WO2023154948A1 - Agents du récepteur 4 de type b de l'éphrine (ephb4) et leur production - Google Patents

Agents du récepteur 4 de type b de l'éphrine (ephb4) et leur production Download PDF

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WO2023154948A1
WO2023154948A1 PCT/US2023/062554 US2023062554W WO2023154948A1 WO 2023154948 A1 WO2023154948 A1 WO 2023154948A1 US 2023062554 W US2023062554 W US 2023062554W WO 2023154948 A1 WO2023154948 A1 WO 2023154948A1
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asn
fusion protein
ephb4
polypeptide
seq
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PCT/US2023/062554
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English (en)
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Valery Krasnoperov
Parkash Gill
Jon COGAN
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Vasgene Therapeutics Inc.
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Publication of WO2023154948A1 publication Critical patent/WO2023154948A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • EPHRIN TYPE-B RECEPTOR 4 (EPHB4) AGENTS AND PRODUCTION THEREOF
  • the present disclosure relates, inter alia, to compositions of fusion proteins of soluble Ephrin type- B receptor 4-HSA (sEphB4-HSA) and methods of manufacturing the same.
  • Cancer is a constellation of diseases characterized by abnormal cell growth with the potential to invade or spread to other parts of the body. While there have been advances in treatment, e.g. using checkpoint inhibition, there remains a need for further agents to allow for more diverse therapies, e.g. for those that do not respond, or respond poorly, to current therapies (indeed, estimates indicate that the majority of cancer sufferers do not respond to checkpoint inhibitor drugs).
  • Soluble Ephrin type-B receptor 4-HSA is a particularly exciting new agent that has shown promise in the clinic across a variety of cancers.
  • sEphB4-HSA is a recombinant fusion protein composed of an extracellular domain (soluble) of human receptor tyrosine kinase ephrin type-B receptor 4 (sEphB4) and human serum albumin (HAS).
  • sEphB4-HSA has potential antineoplastic and anti-angiogenic activities.
  • Efnb2 It functions as a decoy receptor for the membranebound ligand Ephrin-B2 (Efnb2) and interferes with the binding of Efnb2 to its native receptors, including EphB4 and EphA3. This may result in a reduction of angiogenesis and a reduction in cell growth of Efnb2 and/or EphB4 over-expressing tumor cells. In addition, this agent also prevents angiogenic effects of numerous growth factors due to interactions between Efnb2 and EphB4. Efnb2 and EphB4 are overexpressed in a variety of tumor cell types and the bi-directional signaling of Efnb2-EphB4 plays an important role in angiogenesis and tumor cell migration, invasion, and proliferation.
  • Ephrin-B2 Ephrin-B2
  • manufacture of sEphB4-HSA is a multi-step process that could benefit from streamlining to improve economic considerations for the potential pharmaceutical product. Also, manufacturing processes may effect post-translation modifications of the protein biologic, which could be detrimental or beneficial.
  • composition comprising an isolate of a recombinant fusion protein comprising a soluble Ephrin type-B receptor 4 (EphB4) polypeptide and a heterologous polypeptide, wherein the isolate predominantly comprises soluble EphB4 with substantial N-glycosylation at one or more asparagine residues.
  • EphB4 soluble Ephrin type-B receptor 4
  • the glycosylation is or comprises sialylation.
  • the soluble EphB4 polypeptide has substantial glycosylation at Asn 203, Asn 335, Asn 410, and/or Asn 426, or positions equivalent thereto, relative to SEQ ID NO: 1 numbering.
  • the soluble EphB4 polypeptide has increased glycosylation at Asn 203, Asn 335, Asn 410, and/or Asn 426, or positions equivalent thereto, relative to SEQ ID NO: 1 numbering, compared to a equivalent EphB4 fusion protein not produced using a perfusion-based upstream process.
  • the soluble EphB4 polypeptide has increased glycosylation at Asn 203, Asn 335, Asn 410, and/or Asn 426, or positions equivalent thereto, relative to SEQ ID NO: 1 numbering, compared to a equivalent EphB4 fusion protein produced using a fed batch-based upstream process.
  • the fusion protein comprises one or more of the glycan species of FIG. 5. In embodiments, the fusion protein has more of species 2021, 2022, 2121, and/or 2122 of FIG. 5 than an equivalent EphB4 fusion protein produced using a fed batch-based upstream process. In embodiments, the fusion protein has less of species 2020, 2110, 2120, and/or 3130 of FIG. 5 than a comparable EphB4 fusion protein produced using a fed batch-based upstream process.
