NZ623092B2 - Viral vectors purification system - Google Patents
Viral vectors purification system Download PDFInfo
- Publication number
- NZ623092B2 NZ623092B2 NZ623092A NZ62309212A NZ623092B2 NZ 623092 B2 NZ623092 B2 NZ 623092B2 NZ 623092 A NZ623092 A NZ 623092A NZ 62309212 A NZ62309212 A NZ 62309212A NZ 623092 B2 NZ623092 B2 NZ 623092B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- viral vector
- ligand
- cell surface
- vector
- viral
- Prior art date
Links
- 230000003612 virological Effects 0.000 title claims abstract description 93
- 238000000746 purification Methods 0.000 title claims abstract description 62
- 210000004027 cells Anatomy 0.000 claims abstract description 90
- 239000002245 particle Substances 0.000 claims abstract description 56
- 239000006228 supernatant Substances 0.000 claims abstract description 51
- 239000003446 ligand Substances 0.000 claims abstract description 40
- 230000027455 binding Effects 0.000 claims abstract description 38
- 238000004806 packaging method and process Methods 0.000 claims abstract description 37
- 239000003550 marker Substances 0.000 claims abstract description 33
- 102000006240 membrane receptors Human genes 0.000 claims abstract description 15
- 108020004084 membrane receptors Proteins 0.000 claims abstract description 15
- 210000003527 eukaryotic cell Anatomy 0.000 claims abstract description 3
- 102000004965 antibodies Human genes 0.000 claims description 24
- 108090001123 antibodies Proteins 0.000 claims description 24
- 238000000926 separation method Methods 0.000 claims description 22
- 230000005291 magnetic Effects 0.000 claims description 20
- 239000011324 bead Substances 0.000 claims description 18
- 230000014509 gene expression Effects 0.000 claims description 16
- 102000005962 receptors Human genes 0.000 claims description 14
- 108020003175 receptors Proteins 0.000 claims description 14
- 230000001177 retroviral Effects 0.000 claims description 14
- 230000001052 transient Effects 0.000 claims description 10
- 108010032605 Nerve Growth Factor Receptors Proteins 0.000 claims description 8
- 108010045374 CD36 Antigens Proteins 0.000 claims description 7
- 101700039720 DPP4 Proteins 0.000 claims description 7
- 102100012353 DPP4 Human genes 0.000 claims description 7
- 102000035266 SCARB3 Human genes 0.000 claims description 7
- 101700078950 CD44 Proteins 0.000 claims description 6
- 102100003735 CD44 Human genes 0.000 claims description 6
- -1 CD3 Proteins 0.000 claims description 5
- 101700082799 IL2RA Proteins 0.000 claims description 5
- 101700015336 ISG20 Proteins 0.000 claims description 5
- 239000000556 agonist Substances 0.000 claims description 3
- 230000003042 antagnostic Effects 0.000 claims description 3
- 239000005557 antagonist Substances 0.000 claims description 3
- 239000000816 peptidomimetic Substances 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 3
- 102000007339 Nerve Growth Factor Receptors Human genes 0.000 claims 2
- 102100002950 ISG20 Human genes 0.000 claims 1
- 239000002458 cell surface marker Substances 0.000 description 54
- 238000004519 manufacturing process Methods 0.000 description 25
- 238000000034 method Methods 0.000 description 23
- 102000004169 proteins and genes Human genes 0.000 description 17
- 108090000623 proteins and genes Proteins 0.000 description 17
- 238000011084 recovery Methods 0.000 description 17
- 241000700605 Viruses Species 0.000 description 14
- 239000000463 material Substances 0.000 description 10
- 102100008795 NGFR Human genes 0.000 description 9
- 150000001768 cations Chemical class 0.000 description 9
- 239000000356 contaminant Substances 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 9
- 238000001415 gene therapy Methods 0.000 description 9
- 238000002360 preparation method Methods 0.000 description 9
- 241000710961 Semliki Forest virus Species 0.000 description 7
- 241000711975 Vesicular stomatitis virus Species 0.000 description 7
- 230000001413 cellular Effects 0.000 description 7
- 239000011325 microbead Substances 0.000 description 7
- 238000010361 transduction Methods 0.000 description 7
- 230000026683 transduction Effects 0.000 description 7
- 210000000170 Cell Membrane Anatomy 0.000 description 6
- 230000005298 paramagnetic Effects 0.000 description 6
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 5
- 241000580858 Simian-Human immunodeficiency virus Species 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 230000034303 cell budding Effects 0.000 description 5
- 230000002458 infectious Effects 0.000 description 5
- 239000003112 inhibitor Substances 0.000 description 5
- 230000002401 inhibitory effect Effects 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 241000282326 Felis catus Species 0.000 description 4
- 102100006815 IL2RA Human genes 0.000 description 4
- 210000002540 Macrophages Anatomy 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000011143 downstream manufacturing Methods 0.000 description 4
- 239000002609 media Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 101710027066 ALB Proteins 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 241000712431 Influenza A virus Species 0.000 description 3
- 241000713666 Lentivirus Species 0.000 description 3
- 241000714177 Murine leukemia virus Species 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 108091007172 antigens Proteins 0.000 description 3
- 102000038129 antigens Human genes 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 108091006028 chimera Proteins 0.000 description 3
- 229920003013 deoxyribonucleic acid Polymers 0.000 description 3
- 238000003306 harvesting Methods 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 230000003834 intracellular Effects 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 238000011031 large scale production Methods 0.000 description 3
- 239000006148 magnetic separator Substances 0.000 description 3
- 230000035800 maturation Effects 0.000 description 3
- 239000004005 microsphere Substances 0.000 description 3
- 238000003146 transient transfection Methods 0.000 description 3
- 241001430294 unidentified retrovirus Species 0.000 description 3
- 241000710929 Alphavirus Species 0.000 description 2
- 102100005861 CCR3 Human genes 0.000 description 2
- 101700070842 CCR3 Proteins 0.000 description 2
- 102100012080 CCR5 Human genes 0.000 description 2
- 101700043583 CCR5 Proteins 0.000 description 2
- 102100002212 CXCR4 Human genes 0.000 description 2
- 101710003734 CXCR4 Proteins 0.000 description 2
- 102000033180 ERVK-6 Human genes 0.000 description 2
- 101710038044 ERVK-6 Proteins 0.000 description 2
- 101710027967 ERVW-1 Proteins 0.000 description 2
- 241000282324 Felis Species 0.000 description 2
- 102000008100 Human Serum Albumin Human genes 0.000 description 2
- 108091006822 Human Serum Albumin Proteins 0.000 description 2
- 241000282619 Hylobates lar Species 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 2
- 210000004698 Lymphocytes Anatomy 0.000 description 2
- 101710023234 Segment 5 Proteins 0.000 description 2
- 210000002966 Serum Anatomy 0.000 description 2
- 208000003265 Stomatitis Diseases 0.000 description 2
- 210000001744 T-Lymphocytes Anatomy 0.000 description 2
- 229940035295 Ting Drugs 0.000 description 2
- 101700028070 VPX Proteins 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 101700075237 gG Proteins 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 210000004779 membrane envelope Anatomy 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 230000001105 regulatory Effects 0.000 description 2
- 238000011172 small scale experimental method Methods 0.000 description 2
- 230000001225 therapeutic Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 230000010415 tropism Effects 0.000 description 2
- 206010000565 Acquired immunodeficiency syndrome Diseases 0.000 description 1
- 241000432074 Adeno-associated virus Species 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 235000003197 Byrsonima crassifolia Nutrition 0.000 description 1
- 240000001546 Byrsonima crassifolia Species 0.000 description 1
- 102100017562 C5AR1 Human genes 0.000 description 1
- 101700049499 C5AR1 Proteins 0.000 description 1
- 210000003996 CFU-GM Anatomy 0.000 description 1
- 210000000234 Capsid Anatomy 0.000 description 1
- QIVBCDIJIAJPQS-SECBINFHSA-N D-tryptophane Chemical compound C1=CC=C2C(C[C@@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-SECBINFHSA-N 0.000 description 1
- 238000007399 DNA isolation Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102100014610 FCAR Human genes 0.000 description 1
- 101700041688 FCAR Proteins 0.000 description 1
- 102100015540 FCGR1A Human genes 0.000 description 1
- 101710003440 FCGR1A Proteins 0.000 description 1
- 101710044640 FCGR2A Proteins 0.000 description 1
- 101710044641 FCGR2B Proteins 0.000 description 1
- 102100015544 FCGR2C Human genes 0.000 description 1
- 101710044642 FCGR2C Proteins 0.000 description 1
- 101700030336 GLYCP Proteins 0.000 description 1
- 241001663880 Gammaretrovirus Species 0.000 description 1
- RQFCJASXJCIDSX-UUOKFMHZSA-N Guanosine monophosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O RQFCJASXJCIDSX-UUOKFMHZSA-N 0.000 description 1
- 208000005721 HIV Infections Diseases 0.000 description 1
- 206010066476 Haematological malignancy Diseases 0.000 description 1
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 229960000310 ISOLEUCINE Drugs 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000726306 Irus Species 0.000 description 1
- 241000713869 Moloney murine leukemia virus Species 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 102000007312 Recombinant Proteins Human genes 0.000 description 1
- 108010033725 Recombinant Proteins Proteins 0.000 description 1
- 101700082620 Rep78 Proteins 0.000 description 1
- 241000712907 Retroviridae Species 0.000 description 1
- 101710041009 THBS1 Proteins 0.000 description 1
- 102100011242 THBS1 Human genes 0.000 description 1
- 101700087068 VGLG Proteins 0.000 description 1
