EP3676286A1 - Protransducine-d : activateur du transfert de gènes amélioré - Google Patents

Protransducine-d : activateur du transfert de gènes amélioré

Info

Publication number
EP3676286A1
EP3676286A1 EP18769599.4A EP18769599A EP3676286A1 EP 3676286 A1 EP3676286 A1 EP 3676286A1 EP 18769599 A EP18769599 A EP 18769599A EP 3676286 A1 EP3676286 A1 EP 3676286A1
Authority
EP
European Patent Office
Prior art keywords
ala
glu
pro
gly
ile
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP18769599.4A
Other languages
German (de)
English (en)
Inventor
Wolf-Georg Forssmann
Rudolf Richter
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pharis Biotec GmbH
Original Assignee
Pharis Biotec GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pharis Biotec GmbH filed Critical Pharis Biotec GmbH
Publication of EP3676286A1 publication Critical patent/EP3676286A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present application relates to an improved enhancer of gene transfer, protanducin D (PTD-D), an enhancement of the transduction enhancers PTD-A and PTD-B, its polypeptide, an N-terminally protected polypeptide, a pharmaceutical composition containing the polypeptide A polypeptide for use in gene therapy, a method of enhancing the infection of a cell with a genetic engineering construct, a use of the polypeptide for amplification for transfection.
  • PTD-D protanducin D
  • HI viruses preincubated with various concentrations (1-100 pg / ml) of protransduce A show a multiple log-level increase in the rate of reporter cell infection compared to the gold standard "retronectin The mechanism of action was thought to be that EF-C forms fibrillar structures capable of binding, concentrating, and multiplying viruses, in addition to efficiently infecting virus particles Transduction of lentiviral and retroviral particles in various human cell types (T cells, glial cells, fibroblasts, hematopoietic cells)
  • Protransducin A is also the subject of EP 2 452 947 A1.
  • Cysteine is expected to belong to the group of polar neutral amino acids.
  • the cysteine represents an amino acid, its modification or even substitution by another amino acid, especially from another group of amino acids is not considered.
  • the amino acids Tyr, Asp, Ser, Gly, Gin, Thr are counted to the group of polar but neutral amino acids.
  • the skilled person would shy away from an exchange with amino acids from other groups because the effects on the secondary structure of the exchange-modified peptide can not be readily estimated.
  • one skilled in the art would not consider replacing the Cys of protanduzine with amino acids from the group of nonpolar hydrophobic amino acids.
  • a modified protanduzine which on the one hand has a comparable activity to PTD-A as a transduction enhancer, is more stable on storage and ensures easier handling when used as a transduction enhancer, which represents a serious advantage for GMP production.
  • polypeptide according to the invention has at least 80% or 90% sequence agreement, in particular 95% sequence identity to the sequence Z 1 Gln-Ala-Lys-Ile-Lys-Gln-Ile-Ile-Asn-Met-Trp-Gln-Z 2 .
  • Z 1 are each independently the N-terminal end of the polypeptide or independently of one another the amino acids Leu or Ser or the following peptides Ser-Asn, Ser-Asn-Asn, Ser-Asn-Asn-Ile, Ser-Asn-Asn Ile-Thr, Thr-Leu, Ile-Thr-Leu, Asn-Ile-Thr-Leu, Asn-Asn-Ile-Thr-Leu, or Ser-Asn-Asn-Ile-Thr-Leu,
  • Each Z 2 independently represents the C-terminal end of the polypeptide or independently of one another the amino acids Gly or Glu or the following peptides Glu-Val, Glu-Val-Gly, Glu-Val-Gly-Lys, Glu-Val-Gly-Lys-Ala, Glu-Val-Gly-Lys-Ala-Met, Glu-Val-Gly-Lys-Ala Met-Tyr, Glu-Val-Gly-Lys-Ala-Met-Tyr-Ala, Glu-Val-Gly-Lys-Ala-Met-Tyr-Ala-Pro, Glu-Val-Gly-Lys-Ala-Met- Tyr-Ala-Pro-Pro, Glu-Val-Gly-Lys-Ala-Met-Tyr-Ala-Pro-Ile, Glu-Val-Gly-Lys-Ala-Met-Tyr-Ala-Pro Ile-Glu, Glu-Val-G
  • the improvement achievable by the polypeptide according to the invention is accompanied by an increased stability of the transduction enhancer, which can be used for therapeutic purposes.
  • Efficient gene transduction into cells for therapeutic use may e.g. For example, reduce the amount of viral particles needed for gene transduction. Furthermore, the number of infection cycles necessary for efficient transduction can be reduced.
  • the polypeptide according to the invention as a transduction enhancer By means of the polypeptide according to the invention as a transduction enhancer, the duration of in vitro cultivation for the multiplication of the gene-modified cells, the amount of cells to be removed by the patient (eg by leukapheresis) can be reduced and, if appropriate, an efficient and non-toxic in vivo gene transduction-by reduction virus load in vivo. Furthermore, the rapid handling of an efficient transduction enhancer reduces the stress on the cells to be transduced.
  • the polypeptide according to the invention allows a better prediction of the activity as a transduction hen, even after prolonged storage of the substance.
  • the polypeptide of the invention also allows the production of larger peptide batches, which can be stored for a longer time, in comparison to PTD-A.
  • the polypeptide is at least 80% sequence-consistent, more preferably 90% sequence-to-sequence
  • polypeptide according to the invention is also understood to mean those related polypeptides which are formed by variation of the amino acids in the polypeptide chain of the polypeptide according to the invention but which still have a comparable and sufficient activity which can be determined, for example, in the following bioassay.
  • the transduction efficiency can be z. Using primary, activated CD4 + / CD8 + enriched T cells cultured for 3 days with CD3 / CD28 beats as target cells and lentiviral and retroviral vectors encoding Green Fluorescent Protein (GFP). PTD-D can z. B. are tested against PTD-A and retronectin as the gold standard of Transdutechnischsenhencer.
  • PTD-A and D is tested in an assay e.g. B. used in a concentration of 25 pg / ml.
  • the target cells are used in particular in a concentration of 10 3 to 10 6 cells / ml.
  • the batch is incubated and then the cells are washed. Then the cells z. B. cultured for a further 4 days.
