WO2019234021A1 - Anticorps monoclonal glycomodifié - Google Patents

Anticorps monoclonal glycomodifié Download PDF

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Publication number
WO2019234021A1
WO2019234021A1 PCT/EP2019/064484 EP2019064484W WO2019234021A1 WO 2019234021 A1 WO2019234021 A1 WO 2019234021A1 EP 2019064484 W EP2019064484 W EP 2019064484W WO 2019234021 A1 WO2019234021 A1 WO 2019234021A1
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fold
tnfa antibody
tnfa
equal
antibody
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PCT/EP2019/064484
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English (en)
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Manuela Mally
Rikke Christina Nielsen
Amirreza Faridmoayer
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Limmatech Biologics Ag
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Publication of WO2019234021A1 publication Critical patent/WO2019234021A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]

Definitions

  • compositions and methods of producing glycosylated anti-TNFa antibodies in vitro and in vivo include using host cells to produce glycosylated anti-TNFa. Also described herein are glycosylated proteins produced using such methods and uses thereof.
  • Monoclonal antibodies constitute a large portion of marketed biological therapies. In addition to target binding, an antibody also interacts with receptors on immune cells thereby contributing to immune-regulation. The regulation of these effector functions is in part directed by a set of conserved N-linked glycans on the Fc-part of the antibody.
  • an anti-TNFa monoclonal antibody biobetter/with improved functionalities
  • adalimumab protein sequence based on the adalimumab protein sequence, with an engineered glycosylation profile that minimizes, or even prevents, ADA responses and thereby increases sustained treatment response.
  • the proposed invention addresses the unmet medical need of patients suffering from inflammatory bowel disease treated with anti-TNFa. Due to the unwanted immune reactions in the form of anti-drug antibodies against anti-TNFa, up to 38% of patients lose treatment response (Vermeire, Severine, et al. Immunogenicity of biologies in inflammatory bowel disease. Therapeutic advances in gastroenterology. 11, pp. 1756283 X 17750355 (2016)). Elimination of reduction of the ADA response will clearly improve patient care and quality of life.
  • the product and methods described herein generate an improved product by establishing novel modes of action and improved functionalities through glycosylation tailoring. This is shown by using multiple established in vitro assays, patient-derived samples and animal models. The design of the glycosylation profile and the anticipated production of an
  • Lt Leishmania tarentolae
  • N-glycans heterogeneous N-linked glycans
  • the genetically engineered Lt platform holds the promise to reduce production costs due to its use of low-cost of media and ease of fermentation, thereby providing an advantage over the complicated and expensive manufacturing associated with CHO fermentation.
  • the advantages of the platform match with the requirements of the generation of the product concept implemented here. Customizing the adalimumab glycosylation to carry a sialylated, and homogenous N-glycan will likely reduce development anti-drug antibodies. This combination of N-glycans and the associated preclinical testing support the likely improved properties in humans.
  • Described herein are unicellular Kinetoplastida eukaryotic host cells comprising heterologous glycosyltransferases, including N-acetyl glucosamine transferases, galactosyltransferase, and sialyltransferases.
  • the host cells described herein are capable of producing mammalian (e.g., human) therapeutic glycoproteins comprising homogeneous and fully- functional customized N-glycans with a high site occupancy.
  • the host cells provided herein can be used to express full-length therapeutic antibodies (e.g. adalimumab).
  • nucleic acids and combinatorial libraries that can be used to successfully target and express mammalian enzymatic activities (such as those involved in N-acetylglucosamine elongation, galactosylation and sialylation) in intracellular compartments in kinetoplastid eukaryotic host cells.
  • mammalian enzymatic activities such as those involved in N-acetylglucosamine elongation, galactosylation and sialylation
  • the process provides an engineered host cell, which can be used to express and target any desirable gene(s) involved in glycosylation.
  • Design and construction of parental strains comprising a CMP-sialic acid biosynthetic pathway for the production of sialylated glycoproteins is also provided.
  • a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a target protein; and (b) a recombinant nucleic acid encoding a
  • the target protein is an antibody targeting human TNFa. In certain embodiments, the target protein is an anti-TNFa antibody.
  • the recombinant nucleic acid encodes a target protein with an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,
  • the target protein is the heavy chain of anti-TNFa antibody.
  • the recombinant nucleic acid encodes a target protein with an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
  • the target protein is the light chain of anti-TNFa antibody.
  • the recombinant nucleic acid encodes a target protein with an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
  • the recombinant nucleic acid encodes a target protein with the amino acid sequence of the heavy chain of adalimumab.
  • the recombinant nucleic acid encodes a target protein with an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
  • the recombinant nucleic acid encodes a target protein with the amino acid sequence of SEQ ID NO. 54 (adalimumab heavy chain).
  • the recombinant nucleic acid encodes a target protein with an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the SEQ ID NO. 54 (adalimumab heavy chain).
  • the target protein comprises or consists of the amino acid of the heavy chain or light chain of adalimumab.
  • the recombinant nucleic acid encodes a target protein with the amino acid sequence of the light chain of adalimumab. In further embodiments, the recombinant nucleic acid encodes a target protein with an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the light chain of adalimumab.
  • the recombinant nucleic acid encodes a target protein with the amino acid of SEQ ID NO. 53 (adalimumab light chain). In further embodiments, the recombinant nucleic acid encodes a target protein with an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
  • the recombinant nucleic acid encodes a target protein with the amino acid sequence of adalimumab (HUMIRA), AMJEVITA, CYLTEZO, infliximab (REMICADE), golimumab (SIMPONI), or antibody formats such as certolizumab pegol (CIMZIA) or with a circulating receptor fusion protein such as etanercept (ENBREL).
  • the recombinant nucleic acid encodes a target protein with an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,
  • the recombinant nucleic acid encodes a target protein with the amino acid sequence of the heavy chain of adalimumab (HUMIRA),
  • the recombinant nucleic acid encodes a target protein with an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%,
  • adalimumab HUMIRA
  • AMJEVITA AMJEVITA
  • CYLTEZO CYLTEZO
  • infliximab REMICADE
  • golimumab SIMPONI
  • antibody formats such as certolizumab pegol (CIMZIA) or with a circulating receptor fusion protein such as etanercept (ENBREL).
  • the recombinant nucleic acid encodes a target protein with the amino acid sequence of the light chain of adalimumab (HUMIRA), AMJEVITA, CYLTEZO, infliximab (REMICADE), golimumab (SIMPONI), or antibody formats such as certolizumab pegol (CIMZIA) or with a circulating receptor fusion protein such as etanercept (ENBREL).
  • the recombinant nucleic acid encodes a target protein with an amino acid sequence that is at least about 70%
  • HUMIRA adalimumab
  • AMJEVITA CYLTEZO
  • REMICADE infliximab
  • SIMPONI golimumab
  • antibody formats such as certolizumab pegol (CIMZIA) or with a circulating receptor fusion protein such as etanercept (ENBREL).
  • the recombinant nucleic acid encodes a target protein with the amino acid sequence of a biosimilar or functional homologue of adalimumab (HUMIRA), AMJEVITA, CYLTEZO, infliximab (REMICADE), golimumab (SIMPONI), or antibody formats such as certolizumab pegol (CIMZIA) or with a circulating receptor fusion protein such as etanercept (ENBREL).
  • the recombinant nucleic acid encodes a target protein with an amino acid sequence that is at least about 70%, 71%, 72%, 73%,
  • HUMIRA adalimumab
  • AMJEVITA adalimumab
  • CYLTEZO CYLTEZO
  • infliximab REMICADE
  • golimumab SIMPONI
  • antibody formats such as certolizumab pegol (CIMZIA) or with a circulating receptor fusion protein such as etanercept (ENBREL).
  • the recombinant nucleic acid encodes a target protein with the amino acid sequence of the heavy chain of a biosimilar or functional homologue of adalimumab (HUMIRA), AMJEVITA, CYLTEZO, infliximab (REMICADE), golimumab (SIMPONI), or antibody formats such as certolizumab pegol (CIMZIA) or with a circulating receptor fusion protein such as etanercept (ENBREL).
  • the recombinant nucleic acid encodes a target protein with an amino acid sequence that is at least about 70%
  • HUMIRA adalimumab
  • AMJEVITA adalimumab
  • CYLTEZO CYLTEZO
  • infliximab REMICADE
  • golimumab SIMPONI
  • antibody formats such as certolizumab pegol (CIMZIA) or with a circulating receptor fusion protein such as etanercept (ENBREL).
  • the recombinant nucleic acid encodes a target protein with the amino acid sequence of the light chain of a biosimilar or functional homologue of adalimumab (HUMIRA), AMJEVITA, CYLTEZO, infliximab (REMICADE), golimumab (SIMPONI), or antibody formats such as certolizumab pegol (CIMZIA) or with a circulating receptor fusion protein such as etanercept (ENBREL).
  • the recombinant nucleic acid encodes a target protein with an amino acid sequence that is at least about 70%
  • the recombinant nucleic acid encodes a target protein with the amino acid sequence of the heavy chain of
  • the recombinant nucleic acid encodes the amino acid sequence of the heavy chain of HUMIRA, AMJEVITA, CYLTEZO, infliximab (REMICADE), and golimumab (SIMPONI), or antibody formats such as certolizumab pegol (CIMZIA) or with a circulating receptor fusion protein such as etanercept (ENBREL), or a biosimilar thereof.
  • the recombinant nucleic acid encodes a target protein with an amino acid sequence that is at least about 70%, 71%, 72%, 73%,
  • the heterologous glycosyltransferase is an N-acetyl glucosamine transferase; and/or a heterologous galactosyltransferase; and/or a heterologous sialyltransferase.
  • a host cell comprising two or more N-acetyl glucosamine transferases. In other embodiments, the host cell comprising a
  • heterologous sialyltransferase further comprises a heterologous CMP-Sia biosynthetic pathway proteins capable of generating CMP-NeuAc.
  • a host cell wherein one or more endogenous enzymes from the N-glycan biosynthesis pathway have been deleted, mutated and/or functionally inactivated.
  • the amino acid sequence of the N-acetyl glucosamine transferase, galactosyltransferase, and/or sialyltransferase is derived from an N- acetyl glucosamine transferase, a galactosyltransferase, or a sialyltransferase listed in Table 2, or any functional homologue thereof.
  • the CMP-Sia biosynthetic pathway proteins capable of generating CMP-NeuAc are at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,
  • the N-acetyl glucosamine transferase is a GnT-I.
  • the N-acetyl glucosamine transferase is a GnT-II.
  • the N-acetyl glucosamine transferases are GnT-I and GnT-II.
  • the Galactosyltransferase is B4GALT1.
  • provided herein is a host cell wherein the N-acetyl glucosamine transferases are GnT-I and GnT-II and the
  • Galactosyltransferase is B4GALT1.
  • the sialyltransferase is a 2,6-SiaT or a 2,3-SiaT.
  • provided herein is a host cell wherein the N-acetyl glucosamine transferases are GnT-I and GnT-II, the Galactosyltransferase is B4GALT1, and the sialyltransferase is a 2,6-SiaT or a 2,3-SiaT.
  • the sialyltransferase is a 2,6-SiaT.
  • a host cell wherein the N-acetyl glucosamine transferases are GnT-I and GnT-II, the Galactosyltransferase is B4GALT1, the sialyltransferase is a 2,6-SiaT or a 2,3-SiaT, and wherein the sialyltransferase further comprises heterologous CMP-Sia biosynthetic pathway proteins capable of generating CMP-NeuAc.
  • the host cell is a Leishmania tarentolae cell.
  • a Leishmania signal and/or retention sequence is added to the N-acetyl glucosamine transferase, galactosyltransferase, and/or sialyltransferase, wherein the signal sequence targets the N-acetyl glucosamine transferase, galactosyltransferase, and/or sialyltransferase to the endoplasmic reticulum of the Leishmania host cell, and wherein the retention sequence retains the N-acetyl glucosamine transferase, galactosyltransferase, and/or sialyltransferase in the Golgi apparatus.
  • the retention sequence retains the N-acetyl glucosamine transferase and/or galactosyltransferase in the cis Golgi compartment of the host cell. In another embodiment, the retention sequence retains the N-acetyl glucosamine transferase and/or galactosyltransferase in the medial Golgi compartment of the host cell. In another embodiment, the retention sequence retains the galactosyltransferase in the trans Golgi compartment of the host cell. In another embodiment, the retention sequence retains the sialyltransferase in the trans Golgi compartment of the host cell.
  • the retention sequence retains the sialyltransferase and galactosyltransferase in the trans Golgi compartment of the host cell.
  • a host cell wherein one or more endogenous enzymes from the N-glycan biosynthesis pathway have been deleted, mutated and/or functionally inactivated.
  • the signal sequence and/or retention sequence is a signal sequence or retention sequence derived from Leishmania tarentolae.
  • the signal sequence and/or retention sequence is a signal sequence or retention sequence of SEQ ID No. 55.
  • the signal sequence is processed and removed.
  • the retention sequence is a cytoplasmic- transmembrane-stem (CTS) sequence derived from a Leishmania tarentolae protein.
  • CTS sequence is derived from Leishmania tarentolae MAN 1 , NTPDase 1 , or NTPDase 2.
  • the CTS sequence comprises the sequence of SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26 or functionally active fragments thereof.
  • the CTS sequence comprises a sequence that is at least about 70%, 71%, 72%,
  • the CTS sequence comprises the sequence of SEQ ID NO: 55, or functionally active fragments thereof.
  • the CTS sequence comprises a sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,
  • the CTS is derived from Leishmania tarentolae
  • the CTS sequence comprises the sequence of SEQ ID NO: 24 or functionally active fragments thereof.
  • the retention sequence comprises a GRIP sequence derived from Leishmania or functionally active fragments thereof.
  • the GRIP sequence comprises the sequence of SEQ ID NO: 27, or a functionally active fragments thereof.
  • the GRIP sequence comprises a sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
  • the retention sequence comprises a CTS sequence derived from a Leishmania protein, or a functionally active fragment thereof, and a GRIP sequence derived from Leishmania or a functionally active fragment thereof
  • the retention sequence is a cytoplasmic- transmembrane-stem (CTS) sequence derived from a Leishmania tarentolae protein.
  • the signal sequence is a secretion signal peptide.
  • the signal sequence is a secretion signal peptide invertase derived from SEQ ID NO: 28 and any functionally active fragment thereof.
  • the CTS sequence comprises a sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,
  • the retention sequence is a cytoplasmic- transmembrane-stem (CTS) sequence derived from a Leishmania tarentolae protein.
  • the signal sequence is a secretion signal peptide.
  • the signal sequence is a secretion signal peptide invertase derived from SEQ ID NO: 55 and any functionally active fragment thereof.
  • the CTS sequence comprises a sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,
  • the target protein is heterologous to the Leishmania host cell.
  • the target protein has been engineered to comprise a signal sequence from Leishmania.
  • said signal sequence is a signal sequence from Leishmania tarentolae.
  • the signal sequence comprises the sequence of SEQ ID NO: 28, or SEQ ID NO: 29 or functionally active fragments thereof.
  • the signal sequence comprises the sequence of SEQ ID NO: 28 or a functionally active fragment thereof.
  • the signal sequence comprises a sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,
  • the signal sequence is processed and removed from the target protein.
  • the target protein is a therapeutic protein. In other embodiments, the target protein is an Fc-fusion protein. In other embodiments, the target protein is an antibody.
  • the target protein is an antibody against a human protein.
  • the antibody has the amino acid sequence of adalimumab;
  • the antibody is a full length antibody, an Fab, an F(ab') 2 , an ScfV, or a sdAb.
  • the host cell comprises (a) an Stt3
  • oligosaccharyltransferase (OST), and (b) does not have endogenous N-glycan elongation.
  • Another embodiment includes a method for making a glycosylated target protein, wherein the method comprises culturing a host cell, and purifying the target protein from the culture.
  • Another embodiment includes a composition of glycosylated target proteins.
  • composition of glycosylated target proteins have at least about 90% to 100% of the N-linked glycosylation consensus sequences of the target proteins in the composition carry an oligosaccharide comprising the following structure:
  • the square represents an N-acetylglucosamine residue and the grey circle represents a mannose residue; and wherein the Asn is the Asn of the N-linked
  • glycosylation consensus sequence in the target protein is glycosylation consensus sequence in the target protein.
  • the composition of glycosylated target proteins have at least about 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% of the glycosylation on the target protein is GO-Gn glycan, characterized by the following structure:
  • the square represents an N-acetylglucosamine residue and the grey circle represents a mannose residue; and wherein the Asn is an Asn of an N-linked glycosylation consensus sequence in the target protein.
  • composition of glycosylated target proteins have at least about 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%,
  • GO glycan characterized by the following structure:
  • the square represents an N-acetylglucosamine residue and the grey circle represents a mannose residue; and wherein the Asn is an Asn of an N-linked glycosylation consensus sequence in the target protein.
  • the composition of glycosylated target proteins have at least about 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% of the glycosylation on the target protein is Gl-Gn glycan, characterized by the following structure: [0047]
  • the empty circle represents a galactose residue
  • the square represents an N-acetylglucosamine residue
  • the grey circle represents a mannose residue
  • the Asn is an Asn of an N-linked glycosylation consensus sequence in the target protein.
  • composition of glycosylated target proteins have at least about 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%,
  • G2 glycan characterized by the following structure:
  • the empty circle represents a galactose residue
  • the square represents an N-acetylglucosamine residue
  • the grey circle represents a mannose residue
  • the Asn is an Asn of an N-linked glycosylation consensus sequence in the target protein.
  • the glycosylation on the target protein is further modified to optimize the pharmacokinetic properties of the target protein when introduced into a subject.
  • the glycosylation on the target protein is sialylated.
  • composition of glycosylated target proteins have at least about 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%,
  • the diamond represents a sialic acid residue
  • the empty circle represents a galactose residue
  • the square represents an N-acetylglucosamine residue
  • the grey circle represents a mannose residue
  • the Asn is an Asn of an N-linked glycosylation consensus sequence in the target protein.
  • the composition of glycosylated target proteins have at least about 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% of the glycosylation on the target protein is characterized by the following structure:
  • the diamond represents a sialic acid residue
  • the empty circle represents a galactose residue
  • the square represents an N-acetylglucosamine residue
  • the grey circle represents a mannose residue
  • the Asn is an Asn of an N-linked glycosylation consensus sequence in the target protein.
  • the glycosylated target protein purified from a host cell is further glycosylated in vitro by in vitro glycoengineering using enzymes and methods described herein or enzymes and methods known in the art.
  • the glycosylated target protein characterized by any of the structures described herein is purified from a host cell, and is further glycosylated in vitro by in vitro glycoengineering using enzymes and methods described herein or enzymes and methods known in the art.
  • glycosyltransferase enzyme or nucleic acid encoding it, known in the art can be used in accordance with the host cells and methods described herein.
  • glycosylated target protein purified from a host cell is further glycosylated in vitro by in vitro glycoengineering using one or more purified glycosyltransferases.
  • glycosylated target protein purified from a host cell is further glycosylated in vitro by in vitro glycoengineering using one or more purified glycosyltransferases including, but not limited to, a N-acetyl glucosamine transferase a heterologous galactosyltransferase, and a heterologous sialyltransferase.
  • glycosylated target protein purified from a host cell is further glycosylated in vitro by in vitro glycoengineering using one or more purified glycosyltransferases listed in Table 2.
  • the invention also provides an engineered Leishmania tarentolae with paucimannose, glycan extensions performed in vitro with all heterologous glycosyltransferases expressed in Leishmania and purified.
  • the invention provides
  • Leishmania tarentolae with paucimannose further glycan extensions performed in vitro with glycosyltransferases expressed and purified from Leishmania tarentolae or other hosts (E.coli, CHO, HEK, insect).
  • Leishmania tarentolae with paucimannose in vivo glycoengineered with heterologous glycosyltransferase in Leishmania
  • further extensions performed in vitro with heterologous glycosyltransferases expressed in Leishmania and purified further embodiment, provides Leishmania tarentolae with paucimannose in vivo glycoengineered with heterologous glycosyltransferases in
  • Leishmania further extensions performed in vitro with glycosyltransferases expressed and purified from other hosts ⁇ E.coli, CHO, HEK, insect).
  • the invention provides Leishmania tarentolae with paucimannose in vivo glycoengineered with heterologous glycosyltransferases in Leishmania, including all biosynthetic precursor pathway for sialylation.
  • sialylated products could also be produced in recombinant mammalian cell systems like CHO ( e.g ., GlycoDisplays Aps and Glycotope GmbH) as well as non mammalian cells e.g., plants ( Nicotiana tabacum).”
  • N-linked glycosylation consensus sequence is N-linked glycosylation consensus sequence
  • the glycosylated target protein is secreted into the culture media, and wherein the glycosylated target protein is glycosylated.
  • the glycosylated target protein is purified from the culture media. In another embodiment, the glycosylated target protein is purified from the culture media via affinity purification. In another embodiment, the glycosylated target protein contains an FC domain and is affinity purified from the culture media via protein-A. In another embodiment, the
  • glycosylated target protein contains an affinity tag and is affinity purified.
  • hybrid N-acetyl glucosamine transferase comprises (a) catalytic domain of an N-acetyl glucosamine transferase that is not from Leishmania; and (b) amino acid sequence(s) responsible for localization and retention in the Golgi compartment of Leishmania.
  • the hybrid N-acetyl glucosamine transferase is from
  • the hybrid N-acetyl glucosamine transferase has been engineered to comprise a signal sequence and at least one retention sequence, wherein the signal sequence targets the N-acetyl glucosamine transferase to the endoplasmic reticulum of the Leishmania tarentolae host cell, and wherein the retention sequence retains the N-acetyl glucosamine transferase in the Golgi apparatus.
  • the hybrid N-acetyl glucosamine transferase retains the N-acetyl glucosamine transferase in the cis Golgi apparatus. In another embodiment, the hybrid N-acetyl glucosamine transferase retains the N-acetyl glucosamine transferase in the medial Golgi apparatus. In further embodiments, the hybrid N-acetyl glucosamine transferase is a cytoplasmic-transmembrane-stem (CTS) sequence.
  • CTS cytoplasmic-transmembrane-stem
  • the N-acetyl glucosamine transferase is derived from an N-acetyl glucosamine transferase listed in Table 2, or a functional homologue thereof.
  • a hybrid galactosyltransferase wherein the hybrid galactosyltransferase comprises (a) catalytic domain of an
  • galactosyltransferase that is not from Leishmania and (b) amino acid sequence(s) responsible for localization and retention in the Golgi compartment of Leishmania.
  • the hybrid galactosyltransferase is from Leishmania tarentolae.
  • the hybrid galactosyltransferase has been engineered to comprise a signal sequence, wherein the signal sequence targets the
  • the hybrid galactosyltransferase retains the galactosyltransferase in the cis Golgi apparatus. In another embodiment, the hybrid
  • the galactosyltransferase retains the galactosyltransferase in the medial Golgi apparatus. In another embodiment, the hybrid galactosyltransferase retains the galactosyltransferase in the trans Golgi apparatus. In other embodiments, the hybrid galactosyltransferase is a cytoplasmic- transmembrane-stem (CTS) sequence. In further embodiments, the hybrid sialyltransferase is a GRIP sequence. In certain embodiments, the hybrid sialyltransferase is a CTS sequence and a GRIP sequence.
  • CTS cytoplasmic- transmembrane-stem
  • the galactosyltransferase is derived from an galactosyltransferase listed in Table 2, or a functional homologue thereof.
  • hybrid sialyltransferase comprises (a) catalytic domain of an sialyltransferase that is not from Leishmania ; and (b) amino acid sequence(s) responsible for localization and retention in the Golgi compartment of Leishmania.
  • the hybrid sialyltransferase is from Leishmania tarentolae.
  • the hybrid sialyltransferase has been engineered to comprise a signal sequence, wherein the signal sequence targets the sialyltransferase to the endoplasmic reticulum of the Leishmania tarentolae host cell, and wherein the retention sequence retains the sialyltransferase in the Golgi apparatus.
