conjugated to trastuzumab N221, expressed in HEK293S WT, after the second conjugation. Trastuzumab N221 was oxidized overnight with 0.66U/nmol GaOx and 42.9 equivalents aminooxy - MMAF (WT #1 and LN) or with 250 equivalents (WT #2). After PPD catalysis of the conjugation, the antibodies were purified by protein A chromatography and desalted to PBS, and analyzed by SDS PAGE for western blotting using an anti - MMAF detection antibody. The molecular weight ladder used is Precision Plus Protein™ All Blue (BioRad). Abbreviations: LN (HEK293S GlycoDelete LacNAc).
Figure 13. Trastuzumab N221 was conjugated partially with MMAF. Trastuzumab N221 was oxidized overnight with 0.66U/nmol GaOx and 42.9 equivalents aminooxy - MMAF (WT #1 and GDLN), or 250 equivalents - MMAF (WT #2). After PPD catalysis of the conjugation, the antibodies were purified by protein A chromatography and desalted to PBS. The purified proteins were then incubated overnight with aminooxy-PEG-biotin, to check for unreacted aldehydes. As control, non-conjugated trastuzumab N221 (HEK wt and GDLN expressed) ("Stock") was included with and without GaOx ("+GO"). After incubation, the samples were analyzed by SDS PAGE and streptavidin - based western blotting. MMAF conjugation of trastuzumab expressed in HEK293S WT was repeated once more, when a new batch of AmOxMMAF arrived, and was indicated as #2. The molecular weight ladder used is Precision Plus Protein™ All Blue (BioRad).
Figure 14. HER2-affinity of trastuzumab was not drastically reduced upon conjugating to the neo-N- glycosylation site N221. Trastuzumab WT and N221 variants, concerning expression system and MMAF conjugation, were compared in an ELISA setup to assess their binding to coated HER2 (Left). The right panel incorporates the calculated EC5o values.
Figure 15. Trastuzumab - MMAF was only cytotoxic to SK-BR-3 cells when linked through GlycoDelete LacNAc (GDLN) N-glycans. Antibody and antibody-drug conjugate treatments were tested for toxicity at HER2 expressing breast cancer cells (SK-BR-3). Viability was measured after a three day incubation by ATP measurement (CellTiter-Glo) and was plotted in relation to untreated cells. Error bars were calculated for biological repeats. Trastuzumab N221 (WT) was conjugated twice with MMAF (#1 versus #2), with the second repeat more efficiently conjugated which resulted in a higher DAR. Figure 16. Trastuzumab N221 (GDLN)-MMAF induced cytotoxicity at lower concentrations than the clinical grade comparators Kadcyla and Enhertu. ADC treatments were tested for toxicity at HER2 expressing breast cancer cells (SK-BR-3). Viability was measured after a three day incubation by ATP measurement (CellTiter-Glo) and was plotted in relation to untreated cells (upper graph), while the calculated EC5o was plotted (lower graph). Error bars were calculated for biological repeats.
Figure 17. Bystander effect observed only for Enhertu in cocultures of SK-BR-3 and MDA-MB-231 cells. ADC treatments were tested for toxicity at breast cancer cells. Viability was measured after a three day incubation by ATP measurement (CellTiter-Glo) and was plotted in relation to untreated cells. The upper panel shows the lack of toxicity for the HER2 negative MDA-MB-231 cells. The lower panel shows the toxicity in the coculture of SK-BR-3 and MDA-MB-231 cells.
Figure 18. Chemical formula of the Asn-GIcNAc-Gal-oxime-PEG-MMAF portion of the trastuzumab ADC produced in HEK GDLN.
Figure 19. Chemical or enzymatic oxidation approaches can be used to generate aldehydes in glycoproteins. Chemical oxidation with periodate generates aldehydes from vicinol diols such as sialic acid and the backbone hydroxyl groups in, for instance, fucose. Alternatively and more specifically, galactose oxidase can be used to selectively oxidize galactose at its C6 position. The Left part shows the specific oxidation for each monosaccharide, while the Right part shows the impact on antibody glycosylation, with the substrate indicated with a gray background.
Figure 20. Schematic representation of glyco-engineering required to provide for Glycodelete-LacNac (GDLN) production in a mammalian host cells. Left: HEK293S Wild-type (WT) cell N-glycan structures; middle: HEK293S Glycodelete (GD) cell being engineered in lacking GnT-l activity and expression of an exogenously added endoglucosaminidase, such as EndoT, resulting in N-glycan structures composed of GIcNac-Gal-Sia; right: HEK293S Glycodelete-LacNac (GDLN) cell being further engineered in lacking GNE activity resulting in N-glycan structures composed of GIcNac-Gal. GnT-l, N acetylglucosaminyltransferase I; GNE, bifunctional UDP - N - acetylglucosamine 2 - epimerase/N - acetylmannosamine kinase.
Figure 21. Mechanism of action of antibody-drug conjugates generated with GlycoDelete LacNAc. Antibody-drug conjugates (ADCs) bind to targeted cells by interacting with their antigen, expressed on the cell surface. This binding triggers internalization and trafficking of the ADC to the lysosome. Lysosomal degradation of the GDLN-ADC yields the active metabolite Asn-GIcNAc-galactose-Oxime linker-Toxin. This active metabolite escapes from the lysosome to perform its function.
Figure 22. GlycoDelete processing is required for efficient cytotoxicity of MMAF conjugated antibodies in the SK-BR-3 cell line. A. Schematic representation of the GDLN-ADC. B. Chemical representation of the drug-linker compound. C. Viability of SK-BR-3 cells in function of ADC concentration. ADC treatments were tested for toxicity at HER2 expressing breast cancer cells (SK-BR-3). Viability was measured after a five day incubation by ATP measurement (CellTiter-Glo) and was plotted in relation to live cells and dead cells. Error bars were calculated for biological repeats. The proposed active metabolite of the oxime- ligated ADCs is shown on the right. T-DM1 (or Kadcyla) and Enhertu are included as clinical comparators. Treatments E and F are included as unconjugated antibodies.
Figure 23. GlycoDelete processing is required for efficient cytotoxicity of MMAE conjugated antibodies in the SK-BR-3 cell line. A. Schematic representation of the GDLN-ADC. B. Chemical representation of the drug-linker compound. C. Viability of SK-BR-3 cells in function of ADC concentration. ADC treatments were tested for toxicity at HER2 expressing breast cancer cells (SK-BR-3). Viability was measured after a five day incubation by ATP measurement (CellTiter-Glo) and was plotted in relation to live cells and dead cells. Error bars were calculated for biological repeats. The proposed active metabolite of the oxime- ligated ADCs is shown on the right. T-DM1 (or Kadcyla) and Enhertu are included as clinical comparators. Treatments E and F are included as unconjugated antibodies.
Figure 24. GlycoDelete processing is required for efficient cytotoxicity of DM1 conjugated antibodies in the SK-BR-3 cell line. A. Schematic representation of the GDLN-ADC. B. Chemical representation of the drug-linker compound. C. Chemical representation of the conjugation in Kadcyla (T-DM1). D. Viability of SK-BR-3 cells in function of ADC concentration. ADC treatments were tested for toxicity at HER2 expressing breast cancer cells (SK-BR-3). Viability was measured after a five day incubation by ATP measurement (CellTiter-Glo) and was plotted in relation to live cells and dead cells. Error bars were calculated for biological repeats. The proposed active metabolite of the oxime-ligated ADCs is shown on the right. T-DM1 (or Kadcyla) is included as clinical comparator, next to the unconjugated antibodies.
Figure 25. GDLN-ADC with auristatins are as potent in the SK-BR-3 cell line, if not more potent, than conventional ADC conjugated via interchain disulphide cysteines, despite lower DAR. A-B. Schematic representation of maleimide-based conjugation of auristatins to antibodies C-D. Chemical representation of the maleimide-functionalized auristatin-linker compounds. E. Viability of SK-BR-3 cells in function of ADC concentration. ADC treatments were tested for toxicity at HER2 expressing breast cancer cells (SK-BR-3). Viability was measured after a five day incubation by ATP measurement (CellTiter- Glo) and was plotted in relation to live cells and dead cells. Error bars were calculated for biological repeats. The proposed active metabolite of the oxime-ligated ADCs is shown on the right. T-DM1 (or Kadcyla) is included as clinical comparator, next to the unconjugated antibodies.
Figure 26. GlycoDelete processing is required for efficient cytotoxicity of MMAF conjugated antibodies in the BT-474 cell line. A. Schematic representation of the GDLN-ADC. B. Chemical representation of the drug-linker compound. C. Viability of BT-474 cells in function of ADC concentration. ADC treatments were tested for toxicity at HER2 expressing breast cancer cells (BT-474). Viability was measured after a six day incubation by ATP measurement (CellTiter-Glo) and was plotted in relation to live cells and dead cells. Error bars were calculated for biological repeats. The proposed active metabolite of the oxime-ligated ADCs is shown on the right. T-DM1 (or Kadcyla) and Enhertu are included as clinical comparators. Treatments E and F are included as unconjugated antibodies.
Figure 27. GlycoDelete processing is required for efficient cytotoxicity of MMAE conjugated antibodies in the BT-474 cell line. A. Schematic representation of the GDLN-ADC. B. Chemical representation of the drug-linker compound. C. Viability of BT-474 cells in function of ADC concentration. ADC treatments were tested for toxicity at HER2 expressing breast cancer cells (BT-474). Viability was measured after a six day incubation by ATP measurement (CellTiter-Glo) and was plotted in relation to live cells and dead cells. Error bars were calculated for biological repeats. The proposed active metabolite of the oxime-ligated ADCs is shown on the right. T-DM1 (or Kadcyla) and Enhertu are included as clinical comparators. Treatments E and F are included as unconjugated antibodies.
Figure 28. GDLN-ADC with auristatins are more potent in the BT-474 cell line than conventional ADC conjugated via interchain disulphide cysteines, despite lower DAR. A-B. Schematic representation of maleimide-based conjugation of auristatins to antibodies C-D. Chemical representation of the maleimide-functionalized auristatin-linker compounds. E. Viability of BT-474 cells in function of ADC concentration. ADC treatments were tested for toxicity at HER2 expressing breast cancer cells (BT-474). Viability was measured after a six day incubation by ATP measurement (CellTiter-Glo) and was plotted in relation to live cells and dead cells. Error bars were calculated for biological repeats. The proposed active metabolite of the oxime-ligated ADCs is shown on the right. T-DM1 (or Kadcyla) is included as clinical comparator, next to the unconjugated antibodies.
Figure 29. Western blot validation of toxin conjugation. Toxin was successfully conjugated to the antibody of interest as indicated by the anti-toxin specific western blots. Fc-detection was used to normalize the amount of antibody, while different anti-toxin detections were used specifically for each toxin: DM1, MMAF and MMAE. The molecular weight ladder used is the Precision Plus Protein™ All Blue Prestained Protein Standards (Biorad, 1610393). Abbreviations: Mai: maleimide, AmOx: aminooxy, LN: GlycoDelete LacNAc, WT: wild type HEK expressed (oligosaccharide).
DESCRIPTION
The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. Of course, it is to be understood that not necessarily all aspects or advantages may be achieved in accordance with any particular embodiment of the invention. Thus, for example those skilled in the art will recognize that the invention may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other aspects or advantages as may be taught or suggested herein. The invention, both as to organization and method of operation, together with features and advantages thereof, may best be understood by reference to the following detailed description when read in conjunction with the accompanying drawings. The aspects and advantages of the invention will be apparent from and elucidated with reference to the embodiment(s) described hereinafter. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases 'in one embodiment' or 'in an embodiment' in various places throughout this specification are not necessarily all referring to the same embodiment but may.
Definitions
Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments, of the invention described herein are capable of operation in other sequences than described or illustrated herein. The following terms or definitions are provided solely to aid in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016), for definitions and terms of the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g. in molecular biology, biochemistry, structural biology, and/or computational biology).
The terms "protein" and "polypeptide" are interchangeably used further herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. The term "peptide" refers to a polymer of amino acid residues and variants and synthetic analogues of the same, wherein the peptide is of shorter length as compared to a protein or polypeptide, potentially as a result of protein digestion or proteolytic cleavage, preferably with a length of maximally 40-100 amino acids, most preferably not more than 50 amino acids. By "recombinant (poly)peptide " is meant a (poly)peptide made using recombinant techniques, i.e., through the expression of a recombinant or synthetic polynucleotide, which may be obtained in vitro and/or in a cellular context. When the chimeric (poly)peptide or biologically active (i.e. functional) portion thereof is recombinantly produced, it is also preferably purified, or isolated, as used interchangeably herein, or substantially free of culture medium, i.e., the impurities represent less than about 20 %, more preferably less than about 10 %, and most preferably less than about 5 % of the volume of the protein preparation. By "isolated" or "purified" is meant material that is substantially or essentially free from components that normally accompany it in its native state. By "enriched" or "enrichment" is meant herein that material or specific components are present at a substantially higher amount as compared to the non-enriched material, so typically involving a purification or isolation step.
Amino acids are presented herein by their 3- or 1-lettercode nomenclature as defined and provided also in the IUPAC-IUB Joint Commission on Biochemical Nomenclature (Nomenclature and Symbolism for Amino Acids and Peptides. Eur. J. Biochem. 138: 9-37 (1984)); as follows: Alanine (A or Ala), Cysteine (C or Cys), Aspartic acid (D or Asp), Glutamic acid (E or Glu), Phenylalanine (F or Phe), Glycine (G or Gly), Histidine (H or His), Isoleucine (I or He), Lysine (K or Lys), Leucine (L or Leu), Methionine (M or Met), Asparagine (N or Asn), Proline (P or Pro), Glutamine (Q. or Gin), Arginine (R or Arg), Serine (S or Ser), Threonine (T or Thr), Valine (V or Vai), Tryptophan (W or Trp), and Tyrosine (Y or Tyr).
The term "expression vector", as used herein, includes any vector known to the skilled person, including plasmid vectors, cosmid vectors, phage vectors, such as lambda phage, viral vectors, such as adenoviral, AAV or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or Pl artificial chromosomes (PAC). Expression vectors generally contain a desired coding sequence and appropriate promoter sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g. higher eukaryotes, lower eukaryotes). Typically, a vector comprises a nucleotide sequence in which an expressible promoter or regulatory nucleotide sequence is operatively linked to, or associated with, a nucleotide sequence or DNA region that codes for an mRNA, such that the regulatory nucleotide sequence is able to regulate transcription or expression of the associated nucleotide sequence. Typically, a regulatory nucleotide sequence or promoter of the vector is not operatively linked to the associated nucleotide sequence as found in nature, hence is heterologous to the coding sequence of the DNA region operably linked to. The term "operatively" or "operably" "linked" as used herein refers to a functional linkage between the expressible promoter sequence and the DNA region or gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest, and refers to a functional linkage between the gene of interest and the transcription terminating sequence to assure adequate termination of transcription in eukaryotic cells. In addition, the term refers to the linkage (or fusion) between a targeting sequence and the open reading frame of an enzyme. An "inducible promoter" refers to a promoter that can be switched 'on' or 'off' (thereby regulating gene transcription) in response to external stimuli such as, but not limited to, temperature, pH, certain nutrients, specific cellular signals, et cetera. It is used to distinguish between a "constitutive promoter", by which a promoter is meant that is continuously switched 'on', i.e. from which gene transcription is constitutively active.