  • the proportion of GFP + T cells is determined by means of flow centrifugation as well as cell number and vitality.
  • a homologous polypeptide is, in particular, a polypeptide which is related to the sequence of the polypeptide according to the invention and in which exchanges or deletions of amino acids have been carried out to the extent mentioned.
  • substitutions of amino acids having similar properties for example similar polarities, are possible.
  • exchanges of arginine and lysine, glutamic acid and aspartic acid, glutamine, asparagine and threonine, glycine, alanine and proline, leucine, isoleucine and valine, tyrosine, phenylalanine and tryptophan as well as serine and threonine are widely used.
  • Position 1 of the sequence preferably contains the amino acid glutamine.
  • Position 3 and 5 are preferably basic amino acids, preferably lysine.
  • positions 1, 4, 6, 7, 8, 9, 10, 11, and 12 are mostly neutral amino acids.
  • N- and / or C-terminal the sequence may be extended or shortened.
  • N-terminally the sequence of the monomer may be extended by C-terminal portions or the entire amino acid sequence N H2-Ser-Asn-Asn-Ile-Thr-Leu-COOH.
  • C-terminal the sequence of the monomer may be extended by N-terminal portions or the entire amino acid sequence NH 2 -Glu-Val-Gly-Lys-Ala-Met-Tyr-Ala-Pro-Pro-Ile-Glu-Gly-COOH.
  • the polypeptides according to the invention have in common that they form insoluble aggregates in aqueous solutions.
  • the monomers consist of 4 to 25 amino acids, but preferably from 10 to 20 amino acids.
  • homologous molecules have in common that they form insoluble aggregates in aqueous solutions and increase the transduction of target cells with lentiviral or retroviral vectors.
  • the N-terminal end of the amino acid chain forming the polypeptide according to the invention is modified with a chemical group which is selected from the group consisting of one or two alkyl groups, such as methyl, ethyl, propyl or butyl groups, an acyl group such as an acetyl or propionyl group or the amino acid pyroglutamic acid, which forms the N-terminal end.
  • a chemical group which is selected from the group consisting of one or two alkyl groups, such as methyl, ethyl, propyl or butyl groups, an acyl group such as an acetyl or propionyl group or the amino acid pyroglutamic acid, which forms the N-terminal end.
  • the invention also provides a medicament containing at least one polypeptide according to the invention.
  • the invention also provides a polypeptide for use in gene therapy for the treatment of gene therapy-treatable diseases.
  • the subject matter of the present invention is also a method for enhancing the infection of a cell with a virus, comprising the steps:
  • the invention also provides the use of at least one polypeptide according to the invention for enhancing the infection of a cell with a virus.
  • a composition comprising at least one polypeptide according to the invention is likewise an object of the present invention.
  • polypeptide according to the invention can be carried out, for example, by the method according to Merrifilia with Fmoc-protected amino acids.
  • the acylation reactions take in each case 45 minutes, the cleavage of the Fmoc protective group with 20% piperidine takes 15 minutes.
  • protransducine A according to EP 2 452 947 A1 and protransduce D is that in protransduce-D according to the invention Cys 2 is substituted by alanine.
  • protransducin-B according to WO 2014/177635 Al to Protransduzin- D is that Protransduzin-D according to the invention Cys2 is substituted by alanine and N-terminal in exchange for synthetic L-glutamine (Gin), the synthetic L-pyroglutamic acid ( pGlu) is inserted.
  • the original glutamine is modified by a ring closure, the lactam.
  • Cryopreserved, CD4 + / CD8 + enriched T cells were used for transduction. These were thawed and cultured for 3 days with CD3 / CD28 beats. After removal of the beats after 3 days, the T cells were transduced with a GFP vector obtained by the producer cell line HG820 # 4E912 # 4.3. 2 different MOIs were used. The cells were then cultured for an additional 4 days. On day 7, the cell count and vitality of the cells were determined on a NC-200 nucleo counter. The proportion of GFP + T cells was determined by flow centrifugation.
  • the CD4 + / CD8 + T cells were thawed with a Barkey Plasmatherm device on day 0 and washed once with 1 x PBS.
  • Cells were spiked with cell culture medium X-vivo 15 + 2 mM Glutamax + 5% CTS Immune Cell Serum Replacement with addition of 450 IU / ml IL-7 and 50 IU / ml IL-15 at a density of 1 X 10 6 cells / ml were resuspended
  • 3 (three) CD3 / CD28 Dynabeads were added per cell and cultured until day 3.
  • the beats were removed with a MixMate from Eppendorf and a MaxSep magnet from Baxter. After concentration by centrifugation The cells were transduced with a GFP vector at a concentration of MOI 2 or MOI 4.
  • transduction with retronectin and with virus without additive was performed.
  • An untransduced control was used as a negative control.
  • the different protransduce peptides were dissolved with DMSO to 10 mg / ml (stock solution).
  • the stock solution was diluted with 1 ⁇ PBS to a concentration of 1 mg / ml and incubated for at least 10 minutes to allow PTD fibrils to form.
  • the viral supernatant was mixed with the medium and serum replacement to obtain the necessary concentration.
  • the various PTDs were added to give a concentration of 50 pg / ml.
  • the mixture was incubated for a minimum of 5 minutes at room temperature. After incubation, the cells were added so that a cell density of 5 x 10 5 cells / ml and a final PTD concentration of 25 ⁇ / ml was present. Cells were incubated in 6-well plates at 37 ° C, 5% CO 2 and high humidity for 16 +/- 4 hours to day 4.
  • GFP + T cells CD3 +
  • FACSVerse Flow Cytometer 1.5 ⁇ 10 6 viable cells were washed with 1 ⁇ PBS. After centrifugation, the cell pellet was taken up in 300 ⁇ L of 1X PBS and 3 ⁇ M FVS780 solution. Two FACS tubes were stained per test: Tube 1: GFP / FVS 780 / BV421-unstained (FMO control for CD3-BV421)
  • Tube 2 GFP / FVS 780 / CD3-BV421 [5 ⁇ ]