- 208000006110 Wiskott-Aldrich Syndrome Diseases 0.000 description 1
- 102000001392 Wiskott-Aldrich Syndrome Protein Human genes 0.000 description 1
- 108010093528 Wiskott-Aldrich Syndrome Protein Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 230000036436 anti-hiv Effects 0.000 description 1
- 230000000890 antigenic Effects 0.000 description 1
- 229960000070 antineoplastic Monoclonal antibodies Drugs 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000007374 clinical diagnostic method Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 230000001808 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000010192 crystallographic characterization Methods 0.000 description 1
- 230000001086 cytosolic Effects 0.000 description 1
- 230000001419 dependent Effects 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N edta Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 210000002919 epithelial cells Anatomy 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 238000005755 formation reaction Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 230000002068 genetic Effects 0.000 description 1
- 235000013928 guanylic acid Nutrition 0.000 description 1
- 230000003394 haemopoietic Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000001524 infective Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 200000000003 influenza A Diseases 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000011173 large scale experimental method Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000011068 load Methods 0.000 description 1
- 238000007885 magnetic separation Methods 0.000 description 1
- 238000002826 magnetic-activated cell sorting Methods 0.000 description 1
- 230000001404 mediated Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229960000060 monoclonal antibodies Drugs 0.000 description 1
- 102000005614 monoclonal antibodies Human genes 0.000 description 1
- 108010045030 monoclonal antibodies Proteins 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000000737 periodic Effects 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000000750 progressive Effects 0.000 description 1
- 230000000644 propagated Effects 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 230000017610 release of virus from host Effects 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 101710044770 sll1951 Proteins 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 230000002142 suicide Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000002463 transducing Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 230000029302 virus maturation Effects 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
Classifications
-
- 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
- C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
-
- 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
- C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
- C12N2740/13051—Methods of production or purification of viral material
- C12N2740/13052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
-
- 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
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15051—Methods of production or purification of viral material
- C12N2740/15052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
-
- 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
- C12N2740/16051—Methods of production or purification of viral material
- C12N2740/16052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
-
- 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
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
Abstract
Disclosed is a method for the purification of a viral vector comprising: - introducing an exogenous gene encoding a eukaryotic cell surface receptor marker and a gene of interest in a packaging cell line, thus obtaining a producer cell line; - culturing the so obtained producer cell line, so that viral vector particles are released in the supernatant; - collecting the supernatant containing viral vector particles bearing the cell surface receptor marker on their external envelope; - incubating said supernatant with a ligand able to bind to the cell surface receptor marker thus forming a complex ligand-viral vector; - separating complex ligand-viral vector; and - obtaining purified viral vector from complex ligand-viral vector. t viral vector particles are released in the supernatant; - collecting the supernatant containing viral vector particles bearing the cell surface receptor marker on their external envelope; - incubating said supernatant with a ligand able to bind to the cell surface receptor marker thus forming a complex ligand-viral vector; - separating complex ligand-viral vector; and - obtaining purified viral vector from complex ligand-viral vector.
Description
Viral vectors purification system
Field of the invention
The present invention relates generally to a method for efficient purification of viral
vectors (VV) ularly those belonging to the iridae family. More particularly the
invention relates lly to the purification of VV by an immunological method based on
the expression of an exogenous cell surface marker in the packaging cell line.
Background
Viral vectors are commonly used to deliver genetic material into target cells. Nowadays
VV are used in gene therapy applications to vehicle therapeutic genes into patients. In
clinical applications, it is necessary to develop high quality VV in order to meet requisites
imposed by regulatory agencies. Particularly, it is ary to develop safer producer cell
lines, to be used in large-scale production processes in order to obtain large viral stocks. In
the meantime, fficient and scalable purification ses are essential for the
production of al grade viral particles to be stered in humans.
Purification of VV preparations is mandatory to prevent ty, inflammation or immune
response due to vector components, cellular and medium contaminants such as for example
serum (Baekland et al., 2003; Tuschong et al., 2002). Ideally a purification process needs to
assure nance of viral infectivity (stability), high recovery of viral particles, removal of
contaminants such as DNA, proteins and inhibitors of transduction, possibility to concentrate
viral supernatant and, of , scalability of the process (Andreatis et al., 1999; Lyddiatt
and O’Sullivan, 1998).
As of today, different procedures for the purification of retroviral vectors have been
developed based on ent technologies, particularly: centrifugation based methods,
membrane separation processes, chromatographic or other methods based on precipitation
with salts and polymers such as PEG. Currently proposed cation schemes result in low
recovery (approximately 30%) (Rodrigues et al., 2007). All these methods have been
developed originally for protein production and, further, they have been adapted to the
purification of VV. Due to the peculiarity and complexity of viral particles, it is necessary to
improve purification methods in order to obtain high tivity and high hput while
maintaining the biological activity of the final product, particularly in terms of ivity.
A further and more recent example is reported in Merten et al., 2011 that disclose a
process for the production of lentiviral vectors in large scale and under GMP conditions, to
be used in the context of a pilot gene therapy clinical trial for the treatment of Wiskott-
Aldrich syndrome. The disclosed process includes both tion and downstream
processes for the purification of lentiviral particles. Particularly, the purification is based on a
multistep scheme combining several chromatographic and membrane based process steps
including anion exchange and size exclusion chromatography. Notwithstanding the very
good results in respect to the production rate and the absence of DNA and protein
contaminants in the final preparations, the final yield of the purification process is in the
range of previously disclosed methods (below 30%) and the ivity of the viral vectors in
the final sample is reduced.
Both viral and cellular proteins are incorporated into the viral envelope during viral
maturation and release from host cells and, in particular during the so called budding
process (Arthur et al., 1992). It has been shown, for example, that numerous endogenous
host cell proteins are incorporated into the HIV-1 envelope including human lymphocyte
ns, (HLA) classes I and II, CD44, ment control proteins and others whereas
others, such as CXCR4, CCR5 and CCR3, are excluded. On the basis of this observation, it was
suggested that cell type specific antigens may serve as marker of the cellular origin of HIV-1
replication (Roberts et al., 1999). Furthermore, it was developed an immunomagnetic viral
capture assay that was able to distinguish between lymphocytes derived and macrophages
derived ated HIV viruses (Lawn et al., 2000). Particularly, Lawn et al. showed that it
was le to isolate T-cell-derived HIV viruses using an antibody able to bind CD26, and to
discriminate it from macrophage-derived HIV-1 viruses that, in turn, can be captured using
anti CD36 dies. Both CD26 and CD36 are endogenous host cell proteins that are overexpressed
during HIV-1 infection in T-cells and macrophages, tively. Both proteins are
also incorporated in the viral pe thus allowing selective isolation of the virus. Lawn et
al. tested a panel of antibodies able to bind host cell specific antigens before identifying the
sful ones. Interestingly, several dies able to bind antigens endogenously
expressed at high level by macrophages (CD32, CD64, CD88 and CD89) are instead not able
to e the virus, thus showing that over-expression of a certain marker on the surface of
the host cell is a necessary but not sufficient ion for capturing the virus. Lawn et al. do
not show that exogenous proteins expressed by the host can be successfully orated
into the virus envelope, and subsequently used for the purification of functionally active viral
particles.