  • a method of making the fusion protein described herein comprising, in order, obtaining a cell comprising a nucleic acid encoding a recombinant fusion protein comprising a soluble Ephrin type-B receptor 4 (EphB4) polypeptide and a heterologous polypeptide; expanding a culture of the cell; producing the recombinant fusion protein in culture using a perfusion-based method; and isolating the recombinant fusion protein.
  • EphB4 Ephrin type-B receptor 4
  • step (c) comprises alternating tangential -flow (ATF) filtration.
  • step (c) does not comprise a fed batch method.
  • step (c) is conducted in a production bioreactor having a volume of about 1000 L or more.
  • the method maintains glucose concentration at between about 1.5 g/L to about 2.0 g/L in step (c).
  • the method maintains dissolved oxygen at about 25% or greater in step (c).
  • composition comprising a pharmaceutically acceptable excipient or carrier, and the fusion protein described herein.
  • a method of treating or preventing cancer comprising administering an effective amount of the fusion protein described herein to a subject in need thereof.
  • FIG. 1 shows the present upstream production process for manufacture of sEphB4-HSA.
  • FIG. 2 shows a non-limiting schematic of glycosylation events in sEphB4-HSA (for reference, Asn 188 is Asn 203, Asn 320 is Asn 335, and Asn 395 is Asn 410, with reference to SEQ ID NO: 1).
  • FIG. 3 shows a HPLC run of aN-Glycan analysis comparing the product of the present production method (upper curve at SI and S2 on X-axis) as compared to an older production method (bottom curve at SI and S2 on X-axis).
  • FIG. 4 shows a linked Glycan analysis by HPLC of the present purification process (“Perfusion”) and a prior process (“Fed Batch”).
  • FIG. 5 shows a non-limiting schematic of various possible glycosylation patterns. These glycan structures were identified by LC/MS and 2-AA (see Example 2).
  • FIGS. 6A-6B shows pharmacokinetic studies in humans using sEphB4-HSA generated via the present production method.
  • FIG. 7A-7G shows pharmacokinetic studies in humans using sEphB4- HSA generated via a prior production method.
  • the present disclosure relates to the finding that alterations to an upstream manufacturing process of a fusion protein, e.g., sEphB4-HSA, yields a product having different biochemical properties, e.g. glycosylation, that provides for improved pharmacokinetics relative to prior version of the fusion protein.
  • a fusion protein e.g., sEphB4-HSA
  • composition comprising an isolate of a recombinant fusion protein comprising a soluble Ephrin type-B receptor 4 (EphB4) polypeptide and a heterologous polypeptide, wherein the isolate predominantly comprises soluble EphB4 with substantial N- glycosylation at one or more asparagine residues.
  • EphB4 soluble Ephrin type-B receptor 4
  • composition comprising a pharmaceutically acceptable excipient or carrier, and the fusion protein described herein.
  • the soluble EphB4 comprises an extracellular domain of EphB4, or a functional fragment thereof.
  • the soluble EphB4 comprises an amino acid sequence that is at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% or 100% identity to SEQ ID NO: 1, or a functional fragment thereof.
  • a functional variant of a soluble EphB4 polypeptide comprises an amino acid sequence that is at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% or 100% identical to residues 1-197, 29-197, 1-312, 29-132, 1-321, 29-321, 1-326, 29-326, 1-412, 29-412, 1-427, 29-427, 1-429, 29-429, 1-526, 29-526, 1-537 and 29-537 of the amino acid sequence of SEQ ID NO: 1.
  • a soluble EphB4 polypeptide comprises an amino acid sequence that is at least 90%, or at least about 95%, or at least about 97%, or at least about 99% or 100% identical to residues 16-197, 16-312, 16-321, 16-326, 16-412, 16-427, 16-429, 16-526 and 16-537 of the amino acid sequence of SEQ ID NO: 1.
  • a functional variant of a soluble EphB4 polypeptide comprises an amino acid sequence that is at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% or 100% identical to residues 1-197, 16-197, 29-197, 1-312, 16-312, 29-312, 1-321 , 16-321, 29-321, 1-326, 16-326, 29-326, 1-412, 16-412, 29-412, 1-427, 16-427, 29-427, 1-429, 16-429, 29- 429, 1-526, 16-526, 29-526, 1-537, 16-537 and 29-537 of SEQ ID NO: 1
  • the fragment is or comprises an amino acid sequence of residues 1-197, 29-197, 1-312, 29-132, 1-321, 29-321, 1-326, 29-326, 1-412, 29-412, 1-427, 29-427, 1-429, 29-429, 1-526, 29-526, 1-537, or 29-537 relative to SEQ ID NO: 1, or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% or 100% identity thereto.