  • the hybrid sialyltransferase retains the sialyltransferase in the trans Golgi apparatus.
  • the hybrid sialyltransferase is a CTS sequence.
  • the hybrid sialyltransferase is a GRIP sequence.
  • the hybrid sialyltransferase is a CTS sequence and a GRIP sequence.
  • the hybrid sialyltransferase is derived from an sialyltransferase listed in Table 2, or a functional homologue thereof.
  • nucleic acid encoding the hybrid
  • N-acetyl glucosamine transferase N-acetyl glucosamine transferase.
  • a nucleic acid encoding the hybrid galactosyltransferase In certain embodiments, provided herein is a nucleic acid encoding the hybrid sialyltransferase.
  • Leishmania host cells Provided herein are Leishmania host cells, methods for making an anti-
  • TNFa antibody anti-TNFa antibodies
  • compositions comprising a population of glycosylated anti-TNFa antibodies
  • Leishmania host cells comprising: (a) a recombinant nucleic acid encoding an anti-TNFa antibody heavy chain (and optionally a recombinant nucleic acid encoding an anti-TNFa antibody light chain); and (b) a recombinant nucleic acid sequence encoding an a-2,6 sialyltransferase.
  • the host cell further comprises a recombinant nucleic acid sequence encoding a glycosyltransferase capable of synthesizing one glycosyl linkage in one of the following structures:
  • the diamond represents a sialic acid residue
  • the empty circle represents a galactose residue
  • the square represents an N-acetylglucosamine residue
  • the grey circle represents a mannose residue
  • the Asn is an Asn of an N-linked glycosylation consensus sequence in the anti-TNFa antibody heavy chain.
  • the host cell further comprises one or more recombinant nucleic acid sequences encoding glycosyltransferases to synthesize:
  • the diamond represents a sialic acid residue
  • the empty circle represents a galactose residue
  • the square represents an N-acetylglucosamine residue
  • the grey circle represents a mannose residue
  • the Asn is an Asn of an N-linked glycosylation consensus sequence in the anti-TNFa antibody heavy chain.
  • Leishmania host cells comprising a nucleic acid sequence encoding a recombinant anti-TNFa antibody heavy chain; and one or more recombinant nucleic acid sequences encoding glycosyltransferases to synthesize:
  • the diamond represents a sialic acid residue
  • the empty circle represents a galactose residue
  • the square represents an N-acetylglucosamine residue
  • the grey circle represents a mannose residue
  • the Asn is an Asn of an N-linked glycosylation consensus sequence in the anti-TNFa antibody heavy chain.
  • Leishmania host cells comprising a nucleic acid sequence encoding a recombinant anti-TNFa antibody heavy chain; and one or more recombinant nucleic acid sequences encoding glycosyltransferases to synthesize:
  • Leishmania host cells comprising a nucleic acid sequence encoding a recombinant anti-TNFa antibody heavy chain; and a recombinant nucleic acid sequences encoding a glycosyltransferase to synthesize:
  • the diamond represents a sialic acid residue
  • the empty circle represents a galactose residue
  • the square represents an N-acetylglucosamine residue
  • the grey circle represents a mannose residue
  • the Asn is an Asn of an N-linked glycosylation consensus sequence in the anti-TNFa antibody heavy chain.
  • the host cell further comprises one or more nucleic acid sequences encoding a-2,6 sialyltransferase.
  • the anti-TNFa antibody is adalimumab.
  • the Leishmania host cell is Leishmania tarentolae.
  • TNFa antibody comprising culturing the host cell of any one of the preceding claims; and obtaining the anti-TNFa antibody.
  • the method further comprises an in vitro glycosylation step.
  • anti-TNFa antibodies produced by the methods provided herein.
  • the anti-TNFa antibody has maximal a-2,6 sialylation.
  • the“maximal” a-2,6 sialylation is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of N- glycans comprising at least one sialic acid residue per N-glycan or comprising at least two sialic acid residues per N-glycan.
  • N-glycosylation consensus sequences are glycosylated.
  • N-glycans are not mannose 5 (Man5) glycan or mannose 6 (Man6) glycan.
  • anti-TNFa antibodies comprising one or more of the following structures:
  • the diamond represents a sialic acid residue
  • the empty circle represents a galactose residue
  • the square represents an N-acetylglucosamine residue
  • the grey circle represents a mannose residue
  • the Asn is an Asn of an N-linked glycosylation consensus sequence in the anti-TNFa antibody heavy chain.
  • compositions comprising a population of glycosylated anti-TNFa antibodies produced by the method of any one of claims 10 or 11, wherein (a) the population of glycosylated anti-TNFa antibodies is at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homogeneous; or (b) at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 95% of the N-glycosylation sites in the anti-TNFa antibodies carry the same oligosaccharide; or (c) at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 95% of the N-glycosylation sites in the anti-TNFa antibodies are a-2,6 sialylated; or (d) at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or
  • compositions comprising a population of glycosylated anti-TNFa antibodies, wherein the population of anti-TNFa antibodies comprise one or more anti-TNFa antibodies comprising one or more of the following structures:
  • the diamond represents a sialic acid residue
  • the empty circle represents a galactose residue
  • the square represents an N-acetylglucosamine residue
  • the grey circle represents a mannose residue
  • the Asn is an Asn of an N-linked glycosylation consensus sequence in the anti-TNFa antibody heavy chain.
  • compositions wherein (a) the population of glycosylated anti-TNFa antibodies is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homogeneous; or (b) at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 95% of the N-glycosylation sites in the anti-TNFa antibodies carry the same oligosaccharide; or (c) at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 95% of the N-glycosylation sites in the anti-TNFa antibodies are a-2,6 sialylated; or (d) at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 95% of the N-glycosylation sites in the anti-TNFa antibodies carry a biantennary oligosaccharide that is a-2,6 si
  • the provided anti-TNFa antibody herein is adalimumab.
  • TNFa antibody is adalimumab.
  • provided herein are methods of treating or preventing rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, pediatric Crohn’s disease, ulcerative colitis, chronic psoriasis, hidradenitis suppurativa, adult uveitis, pediatric uveitis, plaque psoriasis, or juvenile idiopathic arthritis in a patient, wherein the method comprises administering to the patient an anti-TNFa antibody provided herein or an composition provided herein.
  • the administration step is by subcutaneous injection.
  • the method : (a) requires a lower dose and/or lower administration frequency to achieve the same effect as compared to the same antibody having a different glycosylation profile; and/or (b) can be administered for an extended period of time (at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or at least 12 months, at least 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 years); and/or (c) does not trigger an immune response against the anti-TNFa antibody in the patient.
  • the method comprises treating or preventing rheumatoid arthritis, psoriatic arthritis, or ankylosing spondylitis in the patient, and wherein the method comprises administering to the patient less than or equal to 40 mg of the anti-TNFa antibody on an administration frequency less than or equal to every other week.
  • the method comprises treating or preventing Crohn's disease or ulcerative colitis in the patient, and wherein the method comprises administering to the patient less than or equal to 160 mg of the anti-TNFa antibody on day 1 , less than or equal to 80 mg of the anti-TNFa antibody on day 15, and less than or equal to 40 mg of the anti-TNFa antibody on an administration frequency less than or equal to every other week starting on day 29.
  • the method comprises treating or preventing pediatric Crohn's disease in the patient, and wherein the method comprises administering to the patient: (a) less than or equal to 80 mg of the anti-TNFa antibody on day 1 , less than or equal to 40 mg of the anti-TNFa antibody on day 15, and less than or equal to 20 mg of the anti-TNFa antibody on an administration frequency less than or equal to every other week starting on day 29 in a patient having a body weight between 17 kg and 40 kg, or (b) less than or equal to 160 mg of the anti-TNFa antibody on day 1 , less than or equal to 80 mg of the anti-TNFa antibody on day 15, and less than or equal to 40 mg of the anti-TNFa antibody on an administration frequency less than or equal to every other week starting on day 29 in a patient having a body weight equal to or higher than 40 kg.
  • the method comprises treating or preventing juvenile idiopathic arthritis or pediatric uveitis in the patient, and wherein the method comprises administering to the patient: (a) less than or equal to 10 mg of the anti-TNFa antibody on an administration frequency less than or equal to every other week in a patient having a body weight between 10 kg and 15 kg, (b) less than or equal to 20 mg of the anti-TNFa antibody on an administration frequency less than or equal to every other week in a patient having a body weight between 15 kg and 30 kg, or (c) less than or equal to 40 mg of the anti-TNFa antibody on an administration frequency less than or equal to every other week in a patient having a body weight equal to or higher than 30 kg.
  • the method comprises treating or preventing plaque psoriasis or adult uveitis in the patient, and wherein the method comprises administering to the patient less than or equal to 80 mg of the anti-TNFa antibody on day 1, and less than or equal to 40 mg on an administration frequency less than or equal to every other week starting on day 8.
  • the method comprises treating or preventing hidradenitis suppurativa in a patient, and wherein the method comprises administering to the patient: (a) less than or equal to 80 mg of the anti-TNFa antibody on day 1, and less than or equal to 40 mg of the anti-TNFa antibody on an administration frequency less than or equal to every other week starting on day 8 in an adolescent patient who are 12 years and older having a body weight between 30 kg and 60 kg, or (b) less than or equal to 160 mg of the anti-TNFa antibody on day 1, and less than or equal to 80 mg of the anti-TNFa antibody on day 15, and less than or equal to 40 mg of the anti-TNFa antibody on an administration frequency less than or equal to every week starting on day 29 in an adolescent patient who are 12 years and older having a body weight equal to or higher than 60 kg or an adult patient.
  • TNFa antibody provided herein wherein the single dosage form consists of about 2 mg, about 5 mg, about 7 mg, about 10 mg, about 12 mg, about 15 mg, about 18 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, or about 80 mg of the anti-TNFa antibody.
  • the single dosage form is a prefilled syringe, an injection pen, a vial, a tablet, or a capsule.
  • the single dosage form comprises the anti-TNFa antibody in a lyophilized form or in a liquid solution.
  • anti-TNFa antibodies wherein the anti-TNFa antibody has an antibody-dependent cell mediated cytotoxicity (ADCC) activity that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, lO-fold, l2-fold, 15-fold, 18-fold, 20-fold, 25-fold, or 30-fold higher than that of the same anti-TNFa antibody having a different glycosylation profile.
  • ADCC antibody-dependent cell mediated cytotoxicity
  • the anti-TNFa antibody has a complement- dependent cytotoxicity (CDC) activity that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, lO-fold, l2-fold, l5-fold, l8-fold, 20-fold, 25-fold, or 30-fold lower than or equal to that of the same anti- TNFa antibody having a different glycosylation profile.
  • CDC complement- dependent cytotoxicity
  • the anti-TNFa antibody has a dendritic cells internalization that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5- fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, lO-fold, l2-fold, l5-fold, 18- fold, 20-fold, 25-fold, or 30-fold higher than that of the same anti-TNFa antibody having a different glycosylation profile.
  • the anti-TNFa antibody leads to a B-cell apoptosis that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, lO-fold, l2-fold, l5-fold, l8-fold, 20-fold, 25-fold, or 30-fold higher than that of the same anti-TNFa antibody having a different glycosylation profile.
  • the anti-TNFa antibody has an anti-inflammatory activity that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5-fold, 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, lO-fold, l2-fold, l5-fold, l8-fold, 20- fold, 25-fold, or 30-fold higher than that of the same anti-TNFa antibody having a different glycosylation profile.
  • Figure 1 Schematic representation of three methods (numbers in shaded circles) of generating sialylated mAh, e.g. adalimumab.
  • Left panel indicates method (1) showing the glycan remodeling of mAbs derived from mammalian cells (e.g. CHO).
  • Right panel indicates method (2) representing the Methods combining in vivo and in vitro steps with Lt platform.
  • Right panel labelled with a (3) represents a complete in vivo pathway of sialylation of the Lt platform
  • FIG. 1 Schematic representation of the stepwise sialic acid
  • Figure 3 A Untreated and glyco-engineered HUMIRA were analyzed by
  • Figure 4 Column graph shows the relative percentage of the indicated N- glycan structures from untreated versus glyco-engineered mAh samples based on peak quantification of the UPLC traces.
  • Figure 6A-6C Expression and purification of a full length mAb from recombinant Leishmania tarentolae secreting Adalimumab_LMTB and subsequently in vitro glycoengineering.
  • Figure 6A shows the Coomassie-stained SDS PAGE.
  • Figure 6B shows anti- HC western blot.
  • Figure 6C shows anti-FC western blot of the purified Adalimumab variants under non reducing (“non”) and reducing (“red”) conditions.
  • Adali- Man3 is secreted by recombinant Leishmania and Protein A and HIC purified; Adali- GO is purified after in vitro extension of Man3 to GlcNAc terminated glycan“GO”; Sia- Adali is the in vitro 2,6 sialylated variant. All Leishmania- derived adalimumab glycovariants are compared to HUMIRA, the commercial product from CHO.
  • Figure 6D Functionality tests of Adalimumab FMTB purified from recombinant Leishmania for antigen binding. TNFa was spotted at increasing concentrations onto membranes. Different concentrations of HUMIRA or Adalimumab FMTB or without primary antibody (“negative control”) were used to incubate the membrane strips. After washing, the secondary antibody was anti- human IgG-HRP.
  • Figure 7 The N-glycosylation biosynthesis pathway in Leishmania tarentolae.
  • the N-glycan precursor is assembled as Man3 at the cytoplasmic side of the ER and then flipped to the ER lumen (top left). This Man3 glycan is then directly transferred by the Stt3, the only part of the usually found OST complex.
  • the final glycan on the N-glycosite of the acceptor protein is Man3, which transits to the Cis Golgi. In the cis Golgi (bottom left), the heterologous enzymes catalyze the glycosyltransferase reactions building up the human biantennary N-glycans.
  • Gnt-I and Gnt-II from different organisms that add the first two GlcNAc residues.
  • the corresponding glycoforms are described with their text abbreviation in the boxes below.
  • the GalT and SiaT enzymes from bacterial or eukaryotic sources expressed in Leishmania catalyze the reactions to the completion of the N- glycan, which is then the fucose-free but biantennary sialylated humanized glycan mediating immune-tolerizing functions depending a2,6 linked sialic acid (Neu5Ac) on the secreted protein, represented as mAb molecule, e.g. Adalimumab.
  • CMP-Neu5Ac is also schematically
  • top middle with pro- and eukaryotic enzymes, colored in grey and black, respectively, to be expressed recombinantly in Leishmania for sialylation engineering of the biosynthetic precursor CMP-Neu5Ac for the sialyltransferase reaction.
  • Figure 8A In vitro Glycoengineering on paucimannose glycans of an full length mAb purified from Leishmania with the first step (MGAT1).
  • Figure 8B Coomassie stained SDS PAGE (left) and anti-HC (middle) and anti-LC (right) blots show the purified mAb (Rituximab LMTB) in comparison to commercial MabThera® (therapeutic antibody sold under the trademark MabThera).
  • Figure 8C Purified Rituximab LMTB was used in the in vitro reaction with
  • MGAT1 and cofactors, and glycans were PNGaseF released and analyzed by RapiFluor- mass spectrometry (C).
  • Figure 9 Figure taken from Nitschke et al. (2005) (Nitschke L, et al. The role of CD22 and other inhibitory co-receptors in B-cell activation. Current Opinion in
  • BCR signaling triggers depletion of intracellular Ca2+ stores of the
  • CD22 via SHP-l activates (indicated by +) the Ca2+ pump PMCA4 and thereby controls Ca2+ efflux.
  • IP3R IP3 receptor
  • Figure 10 Schematic overview of B cell ELISPOT assay.
  • Figure 11 Regression analysis for correlation of in vitro ADA and B cell
  • ELISPOT assay (figure taken from Liao K et al. Detection of Memory B Activity against a Therapeutic Protein in Treatment-Naive Subjects. The AAPS journal 20:51 (2016)).
  • a was the intercept (1.00342) the value of dependent variable (y, log(ADA RECL)) at which the value of spot (X) was zero
  • b was the slope (0.36241), when spot increased one unit, the response of
  • Figure 12A Quality parameters for the antibody variants. All antibodies used in preclinical assessment were analyzed for the critical quality attributes (CQA) indicated. “N.d.” means not determined;“n.a.” means not applicable.
  • Figure 12B Glycosylation profiles of commercial HUMIRA (right) and
  • Leishmania produced Adalimumab LMTB (Left).
  • Y axis indicates the relative abundance of a certain glycoform, specific glycoforms are indicated on the x axis. 0% abundance is shown when signals are too small to integrate, or absent.
  • Figure 13A-C Recombinant Leishmania tarentolae cell lines are proficient in 2,6 sialylating N-glycans in vivo.
  • Figure 13 A DMB labelling of biosynthetic Neu5Ac intermediate.
  • the RP-C18-LC trace of DMB-labelled extract from negative control Stl0569 (wt) is displayed as solid line.
  • the traces of DMB-labelled Neu5Ac extracted from Stl7078 and Stl7l64 are shown as dotted and dashed line, respectively.
  • Figure 13B Endogeneous secreted proteins were enriched from culture supernatants using Heparin resin and subjected to PNGaseF release and RF-MS labelling and m/z determination.
  • Figure 14A-B Schematic representation of the mammalian N-glycosylation pathway (A)(adapted from Kellokumpu, S., et al. Glycosyltransferase complexes in eukaryotes: long-known, prevalent but still unrecognized. In Cell. Mol. Life Sci. 73 (2), pp. 305-325 (2016)) in comparison to the engineered Leishmania tarentolae pathway (B).“+” indicates elongation steps; indicates trimming steps.
  • grey arrows indicate the natural pathway
  • black arrows indicate the engineered pathway in Leishmania tarentolae.
  • Figure 15 Results of pH-rodo-labelled adalimumab glyco variant uptake assay by human monocyte-derived dendritic cells. Each bar shows the average mean
  • Figure 17 The Graph shows the mean ECso (pg/ml) of killing in the CDC assay with 95% confidence interval, from triplicate measurements. The star symbol indicates statistical significance (p ⁇ 0.05) for the indicated comparison. All other comparison are not statistically significant.
  • Glycoengineering is a technology that has the potential to increase the tolerance to treatment with biologies and specifically anti-TNFa.
  • IgG (Fc) sialylation has emerged as regulator of anti-inflammatory and immune-tolerizing activity of antibody therapeutics
  • one embodiment of the invention presented here is an monoclonal antibody that is highly sialylated and demonstrates reduced immunogenicity in a set of mechanistic assays.
  • Another embodiment of the invention is that the same product exhibits in addition improved therapeutic function by increased antibody- dependent cell mediated cytotoxicity (ADCC by the defucosylated core glycan), complement dependent cytotoxicity (CDC), or serum half-life (due to sialylation).
  • ADCC antibody- dependent cell mediated cytotoxicity
  • CDC complement dependent cytotoxicity
  • serum half-life due to sialylation
  • a subject refers to an animal (e.g ., birds, reptiles, and mammals).
  • a subject is a mammal including a non-primate (e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, and mouse) and a primate (e.g., a monkey, chimpanzee, and a human).
  • a subject is a non- human animal.
  • a subject is a farm animal or pet (e.g., a dog, cat, horse, goat, sheep, pig, donkey, or chicken).
  • a subject is a human.
  • the terms“subject” and “patient” may be used herein interchangeably.
  • Pfnumbcr [number] refer to glycosidic bonds or glycosidic linkages which are covalent bonds that join a carbohydrate residue to another group. An a-glycosidic bond is formed when both carbons have the same stereochemistry, whereas a b-glycosidic bond occurs when the two carbons have different stereochemistry.
  • a capitalized drug name represents the antibody in the brand- name drug sold under the trademark and the antibody in any biosimilar thereof, for example HUMIRA, AM JE VITA, CYLTEZO, REMICADE, SIMPONI, CIMZIA, and ENBREL.
  • HUMIRA represents the antibody adalimumab in the drug sold under the trademark HUMIRA and the antibody in any biosimilar thereof.
  • Table 1 lists the glycan names and abbreviations used herein.
  • a prominent and well characterized form of protein glycosylation is the linkage of a glycan to the amide in the side chain of an asparagine (N-glycosylation) on newly synthesized proteins.
  • N-glycosylation of proteins starts in the lumen of the endoplasmic reticulum (ER) by transferring a conserved oligosaccharide precursor
  • the two terminal glucose residues are cleaved off by glucosidase I and II and the resulting polypeptide with mono- glucosylated glycan structures (GlciMan9GlcNAc 2 ) can interact with the ER-resident membrane- bound lectins. These lectins support protein folding in a glycan-dependent protein quality control cycle. Secretory glycoproteins that have acquired their native conformation transfer from the ER to the Golgi apparatus.
  • the ER-derived oligomannosidic N-glycans on maturely folded glycoproteins are subjected to further N -glycan modifications, composed of various elongation and trimming steps, which generates the highly diverse complex N-glycans (Stanley, P. Golgi Glycosylation. Cold Spring Harbor Perspectives in Biology 3 (4), a005l99 (2011)).
  • N -glycan modifications composed of various elongation and trimming steps, which generates the highly diverse complex N-glycans.
  • glycosylated biopharmaceuticals produced in such systems contain a highly diverse glycosylation profile with many different glycan forms.
  • these glycan structures there are M4-M9 glyco forms that are composed of different numbers of Mannose residues attached to the paucimannose Man3GlcNAc2 stem (M3).
  • N-glycosylation biosynthesis pathway in Leishmania tarentolae is unique in several aspects.
  • the N-glycan precursor is assembled as Man3 at the cytoplasmic side of the ER and then flipped to the ER lumen ( Figure 7). No further elongation before transferring to M9 or M9G3 is observed, in agreement with the absence of the corresponding enzyme codes in the genome and the lectin folding support.
  • This Man3 glycan is then directly transferred to the acceptor protein.
  • the final glycan on the N-glycosite of the acceptor protein is M3 (not M9 as in mammalian cells), which transits to the Golgi.
  • recombinantly expressed enzymes can catalyze the glycosyltransferase reactions building up the human biantennary N-glycans. All enzymes or homologs responsible for elongation and trimming of the N-glycan in other eukaryotic cells are not found in the genome of Leishmania tarentolae. Our data shows that these diversity-generating activities are absent and biopharmaceuticals produced in Leishmania tarentolae are thus contain a much less complex glycosylation profile. In particular, they do miss all M4-M9 glyco forms, as the enzymes are not encoded in Leishmania and thus the corresponding glyco forms are entirely absent.
  • M5 are major high mannose forms found in antibodies (Goetze AM, et al. High-mannose glycans on the Fc region of therapeutic IgG antibodies increase serum clearance in humans, Glycobiology, Volume 21, Issue 7, 949-959 (2011)), for examples M5 in HUMIRA ( Figure 4). Their relative abundance is variable, and often strongly influenced by process parameters (pH, nutrients) as well as dependent on the specific productivity of the cell. Controlling M4-M9 forms is a current topic of interest (Zalai D et al. A control strategy to investigate the relationship between specific productivity and high- mannose glyco forms in CHO cells. Appl Microbiol Biotechnol. 100(16):7011-24 (2016); Wang Q et al. Antibody gly coengineering strategies in mammalian cells. Biotechnology and
  • PK Serum half-life of high-mannose forms is shorter than of complex glycoforms (Alessandri L et al. Increased serum clearance of oligomannose species present on a human IgGl molecule. mAbs. Volume 4, Issue 4, Pages 509- 520 (2012); Yu M et al.
  • DC-SIGN intercellular adhesion molecule-3 -grabbing nonintegrin
  • MBL2 mannose-binding lectin 2
  • MMR macrophage mannose receptor
  • Figure 14 shows the mammalian N-glycosylation pathway (adapted from
  • oligosaccharide chain Mammalian glycan elongation/trimming is spatially separated in different Golgi compartments (cis, medial, and trans). On the right, the natural (grey arrows) and engineered (black arrows) Leishmania tarentolae pathway is shown. Particularly in Leishmania only elongation steps are occurring, but no trimming. Thus, M4-M9 forms are never formed and biopharmaceuticals made from this system are completely devoid of them.