It has been shown that certain modified cell surface markers can be used for the
purification of transduced cells. Particularly, WO/ 9506723 discloses a process of marking
eukaryotic (mammalian) cells by expressing in these cells the nucleic acid encoding a cell
surface receptor, that is further presented at the cell surface. This cell selection process is
characterized by the use of a c acid in which the region encoding the intracellular
domain of the receptor is completely or partly deleted, or modified so that the receptor
presented at the surface cannot effect any signal transduction after binding to its binding
partner. The cell surface receptor employed in the disclosed process is the Low Affinity
Nerve Growth Factor Receptor (LNGFR), in a truncated form in which the intracellular
domain has been deleted. The resulting ted cell surface receptor is called DLNGFR.
The presence of the DLNGFR n allows the in vitro immunoselection of the genetically
modified cells through the use of monoclonal antibodies and ic beads.
DLNFGR is a truncated cell surface marker that is currently ed in gene therapy for
the selection of transduced cells. For example, it is employed in the HSV-TK gene therapy
ch, which enables safe haploidentical haematopoietic stem cell transplantation
(HSCT) for the treatment of haematological malignancies. The TK therapy employs a
retroviral vector which carries both the suicide gene HSV-TK and the marker gene DLNGFR
(Verzeletti et al. 1998).
So far, the DLNGF receptor has not been employed for the cation of VV.
Due to the necessity of producing purified VV for clinical applications, several attempts
have been made to obtain efficient cation processes that allow good recovery of VV as
well as generation of sufficiently safe vectors that still have good quality in terms of ity.
The methods which are currently employed, allow to obtain low recovery and have some
limits in any case, since they derive from downstream processes developed for the
production of recombinant proteins and adapted to VV purification. Therefore, there is a
need of efficient, fast and le cation methods for VV to be used for large scale
production of vectors for gene therapy, that allow to obtain good recovery and safe viral
particles which maintain high infectivity.
Summary of the invention
The present invention relates generally to the field of purification of VV, particularly those
belonging to the Retroviridae family. Downstream processes currently employed for the
purification of VV are based on s usually applied for recombinant ns. VV are
peculiar particles that are employed in basic research and gene therapy al trials and
that, in the latter case, need al grade production. Purification is therefore mandatory
but, due to the peculiarity of VV, there are several necessities that need to be satisfied. The
purification process needs to be efficient and fast since VV are sensitive to environmental
conditions, and needs to be scalable since large batches are required in clinical applications.
The present invention provides a new strategy for the purification of viral particles, that is
based on the exploitation of the property of such particles to incorporate host cell proteins
ed in the cellular plasma membrane into their external envelope and/or that at least
provides the public with a useful choice. The purification method consists in the expression
of an exogenous gene encoding a cell surface marker in the packaging cell line for the
production of the VV. Such marker is exposed on the cellular membrane of the packaging
cells. In the course of the production of the viral particles, during the maturation phase, the
cell e marker is orated into the viral pe through the budding process.
When incorporated into the viral envelope, the maker is, in fact, a viral surface marker, but
we shall continue to refer to said marker as “cell surface marker”. The viral les can be
then incubated with an antibody able to recognize such marker and purified h
immunological methods. All cell surface proteins that are exogenous to the packaging cells,
particularly to packaging epithelial cells, can be employed as cell surface markers in the
present invention. Cell surface s that can be employed are for example CD26, CD36,
CD44, CD3, CD25 and the Low Nerve Growth Factor Receptor in which the intracellular
domain has been deleted (DLNGFR). In a red embodiment the cell surface marker that
is used for the purification process is DLNGFR.
The developed cation method is extremely ile since it is applicable to any VV
that incorporates the proteins of the host cell membrane during the maturation through the
budding process, such as retroviruses, lentiviruses, alpha viruses [e.g. Semliki Forest virus
(SFV), Sindibis virus (SIN)], rhabdoviruses [e.g. vesicular stomatitis virus , and
orthomyxoviruses [e.g. influenza A virus]. Moreover, the purification method is linked to the
production method because it requires expression of the marker in the packaging cell line
and, ore, allows an integrated approach in which production and downstream
processes are built on the same starting material (the packaging cell line). In this context, it is
le to produce stable packaging cell lines containing all elements necessary for the
tion of VV as well as a further exogenous gene encoding the cell surface marker
necessary for the purification. This aspect is particularly useful for both scalability and
efficiency.
The method is based on the use of a ligand able to bind the cell surface marker, in order
to separate VV from supernatant. Preferably the ligand is an antibody and the separation of
viral particles is obtained by immunological methods. More preferably the method employs
immunomagnetic selection. The method of the invention can be easily scaled-up and
automated since several instruments exist for the performance of immunomagnetic
selection.
Moreover the proposed method is handily, very fast and extremely efficient: the
purification efficiency is higher than that obtained with the currently used s, for
e chromatographic methods such as those employing DEAE and SEC columns.
In addition, it has been found that the purification method of the invention allows high
recovery, since it has been shown that the titer yield of vectors purified through this method
is at least 85% or even higher (120%) in most experiments in small scale. Moreover, a
considerable increase of infectivity of iral vectors purified with the method of the
invention has been ed (43% and 60% for transient and stable production,
respectively), in small scale experiments. Such titer yield and infectivity increase are
achieved with the single main step of the purification method of the invention i.e. the
separation of the complex viral vector-ligand, t taking into consideration other steps
that could affect the final results.
Large scale experiments have also been performed and very good results have been
obtained. In fact, the recovery in terms of virus titer is 60%, with the single step of
separation of the complex viral vector-ligand. Before starting the separation phase, the viral
supernatant is enriched in its viral titer through preliminary filtration and centrifugation
steps, that roughly eliminate contaminants and transduction inhibitors, and also through the
incubation with the ligand of the receptor. The final titer yield after the te multi-step
purification process (harvest viral supernatant vs final purified product) results to be more
than 100%. These are very good s ering that the titer yield of the complete
cation processes disclosed in the literature is around 30% (Rodrigues et al. 2007) or
even below in the case of large scale preparations (Merten et al. 2011). Moreover, VV
ed according to the method of the invention on a large scale result to have a three
times increase of the infectivity, an even higher increase than that obtained with the same
method on a small scale. This is a very important and unexpected advantage since the
literature describes a se of infectivity further to the purification with conventional
methods (Merten et al. 2011).
Statements of the invention
In one aspect, the present invention s to a method for the purification of a viral vector
comprising:
i. Introducing an exogenous gene encoding a eukaryotic cell surface
receptor marker and a gene of interest in a packaging cell line, thus
obtaining a producer cell line
ii. culturing the so obtained producer cell line, so that viral vector particles
are released in the supernatant
iii. collecting the supernatant containing viral vector particles bearing the cell
surface receptor marker on their al envelope
iv. incubating said supernatant with a ligand able to bind to the cell e
receptor marker thus forming a x ligand-viral vector
v. ting complex ligand-viral vector
vi. obtaining purified viral vector from complex ligand-viral vector .
Certain statements that appear below are broader than what appears in the statements of
the invention above. These statements are provided in the interests of ing the reader
with a better understanding of the invention and its practice. The reader is directed to the
anying claim set which defines the scope of the invention.
Also described herein is a method for the purification of a viral vector comprising:
i. introducing an exogenous gene encoding a cell surface marker and a gene
of interest (GOI) in a ing cell line
ii. ing the so obtained producer cell line
iii. collecting the supernatant containing viral vector particles bearing the cell
surface marker on their external envelope
iv. incubating said supernatant with a ligand able to bind to the cell surface
marker
v. ting complex ligand-viral vector
vi. obtaining purified viral vector particles
In another ment the supernatant containing viral vector particles g the cell
surface marker on their external envelope is filtered, optionally concentrated and then
incubated with a ligand able to bind to the cell surface marker.