  • the fragment is or comprises an amino acid sequence of residues 16-197, 16-312, 16-321, 16-326, 16-412, 16-427, 16-429, 16-526, or 16-537537 relative to SEQ ID NO: 1, or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% or 100% identity thereto.
  • the soluble EphB4 polypeptide comprises an amino acid sequence of the globular (G) domain (amino acids 29-197 of SEQ ID NO: 1), the cysteine-rich domain (amino acids 239- 321 of SEQ ID NO: 1), the first fibronectin type 3 domain (amino acids 324-429 of SEQ ID NO: 1), and/or the second fibronectin type 3 domain (amino acids 434-526 of SEQ ID NO: 1).
  • residues 1-15, relative to SEQ ID No: 1 are removed from the soluble EphB4 polypeptide.
  • residues 198-978, 313-978, 322-978, 327-978, 413-978, 428-978, 430-978, 527- 978 or 538-978, relative to SEQ ID No: 1 are removed from the soluble EphB4 polypeptide.
  • residues 1-15, relative to SEQ ID NO: 1 are removed from the soluble EphB4 polypeptide and residues 198-978, 313-978, 322-978, 327-978, 413-978, 428-978, 430-978, 527- 978 or 538-978, relative to SEQ ID NO: 1 are removed from the soluble EphB4 polypeptide.
  • SEQ ID NO: 1 shows SEQ ID NO: 1 with the first 15 amino acids, which are, in embodiments, removed in italics and underlining and four Asn residues (Asn203, Asn335, Asn 410, and Asn426), which are important with regard to glycosylation, in bold and underlining.
  • the soluble EphB4 polypeptide comprises an amino acid sequence that is at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% or 100% identical to SEQ ID NO: 2:
  • one or more amino acids of a sequence described herein is substituted with a naturally occurring amino acid, such as a hydrophilic amino acid (e.g. a polar and positively charged hydrophilic amino acid, such as arginine I or lysine (K); a polar and neutral of charge hydrophilic amino acid, such as asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine I, a polar and negatively charged hydrophilic amino acid, such as aspartate (D) or glutamate I, or an aromatic, polar and positively charged hydrophilic amino acid, such as histidine (H)) or a hydrophobic amino acid (e.g.
  • a hydrophilic amino acid e.g. a polar and positively charged hydrophilic amino acid, such as arginine I or lysine (K); a polar and neutral of charge hydrophilic amino acid, such as asparagine (N), glutamine (Q), serine (
  • a hydrophobic, aliphatic amino acid such as glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), or valine (V)
  • a hydrophobic, aromatic amino acid such as phenylalanine (F), tryptophan (W), or tyrosine (Y) or a non-classical amino acid (e.g. selenocysteine, pyrrolysine, N-formylmethionine P-alanine, GABA and 6-Aminolevulinic acid.
  • 4-Aminobenzoic acid PABA
  • D-isomers of the common amino acids 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, s-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, P- alanine, fluoro-amino acids, designer amino acids such as P methyl amino acids, C a -methyl amino acids, N a -methyl amino acids, and amino acid analogs in general).
  • PABA 4-A
  • the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions. “Conservative substitutions” may be made, for instance, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the amino acid residues involved.
  • the 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.
  • “conservative substitutions” are exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the so modified polypeptide.
  • glycine and proline may be substituted for one another based on their ability to disrupt a-helices.
  • “non-conservative substitutions” are exchanges of an amino acid by another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.
  • heterologous polypeptide is or comprises an albumin protein or fragment thereof.
  • albumin protein or fragment thereof is or comprises mature human serum albumin (HS A) or fragment thereof.
  • the albumin protein or fragment thereof is or comprises an amino acid sequence of SEQ ID NO: 3 or a fragment thereof, or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% or 100% identity thereto:
  • the albumin protein or fragment thereof is or comprises an amino acid sequence of SEQ ID NO: 4 or a fragment thereof, or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% or 100% identity thereto:
  • albumin protein or fragment thereof is or comprises an amino acid sequence of residues 25-609 of SEQ ID NO: 4 or a fragment thereof, or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% or 100% identity thereto:
  • disclosed herein are one or more (e.g. about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or at least about 9, or about 10, or about 15, or about 20, or about 30) substitutions to a sequence described herein or a sequence with at least about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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, 99.5, 99.8, or 99.9% identity to a sequence described herein (or about 70%, or about 75%, or about 80%, or about 85%, or about 90, or about 95%, or about
  • one or more amino acids of a sequence described herein is substituted with a naturally occurring amino acid, such as a hydrophilic amino acid (e.g. a polar and positively charged hydrophilic amino acid, such as arginine I or lysine (K); a polar and neutral of charge hydrophilic amino acid, such as asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine I, a polar and negatively charged hydrophilic amino acid, such as aspartate (D) or glutamate I, or an aromatic, polar and positively charged hydrophilic amino acid, such as histidine (H)) or a hydrophobic amino acid (e.g.