"Binding" means any interaction, be it direct or indirect. A direct interaction implies a contact between the binding partners. An indirect interaction means any interaction whereby the interaction partners interact in a complex of more than two molecules. The interaction can be completely indirect, with the help of one or more bridging molecules, or partly indirect, where there is still a direct contact between the partners, which is stabilized by the additional interaction of one or more molecules. The binding or association maybe non-covalent - wherein the juxtaposition is energetically favoured by for instance hydrogen bonding or van der Waals or electrostatic interactions - or it may be covalent, for instance by peptide or disulphide bonds. By the term "specifically binds," as used herein is meant a binding domain which recognizes a specific target, but does not substantially recognize or bind other molecules in a sample. Specific binding does not mean exclusive binding. However, specific binding does mean that proteins have a certain increased affinity or preference for one or a few of their binders. The term "affinity", as used herein, generally refers to the degree to which a ligand, chemical, protein or peptide binds to another (target) protein or peptide so as to shift the equilibrium of single protein monomers toward the presence of a complex formed by their binding.
The term "antibody" refers to an immunoglobulin (Ig) molecule or a molecule comprising an immunoglobulin (Ig) domain, which specifically binds with an antigen. "Antibodies" can further be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. The term "antigen-binding domain" refers to a portion of any antibody or antibody-like structure that by itself has high affinity for an antigenic determinant, or epitope, and contains one or more complementarity determining regions (CDRs) accounting for such specificity, typically at least 3 CDRs, or in conventional antibodies, such as monoclonal antibodies, defined by 6 CDRs. Non-limiting examples of antigen-binding domains or active antibody fragments include immunoglobulin domains, Fab, F(ab)'2, scFv, heavy-light chain dimers, immunoglobulin single variable domains (ISVDs), Nanobodies (or VHH antibodies), domain antibodies, and single chain structures, such as a complete light chain or complete heavy chain. The term 'antibody' or 'Fc-fusion' as used herein further refers to the genetic linking or fusion of antigenbinding fragments or antigen-binding domains with an Fc constant domain as to obtain dimers forming an antibody structure when expressed in a recombinant host. In particular, antibody fragments, or single domain antibodies such as ISVDs may be C-terminally fused to the N-terminus of an Fc domain, preferably via a linker or hinge region, as known in the art and/or as described herein.
Among the five isotypes of naturally occurring immunoglobulins (Igs), which are IgG, IgA, IgM, IgD, and IgE, IgG comprises the majority, representing 60 % of total serum Igs in humans. Among the human IgG class of antibodies, the IgGl antibody subclass is most abundant, binds the FcR receptor and shows distinct properties compared with the lgG2, lgG3, and lgG4 subclasses and is the most exploited subclass in therapeutic antibodies. The human IgG molecule is composed of two identical fragment antigen binding (Fab) domains and one fragment crystallizable (Fc) domain that make it multivalent and multifunctional. The two Fab fragments each consist of a heterodimer of a light chain and the N-terminal part of the heavy chain, whereas the C -terminal half of the two heavy chains dimerizes to form the Fc fragment of the IgG antibody. The N-terminal domains of the Fab fragment are the variable domains (VL and VH) that are responsible for antigen recognition, whereas the C-terminal part of the heavy chains compose the Fc fragment that is responsible for humoral and cellular effector functions. The two Fabs and the Fc-tail are connected by the hinge region, which facilitates the spatial alignment of the three moieties for binding to antigens and effector ligands.
The "hinge region" of an antibody is defined herein as the amino acid sequence for the antibody region that connects the antigen-binding region (or Fab region) and Fc regions together and contributes flexibility between these regions. The hinge length is linked with IgG functionality, and is generally considered as a three-part structure, an upper, core, and lower section.
"Fc domains" or "Fc-regions" or "Fc-tails", as interchangeably used herein, refer to the single Fc chain and/or the dimeric Fc domain of an Fc-containing proteins. Specifically in antibodies, said Fc domain is thus responsible for antibody function, and Antibody Fc engineering stands for engineering functions of antibodies, which are effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), and controlling serum half-life. Engineered Fc domains may therefore be present in the form of mutants or variants containing amino acid substitutions, insertions or deletions as to allow different modifications of the Fc in post-translational modifications, dimerization behavior, effector function, serum half-life, among others. Fc region sequences often include the hinge region, as to provide for an Fc fragment that can be used for fusion to an antigen-binding domain, such as a Fab, or an ISVD. To indicate the variations present in Fc domains based on the sequence of naturally occurring IgGs, conventional antibody numbering annotations are known in the art, such as for instance IMGT numbering (LeFranc, 2014; Frontiers in Immunology. 5 (22): 1-22), Kabat numbering (Kabat, E.A. et al., Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication n° 91-3242, pp 662,680,689 (1991)), or preferably used herein EU numbering (Edelman et al. (1969). The covalent structure of an entire gammaG immunoglobulin molecule. Proc Natl Acad Sci USA.;63:78-85).
Antibodies can be obtained by immunization of animals or humans, or made recombinantly by introducing a particular exogenous sequence in a host cell for production of an antibody. Most common is the manufacturing of monoclonal antibodies (mAbs) which are human-like antibodies produced from a cell lineage made by cloning a unique white blood cell, and derived from mouse proteins (murine), human proteins, or chimeric (mouse/human parts) or humanized proteins.
The term "glycosylation acceptor site" or "glycan acceptor site", as used interchangeably herein, refers to a position within a polypeptide which can be N- or O-glycosylated. N-linked glycans are typically attached to the side chain of an Asparagine (Asn), while O-linked glycans are commonly linked to the hydroxyl oxygen side chain residue of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline sidechains. Antibody Fc tails conventionally have an Asn at position 297 (EU numbering) functioning as N- glycan acceptor site, though alternative N-glycosylation sites may be present and/or introduced by substitution or mutation of single or multiple amino acids in the original antibody Fc part.
The term "N-glycosylation acceptor site" or "N-glycan acceptor site", as used interchangeably herein, refers to a position within a polypeptide , such as an antibody, which can be N-glycosylated. N-linked glycans are typically attached to Asparagine (Asn) which resides in a consensus site. An "NXT", "NXS", "NXC" or "NXV" motif refers to the consensus sequences Asn-Xaa-Thr/Ser or Asn-Xaa-Cys/Val, wherein Xaa can be any amino acid except proline (Shrimal, S. and Gilmore, R., J Cell Sci. 126(23), 2013, Sun, S. and Zhang, H., Anal. Chem. 87 (24), 2015). It is well known in the art that potential N-glycosylation acceptor sites are specific to the consensus sequence Asn-Xaa-Thr/Ser or Asn-Xaa-Cys/Val. It has been shown in the art that the presence of proline between Asn and Thr/Ser leads to inefficient N- glycosylation.
Glycosylation sites and glycan structures present on proteins, referred to herein as glycoproteins, can be determined in part by enrichment of glycopeptides, or by enzymatically or chemically releasing glycans. The structure of released glycans and remaining peptides can be determined by mass spectrometry and liquid chromatography/mass spectrometry. Fragmentation techniques can be used to obtain glycan structures and amino acid sequences of the peptide backbone of glycopeptides with mass spectrometry, though often a low fragmentation efficiency is obtained with glycopeptides, and customized approaches are often desired to a certain analytical setting. Practitioners are particularly directed to Laue and Wuhrer, High-Throughput Glycomics and Glycoproteomics, Springer Science+Business Media, LLC, part of Springer Nature, Humana New York, NY (2017), for definitions and terms used in glycobiology and glycoproteomic approaches.
A "glycan" generally refers in the art to glycosidically linked monosaccharides, oligosaccharides and polysaccharides. Hence, carbohydrate portions of a glycoconjugate, such as a glycoprotein, glycolipid, or a proteoglycan are referred to herein as a "glycan". Glycans can be homo- or heteropolymers of monosaccharide residues, and can be linear or branched. Generally N-linked glycans may be composed of N-acetylgalactosamine (GalNAc), Galactose (Gal), neuraminic acid (such as sialic acid (Sia), most common being N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Glc), and N-acetyl-9- O-acetylneuraminic acid), N-acetylglucosamine (GIcNAc), Fucose (Fuc), Mannose (Man), and other monosaccharides, as also exemplified further herein.
In eukaryotes, O-linked glycans are assembled one sugar at a time on a serine or threonine residue of a peptide chain in the Golgi apparatus. Unlike N-linked glycans, there are no known consensus sequences but the position of a proline residue at either -1 or +3 relative to the serine or threonine is favourable for O-linked glycosylation.
"Complex N-glycans" in the art refers to structures with typically one, two or more (e.g. up to six) outer branches, most often linked to an inner core structure Man3GlcNAc2. The term "complex N-glycans" is well known to the skilled person and defined in literature. For instance, a complex N-glycan may have at least one branch, or at least two, of alternating GIcNAc and optionally also Galactose (Gal) residues that may terminate in a variety of oligosaccharides but typically will not terminate with a Mannose residue. For the sake of clarity a single GIcNAc, LacNAc (= GIcNAc-Gal; N-acetyllactosamine is a disaccharide that is synthesized from linking GIcNAc and Gal by a pi-4 linkage), sialyl-LacNAc (=GlcNAc-Gal-Sia) present directly on an N-glycosylation site of a glycoprotein (thus lacking the inner core structure Man3GlcNAc2) is not regarded as a complex N-glycan.
'Glycoproteins' as used in the application refers to proteins that, in their normal physiological context and/or their functional form, contain oligosaccharide chains (N-glycans) covalently attached to their polypeptide side-chains. In addition, a glycoprotein comprises also proteins with an artificially introduced glycosylation site, particularly an artificially introduced N-glycosylation site. Typically, a glycoprotein, typically a recombinant glycoprotein, for example a heterologous recombinant glycoprotein (which does not occur normally in the eukaryotic organism) is produced as several glycoforms when it is made in a eukaryotic organism such as a N-glycosylation-engineered eukaryotic organism. The terms '(glyco)protein' and 'enzyme' (e.g. endoglucosaminidase, glycosyltransferase, mannosidase, mannosyltransferase) as used in the application are also intended to cover functionally active fragments and variants of the naturally occurring proteins. Indeed, for many (e.g. therapeutic) proteins, part of the protein may be sufficient to achieve an (e.g. therapeutic, enzymatic) effect. The same applies for variants (i.e. proteins in which one or more amino acids have been substituted with other amino acids, but which retain functionality or even show improved functionality), in particular for variants of the enzymes optimized for enzymatic activity.
In the context of the application, a glycoprotein refers to the protein itself; a glycoprotein may be either in its glycosylated or non-glycosylated form. A 'glycosylated' protein is a (glyco)protein that carries at least one oligosaccharide chain. An N-glycosylated protein, particularly an N-glycosylated recombinant glycoprotein, is a glycoprotein which carries at least one oligosaccharide chain on an N-glycan.
An 'endoglucosaminidase' as used herein refers to enzymes that hydrolyse the bond between the anomeric carbon of a non-terminal beta-linked N-acetylglucosamine residue in an oligosaccharide of a glycoprotein or a glycolipid, and its aglycon, thereby releasing mono- or oligosaccharides from glycoproteins or glycolipids or sugar polymers. Endoglucosaminidases are a subset of the glycosidases, and may or may not have other enzymatic activities (such as e.g. glycosyltransferase activity). A particular class of endoglucosaminidases is formed by the endo-p-N-acetylglucosaminidases or mannosyl- glycoprotein endo-p-/V-acetylglucosaminidases, indicated as EC 3.2.1.96 in the International Union of Biochemistry and Molecular Biology (IUBMB) nomenclature. This particular class of enzymes are capable of catalyzing the endohydrolysis of the N, N '-diacetylchitobiosyl unit in high-mannose glycopeptides and glycoproteins containing the -[Man(GlcNAc)j]Asn- structure. One /V-acetyl-D-glucosamine (GIcNAc) residue remains attached to the protein; the rest of the oligosaccharide is released intact. The result thus is a single GIcNAc-modified N-glycosylation site present on a glycoprotein. A particular preferred class of endoglucosaminidases is formed by the mannosyl-glycoprotein endo-p-/V-acetylglucosaminidases, indicated as EC 3.2.1.96 in the IUBMB nomenclature. These enzymes can remove sugar chains (hybrid N- glycans, high mannose N-glycans and neoglycoforms of N-glycans as shown herein) while leaving one GIcNAc residue on the protein. Examples of these include, but are not limited to Endo A, Endo BH, Endo CE, Endo D, Endo Fl, Endo H, Endo M, Endo T (see also W02006/050584), and ENGase. Other examples are known to the skilled person and can for instance be found on www.cazy.org, in particular under the Glycoside Hydrolase Family 85 and 18. Particularly envisaged is the use of the Endo T enzyme from Hypocrea jecorina (formerly known as Trichoderma reesei) that is described in W02006/050584 (see e.g. SEQ ID NOs: 9-12 therein). A 'glycosyltransferase' as used in the application is any of a group of enzymes that catalyze the transfer of glycosyl groups in biochemical reactions, in particular glycosyl transfer to asparagine-linked sugar residues to give N-linked glycoproteins. Glycosyltransferases fall under EC 2.4 in the IUBMB nomenclature, a particular class of glycosyltransferases are hexosyltransferases (EC 2.4.1). Among the wide variety of these post-translational enzymes that process peptides into glycoproteins are enzymes such as, but not limited to, N-acetylglucosaminyl transferases, N-acetylgalactosaminyltransferases, sialyltransferases, fucosyltransferases, galactosyltransferases, and mannosyltransferases.
Note that exogenous mannosyltransferases are excluded for specific embodiments of N-glycosylation- engineered yeast cells described in the application. 'Mannosyltransferases' as used in the application refers to enzymes that catalyze the transfer of a mannosyl group to an acceptor molecule, typically another carbohydrate, in the Golgi apparatus. Mannosyltransferases are typically endogenous enzymes in fungi and yeast and involved in the synthesis of high-mannose type glycans.
A "higher eukaryotic cell" as used herein refers to eukaryotic cells that are not cells from unicellular organisms. In other words, a higher eukaryotic cell is a cell from (or derived from, in case of cell cultures) a multicellular eukaryote such as a human cell line or another mammalian cell line (e.g. a CHO cell line). Particularly, the term generally refers to mammalian cells, human cell lines and insect cell lines. More particularly, the term refers to vertebrate cells, even more particularly to mammalian cells or human cells. The higher eukaryotic cells as described herein will typically be part of a cell culture (e.g. a cell line, such as a HEK or CHO cell line).
By "lower eukaryotic cell" a filamentous fungus cell or a yeast cell is meant. Yeast cells can be from the species Saccharomyces (e.g. Saccharomyces cerevisiae), Hansenula (e.g. Hansenula polymorpha), Arxula (e.g. Arxula adeninivorans), Yarrowia (e.g. Yarrowia lipolytica), Kluyveromyces (e.g. Kluyveromyces lactis), or Komagataella phaffii (Kurtzman, C.P. (2009) J Ind Microbiol Biotechnol. 36(11) which was previously named and better known under the old nomenclature as Pichia pastoris and further used herein. According to a specific embodiment, the lower eukaryotic cells are Pichia cells, and in a most particular embodiment Pichia pastoris cells. In specific embodiments the filamentous fungus cell is Myceliopthora thermophila (also known as Cl by the company Dyadic), Aspergillus species (e.g. Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus japonicus), Fusarium species (e.g. Fusarium venenatum), Hypocrea and Trichoderma species (e.g. Trichoderma reesei).