Preferably the viral vector is a retroviral vector, a lentiviral vector, an alpha viral vector [e.g.
a vector obtained from Semliki Forest virus (SFV), Sindibis virus (SIN)], a rhabdoviral vector
[e.g. a vector derived from vesicular stomatitis virus (VSV)], and an orthomyxoviral vector
[e.g. a vector derived from influenza A . More preferably the viral vector is a lentiviral
vector or a retroviral .
In one embodiment the cell surface marker is any cell surface marker that is exogenous to
the packaging cell line, which is, preferably, an epithelial packaging cell line. Preferably the
cell surface marker is selected from CD26, CD36, CD44, CD3, CD25 and DLNGFR.
More preferably the cell surface marker is DLNGFR.
In one embodiment the expression of the cell surface marker is transient.
In another embodiment the expression of the cell surface marker is stable.
In one embodiment the GOI and the cell surface marker are expressed in the same transfer
vector.
In another embodiment the GOI and the cell surface marker are expressed in separate
vectors.
Preferably the ligand is a chemical or a biological entity selected from an agonist, an
antagonist, a peptide, a peptidomimetic, an antibody, an antibody fragment, an affibody.
In a further embodiment the ligand is linked to a moiety that can be separated from the
supernatant.
Preferably the ligand is an antibody.
More preferably the antibody is conjugated to magnetic beads and separation is obtained by
applying a magnetic field to a on containing the complex antibody-viral vector.
Preferably the viral vectors are obtained by removing the ic field. More preferably
the tion of the complex antibody-viral vector is performed on a column and the viral
vectors are ed by removing the magnetic field and further eluting them from the
column.
In one embodiment the viral vectors are separated from the antibody by cleaving the cell
surface marker-antibody bond.
Also described herein is an exogenous cell surface marker expressed in a packaging cell line
for use in the purification of viral vectors produced by said ing cell line.
Preferably the viral vector is a retroviral vector, a lentiviral vector, an alpha viral vector [e.g.
a vector ed from i Forest virus (SFV), Sindibis virus (SIN)], a viral vector
[e.g. a vector derived from lar stomatitis virus (VSV)], and an orthomyxoviral vector
[e.g. a vector derived from influenza A virus]. More preferably the viral vector is a lentiviral
vector or a retroviral vector.
The cell surface marker is exogenous to the packaging cell line, preferably exogenous to
epithelial ing cells. Preferably the cell surface marker is selected from CD26, CD36,
CD44, CD3, CD25 and DLNGFR.
More preferably the cell surface marker is DLNGFR.
In one ment the expression of the cell surface marker is transient.
In another ment the expression of the cell surface marker is stable.
Detailed description of the invention
A detailed description of preferred features and embodiments of the invention will be
described by way of non-limiting example.
The invention can be put into practice by a person of ordinary skill in the art who will
employ, unless otherwise indicated, conventional techniques of chemistry, molecular
biology, microbiology, recombinant DNA and immunology. All such techniques are disclosed
and explained in published literature. See, for example, J. Sambrook, E. F. Fritsch, and T.
Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold
Spring Harbor Laboratory Press; Ausubel, F.M. et al. (1995 and periodic supplements;
Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York,
N.Y.); t Protocols in Immunology, ch. 12, John Wiley & Sons, New York, N.Y.); B. Roe, J.
Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential ques, John
Wiley & Sons; J . M. Polak and James O'D. McGee, 1990, In Situ ization: Principles and
Practice; Oxford University Press; M. J . Gait (Editor), 1984, Oligonucleotide Synthesis: A
Practical Approach, IrI Press; and, D. M. J. Lilley and J. E. rg, 1992, Methods of
Enzymology: DNA ure Part A: Synthesis and Physical.
Analysis of DNA Methods in Enzymology, Academic Press. All these publications are
incorporated by reference.
Purification method
Described herein is a new purification method for VV. ably described is a method
for the purification of VV including gamma retroviruses (prototype: Moloney murine
leukemia virus, Mo-MLV), lentiviruses (prototype: HIV), alpha viruses [e.g. Semliki Forest
virus (SFV), Sindibis virus (SIN)], rhabdoviruses [e.g. vesicular stomatitis virus (VSV)], and
orthomyxoviruses [influenza A virus]. The proposed purification method is based on one of
the phases of maturation of viruses. Viral particles are ed from host cells h the
budding s by which process viral capsid is wrapped with the plasma membrane
derived from virus producer cells. In doing so, s incorporate in their external envelope
several host cell proteins which are normally embedded in the cellular plasma membrane.
The VV produced by either a transient or stable packaging system are released from the
packaging cells in an identical manner. The method of the present invention is based on the
hypothesis that VV can be ically purified by using an antibody ed against an
exogenous host plasma membrane n.
Described herein is a method for purification of a VV, that is based on the expression of an
exogenous gene encoding a cell surface marker in a packaging cell line. The cell surface
marker is a protein exogenous to the packaging cells that is expressed on the cellular
membrane. Once expressed, such marker is mounted on the packaging cell membrane and,
therefore, during the tion of the VV, is mounted on the external envelope of the VV
ed by said ing cells. According to the method described, the supernatant
containing viral particles embedding a cell surface marker on their envelope is collected and
incubated with a ligand able to recognize such marker. Optionally, particularly for the
purification of VV on a large scale where large volumes have to be handled and purified, the
supernatant is filtered and concentrated, and then re-suspended and incubated with the
ligand. All these preliminary steps allow a rough removal of contaminants and transduction
inhibitors and, therefore, contribute to the enrichment in viral titer observed in the
intermediate preparations and, consequently, to the final titer of the purified viral particles.
r to these preliminary steps, the complex ligand-VV is separated from the medium and
VV are then ed. The separation phase is the main step of the purification method as
described herein. The VV can be separated from the ligand upon cleavage of the bond
between the ligand and the cell surface marker.
In a preferred embodiment the ligand is an antibody.
The method of the t invention can be summarized in five main phases:
1) Expression of a cell surface marker in a packaging cell line for VV
2) tion of viral particles
3) Incubation with a ligand able to recognize the cell surface marker
4) Separation of the x ligand-receptor
) Recovery of purified viral particles
Expression of the marker and production of viral particles
The purification method of the present invention is based on the expression of an ous
cell surface marker in the packaging cell line for the VV. In a preferred embodiment the cell
surface marker is ous to epithelial ing cells. Preferably the cell surface marker
is selected from CD26, CD36, CD44, CD3, CD25 and the truncated form of Low Nerve Growth
Factor Receptor missing the ellular domain R). In a preferred embodiment the
cell surface marker is DLNGFR. The expression of the cell surface marker can be obtained in
several ways. In one embodiment, the cell surface marker can be transiently expressed in
the packaging cell line. In one embodiment the cell surface marker and the eutic gene
are both expressed in the same transfer . In another embodiment the cell surface
marker and the therapeutic gene are expressed in separate s.
In another embodiment the expression of the cell surface marker is stable. Also described is
a packaging cell line containing all structural elements necessary for the production of VV
such as viral gag/pol, rev, optionally tat and the envelope protein of interest, together with
the cell surface marker. In one embodiment, all these genes are stably integrated into the
stable packaging cell line. In another embodiment the elements necessary for the production
of VV are transiently expressed. The packaging cell line can be used to produce VV further to
the introduction of the transfer vector containing the GOI. This packaging cell line ents
an integrated technical solution that contains all elements necessary for the production of
the VV and allows a rapid, safe and efficient cation method.
Further to the introduction of the transfer vectors, the production is obtained by culturing
the packaging cell line containing the stably integrated or transiently expressed cell surface
marker. The viral particles incorporate the cell surface marker into their envelope during the
budding process and they are ed in the supernatant. Purified viral particles will be
obtained by exploiting the presence of the exogenous cell surface marker as described
above.