  • a hydrophilic amino acid e.g. a polar and positively charged hydrophilic amino acid, such as arginine I or lysine (K); a polar and neutral of charge hydrophilic amino acid, such as asparagine (N), glutamine (Q), serine (
  • a hydrophobic, aliphatic amino acid such as glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), or valine (V)
  • a hydrophobic, aromatic amino acid such as phenylalanine (F), tryptophan (W), or tyrosine (Y) or a non-classical amino acid (e.g. selenocysteine, pyrrolysine, N-formylmethionine P-alanine, GABA and 6-Aminolevulinic acid.
  • 4-Aminobenzoic acid PABA
  • D-isomers of the common amino acids 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, s-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, P- alanine, fluoro-amino acids, designer amino acids such as P methyl amino acids, C a -methyl amino acids, N a -methyl amino acids, and amino acid analogs in general).
  • PABA 4-A
  • the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions. “Conservative substitutions” may be made, for instance, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the amino acid residues involved.
  • the 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.
  • “conservative substitutions” are exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the so modified polypeptide.
  • glycine and proline may be substituted for one another based on their ability to disrupt a-helices.
  • “non-conservative substitutions” are exchanges of an amino acid by another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.
  • the soluble EphB4 polypeptide has substantial glycosylation at one or more of (e.g. about 1 to about 25, or about 5 to about 20, or about 10 to about 15, or about 10 to about 20, or about 15 to about 25, or about 15 to about 20, or about 3, or about 4, or about 5, or about 10, or about 15, or about 20, or about 25): Asn 22, Asn 133, Asn 160, Asn 203, Asn 265, Asn 291, Asn 295, Asn 335, Asn 410, Asn 426, Asn 490, Asn 568, Asn 600, Asn 675, Asn 685, Asn 698, Asn 708, Asn 745, Asn 749, Asn 751, Asn 768, Asn 826, Asn 831, Asn 862, Asn 880, and Asn 891, or positions equivalent thereto, relative to SEQ ID NO: 1 numbering.
  • the soluble EphB4 polypeptide has substantial glycosylation at Asn 203, Asn 335, Asn 410, and/or Asn 426, or positions equivalent thereto, relative to SEQ ID NO: 1 numbering.
  • the soluble EphB4 polypeptide has substantial glycosylation at all of Asn 203, Asn 335, and Asn 410.
  • the soluble EphB4 polypeptide has substantial glycosylation at all of Asn 203, Asn 335, Asn 410, and Asn 426. In embodiments, the soluble EphB4 polypeptide has increased glycosylation at Asn 203, Asn 335, Asn 410, and/or Asn 426, or positions equivalent thereto, relative to SEQ ID NO: 1 numbering, compared to an equivalent EphB4 fusion protein produced without using a perfusion-based upstream process.
  • the soluble EphB4 polypeptide has increased glycosylation at Asn 203, Asn 335, Asn 410, and/or Asn 426, or positions equivalent thereto, relative to SEQ ID NO: 1 numbering, compared to a equivalent EphB4 fusion protein produced using a fed batch-based upstream process.
  • the glycosylation and/or sialylation is increased by at least about 3-fold to at least about to 5-fold at Asn 203, Asn 335, Asn 410, and/or Asn 426, or positions equivalent thereto, relative to SEQ ID NO: 1 numbering, compared to an equivalent EphB4 fusion protein produced using a fed batch-based upstream process
  • the glycosylation and/or sialylation is increased by about 3-fold at Asn 203, or positions equivalent thereto, relative to SEQ ID NO: 1 numbering, compared to an equivalent EphB4 fusion protein produced using a fed batch-based upstream process.
  • the glycosylation and/or sialylation is increased by about 5-fold at Asn 335, or positions equivalent thereto, relative to SEQ ID NO: 1 numbering, compared to a comparable EphB4 fusion protein produced using a fed batch-based upstream process.