Essential to the present invention, the "lower or higher eukaryotic cell" or "eukaryotic cell" of the present invention is a glyco-engineered cell. A "glyco-engineered cell" refers to a cell that has been genetically modified so that it expresses proteins with an altered N-glycan structure and/or O-glycan structure as compared to in a wild type background. Typically, the naturally occurring modifications on glycoproteins have been altered by genetic engineering of enzymes involved in the glycosylation pathway. In general, sugar chains in N-linked glycosylation may be divided in three types: high-mannose (typically yeast), complex (typically mammalian) and hybrid type glycosylation. Besides that, a variety of O-glycan patterns exist, for example with yeast oligomannosylglycans differing from mucin-type O- glycosylation in mammalian cells. The different types of N- and O-glycosylation are all well known to the skilled person and defined in the literature. Considerable effort has been directed towards the identification and optimization of strategies for the engineering of eukaryotic cells that produce glycoproteins having a desired N-and/or O-glycosylation pattern and are known in the art (e.g. De Pourcq, K. et al., Appl Microbiol Biotechnol. 87(5), 2010).
In the present invention the glyco-engineered cells (or the glyco-engineered expression system) which are used are described in patent applications W02010015722 and WO2015032899 (further designated herein as GlycoDelete cells, or cells having a GlycoDelete background) and in Meuris L. et al (2014) Nat. Biotechn. 32(5) 485) and relates to a eukaryotic cell expressing both at least an endoglucosaminidase enzyme and a target protein, which may be an antibody or antibody fragment, and wherein the recombinant secreted target proteins are characterized by a uniform N-glycosylation pattern (in particular one single GIcNAc residue (in lower eukaryotes) or a modification thereof such as GIcNAc modified with Galactose (LacNAc= Gal-GIcNAc) or sialyl-LacNAc (=Sia-Gal-GlcNAc) (in mammalian cells). Particularly preferred in the present invention are higher eukaryotic cells which have a GlycoDelete background. Lower eukaryotic cells having a GlycoDelete background produce N-glycans having one single GIcNAc residue.
The term 'beta-1, 4-galactosyltransferase' refers to an enzyme that has exclusive specificity for the donor substrate UDP-galactose; all transfer galactose in a betal,4 linkage to similar acceptor sugars: GIcNAc, Glc, and Xyl. In the present invention the beta-1, 4-galactosyltransferase adds galactose to N- acetylglucosamine residues that are either monosaccharides or the nonreducing ends of glycoprotein carbohydrate chains. Particularly preferred beta-1, 4-galactosyltransferases are of the mammalian type, even more particularly are of the human type.
The term 'UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase' or abbreviated GNE enzyme in the eukaryotic cell of the present invention refers to a bifunctional enzyme that initiates and regulates the biosynthesis of N-acetylneuraminic acid (NeuAc), a precursor of sialic acids. It is a ratelimiting enzyme in the sialic acid biosynthetic pathway. The enzyme is allosterically regulated and hence is subject to feedback inhibition by cytidine monophosphate-N-acetylneuraminic acid (CMP-Neu5Ac), the end product of neuraminic acid biosynthesis. Hence a eukaryotic cell lacking 'UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase' or GNE in the present invention can refer to a lower eukaryotic cell which - by nature - does not possess this enzyme. Alternatively, it refers to a higher eukaryotic cell which has a gene disruption (or a gene deletion or a mutation which generates a non-functional enzyme) in this enzyme (also see Figure 20).
In a specific embodiment of the present invention, the ADC is thus produced in a Glycodelete background of a higher eukaryotic cell, i.e. an eukaryotic cell deficient in Gntl and expressing an exogenous endoglucosaminidase, resulting mainly in glycoproteins wherein the N-glycans are composed of single GIcNAc-Gal-Sia trisaccharides (=LacNAc-Sia; due to endogenous beta-1, 4-galactosyltransferase and GNE activity). Alternatively, the ADC is produced in a Glycodelete background of a higher eukaryotic cell, i.e. an eukaryotic cell deficient in Gntl and expressing an exogenous endoglucosaminidase, and mutated in its GNE activity, to result mainly in glycoproteins wherein the N-glycans are composed of single GIcNAc- Gal disaccharides (=LacNAc; due to endogenous beta-1, 4-galactosyltransferase activity).
In a further embodiment, the above N-glycan structures may additionally comprise a fucose branched on the Asn-GIcNAc (e.g. as shown for the fucose (triangle) present in the structure of the right panel of Figure 19).
As used herein, a "therapeutically active agent" or "therapeutically active composition" means any molecule or composition of molecules that has or may have a therapeutic effect (i.e., curative or prophylactic effect) in the context of treatment of a disease (as described further herein). Preferably, a therapeutically active agent is a disease-modifying agent, which can be a cytotoxic agent, such as a toxin, or a cytotoxic drug, or an enzyme capable of converting a prodrug into a cytotoxic drug, or a radionuclide, or a cytotoxic cell, or which can be a non-cytotoxic agent. Even more preferably, a therapeutically active agent has a curative effect on the disease. The ADC, or pharmaceutical composition comprising the ADC of the invention may act as a therapeutically active agent, when beneficial in treating patients with a disease related to the target of the antibody part of the ADC its antigen, or at least with a disease that may benefit from a treatment of targeting the cells expressing the antigen of the antibody part of the ADC as described herein, or patients suffering from another disease.
The term "subject", "individual" or "patient", used interchangeably herein, relates to any organism such as a vertebrate, particularly any mammal, including both a human and another mammal, for whom diagnosis, therapy or prophylaxis is desired, e.g., an animal such as a rodent, a rabbit, a cow, a sheep, a horse, a dog, a cat, a lama, a pig, or a non-human primate (e.g., a monkey). The rodent may be a mouse, rat, hamster, guinea pig, or chinchilla. In one embodiment, the subject is a human, a rat or a non-human primate. Preferably, the subject is a human. In one embodiment, a subject is a subject with or suspected of having a disease or disorder, in particular a disease or disorder as disclosed herein, also designated "patient" herein. However, it will be understood that the aforementioned terms do not imply that symptoms are present.
The term "medicament", as used herein, refers to a substance/composition used in therapy, i.e., in the prevention or treatment of a disease or disorder. According to the invention, the terms "disease" or "disorder" refer to any pathological state, in particular to the diseases or disorders as defined herein.
The term "treatment" or "treating" or "treat" can be used interchangeably and are defined by a therapeutic intervention that slows, interrupts, arrests, controls, stops, reduces, or reverts the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve a total elimination of all disease-related signs, symptoms, conditions, or disorders. Therapeutic treatment is thus designed to treat an illness or to improve a person's health, rather than to prevent an illness. Treatment may also refer to a prophylactic treatment which relates to a medication, or a treatment designed and used to prevent a disease from occurring.
Detailed description
The present invention provides for provide a novel innovative Antibody-drug conjugate (ADC) design and manufacturing method for producing the same, and is based on the surprising finding that site-specific coupling of payload via engineered N-glycan chains present in the hinge region. More specifically, by making use of N-glycan structures defined by a 'shortened branch' (GIcNac-Gal or GIcNac-Gal-Sia) homogenous structure obtained using Glycodelete or Glycodelete LacNac production hosts for the antibody of said ADC, the potency in cytotoxicity was positively impacted as compared to the use of wildtype bi-antennary N-glycan structures on the same antibody as conjugation site.
The present application discloses for the first time that ADCs can be efficiently produced with GlycoDelete oxime-ligation-based coupling technology. Terminal galactose residues of the disaccharide N-glycan at the neo-glycan D221N acceptor site in the hinge region are oxidized by galactose oxidase into aldehydes. The resulting aldehydes subsequently react with aminooxy-functionalized toxins in so- called oxime ligation reactions.
This GlycoDelete-ADC technology yields ADCs (also referred to herein as GDLN-ADCs) that are more potent than ADCs that are generated via oxidation and oxime ligation with antibodies that have not been produced in GlycoDelete LacNAc host cells, comprising larger, bulkier and more heterogenous N-glycan chains (WT-ADCs). When comparing the trastuzumab-based GDLN-ADCs with WT-ADCs for in vitro cytotoxicity in SK-BR-3 and BT-474 cells, the superiority of GlycoDelete processing became evident, regardless of toxin compound conjugated. GDLN-ADCs outperformed WT-ADCs when the toxin is conjugated via stable, non-cleavable linkers (exemplified herein with MMAF and DM1), as well as with the MMAE toxin that is conjugated via the valine-citrulline protease cleavable linker. Though, the impact of GlycoDelete processing is most evident for ADCs with non-cleavable linkers, which may stem from a difference in hydrophilicity of the active metabolite. Due to absence of a cleavable site, the active metabolite for DM1- and MMAF-conjugated GDLN-ADCs is the toxin-oxime linker-disaccharide- asparagine. In contrast, the WT-ADCs are assumed to be degraded to an active metabolite carrying an oligosaccharide instead of a disaccharide, with a larger size and an increased hydrophilicity which could delay or prevent its lysosomal escape into the cytosol (see also Figure 21). For GDLN-ADCs employing a cleavable linker (exemplified with MMAE as drug entity herein), the superiority of the in vitro cytotoxicity is less pronounced compared to WT-ADCs, which may be a consequence of the cathepsin cleavage of the dipeptide in the linker which eliminates at least partially the impact of the N-glycan hydrophilicity. Nevertheless, GDLN-ADCs still outperform WT-ADCs with cleavable linkers, leading to the mechanistic assumption that the protease degradation of the linker could be a rate-limiting step in lysosomal ADC maturation, which is less impactful for GDLN-ADCs capable of lysosomal escape, yielding a more potent ADC.
Moreover, the ADC coupling technology presented herein was also benchmarked in vitro with the clinically relevant ADCs applying a conjugation strategy via interchain disulfide bonds and maleimides, with GDLN-ADCs still providing for a superior/non-inferior (depending on cell line and the conjugated toxin) potency, despite the limitation of GDLN-ADCs in terms of lower drug-to-antibody ratio (DAR). The DAR of GDLN-ADCs is limited by the number of hinge N-glycans and the galactosylation efficiency at DAR 1.6-2.0, while the maleimide-generated ADCs can be conjugated with up to a DAR of 8, or at least, as exemplified herein to a conjugation reaction optimized for DAR of 4.
So a first aspect of the invention relates to an antibody-drug-conjugate (ADC) comprising an antibody which has an N-glycan acceptor site in the hinge region and an aminooxy- or oxime-linker-drug-entity coupled to the N-glycan of said N-glycan acceptor site, wherein said N-glycan consists of a Galactose- N- acetylglucosamine (Gal-GIcNAc) or Sialyl-Galactose- N-Acetylglucosamine (Sia-Gal-GIcNAc) glycan chain. More specifically, the antibody-drug-conjugate comprises an antibody format which preferably comprises a monoclonal antibody, which has a GIcNAc-Gal-oxime-linker-drug entity or GIcNAc-Gal-Sia- oxime-linker-drug-entity on an N-glycan acceptor site present in the hinge region.
The latter trisaccharidic glycan entity is providing a slightly higher hydrophilicity as compared to the disaccharidic glycan entity exemplified in the application because of the sialic acid presence, though still much lower in hydrophilicity as compared to the WT native N-glycan branches, making these also potent alternatives for the GDLN-ADC coupling technology disclosed herein.
In a specific embodiment, an antibody-drug-conjugate (ADC) is provided comprising an antibody which has an N-glycan acceptor site in the hinge region and an oxime-linker-drug-entity, wherein said oxime- linker-drug-entity is coupled to the asparagine side chain of said N-glycan acceptor site via an oxidized form (which is presented herein as an aldehyde) of the galactose residue of Galactose-N- Acetylglucosamine (Gal-GIcNAc) or via an oxidized form of the sialic acid residue of Sialyl-Galactose-N- Acetylglucosamine (Sia-Gal-GIcNAc) glycan chain.
N-glycosylation in the hinge region of antibodies for oxidation and conjugation
The present invention relates to the provision of newly generated antibody-drug conjugates by oxidation of galactose or sialyl moieties present in glycan chains obtained from glycol-engineered antibody production hosts, and conjugation to the resulting aldehyde group on said sugar.
As to provide for galactose oxidation on Gal-GIcNAc N-glycans, it was observed that the native N- glycosylation site of IgGl Fes at N297 in the CH2 cavity does not allow very efficient oxidation of galactose. So, to achieve this with moderate galactose oxidase (GaOx) units, we introduced a novel N- glycosylation site in a more accessible region of the IgGl Fc tail hinge region (herein exemplified using the D221N substitution in human IgGl according to EU numbering; residue 6 of the hinge region of human IgGl, as shown in SEQ ID NO:l/2).
SEQ ID NO:1: hinge region of human IgGl
EPKSCDKTHTCPPCPAPELLGGP
SEQ ID NO:2: D221N-mutant hinge region of human IgGl
EPKSCNKTHTCPPCPAPELLGGP
SEQ ID NO:3: human IgGl Fc region (including the hinge region)
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
So with the "hinge region" of an antibody as used herein, we refer to the amino acid sequence for the antibody region that connects the Fab and Fc regions together and contributes flexibility between these regions. More specifically, for the human IgG subclasses IgGl-IgG, the middle hinge regions contain two conserved cysteine residues (Cys226 and Cys229) to form interchain disulfide bonds between the two heavy chains to join these together and the lower hinge sequence is relatively conserved and is responsible for the flexibility and positioning of the Fc region relative to the Fab arms and affects the binding of Fc to Fc_R. However, the upper hinge which determines the arrangement between the two Fab regions and mediates flexibility and reorientations of each Fab arm significantly varies in length and sequence between the four IgGs. As exemplified herein, an N-glycan acceptor site was introduced in the hinge region of human IgGl by substitution of the D221 to an N (Blundell et al. J Immunol (2019) 202 (5): 1595-1611). Said amino acid site is however not conserved in the hinge region of lgG2, 3, or 4 (as described for instance in Rayner et al. (2015; JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 13, pp. 8420-8438; see figure 2)). So the N-glycosylation acceptor site in the hinge region of an ADC with an antibody derived from an lgG3 may for instance be obtained by substituting the D in the upper region (position 8 of the upper hinge) to an N; or alternatively, for antibodies based on the lgG2 or lgG4 hinge region by introducing an Asn in the upper hinge. The latter may be of particular interest when an ADC is desired lacking effector functions. Antibodies without effector function can also be generated by removing the Asn at position N297 (and/or by enzymatically removing the Fc N-glycan).
Alternative antibody formats may also be envisaged for the ADC of the present invention, wherein for instance a VHH-Fc antibody is used for coupling to an oxime-linker-drug-entity. The VHH-Fc fusions as known in the art, known as potent antibodies, are generally composed of the VHH sequence (in monovalent or multivalent/multispecific format), linked (directly or via a linker, such as a GS-linker) to an Fc region, wherein said Fc region sequence may be based on the human IgGl, wherein the EPKSC N- terminal sequence is removed or replaced with EPKSS, thus both allowing to replace the D following to an N, as to obtain a hinge region with an N-glycan acceptor site.
The expression of said recombinant antibody sequence in a higher eukaryotic cell (HEK or CHO) allowed to analyze the galactosylation efficiency of the resulting antibodies. Galactosylation in HEK cells was observed to be higher as compared to ExpiCHO cells initially, and could be further optimized to near completeness by hGalTl and precursors uridine, galactose and MnCL. Additionally, by adapting this expression system for N-glycan trimming with Endo T in the GlycoDelete (GD) platform, and its derivative GlycoDelete LacNAc (GDLN), with absent sialylation, we surprisingly revealed that upon successful oxidation and conjugation with GaOx and aminooxy-functionalized payloads, respectively, the resulting conjugates generated with non-GlycoDelete processed antibodies were incapable of inducing cytotoxicity in antigen expressing cells, in contrast to the alternative conjugate in which the biantennary N-glycan had been shrunk. We even demonstrated a similar, if not improved, anti-tumor efficacy of the GlycoDeleted ADC in comparison to clinically approved HER2-ADCs.