In another embodiment there is provided a producer cell line containing all structural
elements necessary for the production of VV such as viral gag/pol, rev, optionally tat, the
envelope protein of st and the GOI, together with the cell surface marker. Following to
the culturing of the producer cells, viral particles containing the cell surface marker are
released in the supernatant and they are purified exploiting the ce of the exogenous
cell e marker as described above.
Incubation with ligand and separation of the complex ligand-cell e marker
Viral particles containing a cell surface marker incorporated into their envelope can be
isolated. According to the purification method of the present invention, the supernatant
containing VV is incubated with the ligand able to ize the cell surface marker. In
another embodiment the supernatant containing the VV is first filtered and concentrated
and then is ted with the ligand. The ligand is linked to another structure that allows
separation from the supernatant and, consequently, isolation of the ligand-VV complex. The
ligands that can be used in the present invention are chemical or ical entities including
but not limited to agonists, antagonists, es, peptidomimetics, antibodies, antibody
fragments, affibodies.
Preferably the ligand is an antibody and the method of the present invention comprises
immunoselection for the separation of the complex antibody-VV. In this case, VV containing
the cell surface marker on their envelope may be selected on the basis of their reactivity
with the ell surface marker antibodies.
More preferably, the method of the present invention comprises immunomagnetic
selection. Immunomagnetic ion refers to the coupling of dies to paramagnetic
microspheres (beads) enabling a separation of the antigenic structures by means of a
magnet. For example, supernatant containing VV incorporating the cell e marker into
their envelope may be incubated with a primary IgG anti-cell surface receptor antibody. The
iral supernatant may be then incubated with immunomagnetic beads coated with
anti- IgG secondary Ab, and applied to a magnet in order to te the retroviral vectors
carrying the marker. After separation from retroviral supernatant, the isolated vectors may
be recovered by ng the ic field. Alternatively, the anti-cell surface receptor
antibodies can be directly conjugated to magnetic beads. In this case, immediately after the
single incubation phase the magnetic field is d to the solution.
Paramagnetic microsphere to be employed in the present invention are known in the art,
they are polymer particle having small size ranging from 50 nm such as the commercially
available MACS® microbeads from Miltenyi Biotec, to bigger particles of 0.5-500 mm such as
the commercially available Dynabeads®, from Invitrogen. Paramagnetic microsphere can be
directly or indirectly conjugated to the specific antibody of interest able to bind the cell
surface marker incorporated into the VV pe. Method of conjugation of antibody to
paramagnetic beads are known in the art and include, for example, cross linking, formation
of covalent bonds on functional groups, biotin-avidin system and others. tion of the
VV from viral supernatant will be obtained by applying a magnetic field to a solution
ning the complex consisting of the dy conjugated paramagnetic beads and
linked to the cell surface marker.
In a preferred embodiment immunomagnetic selection is performed on a column.
Particularly, the supernatant containing VV may be directly ted with an antibody able
to recognize the cell surface marker, such antibody being conjugated to paramagnetic beads.
In r embodiment the supernatant ning the VV is first filtered and optionally
concentrated and then is incubated as previously described. Following incubation, the
supernatant or the filtered and optionally concentrated solution is applied on a column
placed in a ic separator for the removal of impurities and separation of the viral
particles that remain in the column thanks to the magnetic field.
Recovery of purified particles
According to the method of the invention, the last phase of the process is the recovery of
purified viral particles. Such recovery is obtained removing the viral particles from the
magnetic field. If the purification is performed on a column the recovery is obtained by
removing the ic field. Viral vectors can then be separated from the ligand by cleaving
the bond between the ligand and the cell surface marker. s for cleaving said bond
are known in the art and include the use of displacement ligands or of appropriate solutions
containing enzymes. Depending on the nature of the ligand and of the receptor and their
bond the appropriate method is employed.
Efficiency, scale-up and tion
The method of the invention is ely efficient and is simple and fast. Currently proposed
purification methods allow a recovery around 30%. Remarkably the method of the invention
allows to obtain titer yield of at least 85% or even higher (120%) in most experiments in
small scale. The titer yield, in this case, has been ated referring to the main step of the
method of the invention: the separation of the x viral vector-ligand. The abovementioned
titer yield results from the ratio between the titer of the unpurified particles
incubated with the ligand of the ous receptor and the titer of the purified particles.
The fied particles in ments on a small scale are obtained from the VV
supernatant through preliminary tion step, and are then incubated with the ligand of
the exogenous receptor. The results show that the recovery in terms of viral titer with the
method of the invention is very high.
Viral particles are extremely labile and sensitive to environmental conditions. In particular,
the presence of the cell surface maker on the envelope of VV could, in principle, affect the
composition of such envelope as well as its ure and the availability of viral pe
proteins, affecting in turn the tropism of the vector and, consequently, causing problems to
viral titer and ivity. With the method of the invention, on the contrary, the titer is
unaffected or even increased and, remarkably, the infectivity of lentiviruses purified with
such method is increased (43% and 60% for transient and stable production, respectively) in
small scale. These results are surprising because of the presence of structural elements that
could, ly, negatively affect the tropism and the infectivity of the vectors.
A further advantage of the invention is that the method can be simply scaled-up and
automated. Considering the case in which VV are purified by using immunomagnetic
selection, it is possible to employ machines (e.g. CliniMacs® Plus Instrument, from Miltenyi
Biotec) able to perform automated selection. Such machines must be able to perform liquid
exchange on a solid support such as a column and immunomagnetic selection through the
generation of a magnetic field. Automation helps the production of large VV stocks since it
allows the purification of large quantity of viral supernatant. Purification of VV on a large
scale with the method of the invention allows to obtain at least 60% titer yield in the main
ic tion phase (titer of the unpurified particles incubated with ligand of the
receptor vs titer of the purified les). The unpurified particles, in experiments on a large
scale, are obtained from the VV supernatant through preliminary purification steps:
filtrations, optionally concentration, and are then incubated with the ligand of the
exogenous or. Each of these steps causes a rough removal of contaminants and
transduction inhibitors, with a progressive enrichment in viral titer of the sample that
undergoes the separation. The titer yield of the entire s on a large scale (titer of
harvest viral supernatant vs titer of purified particles) results to be more than 100%. These
are a very important results considering that the titer yield of an entire purification process
of VV is reported to be around 30% (Rodrigues et al. 2007) or even below in the case of large
scale ations (Merten et al. 2011). The purification on a large scale with the method of
the present invention allows an increase of the infectivity of VV that result to be about 3
times more infective than the unpurified particles after the separation. In addition, in order
to have an idea of the quality of the preparation obtained with the purification method of
the t invention, it is le to count the number of the lentiviral infectious particles
(Transfecting Units derivable from the viral titer) vs total physical particle (obtainable from
the conventional equation that 1ng of p24Gag corresponds to 107 physical les, as
ed in Salmon and Trono, 2006). It is very interesting to note that, with the method of
the invention, as shown for example in Experiment 2 of Table 4, starting from a supernatant
containing 1 infectious particle out of 5,318 total physical particles (a very poor ng
material), it was possible to obtain a purified preparation containing 1 infectious particle out
of 250 total physical particles, with a very good enrichment in terms of quality and
functionality of the LV preparation.
In this specification where nce has been made to patent specifications, other external
documents, or other s of information, this is generally for the purpose of providing a
context for discussing the es of the invention. Unless specifically stated otherwise,
reference to such external documents is not to be construed as an admission that such
documents, or such sources of information, in any jurisdiction, are prior art, or form part of
the common general knowledge in the art.
The term “comprising” as used in this specification and claims means “consisting at least in
part of”. When interpreting ents in this specification, and claims which include the
term “comprising”, it is to be understood that other features that are additional to the
features prefaced by this term in each statement or claim may also be present. d
terms such as “comprise” and “comprised” are to be interpreted in similar manner.
pFurther preferred features and embodiments of the present invention will now be
described by way of non-limiting example and with reference to the accompanying drawings
in which:
Description of the s
Figure 1. Schematic representation of the strategy of the anti-DLNGFR-Ab-based purification
process. “Constructs” indicates the plasmids or vectors required to produce either
transiently or stably the VV to be purified (step 1). The cassette of the selection marker
DLNGFR can be incorporated into the transfer vector construct or in a different plasmid
expressed either transiently or stably from the ing cells. The VV are purified by the
anti-DLNGFR Ab coupled to magnetic beads which are then retrained in a magnetic column
(step 2) and y eluted (step 3). The purified VV can be either used with the attached
beads (step 3.1) or after l of the ed beads (step 3.2).