  • the glycosylation and/or sialylation is increased by about 5-fold at Asn 410, or positions equivalent thereto, relative to SEQ ID NO: 1 numbering, compared to an equivalent EphB4 fusion protein produced using a fed batch-based upstream process.
  • the glycosylation comprises the addition of one or more of hexoses (e.g. galactose), mannoses, N-acetylglucosamines, fucoses, and sialic acids.
  • hexoses e.g. galactose
  • mannoses e.g. mannoses
  • N-acetylglucosamines e.g. mannoses
  • fucoses e.g. fucoses
  • the glycosylation comprises the addition of a glycan structure which comprises one or more antennae or lacks one or more antennae.
  • the glycosylation comprises the addition of a glycan structure which comprises one or more core fucose molecules or lacks one or more core fucose molecules. In embodiments, the glycosylation comprises the addition of a glycan structure which comprises one or more terminal hexoses (e.g. galactose) or lacks one or more terminal hexoses (e.g. galactose).
  • a glycan structure which comprises one or more terminal hexoses (e.g. galactose) or lacks one or more terminal hexoses (e.g. galactose).
  • the glycosylation comprises the addition of a glycan structure which comprises one or more terminal mannoses or lacks one or more terminal mannoses.
  • the glycosylation comprises the addition of a glycan structure which comprises one or more terminal N-acetylglucosamines or lacks one or more terminal N-acetylglucosamines.
  • the glycosylation comprises the addition of a glycan structure which comprises one or more terminal fucoses or lacks one or more terminal fucoses.
  • the glycosylation comprises the addition of a glycan structure which comprises one or more terminal sialic acids or lacks one or more terminal sialic acids.
  • the glycosylation is or comprises sialylation.
  • the glycosylation comprises the addition of a glycan structure in which all sugars are saturated by sialic acid.
  • the soluble EphB4 polypeptide has a protein: sialic acid ratio about 1 to about 12, or about 1 to about 10, or about 1 to about 11, or about 1 to about 13, or about 1 to about 14.
  • the fusion protein comprises one or more of the glycan species of FIG. 5.
  • the fusion protein has more of species 2021, 2022, 2121, and/or 2122 of FIG. 5 than an equivalent EphB4 fusion protein produced using a fed batch-based upstream process.
  • the fusion protein has less of species 2020, 2110, 2120, and/or 3130 of FIG. 5 than a comparable EphB4 fusion protein produced using a fed batch-based upstream process.
  • the fusion protein has more of species 2021 and/or 2022 of FIG. 5 at Asn 203, or positions equivalent thereto, relative to SEQ ID NO: 1 numbering, compared to an equivalent EphB4 fusion protein produced using a fed batch-based upstream process.
  • the fusion protein has less of species 2020 of FIG. 5 at Asn 203, or positions equivalent thereto, relative to SEQ ID NO: 1 numbering, compared to an equivalent EphB4 fusion protein produced using a fed batch-based upstream process. In embodiments, the fusion protein has more of species 2121 and/or 2122 of FIG. 5 at Asn 335, or positions equivalent thereto, relative to SEQ ID NO: 1 numbering, compared to an equivalent EphB4 fusion protein produced using a fed batch-based upstream process.
  • the fusion protein has less of species 2120 of FIG. 5 at Asn 335, or positions equivalent thereto, relative to SEQ ID NO: 1 numbering, compared to an equivalent EphB4 fusion protein produced using a fed batch-based upstream process.
  • the fusion protein has more of species 2121 and/or 2122 of FIG. 5 at Asn 426, or positions equivalent thereto, relative to SEQ ID NO: 1 numbering, compared to an equivalent EphB4 fusion protein produced using a fed batch-based upstream process.
  • the fusion protein has less of species 2110, 2120, and/or 3130 of FIG. 5 at Asn 426, or positions equivalent thereto, relative to SEQ ID NO: 1 numbering, compared to an equivalent EphB4 fusion protein produced using a fed batch-based upstream process.
  • the heterologous polypeptide is at the C-terminus and the soluble EphB4 is at the N-terminus.
  • HSA is at the C-terminus and the soluble EphB4 is at the N-terminus.
  • the heterologous polypeptide is at the N-terminus and the soluble EphB4 is at the C-terminus.
  • HSA is at the N-terminus and the soluble EphB4 is at the C-terminus.
  • the heterologous polypeptide (e.g., without limitation, HSA) is covalently attached the soluble EphB4.
  • the covalent attachment is achieved by expression of the sEphB4 polypeptide as a co-translational fusion with the heterologous polypeptide (e.g., without limitation, HSA).