So by combining an improved galactose oxidation through the incorporation of a more accessible N- glycosylation site in the upper hinge region, also the conjugation of an aminooxy-containing toxin to the aldehyde of the oxidized Galactose of said N-glycan, resulted in efficient oxime-ligation to provide for a novel ADC with site-specific conjugation and high potency. Moreover, by targeting this hinge region for oxidation and conjugation, the toxins are located at a similar position as in ADCs in which the interchain disulfide bonds are reduced for conjugation, yet without suffering from the inherent structural instability of interchain disulfide bond removal.
Oxidation of N-qlycosylated antibody
For chemical oxidation, sodium periodate (NalO4) or several other comparable reagents known in the art can be used (also see figure 19).
Enzymatic oxidation targets N-glycans selectively and does not yield unwanted reactions. Galactose oxidase (GaOx) for instance has been shown to oxidize galactose residues uniquely at the C6 position (Solomon et al. Enzymic oxidation of monoclonal antibodies by soluble and immobilized bifunctional enzyme complexes. J Chromatogr. 1990;510:321-329; Angelastro et al. Galactose Oxidase Enables Modular Assembly of Conjugates from Native Antibodies with High Drug-to-Antibody Ratios. ChemSusChem. 2022;15(9)). The resulting aldehydes can subsequently be used in conjugations with aminooxy- and hydrazide-based linkers and reductive amination, among others. This fungal enzyme of 68 kDa, originally isolated from Fusarium graminearum, generates hydrogen peroxide next to oxidized galactose (Parikka et al. Oxidation with galactose oxidase: Multifunctional enzymatic catalysis. J Mol Catal B Enzym. 2015;120:47-59; Whittaker JW. Free Radical Catalysis by Galactose Oxidase. Chem Rev. 2003;103(6):2347-2364). Since GaOx is a naturally secreted protein, both products are generated at the extracellular site. It is therefore hypothesized that the main function of GaOx is the production of peroxide, with which it can combat bacteria in its proximity. GaOx is a multidomain protein, with an N - terminal carbohydrate binding domain (CBD), hypothesized to tether the enzyme to the fungal cell wall, and two C - terminal domains which both contribute to the active site. To catalyze the redox reaction, GaOx requires Cu2+ as cofactor and a tyrosine radical in its active site. In its catalytic cycle, galactose is first bound and deprotonated at its C6 hydroxyl group. This triggers its oxidation into an aldehyde, accompanied by the reduction of the tyrosine radical and Cu2+ to tyrosine and Cu+. Next, the enzyme's oxidation capacity is restored by reduction of oxygen into peroxide. As such, galactose can be oxidized, both as monosaccharide and incorporated as terminal residue in oligosaccharides. Like periodate oxidation of sialic acids, the antibody needs to carry the substrate, here galactose, efficiently also see Figure 19 for oxidation reactions). This may require in vitro processing with GalT and UDP - galactose or host cell engineering for increased galactosylation.
Linkers useful in the ADCs of the present invention
As described herein, the oxime-linker-drug-entity is coupled to the asparagine side chain of said N-glycan acceptor site on the antibody hinge region. In the present invention, the term "linked to", "coupled to", or "fused to", as used herein, and interchangeably used herein as "connected to", "conjugated to", "ligated to" refers, in particular, to "chemical and/or enzymatic conjugation" resulting in a stable covalent link.
In certain embodiments the glycoprotein-conjugates comprise a linker between the LacNAc or Sia- LacNAc N-glycan and the toxin or drug moiety. Certain linkers are more useful than others and the use of a specific linker will depend on the intended application. For example oximes (aminooxy) and hydrazones, in particular derived from aliphatic aldehydes, show less stability over time in water or at lower pH. Aromatically stabilized structures can be more useful to stably link a glycan to a conjugated moiety. Such stabilized linkers are also within the scope of the present application, as they can limit adverse effects due to premature release of the conjugated moiety, particularly when the conjugated moiety is a toxic substance intended for killing of a tumor cell. Of particular interest are BICYCLO[6.1.0]NON-4-YNE REAGENTS as well as aromatically stabilized triazole linkers and sulfamide linkers. It is within common technical knowledge that increased stability of a conjugate can also result from reduced aggregation tendency of any of the moieties comprised within said conjugate. For the production of glycoprotein-conjugates, or in particular ADCs, with increased stability the reader is non- exclusively referred to WO2013036748, WO2014065661, W02015057064 and W02016053107 as well as to other patent applications filed by Synaffix B.V. explicitly mentioned herein.
In general, various linkers known in the art can be used to link the glycosylated antibody and the conjugated moiety according to the invention. As should be clear, cleavable and non-cleavable linkers can be employed to achieve the desired release profile. Non-cleavable linkers are divided into two groups, namely thioether or maleimidocaproyl (MC). They consist of stable bonds that prevent proteolytic cleavage and ensure greater plasma stability than their cleavable counterparts. The most stable linkers currently used are the non-degradable linkers, which incorporate no cleavable functionality, also referred to herein as non-cleavable linkers. As such, they require full degradation of the antibody component in the lysosome, with the final metabolite being the toxin still connected through the linker to the amino acid at which the conjugation was targeted. Likewise, the toxin should allow for this modification to its structure for its toxicity. Next to protease sensitive linkers, two alternative cleavable linkers are incorporated in approved ADCs: acid labile linkers and reducible linkers, exploiting the respectively more acidic (endo)lysosomal environment and higher intracellular concentration of reduced glutathione, compared to serum.
In general, the optimal combination of linker and conjugation chemistry must be uniquely tailored to correlate each unique facet: the antibody, the conjugated moiety, and the profile of the disease to be treated. For reviews on antibody-drug conjugates and linkers used herein see for example Jessica R. McCombs and Shawn C. Owen, AAPS J. 17(2), 2015 and Lu, J. et al., Int J Mol Sci. 17(4), 2016 as well as a recent review by Pillow, T.H., Pharm Pat Anal. 6(1), 2017 describing a novel quaternary ammonium salt linker useful in conjugates for the treatment of cancer and infectious diseases. Still other suitable spacers or linkers will be clear to the skilled person, and may generally be any linker or spacer used in the art. In specific aspects the linkers or spacers are suitable for use in applications which are intended for pharmaceutical use. For example, a linker between the glycan and the drug moiety in the ADC may in certain aspects also be a suitable amino acid sequence, and in particular amino acid sequences of between 1 and 50, or more specifically, between 1 and 30 amino acid residues. Some examples of such amino acid sequences include Gly-Ser (GS) linkers, such as for example (GS)n or (GGGSS)n or (GSS)n, as described in WO 99/42077 and the (G4S)3, GS30, GS15, GSg and GS7 linkers described in the applications by Ablynx mentioned herein (see for example WO 06/040153 and WO 06/122825), as well as hinge-like regions, such as the hinge regions of naturally occurring heavy chain antibodies or similar sequences (such as described in WO 94/04678). Still other suitable linkers generally comprise organic compounds or polymers, in particular those suitable for use in polypeptides for pharmaceutical use. For instance, poly(ethyleneglycol) (PEG) moieties have been used to link antibody domains, see for example WO 04/081026. It is encompassed within the scope of the invention that the length, the degree of flexibility and/or other properties of the linker may have some influence on the properties of the final glycoprotein-conjugate of the invention, including but not limited to the affinity, specificity or avidity for a specific target. Based on the disclosure herein, the skilled person will be able to determine the optimal linker for use in a specific glycoprotein of the invention, optionally after some limited routine experiments. For example, in multivalent glycoproteins of the invention that comprise building blocks, directed against a first and second target, the length and flexibility of the linker is preferably such that it allows each building block to bind to its cognate target. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linker for use in a specific glycoprotein of the invention, optionally after some limited routine experiments. Finally, when two or more linkers are used in the ADC described herein, these linkers may be the same or different. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linkers for use in a specific polypeptide of the invention, optionally after some limited routine experiments. In certain specific embodiments it is desirable to produce ADCs with longer linkers including for example carbohydrates, which can provide the ADCs with higher hydrophilicity and accordingly improved water-solubility. ADCs comprising linkers with more carbohydrates are thus also within the scope of the present application. Also, linkers modified with PEG or consisting of PEG can be useful to increase the hydrophilic properties of a glycoproteinconjugate. Conjugation methods to link drug/toxin moieties to the oxidized N-glycans of the antibody
A comprehensive overview of conjugation or coupling methods is described in https://doi.org/10.1101/2021.06.02.446789 (Van Breedam W. et al (2021) Glyconnect: a glycan-based conjugation extension of the GlycoDelete technology), specifically referred in this reference are pages 6 to 8 which refer to NalO4-based conjugation of LacNAc-Sia-carrying glycoproteins, GaOx-based conjugation on LacNAc-carrying glycoproteins, click-based conjugation on LacNAc-carrying glycoproteins, click-based conjugation on GIcNAc-carrying glycoproteins and GalOx-F2-based conjugation on GIcNAc-carrying glycoproteins.
In view of conjugation strategies, extended chemical adaptations as known in the art may be applied on to provide for optimal customized oxime ligations, such as for example but not limited to : reduction of the oxime bond or application of reverse Pictet-Spengler ligations, both leading to increased stabilization; or alternatively hydrazone formation to reduce the stabilization and inducing lability of the bond in acidic condition (applicable for instance when required in the lysosome).
Toxins suitable for ADCs
The most commonly used toxin in approved ADCs is vedotin (MMAE) which is found in Adcetris, Padcev (enfortumab vedotin), Polivy (polatuzumab vedotin), Tivdak (tisotumab vedotin). Together with mafodotin (monomethyl auristatin F MMAF), found in Blenrep, monomethyl auristatin E (MMAE) belongs to the auristatin toxin family. Both auristatins are synthetic mimetics of dolastatin , a marine tetrapeptide natural product isolated from the sea hare Dolabella auricularia, selected from hundreds of candidates in structure activity relationship (SAR) studies for their high potency, water solubility, stability and possibility for conjugation. Cell death is triggered, with in vitro potencies of 0.1 to 10 nM, respectively for MMAE and MMAF, by inhibiting tubulin binding to GTP and thereby disturbing microtubule dynamics, which results in G2/M cell cycle arrest and apoptosis. The difference in the toxicity between MMAE and MMAF is caused by differential cell penetration, with the non-charged MMAE more efficiently entering cells via diffusion. Nevertheless, upon cell entry, either by methyl- esterification or by conjugation to antibodies, MMAF is lOOx more potent, because of its higher affinity for tubulin due to the additional negative charge.
Methods to produce the ADC of the invention
A further aspect relates to methods to produce the ADCs of the invention. Generally, such methods start by introducing an expression vector comprising a nucleotide sequence encoding the antibody part(s) of the ADC according to the invention in a suitable (higher) eukaryotic cell of choice, followed by expressing the antibody for some time, purifying the N-glycosylated antibody, oxidizing the gal or sia into aldehydes on the N-glycan site of said antibody and linking of a specific aminooxy-conjugation moiety to the purified oxidized N-glycan of the antibody through oxime ligation. The oxidation reaction and the coupling method itself is generally carried out in vitro.
Specifically for the production of the ADCs of the present invention, the antibody expressed in the cells comprises an N-glycan acceptor site in the hinge region, and the eukaryotic cell has a deficiency in the N-acetyl glucosaminyl-transferase I (GNT I) enzyme and comprises a gene encoding for an endoglucosaminidase enzyme, thereby providing for a 'Glycodelete (GD)' eukaryotic cell (and optionally deficient in GNE activity). This results in the production of antibodies with N-glycans on the hinge region which are composed of Sia-Gal-GIcNAc (or Gal-GIcNAc, resp.), which are suitable for oxidation by GaOx and/or sodium periodate (see Figure 19).
Several possibilities exist in the art to link a specific conjugated drug moiety to the glycosylated antibody of the invention. Generally spoken there are chemical, enzymatic and combined chemo-enzymatic conjugation strategies to carry out the coupling reaction.
According to a particular embodiment, the method to produce a ADC comprises the steps of :
- producing the antibody with the N-glycan Asn acceptor site in the hinge region in a 'glycodelete' or 'glycodelete-lacnac' engineered eukaryotic cell, and isolating the antibody from said cell,
- oxidizing the vicinal diol or diols present in LacNAc N-glycans of the antibody produced,
- reacting the obtained free aldehyde groups with hydroxylamine-containing molecules, more specifically aminooxy-linker-drug entities, to result in ADCs with an Asn in the hinge region that links via a GlcNAc-Gla-(Sia-) to an oxime-linker-toxin.
For oxidation, sodium periodate or several other comparable reagents known in the art can be used. In a specific embodiment the diol to be oxidized originates from a LacNAc disaccharide present on the glycoprotein of the invention. Oximes are formed by subsequent reaction of the resulting free aldehyde groups with aminooxy-containing molecules, commonly described as LacNAc oxidation-oxime ligation chemistry. Well-known in the art is the use of catalysts like para-phenylenediamine, 2-aminophenols or 2-(aminomethyl)benzimidazoles. Oxime and hydrazine conjugation are a promising alternative to clickchemistry, a more complex biorthogonal modification strategy. Under reductive amination conditions, the dialdehyde can be reacted with amines, which results in stable oxazepine derivatives.
According to another particular embodiment, said method to produce an ADC optionally comprises the steps of
- oxidizing the C6 hydroxyl group of a Gal residue in the terminal LacNAc N-glycan present on a glycoprotein of the invention - reacting the free aldehyde group with hydroxylamine-containing molecules.
For oxidation, the enzyme Galactose Oxidase (GAO or GaOx or GalOx) can be used. Employing the above mentioned steps typically oxime bonds are formed, optionally in the presence of a catalase, all of this is well-known in the art.
To modulate or to particularly increase stability of oximes and hydrazones, the use of linkers as described before is particularly envisaged herein.
The use of linkers to modulate the stability of the glycoprotein-conjugates as described before is particularly envisaged herein.
The following examples are intended to promote a further understanding of the present invention. While the present invention is described herein with reference to illustrated embodiments, it should be understood that the invention is not limited hereto. Those having ordinary skill in the art and access to the teachings herein will recognize additional modifications and embodiments within the scope thereof. Therefore, the present invention is limited only by the claims attached herein.
EXAMPLES
Example 1. Engineering the antibody for improved oxidation by Galactose oxidase.
For the creation of antibody - drug conjugates (ADCs) wherein the conjugation is made at the N-glycans, we introduced an additional N-glycosylation site besides the conventional N297, said neo-N- glycosylation site being located in the antibody hinge region which is more accessible for enzymatic treatment, especially for galactose oxidation of the N-glycan.