  • the Leishmania tarentolae expression platform (“Engineered and fully- functional customized glycoproteins,” WO 2019/002512) has been validated at the research level that it is suitable for production of therapeutic glycoproteins.
  • the pathways investigated in the proposed preclinical research program have wider applicability.
  • the biological pathways that are modulated by sialylated antibodies have relevance in diseases driven by unwanted immune reactions such as rheumatoid arthritis and allergies (Epp, A., et al. Sialylation of IgG antibodies inhibits IgG-mediated allergic reactions.
  • Epp, A., et al. Sialylation of IgG antibodies inhibits IgG-mediated allergic reactions.
  • Ohmi, Y., et al. Sialylation converts arthritogenic IgG into inhibitors of collagen-induced arthritis.
  • sialylated drug products generated via LMTB’s proprietary expression platform (“Engineered and fully- functional customized glycoproteins,” WO 2019/002512) can be manifold: for example, due to anti-drug antibody issues patients may benefit from treatment with infliximab, pertuzumab, rituximab, to name just a few.
  • the platform generally allows for recombinant glycoprotein production, e.g., vaccine antigens from virus, fungi or parasites.
  • Antibodies are glycoproteins containing two N-glycans, which are present in the Fc portion of all IgG subclasses. Almost all licensed antibodies that are produced in CHO cell lines contain a high structural heterogeneity of N-glycans (more than 10 different glyco- forms observed). Due to the recognized importance of the N-glycan structure for therapeutic glycoproteins in therapy (Liu, L. Antibody Glycosylation and Its Impact on the Pharmacokinetics and Pharmacodynamics of Monoclonal Antibodies and Fc-Fusion Proteins. In J. Pharm. Sci. 104 (6), pp.
  • LMTB uses the native and novel N-glycosylation pathways in non-human pathogenic Leishmania tarentolae (Lt), and exploits a variety of glycosyltransferases from different organisms for glycoengineering.
  • This glycoengineered unicellular Kinetoplastida expression platform enables production of recombinant glycoproteins containing defined N- glycans with terminal a2,6 linked sialic acid.
  • the production of cheaper, faster, safer and more consistent therapeutics with highly homogenous and tailor-made N-glycans will significantly contribute to the development of products with superior quality and functionality.
  • shorter development timelines and faster generation of recombinant strains compared to mammalian cells significantly advances this simple but fully function-customizable expression platform for wide applications on therapeutic proteins.
  • compositions comprising host cells
  • compositions comprising the host cells described herein.
  • Such compositions can be used in methods for generating the glycosylated target proteins described herein, e.g., the compositions comprising host cells can be cultured under conditions suitable for the production of proteins. Subsequently, glycosylated target proteins can be isolated from said compositions comprising host cells using methods known in the art.
  • compositions comprising the host cells provided herein can comprise additional components suitable for maintenance and survival of the host cells described herein, and can additionally comprise additional components required or beneficial to the production of proteins by the host cells, e.g., inducers for inducible promoters, such as arabinose, IPTG.
  • inducers for inducible promoters such as arabinose, IPTG.
  • composition of glycosylated target proteins have at least about 20% to
  • N- glycosylated adalimumab with a 2,6 sialylated biantennary N-glycans (“G2S2”) is sia- adalimumab.
  • TNFa antibody comprising culturing the host cell of any one of the preceding claims; and obtaining the anti-TNFa antibody.
  • the method further comprises an in vitro glycosylation step.
  • the composition of glycosylated target proteins have at least about 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% of the N-linked glycosylation consensus sequences of the target proteins in the composition carry an oligosaccharide comprising the following structure:
  • the diamond represents a sialic acid residue
  • the empty circle represents a galactose residue
  • the square represents an N-acetylglucosamine residue
  • the grey circle represents a mannose residue
  • the Asn is an Asn of an N-linked glycosylation consensus sequence in the target protein.
  • composition of glycosylated target proteins have at least about 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%,
  • glycosylation on the target proteins is characterized by the following structure:
  • the diamond represents a sialic acid residue
  • the empty circle represents a galactose residue
  • the square represents an N-acetylglucosamine residue
  • the grey circle represents a mannose residue
  • the Asn is an Asn of an N-linked glycosylation consensus sequence in the target proteins.
  • the composition of glycosylated target proteins have at least about 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% of the glycosylation on the target proteins is characterized by the following structure:
  • the diamond represents a sialic acid residue
  • the empty circle represents a galactose residue
  • the square represents an N-acetylglucosamine residue
  • the grey circle represents a mannose residue
  • the Asn is an Asn of an N-linked glycosylation consensus sequence in the target proteins.
  • the composition of glycosylated target proteins have at least about 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% of the glycan on the target proteins that is not mannose 5 (Man5) or mannose 6 (Man6) glycan.
  • the glucosylated target proteins are anti-TNFa antibodies.
  • at least about 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% of the glycan on the anti-TNFa antibodies is not mannose 5 (Man5) or mannose 6 (Man6) glycan.
  • one glycosylated target protein may have one or more glycosylation sites, for example one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty glycosylation sites.
  • the one or more glycosylation sites comprises Asn of an N-linked glycosylation consensus sequence in the target protein.
  • the one or more glycosylation sites are sites other than Asn of an N-linked glycosylation consensus sequence in the target protein, for example a serine residue of an O- linked glycosylation consensus sequences in the target protein.
  • the composition of glycosylated target proteins contains a single type of target proteins, for example an anti-TNFa antibody.
  • the single type of target protein may have different glycosylation profiles, for example different number of glycosylation sites, different glycosylation sites, and/or different glycosylation residues (glycans).
  • the single type of target protein may have the same glycosylation profiles.
  • the composition of glycosylated target proteins contains more than one type of glycosylated target proteins, for example glycosylated target proteins having different amino acid sequences.
  • the more than one type of target proteins may have different glycosylation profiles, for example different number of glycosylation sites, different glycosylation sites, and/or different glycosylation residues.
  • the more than one type of target proteins may have the same glycosylation profiles.
  • the percentage of N-glycosylation as used herein represents the percentages of all glycans in a population of glycosylated target proteins. In some embodiments, the percentage of N-glycosylation as used herein represents the percentage of all the glycans on all of the one or more glycosylation sites in the glycosylated target protein. In some embodiments, the percentage of N-glycosylation as used herein represents the percentage of all the glycans on the Asn glycosylation site in the glycosylated target protein.
  • TNFa inhibitors are used to treat inflammatory diseases e.g. rheumatoid arthritis (RA), psoriatic arthritis and inflammatory bowel disease (Crohn’s disease and ulcerative colitis).
  • the inhibitors bind to TNFa and reduce inflammation and thereby halt disease progression.
  • Multiple TNFa inhibitors have been approved, with infliximab and adalimumab being the most widely used.
  • anti-TNFa inhibitors are the first-line treatment in patients with severe IBD.
  • anti-TNFa therapies representing a major advance in the treatment of inflammatory bowel disease, at least 30% of patients fail to respond to treatment and 14-38% of adalimumab treated patients lose response over time (Roda, G., et al. Loss of Response to Anti-TNFs: Definition, Epidemiology, and Management. In Clinical and translational gastroenterology 7, pp. el35 (2016)).
  • the product made by any method described herein and containing different degrees of sialylation and defucosylation may reduce immunogenicity, generate immune tolerization (by sialylation), reduce or conserved CDC, and improve ADCC (through improved FcyRIIIa binding), and improve PK caused by sialylation (Wada, R., et al. Influence of N- glycosylation on effector functions and thermal stability of glycoengineered IgGl monoclonal antibody with homogeneous glyco forms. mAbs 11 (2), pp. 350-372 (2019)). Anti-tumor necrosis factor-a antibodies induce regulatory macrophages in an Fc region-dependent manner (Vos, A., et al.
  • the product may show some but not all of these improvements. In certain embodiments, the product may only show immune tolerization.
  • certain target proteins produced by the methods described herein will have reduced immunogenicity in a human patient.
  • one or more target proteins described herein, made by one or more methods described herein will reduce immunogenicity.
  • one or more target proteins described herein, made by one or more methods described herein will generate immune tolerization.
  • one or more target proteins described herein, made by one or more methods described herein will reduce or conserve CDC.
  • one or more target proteins described herein, made by one or more methods described herein will improve ADCC (e.g. through improved FcyRIIIa binding).
  • one or more target proteins described herein, made by one or more methods described herein will improve PK caused by sialylation. In certain embodiments, one or more target protein described herein, made by one or more methods described herein will improve effect in B-cell apoptosis. In certain embodiments, one or more target protein described herein, made by one or more methods described herein will improve internalization by dendritic cells. In certain embodiments, one or more target protein described herein, made by one or more methods described herein will improve anti-inflammatory activities.
  • one or more target proteins described herein, made by one or more methods described herein will have one or more of the following in vivo effects: reduced immunogenicity, increased immune tolerization (by sialylation), reduced or conserved CDC, improved ADCC, improved PK, improved B-cell apoptosis, improved internalization by Dendritic cells, and improved anti-inflammatory activities.
  • anti-TNFa antibody glycosylated with one or more of the structures described herein, made by one or more methods described herein will have one or more of the following in vivo effects: reduced immunogenicity, increased immune tolerization (by sialylation), reduced or conserved CDC, improved ADCC, improved PK, improved B-cell apoptosis, improved internalization by dendritic cells, and improved anti-inflammatory activities.
  • any target protein described herein glycosylated with any of the structures described herein, made by any method described herein will have one or more of the following in vivo results: reduced immunogenicity, increased immune tolerization (by
  • the present invention relates to unicellular Kinetoplastida eukaryotic host cells, which have been modified to produce homogeneous and fully- function customized N- glycans with a high site occupancy on therapeutic proteins by the properties of the native host cell and the combination with the heterologous expression of a set of glycosyltransferases, including N-acetyl glucosamine transferases, galactosyltransferase, and sialyltransferases, to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins.
  • the Kinetoplastida eukaryotic host cell is a Leishmania tarentolae cell.
  • the Leishmania host cell is a Leishmania tarentolae cell.
  • a Leishmania host cell comprising a recombinant nucleic acid encoding the heavy chain of anti-TNFa antibody (e.g. adalimumab).
  • the Leishmania host cell comprises a recombinant nucleic acid encoding the light chain of anti-TNFa antibody (e.g. adalimumab).
  • a Leishmania host cell comprising a recombinant nucleic acid encoding the heavy chain of anti-TNFa antibody (e.g. adalimumab) and the light chain of anti-TNFa antibody (e.g. adalimumab) (in addition to the glycosylation machinery described in this application).
  • the invention provides an engineered host cell, which can be used to express and target any desirable gene(s) involved in glycosylation.
  • the present invention provides eukaryotic host cells, which have been modified to produce function- customized and homogeneous N-glycans on proteins by the heterologous expression of a set of glycosyltransferases, including N-acetylglucosamine transferases, galactosyltransferase, and sialyltransferases, to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins.
  • the invention also provides an engineered Leishmania tarentolae host cell, which can be used to express and target a full length therapeutic antibody.
  • the novel host cell synthesizes, expresses and secretes homogeneous and function-customized N-glycans on glycoproteins, such as adalimumab.
  • the novel host cell synthesizes, expresses and secretes adalimumab with homogeneous paucimannose N-glycans on the Asn consensus site of adalimumab heavy chain.
  • the invention provides nucleic acid molecules and combinatorial libraries, which can be used to successfully target and express mammalian enzymatic activities (such as those involved in N-acetylglucosamine elongation, galactosylation and sialylation) to intracellular compartments in the kinetoplastid eukaryotic host cell.
  • mammalian enzymatic activities such as those involved in N-acetylglucosamine elongation, galactosylation and sialylation
  • CMP-sialic acid biosynthetic pathway for the production of sialylated glycoproteins are also provided.
  • the invention provides an engineered host cell, which can be used to express and target any desirable gene(s) involved in glycosylation.
  • the present invention provides eukaryotic host cells, which have been modified to produce function- customized and homogeneous N-glycans on proteins by the heterologous expression of a set of glycosyltransferases, including N-acetylglucosamine transferases, galactosyltransferase, and sialyltransferases, to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins.
  • the invention also provides an engineered host cell which can be used to express and target a full length therapeutic antibody.
  • the novel host cell synthesizes, expresses and secretes homogeneous and function-customized N-glycans on glycoproteins, such as anti- TNFa antibody (e.g., adalimumab).
  • the invention described herein is not limited to the use of specific enzymes, genes, plasmids and constructs disclosed herein.
  • a person of skill could use any homologues, variants and derivatives of the genes involved in the synthesis of N-acetyl glucosamine transferase, galactosyltransferase, sialyltransferase, and a CMP-Sia Biosynthetic Pathway Enzyme.
  • a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a target protein; and (b) a recombinant nucleic acid encoding a heterologous glycosyltransferase.
  • the heterologous glycosyltransferase is an N-acetyl glucosamine transferase; and/or a heterologous galactosyltransferase; and/or a heterologous sialyltransferase.
  • a host cell comprising two or more N-acetyl glucosamine transferases.
  • the host cell comprising a heterologous sialyltransferase further comprises a heterologous CMP-Sia biosynthetic pathway proteins capable of generating CMP-NeuAc.
  • Leishmania host cells capable of producing glycosylated proteins
  • the Leishmania host cells comprise (i) a Native OST or a heterologous/recombinant OST; (ii) nucleotides encoding heterologous N-acetyl glucosamine transferase, galactosyltransferase, sialyltransferase, and a CMP-Sia Biosynthetic Pathway enzyme, or modified versions thereof; and (iii) nucleotides encoding recombinant target protein and modified versions of recombinant target protein.
  • sialyltransferase is derived from an N-acetyl glucosamine transferase, a galactosyltransferase, or a sialyltransferase listed in Table 2, or any functional homologue thereof.
  • a host cell wherein one or more endogenous enzymes from the N-glycan biosynthesis pathway have been deleted, mutated and/or functionally inactivated.
  • the endogenous enzyme that has been deleted, mutated and/or functionally inactivated in the said host cell is encoded by the alg genes.
  • a host cell wherein one or more genes encoding endogenous enzymes from the N-glycan biosynthesis pathway have been deleted, mutated and/or functionally inactivated.
  • the gene or genes encoding endogenous enzyme or enzymes from the N-glycan biosynthesis pathway is deleted, mutated and/or functionally inactivated using any of the standard techniques (for example, by site specific homologous recombination or random mutagenesis) known in the art.
  • the host cell is a strain of Leishmania that does not include one or more endogenous enzymes from the N-glycan biosynthesis pathway.
  • the Leishmania host cell is a Leishmania tarentolae cell.
  • a Leishmania tarentolae host cell comprising (a) a recombinant nucleic acid encoding a target protein; and (b) a recombinant nucleic acid encoding a heterologous glycosyltransferase.
  • the heterologous glycosyltransferase is an N-acetyl glucosamine transferase; and/or a heterologous
  • a host cell comprising two or more N-acetyl glucosamine transferases.
  • the host cell comprising a heterologous sialyltransferase further comprises a heterologous CMP-Sia biosynthetic pathway proteins capable of generating CMP-NeuAc.
  • Leishmania tarentolae host cells capable of producing glycosylated proteins
  • the Leishmania tarentolae host cells comprise (i) a Native OST or a heterologous/recombinant OST; (ii) nucleotides encoding heterologous N- acetyl glucosamine transferase, galactosyltransferase, sialyltransferase, and a CMP-Sia
  • Biosynthetic Pathway enzyme or modified versions thereof; and (iii) nucleotides encoding recombinant target protein and modified versions of recombinant target protein.
  • the amino acid sequence of the said N-acetyl glucosamine transferase is not limited to, but not limited to, the amino acid sequence of the said N-acetyl glucosamine transferase,
  • galactosyltransferase, and/or sialyltransferase is derived from an N-acetyl glucosamine transferase, a galactosyltransferase, or a sialyltransferase listed in Table 2, or any functional homologue thereof.
  • a host cell wherein a Leishmania signal and/or retention sequence is added to the N-acetyl glucosamine transferase,
  • the signal sequence targets the N-acetyl glucosamine transferase, galactosyltransferase, and/or sialyltransferase to the endoplasmic reticulum of the Leishmania host cell, and wherein the retention sequence retains the N-acetyl glucosamine transferase, galactosyltransferase, and/or sialyltransferase in the Golgi apparatus.
  • said retention sequence retains the N-acetyl glucosamine transferase and/or galactosyltransferase in the cis Golgi compartment of the host cell. In another embodiment, said retention sequence retains the N-acetyl glucosamine transferase and/or galactosyltransferase in the medial Golgi compartment of the host cell. In another embodiment, said retention sequence retains the galactosyltransferase in the trans Golgi compartment of the host cell. In another embodiment, said retention sequence retains the sialyltransferase in the trans Golgi compartment of the host cell.
  • the retention sequence retains the sialyltransferase and galactosyltransferase in the trans Golgi compartment of the host cell.
  • said signal sequence is processed and removed.
  • said retention sequence is a cytoplasmic-transmembrane-stem (CTS) sequence derived from a Leishmania tarentolae protein.
  • said CTS sequence is derived from Leishmania tarentolae MAN1, NTPDase 1, or NTPDase 2.
  • said CTS sequence comprises the sequence of SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26 or functionally active fragments thereof.
  • said CTS is derived from Leishmania tarentolae MAN1.
  • said CTS sequence comprises the sequence of SEQ ID NO: 24 or functionally active fragments thereof.
  • said retention sequence comprises a GRIP sequence derived from Leishmania or functionally active fragments thereof In another embodiment, said GRIP sequence comprises the sequence of SEQ ID NO: 27, or a functionally active fragments thereof.
  • said retention sequence comprises a CTS sequence derived from a Leishmania protein, or a functionally active fragment thereof, and a GRIP sequence derived from Leishmania or a functionally active fragment thereof
  • a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a target protein; and (b) a recombinant nucleic acid encoding an N-acetyl glucosamine transferase.
  • a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a target protein; and (b) a recombinant nucleic acid encoding a galactosyltransferase.
  • a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a target protein; and (b) a recombinant nucleic acid encoding a sialyltransferase.
  • the host cell comprising a heterologous sialyltransferase further comprises a heterologous CMP-Sia biosynthetic pathway proteins capable of generating CMP-NeuAc.
  • a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a target protein; (b) a recombinant nucleic acid encoding an N-acetyl glucosamine transferase; and (c) a recombinant nucleic acid encoding a galactosyltransferase.
  • a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a target protein; (b) a recombinant nucleic acid encoding an N-acetyl glucosamine transferase; and (c) a recombinant nucleic acid encoding a sialyltransferase.
  • a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a target protein; (b) a recombinant nucleic acid encoding a sialyltransferase; and (c) a recombinant nucleic acid encoding a galactosyltransferase.
  • the host cell comprising a heterologous sialyltransferase further comprises a heterologous CMP-Sia biosynthetic pathway proteins capable of generating CMP-NeuAc.
  • a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a target protein; (b) a recombinant nucleic acid encoding an N-acetyl glucosamine transferase; (c) a recombinant nucleic acid encoding a galactosyltransferase; and (d) a recombinant nucleic acid encoding a sialyltransferase.
  • the host cell comprising a heterologous sialyltransferase further comprises a heterologous CMP-Sia biosynthetic pathway proteins capable of generating CMP-NeuAc.
  • a Leishmania tarentolae host cell comprising (a) a recombinant nucleic acid encoding a target protein; and (b) a recombinant nucleic acid encoding an N-acetyl glucosamine transferase.
  • a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a target protein; and (b) a recombinant nucleic acid encoding a galactosyltransferase.
  • a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a target protein; and (b) a recombinant nucleic acid encoding a sialyltransferase.
  • the host cell comprising a heterologous sialyltransferase further comprises a heterologous CMP-Sia biosynthetic pathway proteins capable of generating CMP-NeuAc.
  • a Leishmania tarentolae host cell comprising (a) a recombinant nucleic acid encoding a target protein; (b) a recombinant nucleic acid encoding an N-acetyl glucosamine transferase; and (c) a recombinant nucleic acid encoding a galactosyltransferase.
  • a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a target protein; (b) a recombinant nucleic acid encoding an N-acetyl glucosamine transferase; and (c) a recombinant nucleic acid encoding a sialyltransferase.
  • a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a target protein; (b) a recombinant nucleic acid encoding a sialyltransferase; and (c) a recombinant nucleic acid encoding a
  • the host cell comprising a heterologous sialyltransferase further comprises a heterologous CMP-Sia biosynthetic pathway proteins capable of generating CMP-NeuAc.
  • a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a target protein; (b) a recombinant nucleic acid encoding an N-acetyl glucosamine transferase; (c) a recombinant nucleic acid encoding a galactosyltransferase; and (d) a recombinant nucleic acid encoding a sialyltransferase.
  • the host cell comprising a heterologous sialyltransferase further comprises a heterologous CMP-Sia biosynthetic pathway proteins capable of generating CMP-NeuAc.
  • the term“heterologous” means from a different species.
  • a“heterologous glycosyltransferase” in a host cell is a
  • glycosyltransferase derived from a species other than the host cell glycosyltransferase derived from a species other than the host cell.
  • the term“heterologous” means from a different strain.
  • glycosyltransferase in a host cell, is a glycosyltransferase derived from a strain other than the host cell.
  • the term“heterologous” means from a different genus.
  • a“heterologous glycosyltransferase” in a host cell is a glycosyltransferase derived from a genus other than the host cell.
  • a glycosyltransferase used with the methods and compositions provided herein is a glycosyltransferase that is genetically modified from its wild type gene.
  • such a glycosyltransferase is of the same species or of the same strain.
  • such a glycosyltransferase is of a different genus, species, or strain.
  • Leishmania host cells comprising: (a) a recombinant nucleic acid encoding an anti-TNFa antibody heavy chain (and optionally a recombinant nucleic acid encoding an anti-TNFa antibody light chain); and (b) a recombinant nucleic acid sequence encoding an a-2,6 sialyltransferase.
  • the host cell further comprises a recombinant nucleic acid sequence encoding a glycosyltransferase capable of synthesizing one glycosyl linkage in one of the following structures:
  • the diamond represents a sialic acid residue
  • the empty circle represents a galactose residue
  • the square represents an N-acetylglucosamine residue
  • the grey circle represents a mannose residue
  • the Asn is an Asn of an N-linked glycosylation consensus sequence in the anti-TNFa antibody heavy chain.
  • the host cell further comprises one or more recombinant nucleic acid sequences encoding glycosyltransferases to synthesize:
  • the diamond represents a sialic acid residue
  • the empty circle represents a galactose residue
  • the square represents an N-acetylglucosamine residue
  • the grey circle represents a mannose residue
  • the Asn is an Asn of an N-linked glycosylation consensus sequence in the anti-TNFa antibody heavy chain.
  • Leishmania host cells comprising a nucleic acid sequence encoding a recombinant anti-TNFa antibody heavy chain; and one or more recombinant nucleic acid sequences encoding glycosyltransferases to synthesize:
  • the diamond represents a sialic acid residue
  • the empty circle represents a galactose residue
  • the square represents an N-acetylglucosamine residue
  • the grey circle represents a mannose residue
  • the Asn is an Asn of an N-linked glycosylation consensus sequence in the anti-TNFa antibody heavy chain.
  • Leishmania host cells comprising a nucleic acid sequence encoding a recombinant anti-TNFa antibody heavy chain; and one or more recombinant nucleic acid sequences encoding glycosyltransferases to synthesize:
  • Leishmania host cells comprising a nucleic acid sequence encoding a recombinant anti-TNFa antibody heavy chain; and a recombinant nucleic acid sequences encoding a glycosyltransferase to synthesize:
  • the diamond represents a sialic acid residue
  • the empty circle represents a galactose residue
  • the square represents an N-acetylglucosamine residue
  • the grey circle represents a mannose residue
  • the Asn is an Asn of an N-linked glycosylation consensus sequence in the anti-TNFa antibody heavy chain.
  • the host cell further comprises one or more nucleic acid sequences encoding a-2,6 sialyltransferase.