  • the heterologous polypeptide (e.g., without limitation, HSA) sequence may be fused at the N-terminus, the C-terminus or at a non-disruptive internal position in the sEphB4 polypeptide.
  • exposed loops of sEphB4 are positions for insertion of the heterologous polypeptide (e.g., without limitation, HSA) sequence.
  • the heterologous polypeptide (e.g., without limitation, HSA) is post-translationally attached to the sEphB4 polypeptide by, for example, chemical cross-linking.
  • the sEphB4 polypeptide is stably associated with more than one heterologous polypeptide (e.g., without limitation, HSA).
  • the fusion protein is a monomer. In embodiments, the fusion protein exhibits enhanced in vivo stability compared to an unfused extracellular domain of an EphB4 protein or an equivalent EphB4 fusion protein lacking substantial N-glycosylation at one of more asparagine residues and/or produced using a fed batchbased upstream process.
  • the fusion protein exhibits enhanced pharmacokinetics compared to an unfused extracellular domain of an EphB4 protein or an equivalent EphB4 fusion protein lacking substantial N-glycosylation at one of more asparagine residues and/or produced using a fed batchbased upstream process.
  • the fusion protein exhibits an enhanced half-life compared to an unfused extracellular domain of an EphB4 protein or an equivalent EphB4 fusion protein lacking substantial N-glycosylation at one of more asparagine residues and/or produced using a fed batchbased upstream process.
  • the fusion protein exhibits an enhanced half-life compared to an unfused extracellular domain of an EphB4 protein or an equivalent EphB4 fusion protein lacking substantial N-glycosylation at one of more asparagine residues and/or produced using a fed batchbased upstream process, without substantial loss of Ephrin B2 binding.
  • the fusion protein exhibits an enhanced half-life compared to an equivalent EphB4 fusion protein lacking substantial N-glycosylation at one of more asparagine residues and/or produced without using a perfusion-based upstream process.
  • the fusion protein exhibits an enhanced half-life compared to an equivalent EphB4 fusion protein lacking substantial N-glycosylation at one of more asparagine residues and/or produced without using a perfusion-based upstream process, without substantial loss of Ephrin B2 binding.
  • the fusion protein exhibits a half-life of greater than about 7 days in humans.
  • the fusion protein exhibits a half-life of about 10 to about 20 days in humans.
  • the fusion protein exhibits a half-life of about 15 to about 20 days in humans.
  • the fusion protein exhibits a half-life of about 17 days in humans.
  • the fusion protein inhibits interaction between EphB4 and Ephrin B2. In embodiments, the fusion protein inhibits or reduces signaling that results from interaction between EphB4 and Ephrin B2.
  • the fusion protein inhibits clustering of Ephrin B2 or EphB4.
  • the fusion protein inhibits phosphorylation of Ephrin B2 or EphB4.
  • a method of making the fusion protein described herein comprising, in order, obtaining a cell comprising a nucleic acid encoding a recombinant fusion protein comprising a soluble Ephrin type-B receptor 4 (EphB4) polypeptide and a heterologous polypeptide; expanding a culture of the cell; producing the recombinant fusion protein in culture using a perfusion-based method; and isolating the recombinant fusion protein.
  • EphB4 Ephrin type-B receptor 4
  • step (b) comprises at least five expansions, of increasing culture volume.
  • step (c) comprises alternating tangential -flow (ATF) filtration. In embodiments, step (c) does not comprise a fed batch method.
  • step (c) is conducted in a production bioreactor having a volume of about 1000 L or more. In embodiments, step (c) is conducted in a single-use bioreactor (S.U.B.). In embodiments, step (c) lasts about 18 to about 22 days (e.g. about 19 to about 21 days, or about 19, or about 20, or about 21 days). In embodiments, in step (c), temperature is shifted from about 37°C to about 34°C on about day 2 of production. In embodiments, the cell viability during step (b) is greater than about 85%.
  • the method maintains glucose concentration at between about 1.5 g/L to about 2.0 g/L in step (c). In embodiments, the method maintains dissolved oxygen at about 25% or greater in step (c).
  • the method comprises one or more, or all of, the steps of FIG. 1. In embodiments, the method comprises one or more, or all of, the steps of
  • cancer is bladder cancer.
  • the cancer is head neck cancer.
  • the cancer is Kaposi’s sarcoma.
  • the cancer is liver cancer, optionally hepatocellular carcinoma (HCC).
  • the cancer is prostate cancer.