As a proof of concept example, we introduced a neo-N-glycosylation site in the hinge region of trastuzumab since Blundell et al. showed efficient N-glycan site occupancy at N221 (D221N) (Blundell PA, et al. J Immunol Author Choice. 2019;202(5):1595-1611; Blundell et al. J Biol Chem. Published online June 15, 2017:jbc.M117.795047), with high galactosylation and sialylation, upon expression in CHO-K1 cells. Figure 1 shows the expression of trastuzumab N221 to a similar extent as trastuzumab WT in both HEK293S cells and ExpiCHO cells, which was confirmed by the similar expression yields for both antibody variants after purification. A high efficiency of the neo-N-glycosylation site was assumed in both expression systems, given that the N221 antibody variant of HEK293S expression ran completely at a higher molecular mass, for trastuzumab N221 expressed in ExpiCHO and HEK293S cells. Moreover, upon Endo F2 treatment to remove N-glycans of both N221 and N297 sites, the trastuzumab variants were detected at similar heights, indicative of additional N-glycosylation contributing to the higher mass of untreated trastuzumab N221 (Figure 1). Yet, more emphasis was given to which glycoforms were transferred to the antibody during expression, since higher galactosylation would result in improved conjugation potential. We, therefore, after protein A and SEC based purification of trastuzumab WT and N221, analyzed the N-glycans by DSA FACE. For antibodies containing N221 N-glycans, the profiles indicated the presence of a mixture of N221 and N297 N-glycans, since no separation of N-glycosylation sites was implemented. Figure 2 shows the lack of galactosylation for antibodies expressed in ExpiCHO cells, in contrast to antibody expressed in HEK293S WT cells, for which a variable galactosylation could be detected.
Example 2. Production of trastuzumab variants in HEK293S wild-type and glyco-engineered 'GlycoDelete LacNac' cells.
Although an increased galactosylation is possible by hGalTl/precursor addition of ExpiCHO cells (data not shown), the trastuzumab (WT and N221-engineered variant) antibodies were recombinantly produced in HEK293S cells. Additionally, by applying the HEK293S cell expression platform, another layer of variation in recombinant production of glyco-engineered antibodies was possible by introducing the 'GlycoDelete' concept (Figure 20). As such, the N-glycan would be reduced in size and homogenized.
HEK293S GlycoDelete (GD) cells have been engineered by knocking out GnT-l and overexpressing EndoT, resulting in N-glycans being trimmed down into GIcNAc residues, possibly extended with galactose to obtain disaccharides (GIcNAc-Gal = LacNAc) and further with neuraminic acid to obtain trisaccharides (GIcNAc-Gal-Sia) (Figure 20) (Meuris et al. GlycoDelete engineering of mammalian cells simplifies N- glycosylation of recombinant proteins. Nat Biotechnol. 2014;32(5):485-489). To limit the glycosylation to 'LacNac' disaccharides, GlycoDelete cells were further manipulated by knocking out GNE (GDLN), in order to abolish the formation of CMP-sialic acid, the precursor for sialylation.
Given the natively higher galactosylation efficiency of antibodies in HEK293S cells as compared to ExpiCHO, we investigated whether including hGalTl DNA during transfection of HEK293S and precursors during expression could further increase this. Dekkers et al. had previously disclosed that hGalTl increased galactosylation in HEK293S cells, yet that DNA contents of the transferase larger than 1% resulted in yield losses (Dekkers et al. Sci Rep. 2016;6(l):36964). We therefore tested whether this improvement could be reproduced for trastuzumab N221. To improve galactosylation of the GlycoDelete GIcNAc stump, we co-transfected a pcDNA3.3 vector encoding bl - 4 human galactosyltransferase 1 (hGalTl), at 10 % of the total DNA content during transfection. Additionally, in an attempt to fully boost the galactose levels, we included precursors during expression at a final concentration of 1 mM MnCI2, 5 mM galactose and 1 mM uridine (Gramer et al. Biotechnol Bioeng. 2011;108(7):1591-1602; Zhong et al. Biotechnol Prog. 2019;35(l):e2724). As indicated in the DSA FACE profiles shown in Figure 3, the galactosylation was efficiently improved upon 1 % hGalTl co-transfection and precursor addition during expression. The main glycan detected for trastuzumab WT and N221 carried two galactose residues, making it an ideal substrate for galactose oxidase and conjugation, as further described.
Next to these fully galactosylated antibodies with biantennary N-glycans, we generated trastuzumab variants processed by EndoT in the HEK293S 'GlycoDelete LacNAc' (GDLN) cells. Trastuzumab WT and N221 were expressed without hGalTl or precursors and analyzed, after purification, by mass spectrometry after FabRICATOR digest (this protease specifically cleaves in the hinge region, conveniently in between the two N-glycosylation sites, yielding the N-terminal fragment VH-CH1 with the N221 glycan (SEQ. ID NO: 4) and the C-terminal fragment CH2-CH3 with the N297 glycan (SEQ ID NO:5)) and TCEP reduction of interchain disulfides. Mass spectrometry results indicated full galactosylation of N221 but a reduced N - glycosylation site occupancy at 84 %. In contrast, the N297 site was efficiently glycosylated, but carried some leftover GIcNAc residue at 9 % (Figure 4). Moreover, the data also illustrated the efficient trimming in the GlycoDelete LacNAc cells, given the lack of N-glycans with higher mass.
Example 3. Binding affinity of trastuzumab after neo-N-glycosylation site incorporation
We verified the binding affinity of Trastuzumab N221 for its antigen human epithelial growth factor (EGF) receptor 2 (HER2). An indirect ELISA was employed with coated HER2 to investigate the binding of serially diluted trastuzumab WT and N221, expressed in HEK293S WT cells. Figure 5 shows limited loss of affinity after N221 N-glycosylation of trastuzumab in HEK293S cells.
Next to binding of trastuzumab to its antigen, we investigated the impact of N221 N-glycosylation on the affinity of CD16a for trastuzumab. As depicted in Figure 6, the interaction is orchestrated at the antibody site by residues of the upper CH2 domain and the hinge. As N221 was situated in the hinge, we aimed to compare affinity of trastuzumab WT and N221. A sandwich ELISA was set up with coated trastuzumab variants, a serially diluted CD16a and anti-His for detection. The data indicated no loss of affinity between the trastuzumab variants WT and N221 (Figure 7). The final goal with the N221 N - glycan is its application in antibody conjugation. Therefore, it may be likely that antibody conjugates may still induce ADCC, if the compound of interest conjugated does not interfere. More specifically for ADCs, this would prove beneficial since the tumor eradication would be driven by those two mechanisms.
Example 4. Conjugating antibody with aminooxy - functionalized linkers: testing the specific oxidation of trastuzumab N221.
Introducing galactosylation at N221 of trastuzumab was optimized in ExpiCHO and HEK293S cells. Yet, high galactose levels by themselves would have been insufficiently useful if their location would prohibit effective oxidation. Based on the higher galactosylation of the N221 N-glycan site, we assumed the site at the hinge region would be more accessible for GaOx-mediated oxidation than N297 in the Fc domain. To investigate oxidation of the N221 N-glycan site, we oxidized a collection of trastuzumab variants overnight with 0.66U/nmol GaOx in the presence of aminooxy-PEG-biotin, which reacts with the aldehyde generated at the galactose residues. The variants differed in two characteristics: (1) only the N297 N-glycan versus N-glycans at N297 and at N221 and (2) the expression system used: ExpiCHO versus HEK293S. More specifically, for the ExpiCHO expressions, the impact of hGalTl and precursors was compared, while for HEK293S, the GDLN platform could be introduced.
The streptavidin signals observed on western blot, indicative of conjugation, were most intense for trastuzumab N221 expressed in HEK293S WT cells (Figure 8). In comparison, the signal of trastuzumab WT expressed in those cells could only be very faintly detected, whereas there was a 30-fold difference between trastuzumab WT and N221 when both expressed in HEK293S WT cells. The lack of galactose in ExpiCHO cells, without hGalTl/precursors optimization, was exemplified by the limited streptavidin signal intensity for trastuzumab WT expressed in ExpiCHO. Nevertheless, trastuzumab N221 expressed in ExpiCHO yielded some biotinylation. As such, the lack of biotin signal for trastuzumab WT is a combination of limited galactose content and reduced activity of GaOx on the Fc N-glycan. Surprisingly, when galactosylation was increased with hGalTl and precursors with trastuzumab N221 in ExpiCHO cells, only a small increase in biotinylation could be observed. A higher biotin signal was expected, since DSA FACE data indicated a larger fraction of trastuzumab N221 that carried galactose residues. The lack of efficient oxidation or conjugation of CHO expressed trastuzumab N221 proved another rationale to switch to HEK293S expression for trastuzumab (next to expression yield loss and the GDLN option).
The reduced GaOx activity at N297 N-glycans of antibodies could be used to our advantage, by combining ADCC with ADC. By selectively oxidizing and conjugating toxin to the neo-N-glycosylation site N221, the Fc N-glycan remained unaltered, which we assume may result in potent ADCC activity. Moreover, as indicated by Angelastro et al., by increasing the oxidase units, the Fc N-glycan became oxidizable (ChemSusChem. 2022;15(9)). Conjugating the galactose residues of the Fc N-glycan next to the N221 N- glycan would increase the drug-to-antibody-ratio (DAR), which could be useful in certain applications. We therefore aimed to titrate the optimal GaOx units required to oxidize either one or both sites, since overoxidation of galactose residues into carboxylic acids could reduce the ultimate conjugation efficiency, since carboxylic acids do not react with aminooxy linkers. Indeed, upon testing variable amounts of GaOx, ranging from the 0.66U/nmol condition optimized in the lab to the 660U/nmol condition disclosed by Angelastro et al., trastuzumab WT (lacking the accessible N221 site) was found to be more efficiently oxidized in conditions with more GaOx units. Yet, for the other antibody variants, the best conditions were found with intermediate amounts of GaOx and differed depending on the number of galactose residues present (Figure 9). For respectively trastuzumab WT versus N221, and HEK293S GDLN (also simply called LN, indicative of the Glycodelete-LacNac engineered background, as described herein and interchangeably used herein) versus WT expressed, the optimal condition resulting in the higher streptavidin signal was 660U/nmol, 330U/nmol, 165U/nmol and 16.5U/nmol.
The difference in the optimal conditions for full oxidation of trastuzumab WT and N221 was assumed to be caused by overoxidation of the N221 site in the latter. To prove its involvement, we aimed to specifically analyze both sites in trastuzumab N221, by FabRICATOR digest. Yet, full digestion could not be obtained. Further experimentation resulted in the conclusion that upon oxidation by GaOx of the N221 site, the digestion by FabRICATOR is inhibited (Figure 10). In the non-reducing, CBB stained gel, F(ab' was only efficiently generated in the condition lacking GaOx. Upon comparing the third and fourth lane, respectively GaOx oxidation present, and GaOx oxidation with biotin conjugation, a decrease in FabRICATOR activity could be observed, indicated by the higher intensity of the band for full length antibody. This observation was most likely explained by the additional steric hindrance introduced by the biotin linker. Increasing the FabRICATOR units during the overnight incubation resulted in increased cleavage though.
Example 5. Generating trastuzumab - drug conjugates with GaOx.
Initially, trastuzumab WT and N221, produced in HEK293S WT cells without hGalTl or precursors, were oxidized overnight at a small scale (50 pg) with varying amounts of aminooxy functionalized toxin (monomethyl auristatin F (MMAF)) (Figure 11). After PPD-based catalysis of the conjugation, the reactions were analyzed by SDS PAGE and western blotting. We initially detected total antibody with anti-hlgG antibody to detect the slight shift in mass after conjugation, since we lacked an anti-MMAF antibody. To minimize warping of the gel during electrophoresis, which would complicate detection of the limited shift in size, we ran the electrophoresis at 100V. A small increase in size could be detected between the control conditions (first and last lane, respectively without MMAF and without GaOx) and the conjugatable conditions with GaOx and an increasing amount of MMAF, indicative of conjugation (Figure 11). Moreover, among the conjugatable conditions, the 250 and 1000 equivalent MMAF conditions were similar, in contrast to the 50 equivalent MMAF condition. Therefore, we assumed the conjugation would be completed with a minimum amount of 250 equivalents. Additionally, for trastuzumab WT, a similar increase in apparent molecular mass could not be detected, indicating specific oxidation of the N221 site in the trastuzumab N221 conditions, as was expected given the use of 0.66 U/nmol GaOx.
We next upscaled the oxidation and conjugation reaction of trastuzumab N221, expressed in HEK293S WT and GDLN cells without hGalTl or precursors, in order to produce sufficient material to test cytotoxicity in vitro with HER2 overexpressing cancer cells. The conjugatable MMAF was lowered to an excess of the toxin, in respect to the antibodies, to 42.9 x. After overnight oxidation and conjugation, the reactions were catalyzed with I mM PPD and purified by protein A chromatography and desalted to PBS. Successful conjugation of MMAF was confirmed by anti-MMAF based western blotting (Figure 12), with the intensity for trastuzumab N221 expressed in HEK293S WT cells very similar to the signal of trastuzumab N221 expressed in HEK293S GDLN, despite its larger galactose content. Moreover, when testing the amount of unreacted aldehydes remaining after MMAF conjugation, by incubating the purified conjugates with aminooxy-PEG-biotin and analyzing the reactions by SDS PAGE and streptavidinbased western blotting (Figure 13), there was no difference to be noted with control antibodies oxidized with GaOx. As such, the conjugation was assumed to be incomplete.
We therefore repeated the oxidation and conjugation of trastuzumab N221, expressed in HEK293S WT cells, to aminooxy-MMAF. The GDLN variant was omitted for this repetition, since this conjugate had been shown to be toxic on HER2 overexpressing cells, as disclosed below. A new batch of toxin was used to conjugation trastuzumab N221 with 250 equivalents during its overnight oxidation. Additionally, a second difference with the first conjugation approach was the use of trastuzumab from a new antibody expression batch, which was expressed in HEK293S WT cells with 1 % hGalTl and precursors, and as such carried more galactose residues. After PPD catalysis, the antibody was purified and analyzed by SDS PAGE identically to the first approach. The resulting anti-MMAF western blot (Figure 12), showed a slight increase in MMAF conjugated to trastuzumab after the second repeat with more equivalents: 1.3x more MMAF was detected for WT #2, compared to WT #1. The larger excess of aminooxy-MMAF resulted in a more efficient conjugation, as indicated by the anti-MMAF detection, and the reduction in unreacted aldehydes (Figure 13).
In contrast to our inability to site-specifical ly determine the number of toxins conjugated, we managed to characterize the DAR of the three ADCs. We first implemented RPLC MS and found that despite their higher galactosylation content, the DAR of WT #1 and #2 was 0.68 and 0.89, respectively. In contrast, the GDLN variant showed a DAR content of 1.26. This data indicates some difficulty of biantennary N- glycans to be efficiently oxidized. The conjugation likely was not problematic since WT #2, with more equivalents only had a slightly increased DAR. Additionally, the profile for conjugated trastuzumab N221 GDLN depicts a peak in the DAR2 section, which could be explained by oxidation of and conjugation at the Fc N-glycan, given that the analysis was run in reduced phase. Additional evidence for Fc conjugation can be found in the DARI section, which shows two peaks, with identical molecular mass after deconvolution, which may be the conjugates with different conjugation sites. If we assume that the largest peak is conjugated at N221, given its more accessible location, the little peak would be Fc N- glycan conjugated. The ratio between the two peaks indicates that about 15 - 20 % would be conjugated at N297, which is more than anticipated based on the aminooxy-biotin conjugation experiment (3.33 %). As a second method for DAR determination, we used native SEC MS. However, the profile after deconvolution was only manageable for annotation for the GDLN variant since the WT variants proved too diverse for peak calling. The drug load varied from DARO to DAR4 with on average 1.61 toxins conjugated to trastuzumab N221. This number is slightly higher than obtained with reduced HPLC MS, which could be due to technical differences between the methods. Additionally, the profiles show DAR3 and DAR4 species which further indicates the oxidation and conjugation of Fc N-glycans.