Figure 2. Schematic representation of the experimental procedure of the invention. The
procedure is divided in three simple steps: 1) the magnetic labelling of the VV consisting in
the incubation of supernatants with the anti-DLNGFR Ab coniugated microbead suspension
for 30 minutes at room temperature; 2) the magnetic separation of the VV consisting in the
application of the sample into the magnetic column placed into the ic separator; all
sample components (i.e contaminants, proteins and excess Abs) go in the flow-through and
further removed by several ; 3) the elution of the VV consisting in the removal of the
column from the magnetic separator and collection of the purified VV. The purified VV can
be either used with the attached beads (step 3.1) or after removal of the attached beads
(step 3.2).
Figure 3. Graph summarizing the experimental data in small scale. The yield of the VV titer
has been calculated as percentage of the titer of the purified VV vs that of the magnetically
labeled VV before loading the sample into the column. The values are the means of 5
experiments for the iral s, produced stably by the RD2-MolPack-Chim3.14
packaging clone, which carry the RD114-TR envelope (Stable LV, RD114-TR); 6 ments
for the lentiviral s, produced by transient transfection of HEK-293T, which carry the
VSV-G envelope (Trans. Transf. LV, VSV-G); 5 experiments for the retroviral vectors,
produced from the AM12-SFCMM-TK clone 48, which carry an Ampho pe (Stable RV,
Ampho).
Examples
e I: Production of VV
Stable production of MLV retroviral vectors (RV)
Murine NIH-3T3-derived, e4070-pseudotyped AM12-SFCMM-TK clone 48 packaging cells
were grown in DMEM (Dulbecco’s Modified Eagle Medium) ittaker™, Cambrex Bio
Science Walkersville, Inc. Walkersville, MD) or X-VIVO 15 supplemented with 10% FBS
(BioWhittaker™) and 2 mM glutamine at 37°C in 5% CO2 atmosphere. The AM12-SFCMM-TK
clone 48 was obtained after transduction of the construct SFCMM-3 Mut2, which encodes a
modified form of the HSV-TK gene characterized by a single silent mutation at nucleotide
330 of the ORF (WO 23912). Transduced cells were immune selected by using the
anti-DLNGFR mAb of the Am12-SFCMM-3 Mut 2 cells and then cloned by limiting dilution
(0.3 cell/well). AM12-SFCMM-TK clone 48 contains two copies of SFCMM-3 Mut2 vector. The
GMP-grade retroviral vector supernatant lots were produced either in roller bottles or in a
packed-bed 32-liter bioreactor in X-VIVO 15 medium in the presence of 1% glutamine, 10%
PBS and isoleucine/tryptophan/Na e.
Stable production of lentiviral vectors (LV)
Human HEK-293T and its derivative RD2-MolPack-Chim3 packaging cells were propagated in
co’s Modified Eagle Medium (DMEM) supplemented with 10% FCS and PSG. RD2-
MolPack-Chim3.14 and Chim3.25 clones stably produce second generation LV for anti-HIV
gene therapy approach. The clones were obtained by sequential integration of the packaging
constructs and the transfer vector using integrating vectors. Briefly, HEK-293T cells were
transiently transfected with a d encoding the adeno-associated virus (AAV) Rep-78
protein and then infected with an hybrid baculovirus-AAV vector, in which the baculoviral
backbone contains an integration cassette expressing the HIV-1 structural gag, pol, the
tory rev and the hygro-resistance genes flanked by the AAV inverted terminal repeats
(ITR) sequences national Patent Application N° ). This , which
allows the Rep78-mediated ation of the ITR-flanked cassette into HEK-293T genome,
generated the first intermediate clone named PK-7. From PK-7 clone, the RD2-MolPack-
14 and Chim3.25 ing clones (International Patent Application N° WO
2012/028681) were obtained through the sequential integration of the SIN-LV sing the
HIV-1 regulatory tat and the chimeric RD114-TR envelope gene and the Tat-dependent LV
vector expressing the IV Vif dominant negative transgene Chim3. Clones were
obtained by seeding the cells at limiting dilution in 96-well plate (0.1 to 0.3 cell/well). For
each cell type cloning experiment, at least 5 to 10 individual clones or more were selected by
visual inspection under l microscope and lly expanded. LV derived from RD2-
MolPack-Chim3 were obtained by small scale culture in either T25 or T75 flasks and by large
scale in T162 flasks
Transient production of LV
Pseudotyped LV produced from HEK-293T cells were obtained by transient co-transfection of
the following plasmids: the packaging constructs CMV-GPRT, the VSV-G construct, and the
2nd-gen. PDN-Chim3 transfer vector (International Patent Application N° ).
The ratio of packaging:envelope:transfer s corresponded to 6.5:3.5:10 mg DNA.
Transient transfections were performed with either the standard Ca2+-PO4 method or the
FugeneTM6 system following the manufacturer’s instruction (Roche Diagnostics
Corporation, Indianapolis, IN) obtaining similar results. Supernatants were harvested 48
hours after transfection and ed through a 0.45-μm .
Example II: Purification of VV by the anti-LNGFR Abs on a small scale
Small scale purification of VV was d out as follows. Supernatants containing VV were
diluted with 1:5 (vol/vol) with PBS ning 0.5% BSA and then filtered with 0.45 mm
filters. From one to five ml of diluted supernatants were incubated with anti-LNGFR Ab
coniugated microbeads suspension (CD271 Microbeads yi Biotec, GmbH, Germany
cat. #130330) at the 1:40 ratio (vol/vol). The samples were then incubated at room
temperature (RT) for 30 minutes on a rotating wheel. The magnetically labelled samples
were loaded on the column placed into the magnetic separator, (Miltenyi, MS Columns cat.
#130201). After the flow-through was collected for analysis and three washes were
performed with 0.5 ml of washing buffer (PBS ning 2% FCS and 0.5% BSA), the column
was removed from the magnetic separator and the purified VV were collected.
Example III: titer calculation
VV titer was calculated on SupT1 cells by transducing them by one cycle of ulation at
1,240 × g for 1 hour in the presence of polybrene 8 μg/ml (Sigma-Aldrich, St Louis, MO).
uction efficiency was monitored by flow cytometry analysis (FACS Calibur BD
Bioscience, San Jose, CA) of DLNFGR expression, as described in Porcellini et al., 2009 &
2010, using the FlowJo software (Tree Star, Inc., Ashland, OR). Only transduction values
ranging from 5 to 20% positive cells were used to calculate the titer according to the
formula: TU = [number of cells × (% positive 100)]/vol sup (in ml).
Example IV: analysis of potency of ed versus fied VV in small scale preparation
Several experiments were performed, as summarized on Table 1, and Table 2, using three
types of VV produced by different modalities and pseudotyped with distinct envelopes. The
2nd generation LV expressing the Chim3 transgene were produced from either the stable
packaging cell line RD2-MolPack-Chim3.14 or by transient transfection of HEK-293T cells as
reported in Example I. In the first ion, the LV were pseudotyped with the chimeric
RD114-TR envelope, made of the ellular and trans-membrane domains of the feline
endogenous retrovirus RD114 envelope and the cytoplasmic tail (TR) of the A-MLVenv 4070A
(Sandrin et al., 2002), whereas in the second condition with the vesicular stomatitis virus
glycoprotein G ) envelope. The gRV were produced from the AM12-SFCMM-TK clone
48 and carried the MLV e4070 envelope.