  • the cancer is pancreatic cancer.
  • the cancer is ovarian cancer.
  • the cancer is glioblastoma.
  • the cancer is parotid gland tumor.
  • the cancer is uveal melanoma.
  • the cancer is myelodysplastic syndrome (MDS).
  • the method further comprises administering one or more checkpoint inhibiting agents.
  • the checkpoint inhibiting agent is an agent that modulates one or more PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, and LAG-3.
  • the checkpoint inhibiting agent is an antibody or antibody format specific for one of PD-1, PD-L1, PD-L2, CTLA-4, TIM- 3, and LAG-3.
  • the antibody or antibody format specific for PD-1 is selected from nivolumab, pembrolizumab, and pidilizumab.
  • the antibody or antibody format specific for PD-L1 is selected from atezolizumab, avelumab, durvalumab, and BMS-936559.
  • the antibody or antibody format specific for CTLA-4 is selected from ipilimumab, tremelimumab, AGEN1884, and RG2077.
  • composition described herein for use in the treatment of one or more of a cancer.
  • composition described herein for the manufacture of a medicament for treating a cancer.
  • kits containing the present compositions, or the compositions and one or more other addition agents that are synergistic in action, in one or more containers.
  • Kits containing the pharmaceutical compositions of the invention are also provided in embodiments.
  • the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the materials, compositions, devices, and methods of this technology.
  • the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.
  • a new cell culture process was developed using MCB50-0019 cells.
  • the cells were cultured and expanded in an animal source-free growth medium containing HyClone Media (Cytiva, Cat# SH30933.06), GS supplement (Cytiva, Cat #SH30586.03), and Cell Boost 2 Supplement (Cytiva, Cat # SH30596.04).
  • HyClone Media HyClone Media
  • GS supplement Cytiva, Cat #SH30586.03
  • Cell Boost 2 Supplement Cell Boost 2 Supplement
  • the production of sEphB4-HSA was done in perfusion mode in 250L single-use bioreactor (S.U.B.).
  • Step 1 Thawing Cells
  • a vial from the master cell bank (approximately l.OxlO 7 cells/mL) was thawed at 37°C in a water bath.
  • the cells were transferred to 10 mL pre-warmed Growth Medium in 15 mL centrifuge tube. Cells were centrifuged at 200g for 5 minutes, re-suspended with 10 mL fresh pre-warmed Growth Medium, and then transferred to a 125 mL Erlenmeyer flask containing 15 mL pre-warmed fresh medium.
  • the flask was gently mixed and incubated at 37°C, 5% CO2, without agitation for a day. On the second day, the flask was shaken at 115 rpm on an orbital shaker for 3 days.
  • the final cell density was approximately > 1.5 x 10 6 cells/mL.
  • Step 2a 1st Expansion, 125 mL Erlenmeyer Flask
  • the culture from Step 1 was aseptically expanded into a 125 mL Erlenmeyer flask to give approximately 40 mL working volume using Growth Medium. Cells were seeded at approximately 4 x 10 5 cells/mL and placed back on orbital shaker for 3 days. The final cell density was approximately 2.9 - 4.0 x 10 6 cells/mL.
  • Step 2b 2nd Expansion, 1 L Spinner Flask
  • the culture from Step 1 was aseptically expanded into a 1 L Erlenmeyer flask to give approximately 250 mL working volume using Growth Medium. Cells were seeded at approximately 4 x 10 5 cells/mL and placed back on stirrer for 3 days. The final cell density 3 was approximately 2.5 - 5.0 x 10 6 cells/mL.
  • Step 3 3rd Expansion, 3L Spinner Flasks
  • the culture from Step 2 was aseptically expanded into two 3 L Spinner flasks to give approximately 1.4 L working volume in each flask using Growth Medium. Cells were seeded at approximately 4 x 10 5 /mL and placed back on stirrer for 2 days. The final cell density was approximately 2.3 - 4.5 x 10 6 cells/mL.
  • Step 4 4th Expansion, 50 L Seed Bioreactor
  • the culture from Step 3 was further expanded into a 50 L Stirred Tank Single-Use bioreactor to give approximately 30 L working volume.
  • Cells were seeded at approximately 0.25 xlO 6 /mL and cultured in the following conditions: temperature: 37°C, aeration: up to 0.3 Lpm; CO2: Auto; pH: 7.1; Agitation: 90 RPM. Cells were allowed to grow to a cell density of approximately 2.5 - 5.0 x 10 6 cells/mL. The entire process duration was about 4 days.