Example 6. Characterizing and testing trastuzumab-MMAF conjugates.
Prior to testing cytotoxicity of the produced trastuzumab-MMAF conjugates on HER2 expressing cancer cell lines, we validated the binding of the conjugates to HER2 with an identical setup as previously used. The indirect ELISA with coated HER2 showed a ~5.5 x loss of affinity upon MMAF conjugation (Figure 14).
Having verified the binding of trastuzumab MMAF conjugates to HER2, we continued the characterization by testing cytotoxicity of the generated conjugates. The HER2 positive breast cancer cell line SK-BR-3 is often used in ADC research and was adopted here as well, for straightforward comparison with efficacies disclosed in literature. Alternatively, the HER2 negative cell line MDA-MB-231 is often used as negative control, to test for non-targeted toxicity. After obtaining both cell lines, their HER2 status was confirmed by fluorescence - assisted cell sorting (FACS) based on staining with trastuzumab and the secondary anti-hlgG antibody coupled to AF633. We next compared the cytotoxicity of trastuzumabMMAF-conjugated derivatives to Kadcyla, the clinically approved ADC consisting of trastuzumab conjugate through lysines to anti - mitotic mertansine (DM-1). SK-BR-3, expressing HER2, were seeded in 96 well plates for 24h to subsequently exchange their growth medium with DMEM medium supplemented with ADCs. The cells were incubated over weekend with the treatments. Next, the cells were washed in PBS and analyzed for cell viability by ATP measurements with CellTiter-Glo. The viability, relative to untreated cells (Figure 15) showed an apparent toxicity of unconjugated trastuzumab for all tested variants (WT versus N221, and HEK WT versus GDLN expressed). This apparent toxicity has been described in literature as caused by the inhibition of HER2 signaling by trastuzumab (Tseng et al. Mol Pharmacol. 2006;70(5):1534-1541; Yang-Kolodji et al. Biomarkers. 2015;20(5):313-322), which resulted in reduced proliferation, in comparison with the non-treated cells. Interestingly, additional toxicity was, apart from positive control Kadcyla, observed when the MMAF toxin was conjugated through GlycoDelete LacNAc sugars. Trastuzumab-MMAF (HEK WT) #1 and #2 generated toxicity at higher concentrations, as viability relative to trastuzumab-treated cells was only reduced at higher dosage.
Next, we further validated the toxicity of trastuzumab-LacNAc (LN) - MMAF for SK-BR-3 cells, compared to the clinically relevant trastuzumab-drug conjugates Kadcyla and Enhertu, which showed the superiority of the conjugate generated with our GaOx/Oxime ligation platform (Figure 16). Trastuzumab N221, expressed in HEK293S GDLN and conjugated to aminooxy-MMAF, induced cytotoxicity in SK-BR-3 cells at lower concentrations than the comparators Kadcyla and Enhertu, with an EC5o of 11 pM versus respectively 51 pM and 149 pM, respectively. In contrast to the anti-mitotic ADCs Kadcyla and trastuzumab N221-GDLN-MMAF, Enhertu was unable to induce cell death to single digit viability percentages.
As alluded to above, novel antibody - drug conjugates are superior in clinical settings to first generation ADCs which can be (partially) attributed to bystander effect. The latter characteristic results in improved solid tumor killing due to the membrane permeability of the active metabolite of the ADC, leading to cell death in cells with a negative or reduced antigen status. The notorious heterogeneity of solid tumors, which can result in incomplete responses in patients, highlights the need for improved ADCs, such as bystander effect-competent variants. We therefore aimed to test bystander effect of the ADC produced applying our novel coupling technology, using trastuzumab N221-GDLN-MMAF. The available clinical ADCs allowed for efficient comparison since Kadcyla is known to lack bystander effect, while the in vivo superiority of Enhertu despite its in vitro inferiority, in respect to Kadcyla, is attributed to its bystander effect. We therefore investigated bystander effect with a coculture toxicity assay. HER2 negative cells, MDA-MB-231, which did not respond to trastuzumab-drug conjugate treatment (Figure 17), were mixed in equal ratio with SK-BR-3 cells. In the Enhertu treatment, the condition with bystander effect, the viability dropped below 50 - 55 %, while in the alternative treatments it did not (Figure 17), indicating a lack of membrane permeability.
In conclusion, we managed to generate an antibody - drug conjugate applying theGaOx - based oxidation and oxime ligation approach, with antigen-specific toxicity when produced under 'GlycoDelete' processing conditions. Likely the short glycan structure reduces hydrophilicity which results in improved lysosomal escape of the active metabolite. Additionally, oxidation efficiency has been 30x increased by introducing the neo-N-glycosylation site at the hinge region as compared to non-modified antibody. Further optimization of the oxidation and conjugation conditions, alongside techniques to accurately analyze the conjugates, will allow for development into products with good manufacturability.
Example 7. Confirmation of the in vitro cytotoxicity of trastuzumab-GaOx/oxime coupling on disaccharide N-glycans in hinge region.
Trastuzumab, with its hinge sequence mutated to include the D221N N-glycosylation site, was oxidized at terminal galactose residues with galactose oxidase (GaOx) and conjugated to aminooxy-functionalized drug-linkers. After purification from the oxidation/conjugation reaction via protein A chromatography and desalting to phosphate buffered saline (pH 7.4), the ADCs were compared with approved trastuzumab-based ADCs (Kadcyla or T-DMl and Enhertu) for in vitro cytotoxicity in the HER2+ cancer cell lines SK-BR-3. To further confirm our previous observation, we tested the difference in cytotoxicity imposed by the N-glycan on the D221N site when being present as wild-type native, biantennary oligosaccharide N-glycan versus when present as (LacNac) disaccharide obtained after GlycoDelete LacNAc (GDLN) processing (Figure 22).
A first ADC was generated with the GaOx/Oxime technology employed MMAF as cytotoxin, with the non- cleavable PEG4 as linker. The linker does not harbour a cleavage site and is classified as a stable linker. The cytotoxicity of Trastuzumab D221N MMAF (HEK GDLN) and Trastuzumab D221N MMAF (HEK WT) was compared in the SK-BR-3 cell line to T-DMl (Kadcyla) and Enhertu (Figure 22). As observed in example 6, the ADCs of which the antibody was produced in HEK GDLN host cells was cytotoxic to these HER2+ breast cancer cells (EC5o of 11 pM), whereas the corresponding ADC construct with WT N-glycan structure did not show cytotoxicity. We hypothesize that this difference is due to differences in hydrophilicity. The active metabolite generated in the SK-BR-3 cell line is believed to be Asn+ N-glycan + Oxime linker + MMAF, when there is no cleavable linker present (such as is the case for MMAF). As such the metabolites differ in hydrophilicity: the GDLN-ADC metabolite carries a disaccharide, while the WT- ADC metabolite carries an oligosaccharide (see right panel Figure 22). Possibly, the increased hydrophilicity of the WT-ADC metabolite is too large to cross the lysosomal membrane. Without escaping the lysosome, the metabolite cannot perform its cytotoxic function (in the cytosol, where the microtubules are).
Example 8. In vitro cytotoxicity for ADC coupling technology with cleavable linkers.
Monomethyl auristatin E (MMAE)-based ADCs are typically constructed using cleavable linkers to obtain the required cytotoxicity, so as a second set of ADCs we conjugated trastuzumab with our GaOx/Oxime technology and MMAE as cytotoxin via the cleavable valine-citrulline-PABC linker. This linker can be digested by cathepsin protease upon lysosomal delivery.
The cytotoxicity of Trastuzumab D221N MMAE (HEK GDLN) and Trastuzumab D221N MMAE (HEK WT) was compared in the SK-BR-3 cell line to T-DMl (Kadcyla) and Enhertu (Figure 23). Due to the cleavable nature of the linker between trastuzumab and MMAE, the impact of the N-glycan was expected to be lower, if not absent. However, when the cytotoxicity of the ADCs, differing in N-glycan composition (oligosaccharide versus disaccharide, see right panel of Figure 23) was compared, only the GlycoDelete- processed molecule showed cytotoxicity (EC5o of 90 pM), despite the mode of action of having a cleaved toxin product (lacking the N-glycans) as active agent, which should at least significantly reduce the impact of the N-glycan on final potency. Still, the conjugated GDLN-ADC molecule is lower in size than the conjugated WT-N-Glycan-ADC, and therefore may escape from the lysosome more easily. Further investigation remains to check whether protease processing of the linker is suboptimal or delayed in SK- BR-3, which would generate a bottleneck, making the WT-ADC metabolite less active, or active on a different time scale.
Example 9. Biosimilar ADC production with GaOx/ oxime hinge coupling technology.
In order to compare our GlycoDelete-ADC coupling technology with the clinical comparator Kadcyla (T- DM1), we generated ADCs with trastuzumab D221N and the toxin used in T-DM1 (toxin = DM1, lysine conjugated N-Hydroxysuccinimide (NHS), Figure 24C). However, we could not obtain aminooxyfunctionalized DM1 compounds. We therefore worked with a two-step protocol. We first conjugated aminooxy-azide linkers to trastuzumab D221N (GaOx based). After purification, we completed the conjugation by adding DBCO-DM, in a SPAAC-based conjugation. As such, we generated ADCs similar to T-DM1, though applying a suboptimal conjugation strategy since the double conjugation protocol lengthens the linker between the toxin and the antibody, which may not be optimal (Figure 24 B).
When analyzing the in vitro cytotoxicity, we only observe cytotoxicity when trastuzumab D221N has the truncated GDLN N-glycan, and not when it carries wild-type biantennary N-glycans (Figure 24 D). Since the linker between DM1 and the antibody cannot be cleaved, coupling to the biantennary N-glycan is likely too hydrophilic for lysosomal escape. Without lysosomal escape, the DM1 toxin cannot inhibit the microtubules and trigger cell death. The GlycoDelete-ADC molecule wherein the N-glycan is limited to a disaccharide does show cytotoxicity, though it is less active than T-DM1 (EC5o for Kadcyla: 51.3 pM versus 259 pM for the GDLN-ADC-DM1 compound). The five-fold difference may reflect the anticipated lower DAR (drug to antibody ratio) of GDLN-ADC (1.6) versus T-DM1 (3.5), in combination with the suboptimal linker chemistry. Still, the novel ADC coupling technology applied in the hinge region with in addition short N-glycan structures (as produced in GDLN cells), allows for an improved effect as compared to applying native N-glycans as site-specific coupling technology.
Example 10. ADC GaOx/oxime-based hinge coupling technology versus maleimide coupling.
The NHS conjugation chemistry used in Kadcyla targets primary amines such as the N-termini and lysine amino acids. Yet, this conjugation strategy is less preferred than targeting cysteines residues with maleimide-functionalized toxins, after partial reduction of the antibody interchain disulfide bonds. To compare the auristatin-based GlycoDelete-ADCs (MMAF and MMAE), we conjugated trastuzumab with maleimide-functionalized MMAF and MMAE, since there is no trastuzumab-based ADC conjugated to auristatins available in the clinic for comparison. To mimic the aminooxy toxins as closely as possible, we selected a non-cleavable linker for MMAF and a protease-cleavable linker for MMAE. Upon testing the cytotoxicity in SK-BR-3 cells (Figure 25), the GDLN-ADCs showed a higher potency than conventional maleimide-based conjugation for MMAF: GDLN-ADC (MMAF) is 5x more potent than its maleimide alternative (EC5o of 11.1 pM versus 50.7 pM), while the GDLN-ADC (MMAE) conjugate is similarly potent as its maleimide alternative (EC5o of 90.6 pM versus 143 pM), despite its lower anticipated DAR (GDLN-ADC at most 1.6-1.8, Mal-MMAE at most 4.4).
Example 11. Confirmation of in vitro cytotoxicity effect in additional cancer cell line.
Next to the SK-BR-3 cell line, we also confirmed cytotoxicity of the oxime-ADCs in a second HER2 positive cell line BT-474, which as compared to SK-BR-3, the BT-474 cell line presents fewer HER2 molecules on its surface (+-70%). Similar conclusions can be drawn for GDLN-ADCs superiority over WT-ADCs, regardless of the stably linked MMAF (Figure 26) and the cleavable linker MMAE (Figure 27). Compared to SK-BR-3, the cytotoxicity is slightly reduced in BT-474, assumed to be due to the lower HER2 density on the cell surface (Figure 28). Another difference is the increased loss of proliferation at higher concentrations with trastuzumab. This is also reflected in the reduced (apparent) viability when cells were treated with non-conjugated trastuzumab.
When comparing the auristatin-conjugated ADCs, differing in conjugation chemistry (GDLN-ADC versus maleimide technology), a similar conclusion can be drawn in the BT-474 cell line compared to the SK-BR- 3 cell line: GlycoDelete-ADCs outperform the maleimide-based ADCs, despite the anticipated lower DAR levels: GDLN-ADC (MMAF) is 14x more potent than its maleimide alternative (EC5o of 24.9 pM versus 351 pM). The GDLN-ADC (MMAE) conjugate is 3.5x more potent as its maleimide alternative (EC5o of 72.5 pM versus 259 pM), despite its lower anticipated DAR (GDLN-ADC at most 1.6-1.8, Mal-MMAE at most 4.4).
In conclusion, the data presented here supports the superiority of GDLN-ADCs over WT-ADCs and the relevance of the GDLN-ADC technology as an alternative to generate clinically relevant ADCs.
Material and methods
Throughout the application, the EU numbering (Edelman et al., 1969; Proc Natl Acad Sci USA.;63:78-85) for antibody residue location was applied.
Construct design, expression and purification of recombinant proteins
Open reading frames encoding the trastuzumab light chain (LC) and heavy chain (HC) were cloned into plasmid pcDNA™3.3-TOPO™ with the TOPO™ TA cloning kit (K830001, Invitrogen). Briefly, cDNA, codon optimized for mammalian expression, was ordered as synthetic DNA fragments (IDT gBIocks). The cDNA contained the full length LC or HC sequences of trastuzumab, with the latter sequence additionally modified with the novel N221 N-glycosylation site (D221N). For secretion, we employed different signal peptides for the light and heavy chain, as optimized for expression in CHO cells by Haryadi et al. (PLoS ONE. 2015;10(2). Upon A-tailing, the cDNA was TA cloned in the pcDNA 3.3 vector. Plasmids were verified by sequencing (Eurofins) and purified to sufficient purity with NucleoBond Xtra Midi (#740410, Macherey-Nagel).
Trastuzumab was expressed in ExpiCHO cells (ThermoFisher Scientific), according to the manufacturer's protocol. Briefly, 6x 106 cells/ml, grown at 37 °C and 8% CO2 were transfected with 0.8 pg DNA/ml using the ExpiFectamine CHO reagent. For efficient antibody expression, LC and HC were transfected at a 2:1 ratio respectively. One day after transfection, ExpiCHO enhancer and ExpiCHO feed were added according to the manufacturer's instructions. Subsequently, the cultures were further incubated at 32 °C and 5% CO2. Cells were fed a second time 5 days post-transfection. Cultures were harvested as soon as cell viability dropped below 70 - 75%.