  • the anti-TNFa antibody is adalimumab.
  • the Leishmania host cell is Leishmania tarentolae.
  • a Leishmania signal and retention sequence is added to the N-acetyl glucosamine transferase, galactosyltransferase, and/or sialyltransferase, wherein the signal sequence targets the N-acetyl glucosamine transferase, galactosyltransferase, and/or sialyltransferase to the endoplasmic reticulum of the Leishmania host cell, and wherein the retention sequence retains the N-acetyl glucosamine transferase, galactosyltransferase, and/o sialyltransferase in the Golgi apparatus.
  • a Leishmania signal and retention sequence is added to the N-acetyl glucosamine transferase and sialyltransferase, wherein the signal sequence targets the N-acetyl glucosamine transferase and sialyltransferase to the endoplasmic reticulum of the Leishmania host cell, and wherein the retention sequence retains the N-acetyl glucosamine transferase and sialyltransferase in the Golgi apparatus.
  • a Leishmania signal and retention sequence is added to the N-acetyl glucosamine transferase and galactosyltransferase, wherein the signal sequence targets the N- acetyl glucosamine transferase and galactosyltransferase to the endoplasmic reticulum of the Leishmania host cell, and wherein the retention sequence retains the N-acetyl glucosamine transferase and galactosyltransferase in the Golgi apparatus.
  • a Leishmania signal and retention sequence is added to the sialyltransferase and galactosyltransferase, wherein the signal sequence targets the sialyltransferase and galactosyltransferase to the endoplasmic reticulum of the Leishmania host cell, and wherein the retention sequence retains the
  • sialyltransferase and galactosyltransferase in the Golgi apparatus sialyltransferase and galactosyltransferase in the Golgi apparatus.
  • a Leishmania signal and retention sequence is added to the N-acetyl glucosamine transferase, wherein the signal sequence targets the N-acetyl glucosamine transferase to the endoplasmic reticulum of the Leishmania host cell, and wherein the retention sequence retains the N-acetyl glucosamine transferase, in the Golgi apparatus.
  • a Leishmania signal and retention sequence is fused to the N-terminal of the N-acetyl glucosamine transferase.
  • a Leishmania signal and retention sequence is fused to the C-terminal of the N-acetyl glucosamine transferase. In a further embodiment, a Leishmania signal and retention sequence is not fused to the N-terminal of the N- acetyl glucosamine transferase. In other embodiments, a Leishmania signal and retention sequence is not fused to the C-terminal of the N-acetyl glucosamine transferase. In other embodiments, a Leishmania signal and retention sequence is fused to one or more amino acids within the polypeptide of the N-acetyl glucosamine transferase.
  • a Leishmania signal and retention sequence is added to the galactosyltransferase, wherein the signal sequence targets the galactosyltransferase to the endoplasmic reticulum of the Leishmania host cell, and wherein the retention sequence retains the galactosyltransferase, in the Golgi apparatus.
  • a Leishmania signal and retention sequence is fused to the N-terminal of the galactosyltransferase.
  • a Leishmania signal and retention sequence is fused to the C-terminal of the galactosyltransferase. In a further embodiment, a Leishmania signal and retention sequence is not fused to the N-terminal of the galactosyltransferase. In other embodiments, a Leishmania signal and retention sequence is not fused to the C-terminal of the galactosyltransferase. In other embodiments, a Leishmania signal and retention sequence is fused to one or more amino acids within the polypeptide of the galactosyltransferase.
  • a Leishmania signal and retention sequence is added to the sialyltransferase, wherein the signal sequence targets the sialyltransferase to the endoplasmic reticulum of the Leishmania host cell, and wherein the retention sequence retains the sialyltransferase, in the Golgi apparatus.
  • a Leishmania signal and retention sequence is fused to the N-terminal of the sialyltransferase.
  • a Leishmania signal and retention sequence is fused to the C-terminal of the sialyltransferase.
  • a Leishmania signal and retention sequence is not fused to the N-terminal of the sialyltransferase. In other embodiments, a Leishmania signal and retention sequence is not fused to the C-terminal of the sialyltransferase. In other embodiments, a Leishmania signal and retention sequence is fused to one or more amino acids within the polypeptide of the
  • the retention sequence retains the N-acetyl glucosamine transferase and/or galactosyltransferase in the cis Golgi compartment of the host cell. In another embodiment, the retention sequence retains the N-acetyl glucosamine transferase and/or galactosyltransferase in the medial Golgi compartment of the host cell. In another embodiment, the retention sequence retains the galactosyltransferase in the trans Golgi compartment of the host cell. In another embodiment, the retention sequence retains the sialyltransferase in the trans Golgi compartment of the host cell. In another embodiment, the retention sequence retains the sialyltransferase and galactosyltransferase in the trans Golgi compartment of the host cell.
  • the signal sequence and/or retention sequence is a signal sequence or retention sequence derived from Leishmania tarentolae.
  • the retention sequence is a cytoplasmic- transmembrane-stem (CTS) sequence derived from a Leishmania tarentolae protein.
  • CTS sequence is derived from Leishmania tarentolae MAN 1 , NTPDase 1 , or NTPDase 2.
  • the CTS sequence comprises the sequence of SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26 or functionally active fragments thereof.
  • the CTS is derived from Leishmania tarentolae
  • the CTS sequence comprises the sequence of SEQ ID NO: 24 or a functionally active fragment thereof
  • the CTS sequence is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the CTS is derived from Leishmania tarentolae MAN1.
  • the CTS sequence comprises a sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:
  • the CTS is derived from Leishmania tarentolae
  • the CTS sequence comprises the sequence of SEQ ID NO:
  • the CTS sequence is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the CTS is derived from Leishmania tarentolae NTPDase 1.
  • the CTS sequence comprises a sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 25 or a functionally active fragment thereof.
  • the CTS is derived from Leishmania tarentolae
  • the CTS sequence comprises the sequence of SEQ ID NO:
  • the CTS sequence is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the CTS is derived from Leishmania tarentolae NTPDase 2.
  • the CTS sequence comprises a sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
  • the retention sequence comprises a GRIP sequence derived from Leishmania or functionally active fragments thereof.
  • the GRIP sequence comprises the sequence of SEQ ID NO: 27, or a functionally active fragment thereof.
  • the retention sequence is at least about 70%, 71%, 72%, 73%,
  • the GRIP sequence comprises a sequence that is at least about 70%, 71%, 72%,
  • the retention sequence comprises a CTS sequence derived from a Leishmania protein, or a functionally active fragment thereof, and a GRIP sequence derived from Leishmania or a functionally active fragment thereof
  • the retention sequence comprises a CTS sequence that is at least about 70%, 71%,
  • the target protein has been engineered to comprise a signal sequence from Leishmania.
  • said signal sequence is a signal sequence from Leishmania tarentolae.
  • the signal sequence comprises the sequence of SEQ ID NO: 28, or SEQ ID NO: 29 or functionally active fragments thereof.
  • the signal sequence comprises the sequence of SEQ ID NO: 28 or a functionally active fragment thereof.
  • the signal sequence comprises a sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,
  • the signal sequence is processed and removed from the target protein. 7.12 N-acetyl glucosamine transferases
  • the hybrid N-acetyl glucosamine transferase comprises (a) catalytic domain of an N-acetyl glucosamine transferase that is not from Leishmania; and (b) amino acid sequence(s) responsible for localization and retention in the Golgi compartment of Leishmania.
  • the hybrid N-acetyl glucosamine transferase is from
  • the hybrid N-acetyl glucosamine transferase has been engineered to comprise a signal sequence and at least one retention sequence, wherein the signal sequence targets the N-acetyl glucosamine transferase to the endoplasmic reticulum of the Leishmania tarentolae host cell, and wherein the retention sequence retains the N-acetyl glucosamine transferase in the Golgi apparatus.
  • the hybrid N-acetyl glucosamine transferase retains the N-acetyl glucosamine transferase in the cis Golgi apparatus. In another embodiment, the hybrid N-acetyl glucosamine transferase retains the N-acetyl glucosamine transferase in the medial Golgi apparatus.
  • the retention sequence is a cytoplasmic- transmembrane-stem (CTS) sequence.
  • CTS sequence comprises the amino acid sequence of MAN 1, NTPDase 1, or NTPDase 2.
  • the CTS sequence comprises the sequence of SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26 or functionally active fragments thereof.
  • the CTS comprises the amino acid sequence of MAN 1.
  • the CTS sequence comprises the sequence of SEQ ID NO: 24 or functionally active fragments thereof.
  • the GRIP sequence comprises the amino acid sequence of SEQ ID NO: 27.
  • the N-acetyl glucosamine transferase is a GnT-I. In another embodiment, the N-acetyl glucosamine transferase is a GnT-II. In another embodiment, the N-acetyl glucosamine transferases are GnT-I and GnT-II. In certain other embodiments, the N-acetyl glucosamine transferase is derived from an N-acetyl glucosamine transferase listed in Table 2, or a functional homologue thereof. Any N-acetyl glucosamine transferase, or nucleic acid encoding it, known in the art can be used in accordance with the host cells and methods described herein.
  • the said N-acetyl glucosamine transferase is N- acetylglucosaminyltransferase 1 of Homo sapiens.
  • the said N-acetyl glucosamine transferase is mannosyl (alpha- l,6-)-glycoprotein beta- l,2-N- acetylglucosaminyltransferase of Homo sapiens.
  • the N-acetyl glucosamine transferase is one that is homologous to a N-acetyl glucosamine transferase of a species of Homo sapiens.
  • the N-acetyl glucosamine transferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to N-acetylglucosaminyltransferase 1 or mannosyl (alpha- l,6-)-glycoprotein beta- l,2-N- acetylglucosaminyltransferase of Homo sapiens.
  • the said N-acetyl glucosamine transferase is N- acetylglucosaminyltransferase 1 of Spodoptera frugiperda.
  • the said N- acetyl glucosamine transferase is N-acetylglucosaminyltransferase 2 of Spodoptera frugiperda.
  • the N-acetyl glucosamine transferase is one that is homologous to a N- acetyl glucosamine transferase of a species of Spodoptera frugiperda.
  • the N-acetyl glucosamine transferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to N-acetylglucosaminyltransferase 1 or N- acetylglucosaminyltransferase 2 of Spodoptera frugiperda.
  • the said N-acetyl glucosamine transferase is N- acetylglucosaminyltransferase 1 of Trypanosoma brucei.
  • the said N- acetyl glucosamine transferase is N-acetylglucosaminyltransferase 2 of Trypanosoma brucei.
  • the N-acetyl glucosamine transferase is one that is homologous to a N- acetyl glucosamine transferase of a species of Trypanosoma brucei.
  • the N-acetyl glucosamine transferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to N-acetylglucosaminyltransferase 1 or N- acetylglucosaminyltransferase 2 of Trypanosoma brucei.
  • the said N-acetyl glucosamine transferase is N- acetylglucosaminyltransferase 2 of Rattus norvegicus.
  • the N-acetyl glucosamine transferase is one that is homologous to a N-acetyl glucosamine transferase of a species of Rattus norvegicus.
  • the N-acetyl glucosamine transferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
  • the said N-acetyl glucosamine transferase is mannosyl (alpha- l,6-)-glycoprotein beta-l,2-N-acetylglucosaminyltransferase of Pan
  • the N-acetyl glucosamine transferase is one that is homologous to a N-acetyl glucosamine transferase of a species of Pan troglodytes.
  • the N-acetyl glucosamine transferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to mannosyl (alpha- l,6-)-glycoprotein beta-l,2-N-acetylglucosaminyltransferase of Pan troglodytes.
  • the said N-acetyl glucosamine transferase is mannosyl (alpha- l,6-)-glycoprotein beta- l,2-N-acetylglucosaminyltransferase of Canis lupus familiaris.
  • the N-acetyl glucosamine transferase is one that is homologous to a N-acetyl glucosamine transferase of a species of Canis lupus familiaris.
  • the N-acetyl glucosamine transferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to mannosyl (alpha- 1,6-)- glycoprotein beta-l,2-N-acetylglucosaminyltransferase of Canis lupus familiaris.
  • the said N-acetyl glucosamine transferase is mannosyl (alpha- 1 ,6-)-glycoprotein beta- 1 ,2-N-acetylglucosaminyltransferase of Bos taurus.
  • the N-acetyl glucosamine transferase is one that is homologous to a N- acetyl glucosamine transferase of a species of Bos taurus.
  • glucosamine transferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to mannosyl (alpha- l,6-)-glycoprotein beta-l,2-N- acetylglucosaminyltransferase of Bos taurus.
  • the said N-acetyl glucosamine transferase is mannoside acetylglucosaminyltransferase 2 of Mus musculus.
  • the N- acetyl glucosamine transferase is one that is homologous to a N-acetyl glucosamine transferase of a species of Mus musculus.
  • the N-acetyl glucosamine transferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
  • the said N-acetyl glucosamine transferase is mannosyl (alpha- l,6-)-glycoprotein beta-l,2-N-acetylglucosaminyltransferase of Rattus norvegicus.
  • the N-acetyl glucosamine transferase is one that is homologous to a N-acetyl glucosamine transferase of a species of Rattus norvegicus.
  • the N-acetyl glucosamine transferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to mannosyl (alpha- 1,6-)- glycoprotein beta-l,2-N-acetylglucosaminyltransferase of Rattus norvegicus.
  • the said N-acetyl glucosamine transferase is mannosyl (alpha- l,6-)-glycoprotein beta- l,2-N-acetylglucosaminyltransferase of Gallus gallus.
  • the N-acetyl glucosamine transferase is one that is homologous to a N- acetyl glucosamine transferase of a species of Gallus gallus.
  • the N-acetyl glucosamine transferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to mannosyl (alpha- l,6-)-glycoprotein beta-l,2-N- acetylglucosaminyltransferase of Gallus gallus.
  • the said N-acetyl glucosamine transferase is mannosyl (alpha- l,6-)-glycoprotein beta-l,2-N-acetylglucosaminyltransferase of Xenopus tropicalis.
  • the N-acetyl glucosamine transferase is one that is homologous to a N-acetyl glucosamine transferase of a species of Xenopus tropicalis.
  • the N-acetyl glucosamine transferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to mannosyl (alpha- 1,6-)- glycoprotein beta-l,2-N-acetylglucosaminyltransferase of Xenopus tropicalis.
  • the said N-acetyl glucosamine transferase is mannosyl (alpha- 1 ,6-)-glycoprotein beta- 1 ,2-N-acetylglucosaminyltransferase of Danio rerio.
  • the N-acetyl glucosamine transferase is one that is homologous to a N- acetyl glucosamine transferase of a species of Danio rerio.
  • the N-acetyl glucosamine transferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to mannosyl (alpha- l,6-)-glycoprotein beta-l,2-N- acetylglucosaminyltransferase of Danio rerio.
  • the said N-acetyl glucosamine transferase is N-acetyl glucosamine transferase
  • the N- acetyl glucosamine transferase is one that is homologous to a N-acetyl glucosamine transferase of a species of Anopheles gambiae.
  • the N-acetyl glucosamine transferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to AgaP_AGAP004397 (GI: 1274542) of Anopheles gambiae.
  • the said N-acetyl glucosamine transferase is N-acetyl glucosamine transferase
  • the N- acetyl glucosamine transferase is one that is homologous to a N-acetyl glucosamine transferase of a species of Anopheles gambiae.
  • the N-acetyl glucosamine transferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to AgaP_AGAP004397 (GI: 1274542) of Anopheles gambiae.
  • the said N-acetyl glucosamine transferase is gly-20
  • the N-acetyl glucosamine transferase is one that is homologous to a N-acetyl glucosamine transferase of a species of Caenorhabditis elegans.
  • the N-acetyl glucosamine transferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to gly-20 (GI: 179562) of Caenorhabditis elegans.
  • the said N-acetyl glucosamine transferase is beta- l,2-N-acetylglucosaminyltransferase II of Arabidopsis thaliana.
  • the N- acetyl glucosamine transferase is one that is homologous to a N-acetyl glucosamine transferase of a species of Arabidopsis thaliana.
  • the N-acetyl glucosamine transferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to gly beta-l,2-N-acetylglucosaminyltransferase II of Arabidopsis thaliana.
  • the said N-acetyl glucosamine transferase is gly-20
  • the N-acetyl glucosamine transferase is one that is homologous to a N-acetyl glucosamine transferase of a species of Caenorhabditis elegans.
  • the N-acetyl glucosamine transferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to gly-20 (GI: 179562) of Caenorhabditis elegans .
  • provided herein is a nucleic acid encoding the hybrid N-acetyl glucosamine transferase. 7.13 Galactosyltransfer ases
  • hybrid galactosyltransferase wherein the hybrid galactosyltransferase comprises (a) catalytic domain of an
  • the galactosyltransferase that is not from Leishmania and (b) amino acid sequence(s) responsible for localization and retention in the Golgi compartment of Leishmania.
  • the hybrid galactosyltransferase is from Leishmania tarentolae.
  • the hybrid galactosyltransferase has been engineered to comprise a signal sequence, wherein the signal sequence targets the
  • the hybrid galactosyltransferase retains the galactosyltransferase in the cis Golgi apparatus. In another embodiment, the hybrid
  • galactosyltransferase retains the galactosyltransferase in the medial Golgi apparatus.
  • the hybrid galactosyltransferase retains the galactosyltransferase in the trans Golgi apparatus.
  • the retention sequence is a cytoplasmic- transmembrane-stem (CTS) sequence.
  • CTS cytoplasmic- transmembrane-stem
  • the hybrid galactosyltransferase is a GRIP sequence.
  • the hybrid galactosyltransferase is a CTS sequence and a GRIP sequence.
  • the CTS sequence comprises the amino acid sequence of MAN1, NTPDase 1, or NTPDase 2.
  • the CTS sequence comprises the sequence of SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26 or functionally active fragments thereof.
  • the CTS comprises the amino acid sequence of MAN1.
  • the CTS sequence comprises the sequence of SEQ ID NO: 24 or functionally active fragments thereof.
  • the GRIP sequence comprises the amino acid sequence of SEQ ID NO: 27.
  • the galactosyltransferase is derived from an galactosyltransferase listed in Table 2, or a functional homologue thereof. Any
  • galactosyltransferase or nucleic acid encoding it, known in the art can be used in accordance with the host cells and methods described herein.
  • the said galactosyltransferase is Beta- 1,4- galactosyltransferase 1 (B4GALT1) of Homo sapiens.
  • the galactosyltransferase is one that is homologous to a galactosyltransferase of a species of Homo sapiens.
  • the galactosyltransferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to Beta-l,4-galactosyltransferase 1 of Homo sapiens.
  • the said galactosyltransferase is Beta- 1,4- galactosyltransferase 1 of Pan troglodytes.
  • the galactosyltransferase is one that is homologous to a galactosyltransferase of a species of Pan troglodytes.
  • the galactosyltransferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%,
  • the said galactosyltransferase is Beta- 1,4- galactosyltransferase 1 of Macaca mulatta.
  • the galactosyltransferase is one that is homologous to a galactosyltransferase of a species of Macaca mulatta.
  • the galactosyltransferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%,
  • Beta-l,4-galactosyltransferase 1 of Macaca mulatta 95%, 96%, 97%, 98%, or 99% homologous to Beta-l,4-galactosyltransferase 1 of Macaca mulatta.
  • the said galactosyltransferase is Beta- 1,4- galactosyltransferase 1 of Canis lupus familiaris.
  • the said galactosyltransferase is Beta- 1,4- galactosyltransferase 1 of Canis lupus familiaris.
  • galactosyltransferase is one that is homologous to a galactosyltransferase of a species of Canis lupus familiaris.
  • the galactosyltransferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to Beta- 1,4- galactosyltransferase 1 of Canis lupus familiaris.
  • the said galactosyltransferase is Beta- 1,4- galactosyltransferase 1 of Bos taurus.
  • the galactosyltransferase is one that is homologous to a galactosyltransferase of a species of Bos taurus.
  • the galactosyltransferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to Beta-l,4-galactosyltransferase 1 of Bos taurus.
  • the said galactosyltransferase is Beta- 1,4- galactosyltransferase 1 of Mus musculus.
  • the galactosyltransferase is one that is homologous to a galactosyltransferase of a species of Mus musculus.
  • the galactosyltransferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%,
  • the said galactosyltransferase is Beta- 1,4- galactosyltransferase 1 of Rattus norvegicus.
  • the galactosyltransferase is one that is homologous to a galactosyltransferase of a species of Rattus norvegicus.
  • the galactosyltransferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to Beta-l,4-galactosyltransferase 1 of Rattus norvegicus.
  • the said galactosyltransferase is Beta- 1,4- galactosyltransferase 1 of Gallus gallus.
  • the galactosyltransferase is one that is homologous to a galactosyltransferase of a species of Gallus gallus.
  • the galactosyltransferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to Beta-l,4-galactosyltransferase 1 of Gallus gallus.
  • the said galactosyltransferase is Beta- 1,4- galactosyltransferase 1 of Xenopus tropicali.
  • the galactosyltransferase is one that is homologous to a galactosyltransferase of a species of Xenopus tropicali.
  • the galactosyltransferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to Beta-l,4-galactosyltransferase 1 of Xenopus tropicali.
  • the said galactosyltransferase is Beta- 1,4- galactosyltransferase 1 of Danio rerio.
  • the galactosyltransferase is one that is homologous to a galactosyltransferase of a species of Danio rerio.
  • the galactosyltransferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to Beta-l,4-galactosyltransferase 1 of Danio rerio.
  • nucleic acid encoding the hybrid galactosyltransferase.
  • hybrid sialyltransferase comprises (a) catalytic domain of an sialyltransferase that is not from Leishmania and (b) amino acid sequence(s) responsible for localization and retention in the Golgi compartment of Leishmania.
  • the hybrid sialyltransferase is from Leishmania tarentolae.
  • the hybrid sialyltransferase has been engineered to comprise a signal sequence, wherein the signal sequence targets the sialyltransferase to the endoplasmic reticulum of the Leishmania tarentolae host cell, and wherein the retention sequence retains the sialyltransferase in the Golgi apparatus.
  • the hybrid sialyltransferase retains the sialyltransferase in the trans Golgi apparatus.
  • the retention sequence is a CTS sequence. In further embodiments, the retention sequence is a GRIP sequence. In other embodiments, the retention sequence is a CTS sequence and a GRIP sequence. In another embodiment, the CTS sequence comprises the amino acid sequence of MAN1, NTPDase 1, or NTPDase 2. In another embodiment, the CTS sequence comprises the sequence of SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26 or functionally active fragments thereof. In another embodiment, the CTS comprises the amino acid sequence of MAN 1. In another embodiment, the CTS sequence comprises the sequence of SEQ ID NO: 24 or functionally active fragments thereof. In another embodiment, the GRIP sequence comprises the sequence of SEQ ID NO: 27, or a functionally active fragments thereof.
  • the sialyltransferase is a 2,6-SiaT or a 2,3-SiaT.
  • the hybrid sialyltransferase is derived from an sialyltransferase listed in Table 2, or a functional homologue thereof.
  • sialyltransferase is Beta-galactoside alpha-
  • sialyltransferase 2, 6-sialyltransferase 1 of Homo sapiens.
  • said sialyltransferase is Beta-galactoside alpha-2, 3 -sialyltransferase 4 of Homo sapiens.
  • the sialyltransferase is one that is homologous to a sialyltransferase of Homo sapiens.
  • the sialyltransferase is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to Beta-galactoside alpha-2, 6-sialyltransferase 1 or Beta- galactoside alpha-2, 3 -sialyltransferase 4 of Homo sapiens.
  • sialyltransferase is Beta-galactoside alpha-
  • sialyltransferase 2, 6-sialyltransferase 1 of Mus musculus.
  • said sialyltransferase is Beta-galactoside alpha-2, 3 -sialyltransferase 3 of Mus musculus.
  • the sialyltransferase is one that is homologous to a sialyltransferase of Mus musculus.