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Virology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Plant Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Polypeptide présentant la séquence Z1-Gln-Ala-Lys-Ile-Lys-Gln-Ile-Ile-Asn-Met-Trp-Gln-Z2. Ce polypeptide est utilisé pour la transduction/transfection rétrovirale.
EP18769599.4A 2017-08-29 2018-08-29 Protransducine-d : activateur du transfert de gènes amélioré Withdrawn EP3676286A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP17188384 2017-08-29
PCT/EP2018/073194 WO2019043037A1 (fr) 2017-08-29 2018-08-29 Protransducine-d : activateur du transfert de gènes amélioré

Publications (1)

Publication Number Publication Date
EP3676286A1 true EP3676286A1 (fr) 2020-07-08

Family

ID=59811073

Family Applications (1)

Application Number Title Priority Date Filing Date
EP18769599.4A Withdrawn EP3676286A1 (fr) 2017-08-29 2018-08-29 Protransducine-d : activateur du transfert de gènes amélioré

Country Status (7)

Country Link
US (1) US20210154327A1 (fr)
EP (1) EP3676286A1 (fr)
KR (1) KR20200038532A (fr)
CN (1) CN111491944A (fr)
AU (1) AU2018326439A1 (fr)
CA (1) CA3080076A1 (fr)
WO (1) WO2019043037A1 (fr)

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6258782B1 (en) * 1998-05-20 2001-07-10 Trimeris, Inc. Hybrid polypeptides with enhanced pharmacokinetic properties
US6656906B1 (en) * 1998-05-20 2003-12-02 Trimeris, Inc. Hybrid polypeptides with enhanced pharmacokinetic properties
EP2452947A1 (fr) 2010-11-16 2012-05-16 Münch, Jan Peptide augmentant l'infection virale
PL2992004T3 (pl) 2013-05-02 2018-10-31 Pharis Biotec Gmbh Protransducyna b - wzmacniacz transferu genów

Also Published As

Publication number Publication date
US20210154327A1 (en) 2021-05-27
WO2019043037A8 (fr) 2020-01-16
CN111491944A (zh) 2020-08-04
KR20200038532A (ko) 2020-04-13
WO2019043037A1 (fr) 2019-03-07
CA3080076A1 (fr) 2019-03-07
AU2018326439A1 (en) 2020-04-16

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