Each experiment was carried out at the ing conditions: 1) diluted supernatant volume
(one ml of supernatant diluted 1:5 with PBS/2% FCS/0.5% BSA); 2) supernatant:microbead
suspension (vol:vol) ratio 1:40; 3) anti-LNGFR Abs directly coupled to magnetic beads (CD271
microbeads). The output of the analysis corresponds to both the percentage of titer yield
(Figure 3A) and the percentage of the increment of infectivity (Figure 3B) of purified VV
ve to that of unpurified ically labelled VV. Titer calculation was med on
SupT1 cells, as detailed in Example III. Remarkably, the yield for LV produced stably was
superior to 100% (121% on average) and that of LV produced transiently was 90%. This
means that the purification of LV, regardless the type of envelope they mount, is highly
effective in removing serum proteins or other contaminants that might decrease titer values.
The purification yield of gRV is slightly inferior to that of LV (85%). Moreover, a considerable
increase of infectivity of lentiviral vectors purified with the method of the invention has
been obtained (43% and 60% for transient and stable production, respectively).
e V: Purification of VV by the NGFR Abs on a large scale
Large scale purification of VV was carried as follows. Filtered supernatants (0.45mm)
containing LV (800 ml) were concentrated 8-fold by centrifugation at low speed (3,400×g) for
16 hours at +4°C in refrigerated bench top centrifuge. VV pellets were resuspended in 100
ml buffer PBS/EDTA 0.5% human serum albumin (HSA) and then incubated with anti-LNGFR
Ab coniugated eads suspension (CD271 Microbeads, Miltenyi Biotec, cat. #130
330) at the 1:40 ratio (vol/vol) in a 150-ml transfer bag (Miltenyi Biotec cat. #183-01). The
s were then incubated at RT for 30 minutes on an orbital shaker. The magnetically
ed samples were loaded on the CliniMacs® Plus Instrument and the automated
separation programme Enrichment 3.2 was started. The purified LV was recovered in 40 ml
and an aliquot was evaluated for cation performance by potency ation.
Example VI: analysis of potency of purified versus unpurified VV in large scale preparation
Two experiments were performed, using the 2nd generation LV expressing the Chim3
transgene produced from the stable packaging cell line RD2-MolPack-Chim3.25. s are
summarized on Table 3 and 4. Each experiment was carried out as described in Example V.
The output of the analysis corresponds to both the percentage of titer yield and the
percentage of the increment of infectivity of purified LV relative to that of unpurified viral
particles bound to NGFR Ab conjugated to the magnetic beads (Table 3) or relative to
that of supernatant (Table 4). Titer calculation was performed on SupT1 cells as sed in
example III. The titer yield of large scale purification (Table 3, EL/Input) was around 60% in
the single step of separation of the complex viral -ligand. Before starting the
separation phase, the viral supernatant is enriched in its viral titer through preliminary
filtration and centrifugation steps, that roughly eliminate contaminants and transduction
inhibitors, and also through the incubation with the ligand of the receptor. The final titer
yield after the complete multi-step cation process (harvest viral supernatant vs final
ed product) (Table 4, EL/Sup), is more than 100%: 118% for experiment 1 and 231% for
experiment 2. Most importantly, the infectivity of purified particles is dramatically increased
of three times in respect to the unpurified, with an even higher ment as compared to
the small scale experiments indicating that large scale and automation further increase the
yield of the process in terms of functionality of VV.
Table 1. Summary of experiments in small scale
DLNGFR
Type of Production Vector Env TU Yield (%)a Infect.(%)b Exp
copy
Stable LV LV 20 RD114-TR 121.4±24SEM 59.8±29SEM 5
Trans. Transf. LVc LV nd VSV-G 90.6±9.3SEM 43.0±12SEM 5
Stable RV RV 2 e4070 85.0±6.4SEM nd 6
iations: LV, lentiviral vector; RV, retroviral vector; RD114-TR, chimeric envelope from the feline endogenous
retrovirus and the TR domain of MLV env; VSV-G, lar stomatitis virus envelope glycoprotein G
aYield has been ated as the % of total TU recovered respect to the total TU input loaded into the magnetic column.
bInfectivity has been calculated as the % of increment of infectivity of purified versus input VV.
cTransient ection has been carried out on HEK293T cells as described in the text.
Table 2. y of all ments in small scale
Stable LV (RD114-TR)
Number of exp. EX1 EX2 EX3 EX4 EX5
Titera p24Gagb Infect.c Titera p24Gagb Infect.c Titera p24Gagb Infect.c Titera b Infect.c Titera p24Gagb Infect.c
Input 3 1,3 3.0×103 4.5×103 1.2 3.8×103 5.2×103 1,5 3.4×103 5.2×103 1,5 3.4×103 7.9×103 1,5 5.2×103
Flow h 0 0,1 nd 1.2×103 nd nd 0 0,3 nd 1.9×103 0,28 6.8×103 3.0×102 0,3 1.0×103
Eluted 8.2×103 1,1 7.5×103 3 0,8 4.6×103 6.3×103 1,2 5.2×103 7.3×103 1,1 6.6×103 5.1×103 1,2 3
YIELD: %recd. & %var.FT/Ine ndd 7.6d nde 26 nd nd nd 20 nd 36 18 50 3.7 20 -80
YIELD: %rec.f & %var.EL/Ing 200f 84f 150g 82 66 21 121 80 53 140 73 94 64 80 -19
Transient Transfection LV (VSV-G)
Number of exp. EX1 EX2 EX3 EX4 EX5
Titera p24Gagb .c Titera p24Gagb Infect.c Titera p24Gagb Infect.c Titera p24Gagb .c Titera p24Gagb Infect.c
Input 1.4×107 165 8.4×104 1.3×107 160 8.1×104 1.4×107 187 7.4×104 1.7×107 196 8.6×104 1.7×107 196 8.6×104
Flow through 2.1×106 44 4.9×104 2.0×106 22 9.0×104 5.0×106 81 6.1×104 7.2×106 91 7.9×104 5.6×106 81 6.9×104
Eluted 7 101 1.6×105 1.0×107 100 1.0×105 7 110 1.0×105 1.3×107 105 1.2×105 1.2×107 108 1.1×105
YIELD: %recd. & %var.FT/Ine 15 26 -41 15 14 10 36 43 -17 42 46 -8 33 41 -20
YIELD: %rec.f & %var.EL/Ing 121 61 90 76 62 23 78 59 35 76 53 39 70 55 28
Stable RV (Ampho)
Number of exp. EX1 EX2 EX3 EX4 EX5 EX6
Titer p19Gag Infect. Titer p19Gag Infect. Titer p19Gag Infect. Titer p19Gag Infect. Titer p19Gag Infect. Titer p19Gag Infect.
Input 6.0×104 8.0×104 9.3×104 9.3×104 7.5×104 7.5×104
Flow through 7.6×103 nd 5.4×103 nd 1.5×104 nd 3.0×104 nd 1.5×104 nd 1.4×104 nd
Eluted 6.9×104 6.8×104 5.8×104 8.1×104 6.1×104 6.0×104
YIELD: %recd. & %var.FT/Ine 12 6.7 16 32 20 19
YIELD: %rec.f & %var.EL/Ing 115 85 62 87 81 80
aTiter of the total amount of loaded and eluted VV
bTotal amount of p24Gag expressed in ng
cInfectivity expressed as TU/ng p24Gag
dPercentage of total titer and p24Gag in the flow thorugh material respect to the total titer and p24Gag of the input VV
ePercentage of variation of the infectivity in the flow through material respect to the ivity of the input VV
fPercentage of total titer and p24Gag in the eluted material respect to the total titer and p24Gag of the input VV
ntage of variation of the infectivity in the eluted material respect to the infectivity of the input VV
nd: not determined
Table 3. Summary of large scale ments
Stable LV (RD114-TR)
Number of exp. EX1 EX2
Titera p24Gagb Infect.c Inf/Totd Titera p24Gagb Infect.c Inf/Totd
Input (102.5 ml) 6.1×107 6,700 7.0×103 1:1,098 8.6×107 6,060 1.4×104 1:704
Eluted (40 ml) 3.9×107 1,480 4 1:358 5.2×107 1,300 4.0×104 1:250
YIELD: %rece & %var.f of EL/IN 64e 22e 371f 60e 21e 285f
aTiter of the total amount of loaded and eluted LV
bTotal amount of p24Gag expressed in ng
cInfectivity expressed as TU/ng p24Gag
dRatio between total infectious vs physical particles
ePercentage of total titer and p24Gag in the eluted material respect to the total titer and p24Gag of the input LV, rec recovery
fPercentage of variation of the infectivity in the eluted material respect to the infectivity of the input LV, var variation
Table 4. Large scale full process yield
Stable LV (RD114-TR)
Number of exp. EX1 EX2
Titera p24Gagb Infect.c Inf/Totd Titera p24Gagb Infect.c Inf/Totd
Sup. (800 ml) 7 11,600 2.8×103 0 2.2×107 11,700 1.8×103 1:5,318
Eluted (40 ml) 3.9×107 1,480 2.6×104 1:358 5.2×107 1,300 4.0×104 1:250
YIELD: %rece & %var.f of EL/SUP 118e 13e 928f 231e 11e 2222f
aTiter of the total amount of starting and eluted LV
bTotal amount of p24Gag expressed in ng
tivity expressed as TU/ng p24Gag
dRatio between total infectious vs physical particles
ePercentage of total titer and p24Gag in the eluted material respect to the total titer and p24Gag of the input LV, rec recovery
fPercentage of variation of the infectivity in the eluted material respect to the infectivity of the input LV, var variation
References
1. Baekeland, V., et al. (2003) Optimized lentiviral vector tion and purification
procedure prevents immune response after transduction of mouse brain. Gene Ther 10:
940.