  • the sialyltransferase is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to Beta-galactoside alpha-2, 6-sialyltransferase 1 or Beta- galactoside alpha-2, 3 -sialyltransferase 3 of Mus musculus.
  • the said sialyltransferase is Beta-galactoside alpha-2, 6-sialyltransferase 1 of Rattus norvegicus.
  • the sialyltransferase is one that is homologous to a sialyltransferase of a species of Rattus norvegicus.
  • the sialyltransferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to Beta-galactoside alpha-2, 6-sialyltransferase 1 of Rattus norvegicus.
  • the said sialyltransferase is alpha-2, 3- sialyltransferase of Campylobacter jejuni.
  • the sialyltransferase is one that is homologous to a sialyltransferase of a species of Campylobacter jejuni.
  • the sialyltransferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to alpha-2, 3 -sialyltransferase of Campylobacter jejuni.
  • the said sialyltransferase is alpha-2, 3/8- sialyltransferase of Campylobacter jejuni.
  • the sialyltransferase is one that is homologous to a sialyltransferase of a species of Campylobacter jejuni.
  • the sialyltransferase or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to alpha-2, 3/8-sialyltransferase of Campylobacter jejuni.
  • a nucleic acid encoding the hybrid sialyltransferase is from an prokaryotic origin, for example bacterium.
  • the sialyltransferase is Beta-galactoside alpha-2, 6- sialyltransferase 1 from Photobacterium damselae.
  • other enzymes involving in the production of the glycosylated target proteins described herein are from prokaryotic origins, for example bacterium.
  • the enzymes comprises CMP-sialic acid synthetase from Neisseria meningitidis.
  • the host cell comprising a heterologous
  • sialyltransferase further comprises a heterologous CMP-Sia biosynthetic pathway proteins capable of generating CMP-NeuAc.
  • the CMP-Sia biosynthetic pathway proteins capable of generating CMP-NeuAc are at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,
  • the said sialic acid biosynthesis enzyme is UDP-
  • the said sialic acid biosynthesis enzyme is CMP-sialic acid transporter of Mus musculus.
  • the sialic acid biosynthesis enzyme is one that is homologous to a sialic acid biosynthesis enzyme of a species of Mus musculus.
  • the sialic acid biosynthesis enzyme is one that is homologous to a sialic acid biosynthesis enzyme of a species of Mus musculus.
  • biosynthesis enzyme or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to UDP-GlcNAc 2-epimerase/ N-acetylmannosamine kinase or CMP-sialic acid transporter of Mus musculus.
  • the said sialic acid biosynthesis enzyme is UDP-
  • the said sialic acid biosynthesis enzyme is N-acetylneuraminic acid phosphate synthase of Homo sapiens.
  • the said sialic acid biosynthesis enzyme is Neu5Ac-9-P phosphatase of Homo sapiens.
  • the said sialic acid biosynthesis enzyme is CMP-sialic acid synthetase of Homo sapiens.
  • the said sialic acid biosynthesis enzyme is CMP-Neu5Ac transporter of Homo sapiens.
  • the sialic acid biosynthesis enzyme is one that is homologous to a sialic acid biosynthesis enzyme of a species of Homo sapiens.
  • the sialic acid biosynthesis enzyme or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
  • the said sialic acid biosynthesis enzyme is UDP-
  • the sialic acid biosynthesis enzyme is one that is homologous to a sialic acid biosynthesis enzyme of a species of Rattus norvegicus.
  • the sialic acid biosynthesis enzyme or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
  • UDP-N- GlcNAc 2-epimerase/N-acetylmannosamine kinase of Rattus norvegicus has point mutations from sialuria patient’s GNE/MNK (Son et al 2011).
  • the said sialic acid biosynthesis enzyme is CMP- sialic acid synthetase of Neisseria meningitidis.
  • the said sialic acid biosynthesis enzyme is UDP-N-acetylglucosamine 2-epimerase of Neisseria meningitidis.
  • the said sialic acid biosynthesis enzyme is CMP-sialic acid synthase of Neisseria meningitidis.
  • the sialic acid biosynthesis enzyme is one that is homologous to a sialic acid biosynthesis enzyme of a species of Neisseria meningitidis.
  • the sialic acid biosynthesis enzyme or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to CMP-sialic acid synthetase, UDP-N-acetylglucosamine 2-epimerase or CMP-sialic acid synthase of Neisseria meningitidis.
  • the said sialic acid biosynthesis enzyme is CMP- sialic acid synthetase of Escherichia coli Kl .
  • the said sialic acid biosynthesis enzyme is UDP-N-acetylglucosamine 2-epimerase of Escherichia coli Kl .
  • the said sialic acid biosynthesis enzyme is CMP-sialic acid synthase of Escherichia coli Kl .
  • the sialic acid biosynthesis enzyme is one that is homologous to a sialic acid biosynthesis enzyme of a species of Escherichia coli Kl.
  • the sialic acid biosynthesis enzyme or a nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to CMP-sialic acid synthetase, UDP-N-acetylglucosamine 2-epimerase or CMP-sialic acid synthase of Escherichia coli Kl.
  • the host cell is a Leishmania cell. In certain embodiments, the host cell is a Leishmania tarentolae cell.
  • the host cell is a Leishmania aethiopica cell. In certain embodiments, the host cell is part of the Leishmania aethiopica species complex. In certain embodiments, the host cell is a Leishmania aristidesi cell. In certain embodiments, the host cell is a Leishmania deanei cell. In certain embodiments, the host cell is part of the
  • the host cell is a Leishmania donovani cell. In certain embodiments, the host cell is a Leishmania chagasi cell. In certain embodiments, the host cell is a Leishmania infantum cell. In certain embodiments, the host cell is a Leishmania hertigi cell. In certain embodiments, the host cell is part of the Leishmania major species complex. In certain embodiments, the host cell is a Leishmania major cell. In certain embodiments, the host cell is a Leishmania martiniquensis cell. In certain embodiments, the host cell is part of the Leishmania mexicana species complex. In certain embodiments, the host cell is a Leishmania mexicana cell. In certain embodiments, the host cell is a Leishmania pifanoi cell.
  • the host cell is part of the Leishmania tropica species complex. In certain embodiments, the host cell is a Leishmania tropica cell.
  • the host cell belongs to the bodonidae family of kinetoplasts.
  • the host cell is a Bodo saltans cell.
  • the host cell belongs to the ichthyobodonidae family of kinetoplasts. In certain embodiments, the host cell belongs to the trypanosomatidae family of kinetoplasts.
  • the host cell belongs to the blastocrithidia family of trypanosomatidae. In certain embodiments, the host cell belongs to the blechomonas family of trypanosomatidae. In certain embodiments, the host cell belongs to the herpetomonas family of trypanosomatidae. In certain embodiments, the host cell belongs to the jaenimonas family of trypanosomatidae. In certain embodiments, the host cell belongs to the lafontella family of trypanosomatidae. In certain embodiments, the host cell belongs to the leishmaniinae family of trypanosomatidae.
  • the host cell belongs to the novymonas family of trypanosomatidae. In certain embodiments, the host cell belongs to the paratrypanosoma family of trypanosomatidae. In certain embodiments, the host cell belongs to the phytomonas family of trypanosomatidae. In certain embodiments, the host cell belongs to the sergeia family of trypanosomatidae. In certain embodiments, the host cell belongs to the strigomonadinae family of trypanosomatidae. In certain embodiments, the host cell belongs to the trypanosoma family of trypanosomatidae. In certain embodiments, the host cell belongs to the wallacemonas family of trypanosomatidae. In certain embodiments, the host cell belongs to the blastocrithidia family of trypanosomatidae.
  • the host cells used to herein are engineered to comprise heterologous nucleic acids, e.g., heterologous nucleic acids that encode one or more carrier proteins and/or heterologous nucleic acids that encode one or more proteins, e.g., genes encoding one or more proteins.
  • heterologous nucleic acids are introduced into the host cells described herein using the methods of insertion provided herein.
  • host cell nucleic acids e.g., genes
  • proteins that form part of a possibly competing or interfering glycosylation pathway (e.g., compete or interfere with one or more heterologous genes involved in glycosylation that are recombinant ly introduced into the host cell)
  • host cell background gene
  • the host cell nucleic acids that are deleted/modified do not encode a functional protein or do not encode a protein whatsoever.
  • nucleic acids when nucleic acids are deleted from the genome of the host cells provided herein, they are replaced by a desirable sequence, e.g., a sequence that is useful for glycoprotein production. Such replacement can be by way of one or more of the methods of insertion described herein, wherein the heterologous insert DNA that is inserted into the host cell may replace the function of the gene(s) deleted from the host cell.
  • the host cells provided herein comprise a gene deletion, wherein a DNA sequence of interest has been inserted into the host cell genome at the site of the gene deletion.
  • a host cell provided herein is Leishmania bearing a gene deletion.
  • nucleic acid e.g., a gene fragment thereof
  • a host cell e.g., Leishmania tarentolae.
  • heterologous nucleic acids are introduced into the host cells described herein using a plasmid, e.g., the heterologous nucleic acids are expressed in the host cells by a plasmid (e.g., an expression vector), and the plasmid is introduced into the modified host cells by transfection, infection, or electroporation, chemical transformation by heat shock, natural transformation, phage transduction, or conjugation.
  • a plasmid e.g., an expression vector
  • said plasmid is introduced into the modified host cells by stable transfection.
  • linearized heterologous nucleic acids are introduced into the host cells described herein using transfection, infection, or electroporation, chemical transformation by heat shock, natural transformation, phage transduction, or conjugation.
  • heterologous nucleic acids are integrated site-specifically into the host cell genome by homologous recombination.
  • provided herein is a method of producing glycosylated target proteins in vivo, using a host cell described herein.
  • a method for producing glycosylated target proteins comprising (i) culturing a host cell provided herein under conditions suitable for protein production and (ii) isolating said target protein.
  • the host cell comprises (a) a recombinant nucleic acid encoding a target protein; and (b) a recombinant nucleic acid encoding a
  • heterologous glycosyltransferase is an N-acetyl glucosamine transferase; or a heterologous galactosyltransferase; or a
  • the host cell is a Leishmania cell. 7.21 Methods of Culturing Cells
  • host cells are cultured using any of the standard culturing techniques known in the art. For example, cells are routinely grown in rich media like Brain Heart Infusion, Trypticase Soy Broth or Yeast Extract, all containing 5 pg/ml Hemin. Additionally, incubation is done at 26 °C in the dark as static or shaking cultures for 2-3 days. In some embodiments, cultures of recombinant cell lines contain the appropriate selective agents. A non-limiting list of selective agents is provided in Table 2.
  • TNFa antibody protein or peptide/polypeptide corresponding to the protein
  • Any TNFa antibody protein or peptide/polypeptide corresponding to the protein
  • a target protein in accordance with the methods described herein.
  • nucleic acid sequence of a known protein, as well as a newly identified protein can easily be deduced using methods known in the art, and thus it would be well within the capacity of one of skill in the art to introduce a nucleic acid that encodes any protein of interest into a host cell provided herein (e.g., via an expression vector, e.g., a plasmid, e.g., a site specific integration by homologous recombination).
  • an expression vector e.g., a plasmid, e.g., a site specific integration by homologous recombination.
  • the target protein used in accordance with the methods and host cells described herein is TNFa antibody.
  • the TNFa antibody is the TNFa antibody of Homo sapiens.
  • the TNFa antibody is a full length antibody, an Fab, an F(ab')2, an Scfv, or a sdAb.
  • the TNFa antibody is a full length antibody, an Fab, an F(ab')2, an Scfv, or a sdAb of Homo sapiens.
  • the target protein comprises the amino acid sequence of adalimumab (HUMIRA); infliximab (REMICADE), and golimumab (SIMPONI), or antibody formats such as certolizumab pegol (CIMZIA) or with a circulating receptor fusion protein such as etanercept (ENBREF).
  • the target protein comprises the amino acid sequence of Amjevita, Cyltezo, HUMIRA or a biosimilar thereof.
  • the target protein comprises the amino acid sequence of full length antibody, an Fab, an F(ab')2, an Scfv, or a sdAb of adalimumab (HUMIRA); infliximab (REMICADE), and golimumab (Simponi), or antibody formats such as certolizumab pegol (CIMZIA) or with a circulating receptor fusion protein such as etanercept (ENBREL), AMJEVITA, CYLTEZO or a biosimilar thereof.
  • the target protein comprises the amino acid sequence of full length antibody, an Fab, an F(ab')2, an Scfv, or a sdAb of any approved drugs that target TNFa or TNFa pathways (e.g. TNFa receptor).
  • the glycosylated TNFa antibody is secreted into the culture media, and wherein the glycosylated TNFa antibody is glycosylated.
  • the glycosylated TNFa antibody is purified from the culture media.
  • the glycosylated TNFa antibody is purified from the culture media via affinity purification.
  • the glycosylated TNFa antibody contains an FC domain and is affinity purified from the culture media via protein-A.
  • the glycosylated TNFa antibody contains an affinity tag and is affinity purified.
  • the population of glycosylated TNFa antibody is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homogeneous. In further embodiments, 90% to 100% of the N-glycosites on the TNFa antibody that are occupied by glycosylation.
  • the TNFa antibody used in accordance with the methods and host cells described herein can be a full length protein, a truncation, a protein domain, a region, a motif or a peptide thereof.
  • the TNFa antibody is an Fc-fusion protein.
  • the TNFa antibody could be modified.
  • the TNFa antibody has been engineered to comprise a signal sequence from Leishmania.
  • the signal sequence is processed and removed from the TNFa antibody.
  • the TNFa antibody has been engineered to comprise one or more tag(s).
  • the tag is processed and removed from the TNFa antibody.
  • compositions in addition to comprising a glycosylated target protein described herein, the compositions (e.g., pharmaceutical compositions) described herein comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeiae for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E.W. Martin.
  • compositions described herein are formulated to be suitable for the intended route of administration to a subject.
  • the compositions described herein may be formulated to be suitable for subcutaneous, parenteral, oral, intradermal, transdermal, colorectal, intraperitoneal, and rectal administration.
  • the pharmaceutical composition may be formulated for intravenous, oral, intraperitoneal, intranasal, intratracheal, subcutaneous, intramuscular, topical, intradermal, transdermal or pulmonary administration.
  • compositions described herein additionally comprise one or more buffers, e.g., phosphate buffer and sucrose phosphate glutamate buffer. In other embodiments, the compositions described herein do not comprise buffers.
  • compositions described herein additionally comprise one or more salts, e.g., sodium chloride, calcium chloride, sodium phosphate, monosodium glutamate, and aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), or a mixture of such aluminum salts).
  • salts e.g., sodium chloride, calcium chloride, sodium phosphate, monosodium glutamate
  • aluminum salts e.g., aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), or a mixture of such aluminum salts.
  • compositions described herein do not comprise salts.
  • TNFa antibody provided herein wherein the single dosage form consists of about 2 mg, about 5 mg, about 7 mg, about 10 mg, about 12 mg, about 15 mg, about 18 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, or about 80 mg of the anti-TNFa antibody.
  • the single dosage form is a prefilled syringe, an injection pen, a vial, a tablet, or a capsule.
  • the single dosage form comprises the anti-TNFa antibody in a lyophilized form or in a liquid solution.
  • the compositions described herein can be administered in a single dosage form, for example a single dosage form of an anti-TNFa antibody.
  • the single dosage form is a prefilled syringe, an injection pen, a vial, a tablet, or a capsule.
  • the single dosage form may consist of about 2 mg, about 5 mg, about 7 mg, about 10 mg, about 12 mg, about 15 mg, about 18 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, or about 80 mg of an anti-TNFa antibody.
  • the single dosage form comprises the anti-TNFa antibody in a lyophilized form or in a liquid solution.
  • compositions described herein can be included in a kit, container, pack, or dispenser together with instructions for administration.
  • compositions described herein can be stored before use, e.g., the compositions can be stored frozen (e.g., at about -20 °C or at about -70 °C); stored in refrigerated conditions (e.g., at about 4 °C); or stored at room temperature.
  • the target protein that can be glycosylated using the methods described herein is a biologic comprising an Fc domain of an IgG.
  • anti-TNFa antibodies produced by the methods provided herein.
  • the anti-TNFa antibody has maximal a-2,6 sialylation.
  • the“maximal” a-2,6 sialylation is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of N- glycans comprising at least one sialic acid residue per N-glycan or comprising at least two sialic acid residues per N-glycan.
  • N-glycosylation consensus sequences are glycosylated.
  • anti-TNFa antibodies comprising one or more of the following structures:
  • the diamond represents a sialic acid residue
  • the empty circle represents a galactose residue
  • the square represents an N-acetylglucosamine residue
  • the grey circle represents a mannose residue
  • the Asn is an Asn of an N-linked glycosylation consensus sequence in the anti-TNFa antibody heavy chain.
  • compositions comprising a population of glycosylated anti-TNFa antibodies produced by the method of any one of claims 10 or 11, wherein (a) the population of glycosylated anti-TNFa antibodies is at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homogeneous; or (b) at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 95% of the N-glycosylation sites in the anti-TNFa antibodies carry the same oligosaccharide; or (c) at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 95% of the N-glycosylation sites in the anti-TNFa antibodies are a-2,6 sialylated; or (d) at least 20%, 30%, 40%, 50%, 60%,
  • the N-glycosylation sites in the anti-TNFa antibodies carry a biantennary oligosaccharide that is a-2,6 sialylated at both termini (e.g., carry G2S2).
  • compositions comprising a population of glycosylated anti-TNFa antibodies, wherein the population of anti-TNFa antibodies comprise one or more anti-TNFa antibodies comprising one or more of the following structures:
  • the diamond represents a sialic acid residue
  • the empty circle represents a galactose residue
  • the square represents an N-acetylglucosamine residue
  • the grey circle represents a mannose residue
  • the Asn is an Asn of an N-linked glycosylation consensus sequence in the anti-TNFa antibody heavy chain.
  • compositions wherein (a) the population of glycosylated anti-TNFa antibodies is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homogeneous; or (b) at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 95% of the N-glycosylation sites in the anti-TNFa antibodies carry the same oligosaccharide; or (c) at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 95% of the N-glycosylation sites in the anti-TNFa antibodies are a-2,6 sialylated; or (d) at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 95% of the N-glycosylation sites in the anti-TNFa antibodies carry a biantennary oligosaccharide that is a-2,6
  • the provided anti-TNFa antibody herein is adalimumab.
  • TNFa antibody is adalimumab.
  • anti-TNFa antibodies wherein the anti-TNFa antibody has an antibody-dependent cell mediated cytotoxicity (ADCC) activity that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, lO-fold, l2-fold, l5-fold, l8-fold, 20-fold, 25-fold, or 30-fold higher than that of the same anti-TNFa antibody having a different glycosylation profile.
  • ADCC antibody-dependent cell mediated cytotoxicity
  • the anti-TNFa antibody has a complement- dependent cytotoxicity (CDC) activity that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, lO-fold, l2-fold, l5-fold, l8-fold, 20-fold, 25-fold, or 30-fold lower than or equal to that of the same anti- TNFa antibody having a different glycosylation profile.
  • CDC complement- dependent cytotoxicity
  • the anti-TNFa antibody has a dendritic cells internalization that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5- fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, lO-fold, l2-fold, l5-fold, 18- fold, 20-fold, 25-fold, or 30-fold higher than that of the same anti-TNFa antibody having a different glycosylation profile.
  • the anti-TNFa antibody leads to a B-cell apoptosis that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, lO-fold, l2-fold, l5-fold, l8-fold, 20-fold, 25-fold, or 30-fold higher than that of the same anti-TNFa antibody having a different glycosylation profile.
  • the anti-TNFa antibody has an anti-inflammatory activity that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5-fold, 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, lO-fold, l2-fold, l5-fold, l8-fold, 20- fold, 25-fold, or 30-fold higher than that of the same anti-TNFa antibody having a different glycosylation profile.
  • provided herein are methods of preventing or treating a disease or disorder in a subject comprising administering to the subject a glycosylated target protein described herein or a composition thereof. Further provided herein are methods of preventing a disease or disorder in a subject comprising administering to the subject a
  • a suitable dose of a glycosylated target protein described herein is the amount corresponding to the lowest dose effective to produce a therapeutic effect.
  • an effective amount of an anti-TNFa antibody may be an amount that inhibits TNFa activity in a subject suffering from a disease to be detrimental TNFa activity.
  • the amount of an anti-TNFa antibody described herein administered to a patient may be not more than the amount listed in the label of a drug product of the same anti-TNFa antibody having a different glycosylation profile from that of the anti-TNFa antibody described herein.
  • the amount of adalimumab produced herein administered to a patient may be not more than the amount listed in the label of the HUMIRA drug product.
  • the frequency of administration of an anti-TNFa antibody described herein administered to a patient may be not more than the frequency list in the label of a drug product of the same anti-TNFa antibody having a different glycosylation profile from that of the anti-TNFa antibody described herein.
  • the frequency of administration of adalimumab produced herein administered to a patient may be not more than the frequency listed in the label of HUMIRA drug product.
  • the accumulated amount of anti-TNFa antibody described herein administered to a patient over a period of time may be not more than the accumulated amount indicated in the label of a drug product of the same anti-TNFa antibody having different glycosylation profile from that of the anti-TNFa antibody described herein.
  • the reduced accumulated amount could be administered in reduced doses on a reduced frequency.
  • the reduced accumulated amount could be administered in one or more doses that are the same or higher than the dose in the label on a reduced frequency.
  • the reduced accumulated amount could be administered in one or more reduced doses on a frequency that is the same or higher than the frequency in the label.
  • the reduced accumulated amount could be administered over a shorter period of time than the period of time for the drug product to achieve the same level of effect in treatment or prevention.
  • the amount of an anti-TNFa antibody described herein in a single dose administered to a patient can be from about 1 to 150 mg, about 5 to 145 mg, about 10 to 140 mg, about 15 to 135 mg, about 20 to 130 mg, about 25 to 125 mg, about 30 to 120 mg, about 35 to 115 mg, about 40 to 110 mg, about 45 to 105 mg, about 50 to 100 mg, about 55 to 95 mg, about 60 to 90 mg, about 65 to 5 mg, about 70 to 80 mg, or about 75 mg.
  • the amount of an anti-TNFa antibody described herein in a single dose administered to a patient can be from about 5 to about 80 mg.
  • the amount of an anti-TNFa antibody described herein in a single dose administered to a patient can be from about 25 to about 50 mg. In certain embodiments, the amount of an anti-TNFa antibody described herein in a single dose administered to a patient can from about 15 mg to about 35 mg.
  • the amount of an anti-TNFa antibody described herein in a single dose administered to a patient can be no more than 40 mg, for example 40 mg, 35 mg, 30 mg, 25 mg, 20 mg, 18 mg, 15 mg, 12 mg, 10 mg, 7 mg, 5 mg, and 2 mg. In certain embodiment, the amount of an anti-TNFa antibody described herein in a single dose
  • administered to a patient can be no more than 80 mg, for example 80 mg, 75 mg, 70 mg, 65 mg, 60 mg, 55 mg, 50 mg, 45 mg, 40 mg, 35 mg, 30 mg, 20 mg, 15 mg, 10 mg, 5 mg and 2 mg.
  • the amount of an anti-TNFa antibody described herein in a single dose administered to a patient can be no more than 160 mg, for example 150 mg, 140 mg, 130 mg,
  • the amount of an anti-TNFa antibody described herein in a single dose administered to a patient can be equal to or more than 160 mg, for example 170 mg, 180 mg, 200 mg, 250 mg, and 300 mg.
  • an anti-TNFa antibody of the disclosure can be administered on a frequency that is every other week, namely every 14 days.
  • an anti-TNFa antibody of the disclosure can be administered on a frequency that is lower than every 14 days, for example, every half a month, every 21 days, monthly, every 8 weeks, bimonthly, every 12 weeks, every 3 months, every 4 months, every 5 months, or every 6 months.
  • anti-TNFa antibody of the disclosure can be administered on a frequency that is the same or higher than every 14 days, for example, every 14 days, every 10 days, every 7 days, every 5 days, every other day, or daily.