  • Step 5 5th Expansion, 250 L Production Bioreactor
  • the culture from Step 4 was transferred into 250 L bioreactor to give approximately 250 L working volume.
  • Cells were seeded at approximately 0.5 x 10 6 cells/mL and cultured in the following conditions: temperature: 37°C, aeration: 4-5 Lpm; CO2: Auto; pH: 7.1; Agitation: 53 - 71. Three days after seeding was designated as day 1 of production.
  • Step 6 Production, 250 L Production Bioreactor
  • Perfusion was conducted with an ATF 6 Unit, with filter 0.2 pm HFM. Temperature was shifted from 37°C to 34°C on day 2 of production. Run was terminated when the viability dropped below 85% (Usually Day 19-21 of production).
  • sEphB4-HSA After upstream processing, downstream (recovery and purification) of sEphB4-HSA was conducted as before. Specifically, the process involved steps for concentration/diafiltration, chromatographic purification steps, viral inactivation, detergent removal, virus filtration, and formulation. Purified sEphB4-HSA was characterized.
  • Example 2 Glycosylation Patterns sEphB4-HSA generated using the upstream process of Example 1 was analyzed to determine a its glycosylation pattern. For comparison, sEphB4-HSA material generated with a fed-batch-based upstream process (and same downstream) was also generated and analyzed.
  • Asn 188 is Asn 203
  • Asn 320 is Asn 335
  • Asn 395 is Asn 410
  • Asn 411 is Asn 426 with reference to SEQ ID NO: 1.
  • FIG. 2 shows a non-limiting schematic of glycosylation events in sEphB4-HSA.
  • FIG. 3 shows a HPLC run of aN-Glycan analysis comparing the product of the present production method (upper curve at SI and S2 on X-axis) as compared to an older production method (bottom curve at SI and S2 on X-axis).
  • FIG. 4 shows a linked Glycan analysis by HPLC of the present purification process (“Perfusion”) and a prior process (“Fed Batch”). N-Glycans were released from sEphB4-HSA by PNGase and analyzed. Note: The longer retention time, the heavier the sialyation and glycosylation pattern of protein. Precise ID of each peak is under mapping.
  • FIG. 5 shows a non-limiting schematic of various possible glycosylation patterns as identified by LC/MS and 2-AA.
  • TABLE E shows the pattern at Asn 188 (Asn 203 with reference to SEQ ID NO: 1), see FIG. 5 to correlate the “Glycan” to a structure.
  • FIGs. 6A-6B show pharmacokinetic studies in humans using sEphB4-HSA generated via the present production method. Specifically, the concentration of sEphB4-HSA in patient serum is shown, using a dose of 10 mg/kg.
  • PK data of two human patients FIG. 6A and FIG. 6B are provided and illustrate level of drug (mg/L) after first intravenous (iv) infusion (from 0.5 h to 96 h), followed by level of drug before second iv (Pre) and right after (Post). The same is shown for the for third, fourth and fifth intravenous (iv) infusion. There is no “29 Days Post” samples of both patients. Both patients demonstrated drug accumulation.
  • FIG. 7A-7G shows pharmacokinetic studies in humans using sEphB4-HSA generated via a prior production method. Specifically, pharmacokinetic studies are shown. The drug dose given was 10 mg/kg for all patients. Dots illustrate the date of intravenous (iv) drug administration and blue bars illustrate the titer of sEphB4-HSA in ug/mL of serum. There were a few skips in drug administration schedules.

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Abstract

La divulgation concerne la fabrication d'une protéine hybride recombinante composée du domaine extracellulaire pleine longueur (soluble) du récepteur 4 de type B de l'éphrine de la tyrosine kinase du récepteur humain (sEphB4) et de l'albumine sérique humaine (HSA) et des formes glycosylées résultantes.
PCT/US2023/062554 2022-02-14 2023-02-14 Agents du récepteur 4 de type b de l'éphrine (ephb4) et leur production WO2023154948A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180339020A1 (en) * 2015-10-27 2018-11-29 The University Of Queensland Method of treatment and agents useful for same
WO2020190977A1 (fr) * 2019-03-17 2020-09-24 Vasgene Therapeutics Inc Traitement de cancers à l'aide des protéines de fusion sephb4-hsa

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180339020A1 (en) * 2015-10-27 2018-11-29 The University Of Queensland Method of treatment and agents useful for same
WO2020190977A1 (fr) * 2019-03-17 2020-09-24 Vasgene Therapeutics Inc Traitement de cancers à l'aide des protéines de fusion sephb4-hsa

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