2. Tuschong, L., et. al. (2002). Immune response to fetal calf serum by two adenosine
deaminase-deficient patients after T cell gene therapy. Hum. Gene Ther. 13: 1605-1610.
3. Andreadis, S.T., et al. (1999). Large scale processing of recombinant retrovirus for
gene therapy. hnolo. Prog. 15: 1-11.
4. tt,A., and O'Sullivan, D.A. (1998). Biochemical recovery and purification of gene
therapy vectors. Curr. Opin Biotechnol. 9: 177-185.
. ues, et al. (2007). Purification of retroviral vectors for clinical application:
biological implications and technological challenges. J. of Biotech. 127: 520-541.
6. Arthur, L.O, et al. (1992). Cellular proteins bound to immunodeficiency viruses:
implication for pathogenesis and es. Science 258: 1935-1938.
7. Roberts, B. D., et al. (1999). Host n incorporation is conserved among e
HIV-1 subtypes. AIDS 13: 425-427.
8. Lawn, S. D., et al. (2000). Cellular compartments of human immunodeficiency virus
type 1 replication in vivo: determination by presence of virion-associated host proteins and
impact of opportunistic infection. J Virol 74 (1): 139-145
9. Verzeletti, S., et al. (1998). Herpes simplex virus thymidine kinase gene transfer for
controlled graft-versus-host disease and graft-versus-leukemia: clinical -up and
improved new vectors. Hum. Gene Ther, 9(15):2243-51
. Sandrin, V., et al. (2002). Lentiviral vectors pseudotyped with a modified RD114
envelope glycoprotein show increased stability in sera and augmented transduction of
primary lymphocytes and CD34+ cells derived from human and nonhuman primates. Blood
100: 823-832.
11. Porcellini, S., et al. (2009). The F12-Vif derivative Chim3 inhibits HIV-1 replication in
CD4+ T cytes and CD34+-derived macrophages by blocking HIV-1 DNA ation.
Blood 113: 3443-3452.
12. Porcellini, S., et al. (2010). Chim3 confers al advantage to CD4+ T cells upon
HIV-1 infection by preventing HIV-1 DNA integration and HIVinduced G2 cell-cycle delay.
Blood 115: 4021-4029.
13. Bastiani Lallos, L., Laal, S., Hoxie, J.A., Zolla-Pazner, S., and Bandres, J.C. (1999)
Exclusion of HIV coreceptor CXCR4, CCR5, and CCR3 from the HIV envelope. AIDS Research
and Human iruses 15: 895-897.
14. s et al., (2011). Large-Scale manufacture and characterization of a lentiviral
vector produced for clinical ex vivo gene therapy application. Hum. Gene Ther 22:343–356.
Patrick Salmon and Didier Trono, (2006) Current Protocols in Neuroscience
Supplement 37: 4.21.1-4.21.24, John Wiley & Sons, Inc.
Claims (3)
1. A method for the purification of a viral vector comprising: i. ucing an exogenous gene encoding a eukaryotic cell surface receptor marker and a gene of st in a packaging cell line, thus 5 obtaining a producer cell line ii. culturing the so obtained producer cell line, so that viral vector particles are released in the supernatant iii. collecting the atant containing viral vector particles bearing the cell surface receptor marker on their external envelope 10 iv. incubating said supernatant with a ligand able to bind to the cell surface receptor marker thus forming a x ligand-viral vector v. separating complex ligand-viral vector vi. obtaining purified viral vector from complex ligand-viral vector .
2. A method according to claim 1, wherein the viral vector is selected from a retroviral 15 , a lentiviral vector, an alpha viral vector, a rhabdoviral vector, and a orthomyxoviral vector. 3. A method according to claim 1 or 2 wherein the cell surface receptor marker is selected from CD26, CD36, CD44, CD3, CD25 or the truncated form of Low Nerve Growth Factor Receptor (DLNGFR). 20 4. A method according to any one of claims 1 to 3 wherein the cell e receptor marker is the truncated form of Low Nerve Growth Factor Receptor (DLNGFR). 5. A method according to any one of claims 1 to 4, n the expression of the cell surface receptor marker is transient. 6. A method according to any one of claims 1 to 4, wherein the expression of the cell 25 surface receptor marker is stable. 7. A method according to any one of claims 1 to 6 wherein the gene of interest and the exogenous gene are sed in the same transfer vector. 8. A method according to any one of claims 1 to 6 wherein the gene of interest and the exogenous gene are sed in separate vectors. 9. A method according to any preceding claim wherein the ligand is a chemical or a biological entity selected from the group consisting of agonists, antagonists, es, peptidomimetics, antibodies, antibody fragments, and affibodies. 10. A method according to any preceding claim wherein the ligand is linked to a moiety 5 that can be separated from the supernatant. 11. A method according to claim 9 or 10 wherein the ligand is an antibody conjugated to magnetic beads and separation is obtained by applying a magnetic field to a solution containing the complex antibody-viral vector. 12. A method according to claim 11 wherein purified VV are obtained by removing the 10 magnetic field. 13. A method according to any preceding claim n the separation of the complex ligand-viral vector is performed on a column. 14. A method as defined in any one of claims 1 to 13 ntially as herein described with nce to any example thereof. 1 2 Magnetic column Constructs Viral Vector (VV) Envelope Transfer vector+ ∆LNGFR ∆LNGFR ∆LNGFR
3.1 Purified VV with 3.2 Purified VV with Packaging cell bead attached bead removed
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP11183937 | 2011-10-05 | ||
EP11183937.9 | 2011-10-05 | ||
PCT/EP2012/069713 WO2013050523A1 (en) | 2011-10-05 | 2012-10-05 | Viral vectors purification system |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ623092A NZ623092A (en) | 2016-07-29 |
NZ623092B2 true NZ623092B2 (en) | 2016-11-01 |
Family
ID=
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20220243223A1 (en) | Methods, kits, agents and apparatuses for transduction | |
CN107002084B (en) | Co-stimulatory chimeric antigen receptor T cells targeting IL13R alpha 2 | |
AU2016355323B2 (en) | Buffers for stabilization of lentiviral preparations | |
CN116063574A (en) | HER 2-targeting chimeric antigen receptor | |
US11957715B2 (en) | Method for NK cell transduction | |
EP3186382B1 (en) | Method and means for purifying retroviral vectors | |
CN115074331A (en) | Chimeric antigen receptor targeting PSCA | |
US20140248695A1 (en) | Viral Vectors Purification System | |
CA3110926A1 (en) | Improved lentiviral vector | |
CN113045675A (en) | Antibody for resisting CD22 protein molecule and application thereof | |
NZ623092B2 (en) | Viral vectors purification system | |
TW202321443A (en) | Cell lines for producing a retroviral vector encoding a car | |
Schatz | Development of target antigen-displaying virus-like particles (VLPs) for the generation of antibodies using hybridoma technology |