  • the administration of an anti-TNFa antibody of the disclosure can comprise an induction dose that is higher than the following doses, for example the following maintenance doses.
  • the administration of an anti-TNFa antibody of the disclosure can comprise a second dose that is lower than the induction dose and higher than the following maintenance doses.
  • the administration of an anti-TNFa antibody of the disclosure can comprise the same amount of the anti-TNFa antibody in all the doses throughout the treatment period.
  • the an anti-TNFa antibody of the disclosure can be administered in a single dosage form.
  • the single dosage form consists of about 2 mg, about 5 mg, about 7 mg, about 10 mg, about 12 mg, about 15 mg, about 18 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, or about 80 mg of the anti- TNFa antibody.
  • the single dosage form can be a prefilled syringe, an injection pen, a vial, a tablet, or a capsule.
  • the single dosage form can comprise the anti-TNFa antibody in a lyophilized form or in a liquid solution.
  • a method of treating or preventing rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, pediatric Crohn’s disease, ulcerative colitis, chronic psoriasis, hidradenitis suppurativa, adult uveitis, pediatric uveitis, plaque psoriasis, or juvenile idiopathic arthritis in a patient may comprise administering to the patient an anti-TNFa antibody of the disclosure.
  • an anti-TNFa antibody of the disclosure can be administered at an accumulated amount over a period of time that is no more than the accumulated amount indicated in the label of a drug product of the same anti-TNFa antibody having different glycosylation profile from that of the anti-TNFa antibody described herein.
  • an anti-TNFa antibody of the disclosure can be administered at a dose of 10 to 50 mg, for example 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg or 50 mg every other week.
  • an anti-TNFa antibody of the disclosure can be administered on a frequency that is lower than every 14 days, for example, every half a month, every 21 days, monthly, every 8 weeks, bimonthly, every 12 weeks, every 3 months, every 4 months, every 5 months, or every 6 months.
  • an anti- TNFa antibody of the disclosure can be administered on a frequency that is the same or higher than every 14 days, for example, every 14 days, every 10 days, every 7 days, every 5 days, every other day, or daily.
  • methotrexate to treat or prevent rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis in a patient
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • DMARDs disease-modifying anti rheumatics drug
  • some patients not taking concomitant methotrexate can derive additional benefit from increasing the dosing frequency from biweekly to weekly.
  • an anti-TNFa antibody of the disclosure can be administered in certain non-limiting embodiments at a dose of 40-280 mg, for example 40 mg,
  • the amount of the anti-TNFa antibody in the initial dose and following doses depends on the body weight of the patient.
  • the method of treating or preventing pediatric Crohn's disease in the patient may comprise administering to the patient less than or equal to 80 mg of the anti-TNFa antibody on day 1, less than or equal to 40 mg of the anti-TNFa antibody on day 15, and less than or equal to 20 mg of the anti-TNFa antibody on an administration frequency less than or equal to every other week starting on day 29 in a patient having a body weight between 17 kg and 40 kg, or less than or equal to 160 mg of the anti-TNFa antibody on day 1, less than or equal to 80 mg of the anti-TNFa antibody on day 15, and less than or equal to 40 mg of the anti-TNFa antibody on an administration frequency less than or equal to every other week starting on day 29 in a patient having a body weight equal to or higher than 40 kg.
  • the frequency of the maintenance dose may be lower than every 14 days, for example, every half a month, every 21 days, monthly, every 8 weeks, bimonthly, every 12 weeks, every 3 months, every 4 months, every 5 months, or every 6 months.
  • the frequency of the maintenance dose may be the same or higher than every 14 days, for example, every 14 days, every 10 days, every 7 days, every 5 days, every other day, or daily.
  • aminosalicylates e.g., corticosteroids, and/or immunomodulatory agents (e.g., 6- mercaptopurine and azathioprine) can be continued during treatment with the anti-TNFa antibody.
  • immunomodulatory agents e.g., 6- mercaptopurine and azathioprine
  • an anti-TNFa antibody of the disclosure is administered at a dose that depends on the patient's weight.
  • the dose for pediatric patients weighing 10 kg (22 lbs) to under 15 kg (33 lbs) ranges from 2 to 20 mg, for example 2 mg, 4 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, or 20 mg, every other week.
  • the dose for pediatric patients weighing 15 kg (33 lbs) to under 30 kg (66 lbs) ranges from 5 to 40 mg, for example 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg every other week.
  • the dose for pediatric patients weighing greater than 30 kg (66 lbs) ranges from 5 to 80 mg, for example 5 mg, 8 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg, 70 mg or 80 mg, every other week.
  • the frequency of the maintenance dose may be lower than every 14 days, for example, every half a month, every 21 days, monthly, every 8 weeks, bimonthly, every 12 weeks, every 3 months, every 4 months, every 5 months, or every 6 months.
  • the frequency of the maintenance dose may be the same or higher than every 14 days, for example, every 14 days, every 10 days, every 7 days, every 5 days, every other day, or daily.
  • methotrexate to treat or prevent juvenile idiopathic arthritis or pediatric uveitis, methotrexate, glucocorticoids, salicylates, NSAIDs or analgesics can be continued during treatment with the anti-TNFa antibody.
  • an anti-TNFa antibody of the disclosure can be administered at a dose of 40-160 mg, for example 40 mg, 60 mg, 80 mg, 100 mg, 120 mg, 140 mg, 120 mg, or 160 mg given initially (on Day 1 or divided between Day 1 and Day 2), followed by a maintenance dose of approximately 20% to 60% (for example, 20%, 30%, 40%, and 50%) given every other week starting one week after the initial dose (on Day 8).
  • the frequency of the maintenance dose may be lower than every 14 days, for example, every half a month, every 21 days, monthly, every 8 weeks, bimonthly, every 12 weeks, every 3 months, every 4 months, every 5 months, or every 6 months.
  • the frequency of the maintenance dose may be the same or higher than every 14 days, for example, every 14 days, every 10 days, every 7 days, every 5 days, every other day, or daily.
  • an anti-TNFa antibody of the disclosure is administered at a dose that depends on the patient's weight and age.
  • an anti-TNFa antibody of the disclosure can be administered at a dose of 20 - 160 mg, for example 20 mg, 30 mg, 40 mg, 60 mg, 80 mg, 100 mg, 120 mg, 140 mg, 120 mg, or 160 mg given initially (on Day 1 or divided between Day 1 and Day 2), followed by a maintenance dose of approximately 20% to 60% (for example, 20%, 30%, 40%, and 50%) given every other week starting one week after the initial dose (on Day 8).
  • an anti-TNFa antibody of the disclosure can be administered at a dose of 20 to 280 mg, for example 20 mg, 30 mg, 40 mg, 60 mg, 80 mg, 100 mg, 120 mg, 140 mg, 120 mg, 160 mg, 180 mg, 200 mg, 240 mg, or 280 mg given initially (on Day 1 or divided between Day 1 and Day 2), followed by a dose of approximately 10% to 60% (for example, 20%, 30%, 40%, and 50%) of the initial dose two weeks later (on Day 15). Two weeks later (on Day 29), a maintenance dose of 10% to 30% (for example, 10%, 20% and 25%) of the initial dose is administered every week.
  • the frequency of the maintenance dose may be lower than every 14 days, for example, every half a month, every 21 days, monthly, every 8 weeks, bimonthly, every 12 weeks, every 3 months, every 4 months, every 5 months, or every 6 months, or higher than every 14 days, for example, every 14 days, every 10 days, every 7 days, every 5 days, every other day, or daily.
  • the frequency of the maintenance dose may be lower than every 7 days, for example, every 14 days, every half a month, every 21 days, monthly, every 8 weeks, bimonthly, every 12 weeks, every 3 months, every 4 months, every 5 months, or every 6 months, or higher than every 7 days, for example, every 5 days, every other day, or daily.
  • provided herein are methods of treating or preventing rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, pediatric Crohn’s disease, ulcerative colitis, chronic psoriasis, hidradenitis suppurativa, adult uveitis, pediatric uveitis, plaque psoriasis, or juvenile idiopathic arthritis in a patient, wherein the method comprises administering to the patient an anti-TNFa antibody provided herein or an composition provided herein.
  • the administration step is by subcutaneous injection.
  • the method : (a) requires a lower dose and/or lower administration frequency to achieve the same effect as compared to the same antibody having a different glycosylation profile; and/or (b) can be administered for an extended period of time (at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or at least 12 months, at least 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 years); and/or (c) does not trigger an immune response against the anti-TNFa antibody in the patient.
  • the method comprises treating or preventing rheumatoid arthritis, psoriatic arthritis, or ankylosing spondylitis in the patient, and wherein the method comprises administering to the patient less than or equal to 40 mg of the anti-TNFa antibody on an administration frequency less than or equal to every other week.
  • the method comprises treating or preventing Crohn's disease or ulcerative colitis in the patient, and wherein the method comprises administering to the patient less than or equal to 160 mg of the anti-TNFa antibody on day 1 , less than or equal to 80 mg of the anti-TNFa antibody on day 15, and less than or equal to 40 mg of the anti-TNFa antibody on an administration frequency less than or equal to every other week starting on day 29.
  • the method comprises treating or preventing pediatric Crohn's disease in the patient, and wherein the method comprises administering to the patient: (a) less than or equal to 80 mg of the anti-TNFa antibody on day 1 , less than or equal to 40 mg of the anti-TNFa antibody on day 15, and less than or equal to 20 mg of the anti-TNFa antibody on an administration frequency less than or equal to every other week starting on day 29 in a patient having a body weight between 17 kg and 40 kg, or (b) less than or equal to 160 mg of the anti-TNFa antibody on day 1 , less than or equal to 80 mg of the anti-TNFa antibody on day 15, and less than or equal to 40 mg of the anti-TNFa antibody on an administration frequency less than or equal to every other week starting on day 29 in a patient having a body weight equal to or higher than 40 kg.
  • the method comprises treating or preventing juvenile idiopathic arthritis or pediatric uveitis in the patient, and wherein the method comprises administering to the patient: (a) less than or equal to 10 mg of the anti-TNFa antibody on an administration frequency less than or equal to every other week in a patient having a body weight between 10 kg and 15 kg, (b) less than or equal to 20 mg of the anti-TNFa antibody on an administration frequency less than or equal to every other week in a patient having a body weight between 15 kg and 30 kg, or (c) less than or equal to 40 mg of the anti-TNFa antibody on an administration frequency less than or equal to every other week in a patient having a body weight equal to or higher than 30 kg.
  • the method comprises treating or preventing plaque psoriasis or adult uveitis in the patient, and wherein the method comprises administering to the patient less than or equal to 80 mg of the anti-TNFa antibody on day 1, and less than or equal to 40 mg on an administration frequency less than or equal to every other week starting on day 8.
  • the method comprises treating or preventing hidradenitis suppurativa in a patient, and wherein the method comprises administering to the patient: (a) less than or equal to 80 mg of the anti-TNFa antibody on day 1, and less than or equal to 40 mg of the anti-TNFa antibody on an administration frequency less than or equal to every other week starting on day 8 in an adolescent patient who are 12 years and older having a body weight between 30 kg and 60 kg, or (b) less than or equal to 160 mg of the anti-TNFa antibody on day 1, and less than or equal to 80 mg of the anti-TNFa antibody on day 15, and less than or equal to 40 mg of the anti-TNFa antibody on an administration frequency less than or equal to every week starting on day 29 in an adolescent patient who are 12 years and older having a body weight equal to or higher than 60 kg or an adult patient.
  • Example 1A Maximally sialylated adalimumab is produced by an in vitro N-glycan remodeling of commercial HUMIRA
  • Such a-2,6 sialylated adalimumab was synthesized by in vitro glyco-engineering as described above, demonstrating a maximal Fc a-2,6 sialylation (of 80% of N-glycans contain at least one sialic acid residues per N-glycan whereby 60% is biantennary sialylated (G2FS2) and 20% is mono-sialylated (G2FS1), Figure 4).
  • G2FS2 biantennary sialylated
  • G2FS1 mono-sialylated
  • glycosyltransferases to re-model the N-glycan profile of HUMIRA, the CHO produced adalimumab commercial product to be used for the proposed preclinical assays.
  • Method number 1 ( Figures 1 and 2).
  • Glyco-engineered (sialylated) full length HUMIRA migrates at higher molecular weight compared to untreated mAh.
  • the heavy chain (HC) and the light chain (LC) of the full length antibody were separated from each other.
  • the HC of the glyco-engineered antibody migrates at higher molecular weight compared to the HC of the untreated antibody.
  • PNGaseF removal of N-glycans
  • N-glycan profile of untreated HUMIRA mAh displays > 80% agalactosylated and asialylated structures ( Figure 4, upper panel, grey dashed bars).
  • N-glycan profile of the mAh was monitored during the IVGE process by N-glycan profiling using
  • H-S Maximally sialylated Hurmira is to be referred to as H-S; a Humira control sample treated the same way as H-S but lacking any glycosyltransferases in all in vitro reaction, is referred to as H-M, or Humira mock treated.
  • Method number 2 depicts the concept of in vitro extension of mAbs generated in vivo in Leishmania tarentolae (Ft).
  • Example ID The product is completely produced in vivo in engineered Leishmania tarentolae cells
  • Method number 3 ( Figures 1 and 2) describes the desired way to produce the target product completely in vivo by engineering the Lt platform with a sialylated defucosylated glycan.
  • the Lt platform can express and secrete a full length functional monoclonal antibody, passing all Critical Quality Attributes (“Engineered and fully- functional customized
  • the Ft platform has been genetically engineered to modify the native glycan to desired glycans in vivo.
  • Recombinant proteins were synthesized in vivo, with desired glycans, completely within the host’s secretory pathway as described (Detailed Description and “Engineered and fully- functional customized glycoproteins,” WO 2019/002512).
  • Example IE In vitro glycoengineering on paucimannose glycans of a full length mAh purified from Leishmania
  • An in vitro glycan extension strategy can be employed, for any antibody as shown in Figure 8.
  • the native glycan of a mAh (Rituximab FMTB) expressed in Feishmania tarentolae can be efficiently elongated in vitro with more than 95%.
  • the remaining enzymatic steps can be used in in vitro reactions leading to fully sialylated adalimumab ( Figure 12A).
  • Adalilumab glyco forms produced in vivo in Leishmania and/or using enzymatic in vitro elongation steps are herein referred to as“adalilumab-xx” wherein xx indicates the glyco form (i.e. Man3, GO, G2S2, etc.) or, in a short version, as“A-yy” wherein yy indicates the glyco forms according to decoding given in Fig. 12A and Tables 3 and 4.
  • In vitro modified commercial HUMIRA preparations are described in analogy.
  • Adalimumab anti-drug antibodies found in patients are of the IgG type (van
  • sialylated adalimumab potentially has the following benefits: (a) decreased immunogenicity due to altering the antigen presenting cell competency of dendritic cells (generating lower T Helper cell activation and induction of regulatory T cells) and B-cell silencing;
  • Dendritic cells are decisive regulators of the immune response specialised in antigen presentation with the aim of directing T-cell differentiation. Dendritic cells can both drive an immune response by activating T-cell immune responses, and have tolerogenic functions and contribute to reduction of immune responses (van Kooyk Y (2008) C- type lectins on dendritic cells: key modulators for the induction of immune responses. Biochem. Soc. Trans 36:1478-1481).
  • the antibody internalization assay evaluates if sialylation of adalimumab will reduce the amount of antibody internalized (receptor-mediated internalization and pinocytosis) by dendritic cells, thereby reducing the amount of immunogenic adalimumab peptides displayed by human dendritic cells (“Glycoengineered binding protein compositions” WO2015/073884 - US9550826).
  • sialylation of adalimumab could also trigger higher detection and internalization of the antibody as described in Perdicchio et al., which reports that a sialylated antigen (sialylated-ovalbumin) showed an increased uptake compared to the non-sialylated ovalbumin. This increased uptake lead to acquisition of a tolerogenic phenotype by the dendritic cells (Perdicchio, M., et al. Sialic acid-modified antigens impose tolerance via inhibition of T- cell proliferation and de novo induction of regulatory T cells. In Proceedings of the National Academy of Sciences 113 (12), pp. 3329-3334 (2016)). Therefore, the antibody internalisation assay also evaluates if sialylation of adalimumab can increase the amount of antibody internalized.
  • the protocol provided herein is based on the patent“Glycoengineered binding protein compositions” WO2015/073884 - US9550826, where human monocyte-derived dendritic cells that express membrane-bound TNF are generated as a target for adalimumab and sia-adalimumab.
  • Monocytes are isolated from PBMC (derived from leukopack from healthy donors) via CD 14 magnetic microbeads and dendritic cells are generated by addition of GM-CSF and IL-4 to the culture medium for 4 days. TNFa-surface expression was induced by stimulation with LPS and checked by staining with a fluorophore conjugated anti-TNFa antibody.
  • Example 2C Staining cells and flow cytometric analysis
  • Example 2D The induction of tolerogenic dendritic cells
  • tolerogenic dendritic cells tests whether exposure of immature dendritic cells to sia-adalimumab induces a tolerogenic phenotype that can direct the maturation of T-cells into Treg cells.
  • These regulatory T-cells are a subpopulation of T cells that modulate the immune system and maintain tolerance. It has been previously shown that sialylated antigens induce Tregs (Perdicchio, M., et al. Sialic acid-modified antigens impose tolerance via inhibition of T-cell proliferation and de novo induction of regulatory T cells. In Proceedings of the National Academy of Sciences 113 (12), pp. 3329-3334 (2016)) and we aim to investigate if this effect is connected with a tolerogenic dendritic cell phenotype that can be induced by sialylation of antibodies.
  • Tolerogenic dendritic cells can be identified and compared with classical dendritic cells based on the following characteristics: increased secretion of IL-10, IL-6, IDO, TRIAL (Tumor necrosis factor-related apoptosis inducing ligand), PD-L1 and PGE2 plus decreased expression of CD40. Dendritic cells will be incubated with adalimumab and sia- adalimumab and membrane-bound markers will be measured by flow cytometry and secreted cytokines by ELISA in the supernatant.
  • Dendritic cells incubated with sia-adalimumab express the markers specific for the tolerogenic phenotype. This can be done using an in vitro assay as described by Volchenkov et al. or by Torres- Aguilar et al.
  • dendritic cells are incubated in vitro with adalimumab and sia- adalimumab plus relevant controls (activated form of vitamin D plus dexamethasone as a positive control) and the production of IL-10, IL-6 and IDO is measured by ELISA while the expression of PD-L1 and CD40 can be checked using flow cytometry.
  • the dendritic cells can be screened for their T cell stimulatory capacity. This can be done by incubating the antigen-loaded dendritic cells (with PPD or TT) with CFSE or eFluor labelled autologous T cells and analysis of proliferation after an optimized in vitro culture period.
  • Dendritic cells are generated using controls (medium or Vitamin
  • D/dexamethasone and adalimumab and sia-adalimumab and used to prime autologous naive T cells for 5 days.
  • the non-adherent T cells are harvested and washed following a rest period and the percentage of regulatory T cells can be evaluated using flow cytometry.
  • the DC-primed T cells are incubated with CFSE-labelled autologous CD4+CD25- T cells on CD3 coated well plates in the presence of soluble CD28. After an optimized culture period, the percentage of T cell proliferation can be analyzed and the potential suppression of the tolerogenic primed T cell fraction can be evaluated. [00383] Results.
  • Dendritic cells co-incubated with sia-adalimumab with a tolerogenic phenotype will result in reduced T cell proliferation.
  • These adalimumab-specific T cells express markers of regulatory T cells such as FoxP3 and secrete IL-10.
  • Example 2F Reduced recognition of sia-adalimumab by adalimumab-specific T cells from healthy donors
  • PBMCs from healthy donors and/or adalimumab-treated IBD patients will be screened for adalimumab-specific T cells. Positive donors will be stimulated with increasing concentrations of adalimumab and sia-adalimumab. Frequencies of adalimumab-specific T cells will be determined.
  • PBMCs or CD4+ T cells + DCs from IBD patients with detectable anti-adalimumab antibodies will be stimulated in vitro with adalimumab and sia-adalimumab to measure the frequencies of adalimumab-specific T cells in several donors.
  • sia-adalimumab is expected to result in lower frequencies of adalimumab-specific T cells due to impaired antigen presentation in the assay. It may be important to control the tests with de-glycosylated sia- adalimumab.
  • BCR B cell receptor
  • B cell apoptosis assay in absence of BCR costimulation, was performed.
  • B cells isolated from PBMCs from 3 healthy blood donors were incubated for 16 hours with sia- HUMIRA (H-S), sia-adalinumumab (A-S), mock-treated HUMIRA (H-M), or IVIg, or left untreated for 16 hours.
  • Apoptosis and cell death was evaluated by flow cytometry using Hoechst 33342 and propidium iodide (PI) staining. Early apoptoctic cells would stain positive for Hoechst 33342 but negative for PI, while dead cells are double positives.
  • PI propidium iodide
  • HUMIRA-treated B cells showed a level of apoptotic cells (Hoechst 33342+ PI-) similar to untreated B cells ( Figure 16).
  • IVIg resulted in a decreased proportion of apoptotic cells in 2 out of 3 donors ( Figure 16) but an increased proportion of dead cells (not shown).
  • Sia-adalimumab triggered a statistically significant increase of apoptosis compared to no treatment and mock-treated HUMIRA (H-M) for the 3 donors tested (p ⁇ 0.0001).
  • Sia- HUMIRA triggered an increased apoptosis compared to mock-treated HUMIRA (H-M) on the 3 donors tested (p ⁇ 0.01) ( Figure 16).
  • CD22 binds to sialic acids in a2,6 linkages in cis, on the surface of the same B cell or in trans, on other cells or potentially the antigen molecule (sia-adalimumab).
  • Sialic acid-binding receptors CD22 and siglec-G described that the latter exerts its function mainly on Bl cells and influences their lifespan and antibody production while CD22 is the dominant regulator of calcium signaling on conventional B2 cells but also plays a role on marginal zone B cells (Jellusova J and Nitschke L. Regulation of B cell functions by the sialic acid-binding receptors siglec-G and CD22. Front. Immunol. 2:96 (2011)).
  • Detection of anti-drug-antibody specific cells can be done via ELISPOT but it should be noted that in the set-up described below, the in vivo effect of sialylated adalimumab is missing.
  • the frequency of anti-drug-antibody producing cells can be determined using B cells from IBD patients. To do this, a short in vitro activation of the memory B cells using the TLR7/8 agonist R484 is required. After that, antigen-specific B cells can be quantified using the ELISPOT method.
  • the adalimumab and sia-adalimumab can be either coated directly (Figure 10A) or used as a biotinylated antigen ( Figure 10B). The latter is preferred as with option A, a very high coating concentration is needed and the outcome depends on the coating efficiency of the test antigens which could be different for the normal and sia-adalimumab.
  • the ELISPOT wells are coated with an anti IgG antibody and incubated with the test cells. After that, the specific B cells are quantified after incubation with biotinylated test antigens, followed by incubation with Streptavidin- enzyme.
  • Example 2J Study with B cells from adalimumab-immunized mice
  • BALB/c and/or C57BE6 mice will be immunized with adalimumab and sia- adalimumab.
  • B-cell ELISPOTs will be performed to measure the frequency of anti-drug- antibody secreting B cells in peripheral blood and/or splenocytes by stimulation with
  • the B cell ELISPOT has been used frequently to assess antibody responses, mainly in the field of vaccination but also in the field of therapeutic antibodies. This technology has also been evaluated to compare with ADA responses in a therapeutic antibody setting. As shown in Figure 11, (Liao K., et al. Detection of Memory B Activity against a Therapeutic Protein in Treatment-Naive Subjects. AAPS J. 16;20(3):51 (2016)) showed a correlation between the frequency of Antibody Secreting Cells (ASC) detected by ELISPOT and the ADA levels.
  • ASC Antibody Secreting Cells
  • the T-cell recall assay aims at evaluating the reduction of immunogenicity by sialylated adalimumab in IBD patients in which adalimumab-specific T-cells have developed.