Alternatively, trastuzumab was also expressed in HEK293S cells (WT and 'GlycoDelete LacNAc' (abbreviated herein as GDLN or LN) modified). Prior to transfection, cells (ThermoFisher Scientific) were cultured in FreeStyle293 expression media (Life Technologies) supplemented with equal volumes of Ex- Cell-293 (Sigma), at 37 °C with 8% CO2 while shaking at 130 rpm. Mammalian expression plasmids encoding the LC and HCs of trastuzumab were transfected, at a 2:1 ratio respectively, into FreeStyle293 cells using polyethylenimine (PEI). Briefly, suspension-adapted and serum-free HEK293S cells were seeded at 3x 106 cells/mL in Freestyle-293 medium (ThermoFisher Scientific). Next, 2.25 pg DNA/ml cells was added to the cells and incubated on a shaking platform at 37 °C and 8% CO2, for 5 min. Next, 4.5 pg PEI/ml cells was added to the cultures, and cells were further incubated for 5 h, after which an equal culture volume of Ex-Cell-293 was added to the cells. Transfections were incubated until cell viability dropped below 70 - 75%, after which cells were pelleted (10', 300 g) and supernatants were filtered before purification.
For purification of the recombinant antibody, supernatant was loaded on a 5 mL MabSelect SuRe column (Cytiva). Unbound proteins were washed away with Mcllvaine (phosphate/citrate) buffer77 pH 7.2, and bound proteins were eluted using Mcllvaine buffer pH 3. Immediately after elution, protein-containing fractions were neutralized using 0.4M NasPC buffer. These neutralized fractions were then pooled and loaded onto a HiLoad 16/600 200 pg column (Cytiva) for size exclusion chromatography (SEC) into lx PBS (phosphate buffered saline) or 100 mM NazHPC pH 7, for further oxidation and conjugation. The PBS buffer was used for in vitro cellular toxicity tests, while the sodium phosphate buffer was used for conjugation purposes.
To boost the galactosylation, required mostly in during ExpiCHO expressions, we co-transfected a pcDNA3.3 vector encoding pi-4 human galactosyltransferase 1 (hGalTl), at variable amounts of the total DNA content during transfection (Gramer et al. Biotechnol Bioeng. 2011;108(7):1591-1602; Zhong et al. Biotechnol Prog. 2019;35(l):e2724). Additionally, when completing the expression medium with equal volume of Ex-Cell-293 (HEK293S) or ExpiCHO Enhancer/Feed (CHO), galactosylation-related additives were added at a final concentration of 1 pM MnCL, 5 mM galactose and 1 mM uridine. HEK293S transfection were ultimately also boosted for improved galactosylation with 1% hGalTl, 1 pM MnCL, 5 mM galactose and 1 mM uridine added.
Full length GaOx was cloned previously, from a gBIock encoding the enzyme (GAOA - Galactose oxidase - Gibberella zeae (strain ATCC MYA-4620 / CBS 123657 / FGSC 9075 / NRRL 31084 / PH-1) (Wheat head blight fungus) | UniProtKB | UniProt. Accessed May 8, 2023. https://www.uniprot.org/uniprotkb/HS2N3/entry) with the N-terminal 0MF and C-terminal HisTag, into an expression plasmid (Breedam WV, Thooft K, Santens F, et al. GlyConnect: a glycan-based conjugation extension of the GlycoDelete technology. Published online June 4, 2021:2021.06.02.446789. doi:10.1101/2021.06.02.446789).
Upon NGS verification of the sequence, all constructs were linearized in the promoter region for targeted integration in the Pichia genome. Subsequently, lithium/acetate competent Pichia pastoris were transformed with the expression plasmids. Protein expression was induced with methanol in a two-step procedure. First, the transformants were incubated for 48h at 28 °C in BMGY medium (100 mM potassium phosphate pH 6, 2% peptone, 1% yeast extract, 1% yeast nitrogen base without amino acids and 1% glycerol). Next, the cells were pelleted and resuspended in BMMY medium (100 mM potassium phosphate pH 6, 2% peptone, 1% yeast extract, 1% yeast nitrogen base without amino acids and 1% methanol), supplemented with 0.5 mM CuSO4. Protein production was induced with methanol, 1% methanol was added every 10 - 14h, for a total incubation of 48h at 28 °C. Next, the supernatant, containing the recombinant proteins, was collected by centrifugation and analyzed directly on SDS PAGE.
Clinically approved ADCs were provided by AZ Sint-Lucas & Volkskliniek (Belgium) and by Maria Middelares (Belgium).
Characterization of recombinant proteins
Proteins were analyzed on SDS PAGE. Resulting gels were either stained with Coomassie Brilliant Blue R250 (CBB) or transferred to nitrocellulose membranes by wet blotting in Towbin buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol (pH 8.3)) for lh at 100V for specific detection with DyLight800 conjugated anti-hlgG antibody (609-145-002, Rockland) for antibody detection or anti-HisTag antibody (ROCK200-345-382, Rockland) for GaOx detection through its HisTag.
N-glycosylation analysis of wild-type N-glycans was performed as described previously by DNA- sequencer assisted fluorophore assisted capillary electrophoresis (DSA FACE). Isolation and analysis of N-glycans was performed as described previously (Laroy W, Contreras R, Callewaert N. Nat Protoc. 2006;l(l):397-405). Briefly, glycoproteins were denatured and immobilized on 96-well PVDF plates (Merck). After reduction and alkylation of disulfide bonds and blocking in 1% PVP, N-glycans were released by employing in-house produced PNGase F (15.4 IUB mU/pl) for 3h at 37 °C. The resulting reducing end of the anomeric oxygen was subsequently derivatized with APTS (10 mM final concentration) by reductive amination with 2-picoline borane (2-PB). Before analysis on an ABI3130 Genetic Analyzer (Applied Biosystems) with a 50 cm capillary, excess label was removed by size exclusion chromatography over Sephadex GIO resin (GE Healthcare).
APTS-labeled N-glycans were treated overnight in 5 mM ammonium acetate pH 5 with exoglycosidases to verify the N-glycan composition. The following enzymes were used: a-fucosidase O (P0749, NEB), a- mannosidase (P0768, NEB), p-hexosaminidase (GKX-5003, Agilent), -galactosidase (GKX-5014, Agilent), a-sialidase (In house produced, Arthrobacter ureafaciens). Non-PNGase F treated samples were taken along as controls.
Endo F2 digest was performed on purified trastuzumab variants for SDS PAGE analysis of treated samples, to verify the high occupancy of the N221 site. Antibody and Endo F2 (P0772, NEB) were incubated at 1U/10 ug trastuzumab overnight at 37 °C.
In order to determine the N-glycosylation for the specific sites in trastuzumab N221, we digested 750 pg trastuzumab N221 overnight at 37 °C in PBS with 70 units FabRICATOR (A0-FR1-020, Genovis). This protease cleaves antibodies specifically below the hinge region, successfully separating the N221 glycan from the N297 N-glycan, respectively located at the F(ab')j and Fc/2 fragments. We next separated the fragments based on their size with SEC (HiLoad 16/600 200 pg column) and subjected the pooled elution fractions to DSA FACE.
N-glycosylation analysis of GlycoDelete truncated trastuzumab by intact protein mass spectrometry (MS) was included to analyze the recombinant proteins more accurately. LC-MS was performed on an Ultimate 3000 HPLC (Thermo Fisher Scientific, Bremen, Germany) equipped with a Poroshell 300SB-C8 column (Thermo Scientific 1.0 mm of LD. x 150 mm), in-line connected with an ESI source to an LTQ XL mass spectrometer (Thermo Fischer Scientific). Mobile phases were 0.1% formic acid and 0.05% trifluoroacetic acid (TFA) in H2O (solvent A) and 0.1% formic acid and 0.05% TFA in acetonitrile (solvent B). The proteins were separated using a gradient ranging from 10% to 80% solvent B at a flow rate of 100 pl/min for 15 min. The mass spectrometer was operated in MSI profile mode in the orbitrap analyzer at a resolution of 60,000 (at m/z 400) and a mass range of 600-4000 m/z. The following ESI parameters were used: a surface-induced dissociation of 30 V, a spray voltage of 5.0 kV, capillary temperature of 325 °C, capillary voltage of 35 V and a sheath gas (N2) flow rate setting of 7. Binding of trastuzumab variants to antigen HER2 was validated by ELISA. Briefly, 75 ng HER2 (10004- H08H, Sino Biological) was coated overnight at 4 °C. After one hour blocking in 4% milk (in PBS, 0.5% Tween20), a serial dilution of the trastuzumab variants was incubated for one hour at room temperature. Next, antibody was detected with anti-hlgG antibody coupled to HRP (AQ112P, Millipore). In between each step, the ELISA plate was washed three times in 0.05% PBS-Tween20. Finally, HRP activity was analyzed by incubating TMB substrate (BD OptEIA) in the wells for 10 minutes, followed by acidification with IM H2SO4. Absorbance was measured at 450 nm and 655 nm and plotted from duplicated experiments.
Binding of trastuzumab variants to CD16a 158V was validated by ELISA. 200 ng trastuzumab was coated overnight at 4 °C. After one hour blocking in 4% milk (in PBS, 0.5% Tween20), a serial dilution of the CD16a 158V (#1O389-H27H1, Sino Biological) was incubated for two hours at room temperature. Next, antibody was detected with anti-HisTag antibody coupled to HRP (R931-25, Invitrogen). In between each step, the ELISA plate was washed three times in PBS-Tween20. Finally, HRP activity was analyzed by incubating TMB substrate (BD OptEIA) in the wells for 10 minutes, followed by acidification with 2N H2SO4. Absorbance was measured at 450 nm and 655 nm, and plotted from duplicated experiments.
Oxidation, conjugation and purification of trastuzumab - aminooxy-PEG4-MMAF drug conjugates
Trastuzumab was oxidized with 0.66U GaOx/nmol antibody overnight in 100 mM sodium phosphate at pH 7 at 28 °C, with 100 equivalents aminooxy-PEG-biotin. After oxidation, conjugation was catalyzed for one hour with para-phenylenediamine (PPD). Successful conjugation was verified with SDS PAGE and western blotting for streptavidin-based detection (#21851, Invitrogen). No GaOx unit adjustments for the number of galactose residues were implemented.
In a first oxidation and conjugation, 4 mg of trastuzumab N221 (HEK293S WT and HEK293S GDLN expressed with hGalTl or precursors) was oxidized with 0.66U GaOx/nmol overnight in 100 mM sodium phosphate at pH 7 at 28 °C. During oxidation, the remainder of available aminooxy-functionalized MMAF toxin was divided over the two reactions, for 42.9 molar equivalents of toxin compared to 4 mg antibody. Next, the conjugation was catalyzed by 1 mM PPD for lh. Antibodies were then purified by protein A chromatography as above and desalted over HiPrep 26/10 Desalting columns (Cytiva) to PBS.
In a second oxidation and conjugation, 2 mg of trastuzumab N221, expressed in HEK293S WT cells, with 1% GalT and precursors (1 ISM MnCI2, 5 mM galactose and 1 mM uridine), was oxidized with 0.66U GaOx/nmol overnight in 100 mM sodium phosphate at pH 7 at 28 °C. In contrast to the first conjugation, the molar equivalents of MMAF toxin were increased to 250 equivalents. After the overnight incubation, the conjugation reaction was treated with PPD and purified, identically to the first oxidation and conjugation. The purified trastuzumab - drug conjugates were analyzed SDS PAGE and western blotting for specific detection with anti-MMAF antibody (MA542538, Fisher Scientific) and anti-mouse antibody conjugated to DyLight800 (SA5-35521, ThermoFisher Scientific).
Digest with 1U FabRICATOR/0g antibody showed the lack of activity of the enzyme on MMAF-conjugated trastuzumab N221.
To determine the DAR, peptide MS was performed after digest with 0.5 pg GluC (P8100, NEB), or with the combination of 0.5 pg Trypsin (V5111, Promega) and 0.5 pg LysC (P8109, NEB) in triethylammonium bicarbonate (TEAB). After heat denaturation in 8M urea at 95°C for 10 minutes, reduction in 5 mM DTT at 55°C for 30 minutes and alkylation in 10 mM IAA at RT for 15 minutes, proteases GluC or trypsin and LysC were incubated with antibodies and MMAF conjugates for 4h to overnight. Next, the resulting peptides were analyzed by MS.
DAR determination of MMAF conjugates was performed in collaboration with RIC (Kortrijk, Belgium) by implementing reduced RPLC MS and native SEC MS. The former method was run with a Q-TOF (6540) setup using 0.1% TFA in H2O (buffer A) and 0.1% TFA (buffer B) in acetonitrile as mobile phases in a gradient mode. The native SEC MS technique was run with 100 mM ammonium acetate in isocratic mode on an Orbitrap Q - Exactive Plus.
Conjugation of antibody with aminooxy-valine-citruline-PABC-MMAE
Trastuzumab N221 was oxidized and conjugated with the MMAE drug-linker compound (MedChemExpress, HY-153263), as described above for the MMAF drug-linker. Antibody was oxidized overnight with 0.66U GaOx per nmol antibody, in the presence of 50 molar equivalents of aminooxytoxin linker (in 50-100 mM sodium phosphate buffer pH 7). After overnight incubation, the oxime ligation was catalyzed by addition of PPD. After an incubation of two hours, the reaction mixture was diluted in protein A binding buffer and subjected to protein A affinity chromatography. The eluted antibody-drug conjugate was subsequently desalted to phosphate buffered saline pH 7.4.
Conjugation of antibody with DM1
Trastuzumab N221 was oxidized and conjugated to aminooxy-PEG4-azide (BroadPharm, BP-23595), as described above for the MMAF and MMAE conjugations. After protein A chromatography and desalting to phosphate buffered saline pH 7.4, the azide-modified antibody was then conjugated with 15 molar equivalents DBCO-PEG4-DM1 (MedChemExpress, HY-136261). The conjugation was terminated after 72h and purified by protein A chromatography. The final antibody-drug conjugate was subsequently desalted to phosphate buffered saline pH 7.4. Conjugation of antibody with maleimide-MMAF and maleimide-MMAE
Trastuzumab was reduced with 2.2 molar equivalents TCEP for 90 minutes at 37 °C in 50 mM sodium phosphate pH 7. Subsequently, the reduced cysteines were conjugated with 10 molar equivalents maleimide-functionalized toxins for lh at 25 °C. Maleimide-functionalized toxins were bought from MedChem Express: Maleimidocaproyl MMAF (HY-15578) and MC-Val-Cit-PAB-MMAE (HY-15575). Next, the reactions were diluted in protein A binding buffer and purified with protein A chromatography. The final antibody-drug conjugate was subsequently desalted to phosphate buffered saline pH 7.4.
Verifying conjugation of toxins
To verify whether the toxin was properly conjugated toxin-specific detection was performed through western blotting of the ADC samples. DM1 and MMAE were detected as expected on both clinical material (Kadcyla and Padcev (Enfortumab conjugated to MMAE)) and on the produced biosmilar ADCs (Figure 29). MMAF detection was successful, yet the detection of maleimide-MMAF was superior compared to aminooxy-PEG4-MMAF detection. In the western blot without the maleimide-conjugated ADCs, GDLN-ADC and WT-ADC are clearly detected, while in the other MMAF-specific western blot, limited signal is detected for the Oxime-ADCs (Figure 29).
In vitro Toxicity of trastuzumab - aminooxy-PEG4-MMAF in SK-BR-3 cells (Example 6)
In vitro cytotoxicity of the trastuzumab - drug conjugates was verified with the HER2+ breast cancer cell line SK-BR-3 (ATCC) or BT-474, and the HER2- breast cancer cell line MDA-MB-231 (ATCC). Cell lines were cultured at 37°C and at 5 % CO2, in DMEM, supplemented with 10 % heat-inactivated FBS, non-essential amino acids (Invitrogen), 2 mM L-glutamine and 1 mM sodium pyruvate.