  • the assay will determine the frequency of adalimumab-specific T cells in blood samples from IBD patients with detectable anti-adalimumab antibodies. Treatment with sialylated adalimumab is expected to result in lower frequencies of adalimumab-specific T cells.
  • the assay has the potential to be translated into the clinic for future clinical studies.
  • Example 2N In vivo evaluation of the immune response after injection of sia- adalimumab
  • Adalimumab-specific ADA can be detected in WT mice after a single injection, showing the strong immunogenicity of adalimumab in WT mice.
  • the following experimental set-up will be performed:
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • FcRs Fc receptors
  • Effector cells that mediate ADCC includes natural killer (NK) cells, monocytes, macrophages, neutrophils, and eosinophils.
  • the Fc receptors displayed on the surface of effector cells bind the Fc region of immunoglobulins in a manner that may be influenced by the glycosylation profile of the Fc region and by the polymorphism of Fey receptors.
  • Two N-linked biantennary glycans are bound to the Fc region and the glycans in part direct ADCC through interaction with leukocyte receptors of the Fcgamma Receptor Ilia (Fcyllla).
  • ADCC is important for the efficacy of antibodies. In a therapeutic setting
  • ADCC levels may be suboptimal for treatment due to competition of nonspecific IgG for binding Fcyllla on effector cells.
  • Afucosylated monoclonal antibodies have improved Fcyllla binding (Luo C., et al. Glycoengineering of pertuzumab and its impact on the
  • the increase in binding to Fcyllla and ADCC stimulation may improve efficacy of anti-TNLa IgG in a therapeutic setting.
  • a hypo-fucosylated anti-TNLa showed increased efficacy (Bloemendaal LM et al. (2017) Anti-Tumor Necrosis Lactor with a Glyco-engineered Lc Region Has Increased Efficacy in Mice with Colitis.
  • Lurthermore, anti-TNLa antibodies induce regulatory macrophages in an Lc- dependent manner that supports gastro -intestinal wound healing (Vos, A., et al. Regulatory macrophages induced by infliximab are involved in healing in vivo and in vitro. Inflammatory bowel diseases 18:401-408 (2012)).
  • the improved efficacy and wound healing are both Fc- dependent effects that may be improved by glycoengineering.
  • a glycoengineered anti-TNFa could improve treatment for patients by multiple modes of actions.
  • ADCC method Standardized laboratory methods exist for measuring ADCC of TNFa-binding antibodies with transmembrane TNFa (tmTNFa)-expressing target cells (Arora T et al. Differences in binding and effector functions between classes of TNF antagonists. Cytokine 45:124-131 (2009)).
  • a standard ADCC assay consists of two steps, one where the ratios for the use of effector (E) and target cells are optimized, and an actual antibody dose study under optimized conditions. For the optimization, target antigen (mTNFa) positive cells (cells transfected with a non-cleavable form of mTNFa) are incubated with 10 pg/ml mAB test sample for 30 minutes at 37 °C.
  • Adalimumab elicited ADCC lysis on transmembrane TNFa expressing stable cell lines was used as positive control in each plate.
  • a colorimetric lactate- dehydrogenase (LDH) release assay (Roche-applied-science) can be used to collect data.
  • the optimized effector to target ratio was chosen based on the highest level of LDH release. With the optimized E/T ratio, a dose response study for the test sample was performed. The target cells were incubated with varying concentration of each test sample for 30 minutes at 37 °C. Effector cells (PBMCs or NK cells) were added to the target cell cultures followed by 6 hours incubation, after which supernatants were collected for measuring released LDH to calculate the specific ADCC lysis of target cells. Data analysis was be performed as described in the section of ADCC and CDC assays in the material and method.
  • ADCC Results We produced a sia-adalimumab antibody which is 100% afucosylated.
  • the afucosylated glycovariants A-M, A-G and A-S produced in Leishmania ( Figure 12A), all triggered significantly enhanced ADCC ascertainised by a lower ECso value compared to HUMIRA or H-M (mock treated HUMIRA) which as a high fucose content (Table 3 and Table 4).
  • the IgGl control (mabthera, anti-CD20) did not trigger ADCC.
  • the fold increased potency over the H-M reference was 9-fold for the Man3-adalimumab (A-M) and 13-fold with sialylated adalimumab (A-S) (Table 3).
  • the sialylation of adalimumab or of HUMIRA did not impair ADCC potency since sialylated HUMIRA (H-S) showed a similar ADCC potency compared to HUMIRA and A-S showed the highest increase in potency of the glycovariant tested in presence of NK cells (Table 4).
  • the increase in ADCC may improve efficacy especially in Inflammatory Bowel Diseases where the response to HUMIRA treatment has been proven to be Fc-dependent.
  • CDC Complement-dependent cytotoxicity
  • Clq initiates a cascade involving multiple complement proteins; C2, C3, C4, C5, C6, Cl, C8, C9 as well as cleaved forms and complexes of these.
  • Cl, C8 and C9 forms membrane attack complexes (MAC) that is inserted as a pore into the lipid bi-layer thereby lysing the cell.
  • MAC membrane attack complexes
  • Transmembrane TNFa expressing target cells were incubated with test samples and pooled normal human serum as the source of complement proteins. The CDC reaction was continued for 4 hours after which cell viability was quantified to analyze the specific CDC lysis of target cells. CDC elicited by HUMIRA mock treated for in vitro glycosylation (H-M) was used as the reference.
  • ADCP antibody-dependent cell-mediated phagocytosis
  • MO A Antibody-dependent cell-mediated phagocytosis
  • ADCP can be mediated by monocytes, macrophages, neutrophils and dendritic cells via FcyRIIa (CD32a), FcyRI (CD64), and FcyRIIIa (CDl6a).
  • FcyRIIa CD32a
  • FcyRI CD64
  • FcyRIIIa CDl6a
  • FcyRIIa is the predominant FcyR receptor involved in this process (Richards, JO., et al. Optimization of antibody binding to FcyRIIa enhances macrophage phagocytosis of tumor cells. Mol. Cancer Ther. 7, 2517-27 (2008); Dugast, A.S., et al.
  • Fc-Receptor expression on innate immune cells is associated with impaired antibody mediated cellular phagocytic activity in chronically HIV-l infected individuals.
  • ADCP ADCP-derived neurotrophic factor
  • ADCP Reporter Bioassay is a bio luminescent cell-based assay that overcomes the limitations of existing assays and can be used to measure the potency and stability of antibodies and other biologies with Fc domains that specifically bind and activate FcyRIIa. [00413] Results. In ADCP assays performed with afucosylated sialylated adalimumab higher levels of cell killing on target cells expressing mTNFa will be observed compared to adalimumab.
  • mAbs Monoclonal antibodies
  • PK pharmacokinetics
  • the PK of mAbs can be influenced by various factors.
  • One such factor is the glycosylation profile of the antibody. The glycosylation status can impact the half-life of the antibody (Luo C., et al. Glycoengineering of pertuzumab and its impact on the pharmacokinetic/pharmacodynamic properties. Scientific Reports. 11, 46347 (2017),“Glycoengineered binding protein compositions,” WO2015/073884 - US9550826).
  • Capping of glycans with sialic acid can extend the half-life. It is believed that the capping reduces the amount of protein removed from the circulation via a glycan dependent interaction with asialoglycoprotein receptors (ASGPRs) on hepatic cells (Seested T., et al. The unsialylated subpopulation of recombinant activated factor VII binds to the asialo glycoprotein receptor (ASGPR) on primary rat hepatocytes. Thromb Haemost. 104, 1166-73 (2010)). Improvement of PK and circulating half-life of a sialylated antibody is a benefit for patients that may allow a reduction of dosing frequency or dose compared with HUMIRA and other commercially available anti-TNFa antibodies.
  • ASGPRs asialoglycoprotein receptors
  • PK method Standardized in vivo methods exist for measuring PK (Deng R et al. (2010) Pharmacokinetics of humanized monoclonal anti-tumor necrosis factor- ⁇ alpha ⁇ antibody and its neonatal Fc receptor variants in mice and cynomolgus monkeys. Drug metabolism and disposition: the biological fate of chemicals 38:600-605). The objective is to compare in mice the serum pharmacokinetics of Adalimumab glyco variants. Mice receive a single i.v. bolus administration of equal amounts of different mAB test items followed by blood sampling at various time points. The collected serum samples will subsequently be tested for the remaining concentrations of the injected mAB using a quantitative ELISA.
  • mice are a commonly used species for PK studies and due to their lower bodyweight compared to other species require less material to achieve high drug quantity/unit bodyweight ratios.
  • the test dose will be selected to allow for optimal detection of the test items in the quantitative ELISA and to be in the same range as published in PK studies in mice with anti-TNF antibodies (Deng R., et al. Pharmacokinetics of humanized monoclonal anti-tumor necrosis factor- ⁇ alpha ⁇ antibody and its neonatal Fc receptor variants in mice and cynomolgus monkeys. Drug metabolism and disposition: the biological fate of chemicals 38:600-605 (2010)). The endotoxin level will be at a maximum of 50 EU/kg for the antibody variants. Since the serum half-life is considered independent of the dose, the comparison of determined half-lives remains valid.
  • Animal numbers and sampling for example, 9 animals per group will be needed for 3 time points, selected to allow for 3 animals per sampling time point with 2 sampling times per animal. From 3 animals of each group a blood sample of at least 300 m ⁇ will be drawn approximately 60 min prior to administration of the test items. Then, ah animals will receive a single i.v. bolus injection into the tail vein of 150 m ⁇ test item. 5 minutes, 60 minutes, 24 hours,
  • a blood sample from three animals per group will be drawn. Sufficient whole blood will be collected from the animals in order to obtain at least 2 x 75 pL serum/animal/sampling time. Blood is withdrawn from the retrobulbar venous plexus under light isoflurane anesthesia. After collection in serum separator tubes (Sarstedt), blood samples will be allowed to clot for approximately 0.5 hours at room temperature; then they will be centrifuged in order to obtain serum. Immediately after centrifugation the serum will be collected into micro tubes (0.5 mL with cap, Sarstedt) and stored at -80 °C until analysis. The collected serum samples will subsequently be tested for the concentrations of the test items by a quantitative ELISA.
  • the quantitative ELISA to determine adalimumab will be performed as described here at the website of assets.thermofisher.com/TFS-Assets/LSG/manuals/5l885.pdf.
  • the pharmacokinetic evaluation will be based on mean serum concentration values per time- point.
  • Non-compartmental analysis of serum adalimumab concentration over time following single exposure will be performed and the following Pk parameters will be determined:
  • PK results The high sialylation level of Sia-adalimumab prolongs PK compared with commercially available HUMIRA. This is a benefit for patients as dosing frequency and dose may be reduced with a longer acting antibody. 8.23 Example 4: Approach to address the reduced immunogenicity and antiinflammatory effects in a Phase I
  • Tumor necrosis factor alpha (TNFa) antibodies are successfully used in the therapy of inflammatory bowel disease (IBD). Nevertheless, 10-30% of patients are primary non responders and 20-50% of patients have a secondary loss of response (Lin N., et al. Chinese hamster ovary (CHO) host cell engineering to increase sialylation of recombinant therapeutic proteins by modulating sialyltransferase expression. Biotechnology progress 31 :334-346 (2015)).
  • One mechanism underlying a secondary loss of response is characterized by sub- therapeutic drug concentrations due to increase immune clearance. This is attributed to immunogenicity and to the development of antidrug antibodies (ADA)(Lin N., et al.
  • the groups will be compared for the generation of several relevant inflammation biomarkers such as CRP (C-reactive protein levels) and/or immune cell profiles (such as macrophage Ml -type in comparison to macrophage M2 -type, and/or T cells from blood or inflamed tissue).
  • CRP C-reactive protein levels
  • immune cell profiles such as macrophage Ml -type in comparison to macrophage M2 -type, and/or T cells from blood or inflamed tissue.
  • CRP C-reactive protein levels
  • immune cell profiles such as macrophage Ml -type in comparison to macrophage M2 -type, and/or T cells from blood or inflamed tissue.
  • sialylated afucosylated adalimumab may indicate an anti inflammatory activity of the sialylated afucosylated adalimumab.
  • Neu5Ac N-acetylneuraminate lyases
  • D-ManNAc N-acetylmannosamine
  • Neu5Ac lyase from E. coli can be used for the large-scale production of Neu5Ac from D-GlcNAc.
  • Neu5Ac synthases such as NeuB, can be used to catalyze the condensation of ManNAc onto phosphoenol pyruvate (PEP) and are directly involved in the biosynthesis of sialic acids (reviewed in Tanner, Martin E. The enzymes of sialic acid biosynthesis. Bioorganic chemistry 33 (3), pp. 216-228 (2005)).
  • the initial step in the mammalian sialylation pathway is the biosynthesis of the activated sugar nucleotide precursor CMP-Neu5Ac.
  • the action of at least four enzymes can be used: (1) GNE, a bifunctional enzyme, which catalyzes the conversion of UDP-GlcNAc to ManNAc and the phosphorylation of ManNAc to ManNAc-6-phosphate; (2) NANS, which condenses ManNAc-6-phosphate and phosphoenolpyruvate, resulting in Neu5Ac-9-phosphate; (3) a specific phosphatase acting on Neu5Ac-9-phosphate; and (4) a CMAS that activates the resulting primary sialic acid in the nucleus to CMP-Neu5Ac (Castilho, A., et al. I Planta Protein
  • Suitable sialyltransferases (from Table 2) can be recombinant ly expressed in
  • the semi-purified enzymes can be tested in vitro for their proper activity with co-factors and the activated sugar nucleotide precursor CMP-Neu5Ac.
  • the host cell can be engineered to express a functional CMP-sialic acid
  • CMP-Sia Mammalian biosynthesis and/or bacterial biosynthesis (Table 5) can be employed in L. tarentolae host cells. Once CMP-NeuAc is available in the Golgi of the L. tarentolae host cell, specific sialyltransferases can transfer sialic acid to the acceptor substrates (e.g. b 1 ,4-galactosylatcd, diantennary N-glycan). Table 2 indicates the mammalian and bacterial Sialyltransferase candidates.
  • the host cell was engineered to express a functional CMP-sialic acid biosynthesis pathway (Table 5) and a monoantennary galactosylated N glycan (“Gl-N”, names see Table 1), present on endogeneous proteins.
  • Strain Stl7l64 contains the following genetic elements encoding for human MGAT1 (C terminal 3x HA tag) and B4GalTl (C terminal strep tag) (Table 2) integrated into PTR1 locus with selection marker hyg, resulting into Gl-N as host N-glycans.
  • coding sequences for murine ST6 C terminal 3x HA tag
  • human CMAS N-terminally Strep tagged
  • human CST C terminal 3x flag tag
  • selection marker bsd Blasticidin S deaminase
  • coding sequences for human GNE sialuria point mutant N terminal 3x flag tag
  • human NANS C terminal 3x myc tag
  • human NANP N terminal 3x HA tag
  • Neu5Ac and CMP-Neu5Ac were harvested (4 OD per sample) and subjected to methanol-chloroform treatment (MeOH/chloroform/H20; 1 :0.54: 1) before the mixture was spun at 2200g and RT for 20 min. Half of the material of the aqueous-phase was collected and dried in a SpeedVac. Next, dried metabolites were resuspended in water and Neu5Ac was fluorescently labelled with 1, 2-diamino-4, 5methyleneoxybenzene (DMB) for 3 h at 50°C according to the manufacturer’s protocol (Takara #4400).
  • DMB 2-diamino-4, 5methyleneoxybenzene
  • the reaction was stopped through a 1 :20 dilution in water and subjected to RP-C18-LC analysis according to the application note from Waters using a Acquity UPLC BEH C18 column.
  • the RP-C18-LC trace of DMB-labelled extract from SU0569 (wt) is displayed as solid line and serves as negative control.
  • the traces of DMB-labelled Neu5Ac extracted from SU7078 and Stl 7164 are shown as dotted and dashed line, respectively.
  • Stl 7078 contains prokaryotic CMP-Neu5Ac biosynthesis components (Table 5) NeuC (C terminal 3x myc tag), NeuB (C terminal 3x myc tag), NeuA (C terminal 3x HA tag) coding sequences integrated into gp63 locus using pac selection marker. Furthermore the strains contains coding sequences for murine ST6 (C terminal 3x HA tag, Table 2) and human CST (C terminal 3x flag tag, Table 5) that were integrated with selection marker bsd into alpha tubulin locus, but no other recombinant glycosylatransferases (retaining an Man3 native N-glycan).
  • Stl 7078 surprisingly synthesizs 3x more Neu5Ac using prokaryotic enzymes than Stl 7164 containing eukaryotic biosynthesis enzymes. This trend was also observed in other strains where only Neu5 Ac biosynthesis genes were expressed without any N- glycosyltransferases (data not shown).
  • a method of engineering a CMP-Sia biosynthetic pathway into a non-human eukaryotic cell involves the cloning and expression of several enzymes of mammalian origin, bacterial origin or both, in a L. tarentolae host cell that lacks endogenous sialylation.
  • the engineered CMP-Sia biosynthetic pathway is useful for producing sialylated glyco lipids, O-glycans and N-glycans in vivo. This is thus useful for facilitating the generation of sialylated therapeutic glycoproteins in non-human host cells lacking endogenous sialylation.
  • the a 2,3- or a 2,6-sialyltransferase caps galactose residues with sialic acid in the trans-Golgi and trans Golgi network (TGN) of humans leading to a mature form of the glycoprotein.
  • TGN trans-Golgi and trans Golgi network
  • the following can be incorporated into the host cell (1) a 2,3- or a 2,6-sialyltransferase activity and (2) a sufficient supply of CMP-N-acetyl neuraminic acid, in the late Golgi.
  • the catalytic domain of a known sialyltransferase e.g. from mammalian or bacterial origin
  • transporters can be engineered to allow the transport of CMP-N-acetyl neuraminic acid into the same location of the secretory pathway (e.g. late Golgi). Consequently, to ensure the adequate supply of substrate for the corresponding glycosyltransferases, one can metabolically engineer the production of CMP-sialic acid into these host cells. All analysis can be done as described previously herein and in“Engineered and fully- functional customized glycoproteins,” WO 2019/002512. 8.25 Example 6: Glycosylation profile of commercial HUMIRA and Leishmania- produced glycoengineered adalimumab
  • Leishmania cells producing the natural, wild type Man3 and the in vivo engineered GO glycosylation profiles were used to produce glycosylated Adalimumab as described in Example 1E and Section 8.28. Resulting purified protein preparations were analyzed for their glycosylation profile, in particular for the presence/absence of high mannose glyco forms, and compared to commercial Hurmira produced in CHO cells ( Figure 12A-B, samples A-M, A-GO missing, H-M). N-glycan profiles of Leishmania derived- Adalimumab variants are all completely afucosylated, whereas the HUMIRA commercial comparator is majority core fucosylated.
  • the monoclonal antibody (mAh, e.g. HUMIRA (AbbVie) or Leishmania tarentolae derived adalimumab with GO N-glycans) was rebuffered to 30 mM MES buffer pH 6.5 using ZebaSpin columns (Thermo Fischer, US). The galactosylation and siatylation were performed in an l-pot reaction at 37 °C under mild rotation.
  • the galactosylation was carried out using a final concentration of 5 pg/pL mAh, 63 ng/pL human GALT1, 4mM MnCl2 (Sigma, Germany), 8mM UDP-Gal (Merck, Germany), 35 mM MES (Sigma, Germany) pH 6.5 for 14 hours. Thereafter, the siatylation was carried out supplementing the galactosylation reaction to a final concentration of 350 ng/pL human ST6, 21 ng/pL recombinant shrimp alkaline
  • phosphatase (NEB, UK), 0.1 mM ZnCl2 (Sigma, Germany), 4 mM CMP -NANA (Sigma, Germany), 22.3 mM MES (Sigma, Germany) pH 6.5.
  • Siatylation was carried out for 10 hours and subsequently boosted by supplementation with additional co-factors.
  • the volume of the boost is 19 % of the siatylation reaction and consists of co-factors adjusting the reaction to a final concentration of 5.3 mM CMP -NANA, 0.1 mM ZnCl2, 21 ng/pL recombinant shrimp alkaline phosphatase.
  • Boosted IVGE reaction is incubated for 14 h followed by Protein A capturing of the sialylated mAh.
  • any Leishmania derived antibody with Man3 N-glycans can be used for in vitro GlcNAc- extension.
  • This is exemplified by Rituximab containing Man3, which has been in vitro engineered, using the first step modification by human MGAT1. Reactions contained 1 mg/mL mAh, 25 pg/mL human MGAT1, 40 mM MnCl2, 12 mM UDP-GlcNAc, 25 mM Tris-HCl, 150 mM NaCl, pH 7.5 for 16 hours ( Figure 8B).
  • ProteinA sepharose (MabSelectSuRe, GE Healthcare) using FPLC (Bio-Rad NGC, Germany). Bound protein was washed with 25 CV binding buffer (20 mM phosphate buffer pH 7.2, 150 mM NaCl, all Sigma, Switzerland) and eluted with 10 CV elution buffer (100 mM acetic acid, 100 mM NaCl, pH 3.2, all Sigma, Switzerland). Elution fractions were neutralized immediately using 1M TrisHCl, pH 8. Alternatively HiTrap MabSelect PrismA column (GE) was used according to manufacturer’s recommendation.
  • Heparin was derived from an unpacked HiTrapTM Heparin HP, lml column
  • Heparin Sepharose 6 Fast Flow Heparin Sepharose 6 Fast Flow, GE Healthcare, .Resin was equilibrated 3x with Buffer A (10 mM sodium phosphate, pH 7) 100 m ⁇ Heparin resin (in 0.5ml Buffer A) was added to 45ml culture supemantant in batch and incubated at RT (rotating) for either 2 hours or overnight. The sample was centrifuged at 500 xg, 5 minuites and the SN was removed (flow-through).
  • Buffer A 10 mM sodium phosphate, pH 7
  • Heparin resin in 0.5ml Buffer A
  • the resin was then transferred to spin columns and washed with 3 x 5 CV of Buffer A, by centrifugation at 1000 xg, RT, with lmin between each step.
  • the first wash fraction was used to rinse remaining resin from the incubation tube. Elution was performed with 3 X 1 CV of Buffer B (10 mM sodium phosphate, 2M NaCl, pH 7). All elution fractions were pooled and re-buffered to PBS, pH 6 using a
  • N-glycan profile 20pg IgG were sampled and processed using the GlycoWorks RapiFluor-MS N-Glycan Kit (Waters, US) according to the manufacturer's instructions. Identification and quantification of the individual N-glycan structures was assessed based on mass confirmation (m/z) and peak area calculation, respectively.
  • (sialylated) mAh was diluted 1 : 10 in 20 mM ammonium bicarbonate buffer pH 8, supplemented with 5 units PNGaseF (Sigma, Switzerland) and incubated at 37 °C for 16 hours. Afterwards, 1 pg mAh from the control and the PNGaseF reaction were loaded on a precast 10% SDS-PAGE gel (ThermoFisher, US) and run under reducing conditions. The HC and the LC were separated in MES buffer at 200 V for 55 min.
  • TNFa 10-1000 ng TNFa (Abeam plc, AB 155699) was spotted and air dried on a nitrocellulose membrane and blocked o/n at 4 °C in 10% milk/PBST. Lanes were cut and incubated with 2 pg/ml Adalimumab LMTB or 2 pg/ml HUMIRA in 1% milk in PBST 3h 30°C. Negative control was incubated with 1 :2000 AntiHuman-IgG-HRP (#239) in 1% milk in PBST 3h 30°C.
  • the expression cassettes contain 1.) Homology sites for site specific integration by homologous recombination, 2.) an 5’ untranslated terminal repeats containing a splice leader acceptor sequence, 3.) the gene of interest as ORF that was codon usage optimized for Leishmania (either L. major or L. tarentolae ) 4.) an intergenic region that contains a 3’UTR that contains the polyadenylation sequence and a 5’ UTR for the downstream gene, for example 5.) the resistance marker, that is followed by 6.) its 3’ UTR and 7.) the 3’ homology region for site specific recombination into the genome.