HER2 status was verified by FACS analysis. Cells were stained with live/dead stain LDeFI506 (65-0866-18, eBiosciences), fixed in 4 % PFA and stained with 5 g/ml trastuzumab WT (HEK293S WT or ExpiCHO produced) in FACS buffer (PBS, 1 % BSA, 0.5 mM EDTA) for 30 minutes. After washing, the AF633 conjugated anti-hlgG antibody (A21091, Invitrogen) was incubated at 4 g/ml on the cells for 30 minutes. After a final PBS wash, the cells were analyzed in the LSRII flow cytometer (BD).
To test toxicity in vitro, SK-BR-3 or BT-474cells were seeded at 5.000 or 10.000 cells/well at day 0. At day 1, the medium was exchanged for DMEM medium, supplemented with the treatment (a serial dilution of antibody or the toxin conjugates). The cells were then incubated for 5-6 days (for 5.000 cells/well) or 2.5 - 3 days (for the 10.000 cells/well). Next, dead cell debris and free ATP was removed by washing the cells three or two times, resp. with PBS. Cells were then analyzed for viability with the Cel ITiter Gio assay (G7572, Promega). Briefly, equal amounts of CellTiter-Glo reagent were mixed with the cell culture. Cell lysis was induced for two minutes on an orbital shaker, after which the luminescence signal was read for Is using a plate reader (GloMax, ProMega).
To assess bystander effect of the ADCs, the in vitro toxicity assay, described above, was adapted at day 0 with the seeding of 5000 SK-BR-3 cells with 5000 MDA-MB-231 cells per well, premixed for all wells.
As control for live cells, non-treated cells were included. Additionally, for BT-474 cells, as control for dead cells, non-treated cells were treated with 30 % ethanol after the PBS wash. The viability of treated cells was analyzed relative to the live and dead cells. Finally, in BT-474 cells, a longer incubation period was required when alternative (weaker, less potent) toxins were used as compared to MMAF.
Comparing drug-to-antibody ratio (PAR)
The generated ADCs were compared to clinically approved ADCs for drug loading with SDS PAGE and western blotting with anti-toxin based detection. Briefly, 50 - 150 ng antibody was loaded for SDS PAGE (140V, lh) and subsequently wet blotted to nitrocellulose membrane in Towbin buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol (pH 8.3)) for lh at 100V. After blocking in 4% milk (in PBS), primary antibodies were added: 1/5000 anti-toxin murine antibodies and 1/15000 anti-Fc rabbit antibody. The anti-toxin antibodies used were anti-DMl (Invitrogen, MA5-42527), anti-MMAE (AcroBiosystems, ACRBMME-M5252) and anti-MMAF (Invitrogen, MA5-42538). After overnight binding, membranes were washed in PBS and incubated for lh with 1/15000 secondary antibodies (anti-murine lgG-DyLight800 and anti-Rabbit IgG DyLight680).

Claims

1. An antibody-drug-conjugate (ADC) comprising an antibody which has an N-glycan acceptor site in the hinge region and comprising an oxime-linker-drug-entity, wherein said oxime-linker-drug-entity is coupled to said N-glycan acceptor site via the galactose residue of Galactose-N-acetylglucosamine (Gal-GIcNAc) or via the sialic acid residue of Sialyl-Galactose-N-acetylglucosamine (Sia-Gal-GIcNAc) glycan chain.
2. The ADC of claim 1, wherein the antibody is an antibody format comprising an Fc-tail, preferably a monoclonal antibody.
3. The ADC of any one of claims 1 or 1, wherein the N-glycan acceptor site in the hinge region is located at the position corresponding to the amino acid 221 (according to EU numbering) from the human IgGl hinge region sequence, preferably wherein the amino acid at said position is an asparagine.
4. The ADC of any one of claims 1 to 3, wherein the drug-entity comprises an auristatin-based drug, an anti-mitotic drug, or a DNA topoisomerase I inhibitor drug.
5. The ADC of any one of claims 1 to 4, wherein the linker comprises a non-cleavable linker.
6. The ADC of any one of claims 1 to 5, wherein the N-glycan-oxime-linker-drug-entity is Asn-GIcNAc- Gal-oxime-linker-MMAF, Asn-GIcNAc-Gal-oxime-linker-MMAE, or Asn-GIcNAc-Gal-oxime-linker- DM1.
7. A method to produce an ADC of any one of claims 1 to 6 comprising the steps of: a. expressing an antibody with a binding specificity for a cellular target of an eukaryotic cell, wherein said antibody has an N-glycan acceptor site in the hinge region, b. oxidizing the galactose residue of the Galactose-N-acetylglucosamine (Gal-GIcNac) N-glycan or oxidizing the sialic acid of the Sialyl-Galactose-N-acetylglucosamine (Sia-Gal-GIcNAc) N- glycan present on the asparagine of said N-glycan acceptor site in the hinge region of said antibody of step a) to obtain an aldehyde for coupling, and c. connecting an aminooxy-linker-drug-entity by ligation to said aldehyde of the oxidized N- glycan, d. obtaining the antibody-N-glycan-oxime-linked ADC, wherein said eukaryotic cell has a deficiency in the N-acetyl glucosaminyl-transferase I (GNT I) enzyme and said eukaryotic cell comprises a gene encoding for an endoglucosaminidase enzyme.
8. The method of claim 7 , wherein said eukaryotic cell further has a deficiency in bifunctional UDP-N- acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) activity.
9. The method of any one of claims 7 or 8, wherein the N-glycan acceptor site is Asn221 (according to EU numbering) from the human IgGl hinge region sequence.
10. The method of any one of claims 7 to 9, wherein the antibody is an antibody format comprising an
Fc-tail, preferably a monoclonal antibody.
11. The method of any one of claims 7 to 10, wherein the antibody is specifically binding a protein allowing selectivity for tumor targeting, or the antibody is specifically binding to tumor cells.
12. The method of any one of claims 7 to 11, wherein the linker of the aminooxy-linker-drug-entity is a non-cleavable linker.
13. The method of any one of claims 7 to 12, wherein the oxidation is performed using enzymatic or chemical oxidation, preferably using Galactose oxidase or sodium periodate, respectively.
14. An antibody-drug conjugate obtainable by the method of any one of claims 7 to 13.
15. The ADC of any one of claims 1 to 6, or 14, for use as a medicament.
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Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994004678A1 (en) 1992-08-21 1994-03-03 Casterman Cecile Immunoglobulins devoid of light chains
WO1999042077A2 (en) 1998-02-19 1999-08-26 Xcyte Therapies, Inc. Compositions and methods for regulating lymphocyte activation
WO2004081026A2 (en) 2003-06-30 2004-09-23 Domantis Limited Polypeptides
WO2006040153A2 (en) 2004-10-13 2006-04-20 Ablynx N.V. Single domain camelide anti -amyloid beta antibodies and polypeptides comprising the same for the treatment and diagnosis of degenarative neural diseases such as alzheimer's disease
WO2006050584A1 (en) 2004-11-10 2006-05-18 Universiteit Gent Endo-n-acetyl-beta-d-glucosaminidase enzymes of filamentous fungi
WO2006122825A2 (en) 2005-05-20 2006-11-23 Ablynx Nv Single domain vhh antibodies against von willebrand factor
WO2010015722A1 (en) 2008-08-08 2010-02-11 Vib Vzw Cells producing glycoproteins having altered glycosylation patterns and methods and use thereof
WO2013036748A1 (en) 2011-09-09 2013-03-14 Berry, Lana, L. Bicyclo[6.1.0]non-4-yne reagents for chemical modification of oligonucleotides
WO2014065661A1 (en) 2012-10-23 2014-05-01 Synaffix B.V. Modified antibody, antibody-conjugate and process for the preparation thereof
WO2015032899A1 (en) 2013-09-05 2015-03-12 Vib Vzw Cells producing fc containing molecules having altered glycosylation patterns and methods and use thereof
WO2015057064A1 (en) 2013-10-14 2015-04-23 Synaffix B.V. Modified glycoprotein, protein-conjugate and process for the preparation thereof
US20160060354A1 (en) * 2014-03-19 2016-03-03 Genzyme Corporation Site-specific glycoengineering of targeting moieties
WO2016053107A1 (en) 2014-10-03 2016-04-07 Synaffix B.V. Sulfamide linker, conjugates thereof, and methods of preparation

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994004678A1 (en) 1992-08-21 1994-03-03 Casterman Cecile Immunoglobulins devoid of light chains
WO1999042077A2 (en) 1998-02-19 1999-08-26 Xcyte Therapies, Inc. Compositions and methods for regulating lymphocyte activation
WO2004081026A2 (en) 2003-06-30 2004-09-23 Domantis Limited Polypeptides
WO2006040153A2 (en) 2004-10-13 2006-04-20 Ablynx N.V. Single domain camelide anti -amyloid beta antibodies and polypeptides comprising the same for the treatment and diagnosis of degenarative neural diseases such as alzheimer's disease
WO2006050584A1 (en) 2004-11-10 2006-05-18 Universiteit Gent Endo-n-acetyl-beta-d-glucosaminidase enzymes of filamentous fungi
WO2006122825A2 (en) 2005-05-20 2006-11-23 Ablynx Nv Single domain vhh antibodies against von willebrand factor
WO2010015722A1 (en) 2008-08-08 2010-02-11 Vib Vzw Cells producing glycoproteins having altered glycosylation patterns and methods and use thereof
WO2013036748A1 (en) 2011-09-09 2013-03-14 Berry, Lana, L. Bicyclo[6.1.0]non-4-yne reagents for chemical modification of oligonucleotides
WO2014065661A1 (en) 2012-10-23 2014-05-01 Synaffix B.V. Modified antibody, antibody-conjugate and process for the preparation thereof
WO2015032899A1 (en) 2013-09-05 2015-03-12 Vib Vzw Cells producing fc containing molecules having altered glycosylation patterns and methods and use thereof
WO2015057064A1 (en) 2013-10-14 2015-04-23 Synaffix B.V. Modified glycoprotein, protein-conjugate and process for the preparation thereof
US20160060354A1 (en) * 2014-03-19 2016-03-03 Genzyme Corporation Site-specific glycoengineering of targeting moieties
WO2016053107A1 (en) 2014-10-03 2016-04-07 Synaffix B.V. Sulfamide linker, conjugates thereof, and methods of preparation

Non-Patent Citations (34)

* Cited by examiner, † Cited by third party
Title
"Nomenclature and Symbolism for Amino Acids and Peptides", EUR. J. BIOCHEM., vol. 138, 1984, pages 9 - 37
ANGELASTRO ET AL.: "Galactose Oxidase Enables Modular Assembly of Conjugates from Native Antibodies with High Drug-to-Antibody Ratios", CHEMSUSCHEM, vol. 15, no. 9, 2022
BLUNDELL ET AL., J BIOL CHEM., 15 June 2017 (2017-06-15)
BLUNDELL ET AL., J IMMUNOL, vol. 202, no. 5, 2019, pages 1595 - 1611
BLUNDELL PA ET AL., J IMMUNOL AUTHOR CHOICE, vol. 202, no. 5, 2019, pages 1595 - 1611
BREEDAM WVTHOOFT KSANTENS F ET AL., GLYCONNECT: A GLYCAN-BASED CONJUGATION EXTENSION OF THE GLYCODELETE TECHNOLOGY, 4 June 2021 (2021-06-04)
BUECHELER ET AL., J PHARM PHARMACOL., vol. 70, no. 5, 2018, pages 625 - 635
DE POURCQ, K ET AL., APPL MICROBIOL BIOTECHNOL., vol. 87, no. 5, 2010
DEKKERS ET AL., SCI REP, vol. 6, no. 1, 2016, pages 36964
EDELMAN ET AL., PROC NATL ACAD SCI USA., vol. 63, 1969, pages 78 - 85
EDELMAN ET AL.: "The covalent structure of an entire gammaG immunoglobulin molecule", PROC NATL ACAD SCI USA., vol. 63, 1969, pages 78 - 85, XP055122293, DOI: 10.1073/pnas.63.1.78
EPENETOS ET AL., CANCER RES., vol. 46, no. 6, 1986, pages 3183 - 3191
GRAMER ET AL., BIOTECHNOL BIOENG, vol. 108, no. 7, 2011, pages 1591 - 1602
HARYADI ET AL., PLOS ONE., vol. 10, no. 2, 2015
JESSICA R. MCCOMBSSHAWN C. OWEN, AAPS J., vol. 17, no. 2, 2015
KABAT, E.A. ET AL.: "Sequences of proteins of immunological interest", 1991, US DEPARTMENT OF HEALTH AND HUMAN SERVICES, pages: 662,680,689
KURTZMAN, C.P., J IND MICROBIOL BIOTECHNOL, vol. 36, no. 11, 2009
LAROY WCONTRERAS RCALLEWAERT N, NAT PROTOC., vol. 1, no. 1, 2006, pages 397 - 405
LEFRANC, FRONTIERS IN IMMUNOLOGY, vol. 5, no. 22, 2014, pages 1 - 22
LU, J. ET AL., INT J MOL SCI., vol. 17, no. 4, 2016
MEURIS ET AL.: "GlycoDelete engineering of mammalian cells simplifies N-glycosylation of recombinant proteins", NOT BIOTECHNOL, vol. 32, no. 5, 2014, pages 485 - 489, XP055207876, DOI: 10.1038/nbt.2885
MEURIS L. ET AL., NAT. BIOTECHN., vol. 32, no. 5, 2014, pages 485
PARIKKA ET AL.: "Oxidation with galactose oxidase: Multifunctional enzymatic catalysis", J MOL CATAL B ENZYM, vol. 120, 2015, pages 47 - 59, XP093135729, DOI: 10.1016/j.molcatb.2015.06.006
PILLOW, T.H., PHARM PAT ANAL., vol. 6, no. 1, 2017
RAYNER ET AL., JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 290, no. 13, 2015, pages 8420 - 8438
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 2012, COLD SPRING HARBOR PRESS
SHRIMAL, S.GILMORE, R., J CELL SCI., vol. 126, no. 23, 2013
SOLOMON ET AL.: "Enzymic oxidation of monoclonal antibodies by soluble and immobilized bifunctional enzyme complexes", J CHROMATOGR, vol. 510, 1990, pages 321 - 329, XP026656328, DOI: 10.1016/S0021-9673(01)93766-5
SUN, S.ZHANG, H., ANAL. CHEM., vol. 87, no. 24, 2015
TSENG ET AL., MOL PHARMACOL., vol. 70, no. 5, 2006, pages 1534 - 1541
VAN BREEDAM W. ET AL., GLYCONNECT: A GLYCAN-BASED CONJUGATION EXTENSION OF THE GLYCODELETE TECHNOLOGY, 2021, Retrieved from the Internet <URL:https://doi.org/10.1101/2021.06.02.446789>
WHITTAKER JW: "Free Radical Catalysis by Galactose Oxidase.", CHEM REV., vol. 103, no. 6, 2003, pages 2347 - 2364, XP055718270, DOI: 10.1021/cr020425z
YANG-KOLODJI ET AL., BIOMARKERS, vol. 20, no. 5, 2015, pages 313 - 322
ZHONG ET AL., BIOTECHNOL PROG, vol. 35, no. 1, 2019, pages e2724

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