  • restriction digest was performed until completion or o/n at 30°C and purified DNA by EtOH precipitation (2 volume 100% ice cold EtOH was added to 1 volume digested DNA, incubated 30 minutes on ice, centrifuged for 30 minutes l7’500 x g at 4°C. Pellet was washed with 70% EtOH, pellet was dried for maximum 15 minutes and resuspended in ddH20.
  • EtOH precipitation 2 volume 100% ice cold EtOH was added to 1 volume digested DNA, incubated 30 minutes on ice, centrifuged for 30 minutes l7’500 x g at 4°C. Pellet was washed with 70% EtOH, pellet was dried for maximum 15 minutes and resuspended in ddH20.
  • 1 or 2 restriction enzymes with recognition sites in the vector backbones were chosen and digest was done for around 1 hour at 37°C and purified by EtOH as described above.
  • Transformation of lOOng digested DNA, undigested control and ddH20 control in E. coli DH5 alpha was done by heat shock to check if there was remaining intact plasmid DNA.
  • Chemically competent DH5 alpha thawed on ice for 10-15 minutes were carefully mixed with 100 ng digested plasmid DNA, incubated on ice for 25-30 minutes followed by a heat shock at 42 °C for 90 seconds, and incubated on ice for 5 minutes lml LB or SOC media was added with incubation at 37 °C for 1 hour. Aliquots were then plated on LB with ampicillin and incubated o/n at 37 °C upside down.
  • the SN was removed and pellet resuspended in 5ml transfection buffer (200 mM Hepes pH 7.0, 137 mM NaCl, 5mM KC1, 0.7 mM Na2HP04, 6mM dextrose, anhydrous (glucose), sterile filtered 0.22um). Cells were centrifuged again and the pellet was resuspended in 400ul transfection buffer. 400ul of cells were added to the DNA and transferred into the cuvettes and incubate on ice for lOmin. Electroporation was performed with a Gene Pulser XcellTM (Biorad) using a low voltage protocol, exp.
  • 5ml transfection buffer 200 mM Hepes pH 7.0, 137 mM NaCl, 5mM KC1, 0.7 mM Na2HP04, 6mM dextrose, anhydrous (glucose), sterile filtered 0.22um. Cells were centrifuged again and the pellet was resus
  • Plasmids were derived from a pUC57 vector backbone for E. coli propagation and contained an ampicillin section marker.
  • the expression cassettes are flanked by restriction sites suitable for excision.
  • the cassettes contain 1.) Homology sites for site specific integration by homologous recombination, 2.) an 5’ untranslated terminal repeats containing a splice leader acceptor sequence, 3.) the gene of interest as ORF that was codon usage optimized for Leishmania (either L. major or L.
  • tarentolae 4.) an intergenic region that contains a 3’UTR that contains the polyadenylation sequence and a 5’ UTR for the downstream gene, for example 5.) the resistance marker, that is followed by 6.) its 3’ UTR and 7.) the 3’ homology region for site specific recombination into the genome.
  • GCA 22.791; GCC: 29.220; GCG: 34.130; GCT: 19.986; TGC: 13.986;
  • TGT 6.059; GAC: 30.120; GAT: 17.499; GAA: 15.444; GAG: 43.653; TTC: 17.287; TTT: 12.657; GGA: 8.405; GGC: 27.204; GGG: 11.971; GGT: 14.274; CAC: 19.637; CAT: 8.559; ATA: 3.929; ATC: 17.100; ATT: 10.524; AAA: 18.838; AAG: 26.306; CTA: 6.293; CTC: 23.191; CTG: 32.564; CTT: 14.172; TTA: 2.836; TTG: 12.837; ATG: 23.282; AAC: 20.217; AAT: 7.912; CCA: 13.041; CCC: 12.627; CCG: 20.543; CCT: 10.758; CAA: 9.847; CAG: 30.402; AGA: 3.857; AGG: 6.046; CGA: 8.962; CGC: 26.3
  • Samples were prepared using the Waters RapiFluor labelling kit, mostly following the Waters Application Note: «Quality control and Automation Friendly Glyco Works RapiFluor-MS N-Glycan Sample Preparation». Briefly, for purified protein samples 10 m ⁇ of sample at a concentration of 1.5 mg/ml (15 pg) were used. To each sample 10 m ⁇ of 3% RapiGest were added before denaturing them for 3 minutes at 100 °C, kept for 3 minutes at RT before adding 10 m ⁇ PNGase F (30 m ⁇ PNGase + 220 m ⁇ water) and incubated for 5 minutes at 50°C for glycan release.
  • PNGase F 30 m ⁇ PNGase + 220 m ⁇ water
  • the UPLC was directly connected to a Waters ESI-Q-TOF Synapt G2Si for mass determination that was run in resolution mode. Masses were acquired in the positive ion mode between m/z 300 and 3500 applying a lock mass spray (Leucine Enkephaline) for lock mass correction.
  • the source was set to a capillary voltage of 2.2 kV, cone voltage of 75V, Source offset of 50 V, source temperature of l20°C and desolvation temperature of 500°C, with a desolvation gas flow of 600 Eh. Data were analyzed using MassLynx and UNIFI (both
  • Samples were prepared following the Waters Application Note: «Quality control and Automation Friendly GlycoWorks RapiFluor-MS N-Glycan Sample Preparation». Briefly, sample amount was 10 m ⁇ , 1.5 mg/ml (15 pg) for purified protein samples or 10*8 cells as pellets. 3% RapiGest was added for 3 minutes at 100 °C, 3 minutes at RT before adding 10 m ⁇ PNGase F (Sigma, diluted 30 m ⁇ PNGase + 220 m ⁇ water) and incubated for 5 minutes at 50°C. After 3 minutes at RT 10 m ⁇ RFMS (9 mg in 110 m ⁇ DMF anhydrous) was added and incubated for 5 minutes at RT.
  • PNGase F Sigma, diluted 30 m ⁇ PNGase + 220 m ⁇ water
  • the UPLC was directly connected to a Waters Q-TOF Synapt HDMS for mass determination in ESI mode. Masses were acquired in the positive ion mode between m/z 300 and 3500 using a lock mass spray (Leucine Enkephaline) for lock mass correction.
  • a lock mass spray Leucine Enkephaline
  • Buffers A: 50 mM AmFor (H20) pH 4.5, B: CAN; Flow: 0.5ml/min; Temp: 45°C; Inj.: 10 m ⁇ (Full Loop Injection); Gradient: 80-73%B in 3’, 73-63% B in 32’ (total 55min), LC method ESI_RFMS_mAB_55_FLR; Synapt settings: 20l6l006_uba284_esi_RFMS), MS method ESI_RFMS_mAB_300_3500_vpos_55; Cap voltage: 3kV; Cone voltage: 80V; Source temp: l20°C; Desolv temp: 350°C; Desolv gas: 800l/h; Lock Mass: Leucine Enkephaline lng/ul at flow rate 4uL/min; Fluorescence detection: Ex 265/Em 425 nm (RapiFluor-MS) (2Hz). 8.26.15 N-glycan profiling of crude cell pellets by per
  • Proteins of the cell pellets were extracted by 3 x 90 sec of sonication in 200 m ⁇ 50 mM ammonium bicarbonate buffer containing 0.1% Rapigest® SF (WATERS) and 10 mM DTT. Proteins were denatured at 56 °C for 45 minutes and alkylated by iodoacetamide (50 mM) in the dark for 60 minutes at room temperature. The samples were then incubated for 16 h, at 37°C with 10 pg of trypsin/Lys-C Mix Mass Spec Grade (PROMEGA) to obtain the digested proteins.
  • WATERS Rapigest® SF
  • N-glycans were purified on Ultra Clean SPE Carbograph (ALLTECH). The
  • the purified permethylated glycans were solubilized in 20 m ⁇ of 50:50 methanol/water. 2 m ⁇ of non-diluted and 1/2 diluted N-glycans were mixed with 2 m ⁇ of 2,5 DHB (LaserBiolabs) matrix solution (10 mg/ml 50:50 methanol/water). Positive ion reflectron MALDI-TOF mass spectra were acquired using an Autoflex III mass spectrometer (Bruker). The spectra were obtained by accumulation of 4000 shots and were calibrated with an external standard (Pepmix4 LaserBiolabs). The acceleration and reflector voltage conditions were set up as follows: voltage 10.3 x 1954V, 80% laser.
  • HA-Purification procedure was as follows. Culture of recombinant cells expressing heterologous Gnts was first analyzed for intracellular expression of the target HA- tagged Gnt enzyme lxl 0 9 cells were harvested and centrifuged (2000 x g / 5 min) to separate cell pellets for affinity purification. Pellet was resuspended in 1 mL Extraction Buffer (25 mM Tris pH 7.5, 100 mM NaCl, 1% v/v Triton X-100, Protease inhibitors without EDTA [Roche], 1 mM PMSF) containing 1% (v/v) Triton.
  • Extraction Buffer 25 mM Tris pH 7.5, 100 mM NaCl, 1% v/v Triton X-100, Protease inhibitors without EDTA [Roche], 1 mM PMSF
  • the lysate was mixed 1 :2 with cold PBS (containing lx Protease inhibitor tablets) and incubated with lOOpL anti-HA-magnetic beads [Thermo Scientific] at RT, 600rpm for 30min.
  • HA-magnetic beads were harvested in 2mL tube using magnetic rack [Thermo Scientific] and washed twice with 2mL ice cold TBS.
  • the elution was carried out with 70 pL Elution buffer (2mg/mL HA peptide [Thermo Scientific] in TBS). The elution fractions were analyzed on SDS-PAGE followed by anti-HA WB and used for in vitro activity assay.
  • StrepTactin-Purification procedure was as follows: Culture of recombinant cells expressing heterologous Gnts was analyzed for intracellular expression of the target Strep- tagged Gnt enzyme. 1x109 cells were harvested and centrifuged (2000 x g / 5min) to separate cell pellets for affinity purification. Pellet was resuspended in 1 mL Extraction Buffer containing 1% (v/v) Triton. Resuspended cells were sonicated on ice avoiding any kind of overheating! Sonication [Bandelin Sonopuls] was carried out in 3 steps for 20 sec, 70% power input, 7 cycles. Mix vigorously on vortexer for 10 sec.
  • the suspension of disrupted cells was centrifuged at 13’ 000 x g at 4 °C for lh. Supernatant (lysate) was carefully removed and used for purification. The remaining solid fraction was resuspended in lx Laemmli and used for SDS-PAGE analysis. The lysate was mixed 1 :5 with cold PBS (containing lx Protease inhibitor tablets) and incubated with lOOpL StrepTactin sepharose [VWR] at RT, 600rpm for 30min. 5-fold dilution in PBS was performed to reduce the interfering effect of cellular/media biotin on the StrepTactin purification efficacy.
  • StrepTactin sepharose was collected in 2mL tubes through centrifugation (2000 x g / lmin) and washed twice with 2mL cold TBS. The elution was carried out with 70 pL Elution buffer (2.5mM Desthiobiotin in lxTBS). The elution fractions were analyzed on SDS-PAGE followed by anti-Strep WB and used for in vitro activity assay.
  • SDS PAGE and Capillary Gel Electrophoresis SDS PAGE was performed under reduced or non-reducing conditions using lOug for Coomassie, 2.5ug for WB, separated on 4-12% Gel with MOPS buffer for 55min. Determination of Protein purity was determined by Coomassie Stained SDS-PAGE with 10 ug protein sample and compared to a BSA standard curve. Impurities were quantified by ImageQuant. Capillary Gel Electrophoresis (CGE) was performed using an Agilent Protein 230 Kit (5067-1518), according to protocol.
  • Analytical SEC MAbPac SEC-l (4x300 mm) is a size exclusion chromatography (SEC) column specifically designed for separation and characterization of monoclonal antibodies (mAbs) and was used according to manufacturer’s recommendation (Temperature: 30 °C; Eluent: PBS 50mM NaP0 4 , 300 mM NaCl pH 6.8; Elution: isocratic, 30 min; Flow: 0.2 mL/min; Detection: 2l5nm; Injection V: 5 uL corresponding to 5 ug protein).N- glycan profiling using Rapifluor (RF) labelling and UPLC-MS
  • RF Rapifluor
  • the UPLC was directly connected to a Waters ESI-Q-TOF Synapt HDMS for mass determination. Masses were acquired in the positive ion mode between m/z 300 and 3500 applying a lock mass spray (Leucine Enkephaline) for lock mass correction.
  • the source was set to a capillary voltage of 2.2 kV, cone voltage of 75V, source temperature of l20°C and desolvation temperature of 350°C, with a desolvation gas flow of 800 l/h. Data were analyzed manually using MassLynx.
  • the IgG (200pg/40pl) was suspended in 200 m ⁇ of 50 mM phosphate buffer pH 7.5 and denatured in 0.5% sodium dodecyl sulfate (SDS Sigma L4509) and 1% b
  • Deglycosylation was controlled by electrophoresis. 6.5 m ⁇ of sample before and after
  • N-glycans were purified on Hypercarb Hypersep 200 mg (Thermo Fisher).
  • the SPE was equilibrated in 0.1% TFA before loading the PNGase released N-glycans and washed with 0.1% TFA. After elution with 3 ml of 25% acetonitrile, 0.1% TFA, the N-glycans were lyophilized before permethylation. Permethylation using about 25 mg of sodium hydroxide, 500 m ⁇ DMSO and 300 m ⁇ ICH3 was performed on the lyophilized samples during 40 min. After quenching the reaction with 1 ml of water, 3 x 500 m ⁇ of chloroform was used for the extraction of the permethylated glycans. The chloroform phase was washed with equal volumes of water then dried. The reaction products were loaded on C18 SepPak 200 mg (WATERS) and eluted in 2 ml 80% acetonitrile and lyophilized before MALDI-TOF MS analysis.
  • WATERS C18 SepPak 200 mg
  • the purified permethylated glycans were solubilized in 20 m ⁇ of 50:50 methanol/water. 2 m ⁇ of non-diluted and 1/2 diluted N-glycans were spotted with 1 m ⁇ of CHCA (LaserBiolabs) matrix solution (7 mg/ml 50:50 acetonitrile/water). Positive ion reflectron MALDI-TOF mass spectra were acquired using an Autoflex III mass spectrometer (Bruker). The spectra were obtained by accumulation of 4000 shots and were calibrated with an external standard (Pepmix4 LaserBiolabs). The acceleration and reflector voltage conditions were set up as follows: voltage 14.7 x 2008V, 80% laser.
  • [M+Na]+ was performed using EXPAZY GlycoMod tool. Relative intensities (%) of N-glycans were calculated to establish N-glycan profiles for each spectrum. For this the sum of intensities of deisotoped N-glycan peaks was determined and set as 100%. The relative intensity (%) of each glycan was then determined in relation to this value.
  • Raji cells were used for anti-CD20 antibody test. There were 5 different concentrations foreseen, using 10 microgram/ml as the expected optimal concentration for flow cytometry (Table 8). Staining sequence: - FcR blocking, - IgGl blocking, - Primary antibody, - Secondary antibody anti-IgGl APC. After gating, each sample represents analysis of 10000- 13000 cells. Control IgGl antibody also gives some signal. Reasons could be insufficient blocking and binding through Fc-receptors, or binding through its specificity - the specificity of the control antibody is unknown, although it is commonly used for this purpose. Blocking controls show that FcR blocking on its own actually causes a low background signal, reversed again by the second blocking step with unlabeled anti-human-IgGl (not shown).
  • Cells were resuspended in 20 m ⁇ wash buffer per 2 x 10 6 cells, 2 m ⁇ secondary antibody anti-human-IgGl -APC was added according to samples schedule below, incubated for 10 minutes in the refrigerator in the dark, washed by adding 0.7 ml wash buffer per 10 7 cells, cells spun 5 minutes at 250 x g, supernatant was removed completely. Cells were resuspended in 300 m ⁇ wash buffer, kept on ice until analysis by flow cytometry.
  • Reagents were used as follows: FcR Blocking Reagent, Miltenyi 130-059-901, Lot 5170330778; anti-human-IgGl pure (100 pg in 1 ml), Miltenyi 130-093-197, Lot
  • Lanes washed, 3x5min PBST and incubated with Anti-Human- IgG-HRP (1 :2000) or anti-mouse for lh at RT in PBST 1% Milk. Lanes were washed for 3x5min with PBST and blot was developed using TMB.
  • Intact mass determination The deglycosylated and reduced antibody was used for intact mass determination of its subchains after dilution to ca. 1 pmol/pl (1 :50) with 0.5% ACN, 0.5% FA and 5 m ⁇ were applied to the mass spectrometer by a LC-system. Detection was done with the LTQ and FT detector of a Thermo Scientific Orbitrap XL mass spectrometer. Charge deconvolution was done using the Znova algorithm.
  • Adalimumab pools were adjusted with Tris and (NH 4 ) 2 S0 4 and loaded on a
  • SDS PAGE and Capillary Gel Electrophoresis SDS PAGE was performed under reduced or non-reducing conditions using lOug for Coomassie, 2.5ug for WB, separated on 4-12% Gel with MOPS buffer for 55minutes . Determination of Protein purity was determined by Coomassie Stained SDS-PAGE with 10 ug protein sample and compared to a BSA standard curve. Impurities were quantified by ImageQuant. Capillary Gel Electrophoresis (CGE) was performed using an Agilent Protein 230 Kit (5067-1518), according to protocol.
  • Analytical SEC MAbPac SEC-l (4x300 mm) is a size exclusion
  • SEC chromatography
  • Kd equilibrium dissociation constant
  • Anti-TNF alpha antibodies derived from Leishmania either with native glycan (Man3) and in vitro extended sialylated N-glycan structure were compared to HUMIRA and the in vitro sialylated HUMIRA. Additionally pHrodo labelled antibodies were tested for its affinity to TNFa.
  • a Biacore T200 instrument was used at analysis temperature of 25°C, with a flow rate of 50 m L/m i n for kinetic interaction analyses, using an analysis buffer consisting of 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween.
  • an analysis buffer consisting of 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween.
  • covalent immobilization of rh-TNFa to sensor surface was performed with a qualitative test of binding of the anti-TNF mAbs in flow phase.
  • For the quantitative analyses sample were measured three times in two weeks on 2 different chips.
  • CHO/DG44-tmTNFa cells were used as target cells and primary NK cells or PBMCs were effector cells.
  • Samples A-M, A-G, A-S, H-M, H-S, MabThera (Rituxan) were used for ADCC potency evaluation at 10 pg/ml (Saturation) with different E/T Ratio (10:1, 5:1 and 1 :1 with NK cells and 10:1, 25:1 and 50:1 for PBMCs)) at first.
  • the dose responses for ADCC potency were evaluated with E:T Ratio of 5 : 1 for NK cells and 25 : 1 with PBMCs, according to the E:T ratio optimized assay results.
  • the mTNF expressing cells were incubated with antibody dilutions, prepared in ADCC medium for about 0.5 hour in a cell incubator (37°C, 5% C0 2 ).
  • the effector cells were added to reach the desired E:T ratiovand the plates were further incubated for 6 hours in a cell incubator.
  • Maximum release and minimum release wells were included. Maximum release indicates conditions where the target cells were treated with Triton lysis buffer. The minimum release indicates condition in which the target cells were treated only with ADCC buffer.
  • the plates were then centrifuged and the assay supemantants were transfered to a new plate.
  • the FDH content in the supernatants was measured using FDH cytotoxicity kit (Roche, ref 11644793001) according to manufacturer’s instructions.
  • the Optical density (OD) was read at 492 (detection) and 650 nm (background wavelength).
  • the background (OD650nm) was subtracted from the OD492nm.
  • the target cells (CHO/DG44-tmTNFa cells) were transfered in 384-well plates in assay medium.
  • the test antibody dilutions (performed in assay medium) were added and the plates were incubated for about 0.5 hour in a cell incubator (37°C, 5% C0 2 ).
  • the test antibodies were evaluated at 50, 10, 2, 0.4, 0.08, 0.016, 0.0032 and 0.00064 pg/mL.
  • a solution of pooled human normal serum (PNHS) was added to reach a final concentration of 10% PNHS (vol/vol).
  • PNHS is the source of human complement.
  • Sample represents the condition target cells + PNHS + antibody.
  • B cells were isolated from PBMCs by negative magnetic selection and seeded at 50 000 cells/ wells in RPMI1640 supplemented with 10% heat- inactivated FCS, 2 mmol/L L-glutamine, 200 U/mL penicillin, and 1 mg/mL streptomycin in a 96-well round-bottom well. Cells were incubated 30 minutes before addition of HUMIRA, Sia- HUMIRA and Sia-Adalimumab at 100 Lig/ml. Test products were then added, and cells were incubated for 16 hours at 37°C and 5% C02.
  • Monocytes were isolated by positive CD 14+ magnetic separation and cultured for five days in serum- free medium supplemented with interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) and 5% of inactivated human AB serum.
  • IL-4 interleukin 4
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • TAPI-2 granulocyte-macrophage colony-stimulating factor

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Abstract

La présente invention concerne des procédés de production et le produit résultant d'un anticorps monoclonal ayant un profil de glycosylation spécifique permettant d'obtenir une efficacité améliorée. L'impact des nouvelles caractéristiques structurelles et de l'homogénéité sur l'efficacité est obtenu par une sélection d'essais mécanistiques in vitro, in vivo et ex vivo, en particulier par la mesure de la capacité à réduire la quantité d'anticorps anti-médicament provoquée par l'anticorps monoclonal, et d'autre part, par la capacité à induire d'autres effets connus dans l'état de la technique pour l'anticorps particulier. De telles modifications de glycane améliorent les traitements actuels et permettent une meilleure qualité de vie pour des patients.
PCT/EP2019/064484 2018-06-04 2019-06-04 Anticorps monoclonal glycomodifié WO2019234021A1 (fr)

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Cited By (7)

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WO2021140143A1 (fr) * 2020-01-07 2021-07-15 Limmatech Biologics Ag Glyco-ingénierie à l'aide de cellules de leishmania
WO2022053673A1 (fr) 2020-09-14 2022-03-17 Limmatech Biologics Ag Sialylation fab d'anticorps
WO2022200388A1 (fr) * 2021-03-23 2022-09-29 Glycoera Ag Dégradation de protéine médiée par le mannose 3 glycane
WO2022221163A1 (fr) * 2021-04-16 2022-10-20 Merck Sharp & Dohme Llc Remodelage glycane en phase solide de glycoprotéines
WO2022229854A1 (fr) * 2021-04-29 2022-11-03 Novartis Ag Cellules hypersialylées
WO2022200390A3 (fr) * 2021-03-23 2022-11-10 Glycoera Ag Dégradation de protéine à médiation par glycane
WO2024218743A1 (fr) * 2023-04-21 2024-10-24 Glycoera Ag Molécules multifonctionnelles comprenant des glycanes et leurs utilisations

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021140143A1 (fr) * 2020-01-07 2021-07-15 Limmatech Biologics Ag Glyco-ingénierie à l'aide de cellules de leishmania
WO2022053673A1 (fr) 2020-09-14 2022-03-17 Limmatech Biologics Ag Sialylation fab d'anticorps
WO2022200388A1 (fr) * 2021-03-23 2022-09-29 Glycoera Ag Dégradation de protéine médiée par le mannose 3 glycane
WO2022200390A3 (fr) * 2021-03-23 2022-11-10 Glycoera Ag Dégradation de protéine à médiation par glycane
WO2022221163A1 (fr) * 2021-04-16 2022-10-20 Merck Sharp & Dohme Llc Remodelage glycane en phase solide de glycoprotéines
WO2022229854A1 (fr) * 2021-04-29 2022-11-03 Novartis Ag Cellules hypersialylées
WO2024218743A1 (fr) * 2023-04-21 2024-10-24 Glycoera Ag Molécules multifonctionnelles comprenant des glycanes et leurs utilisations

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