WO2001011069A1 - Complexes de lipide congeles/decongeles et leur preparation - Google Patents

Complexes de lipide congeles/decongeles et leur preparation Download PDF

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Publication number
WO2001011069A1
WO2001011069A1 PCT/GB2000/003040 GB0003040W WO0111069A1 WO 2001011069 A1 WO2001011069 A1 WO 2001011069A1 GB 0003040 W GB0003040 W GB 0003040W WO 0111069 A1 WO0111069 A1 WO 0111069A1
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Prior art keywords
freeze
complex
lipid
cells
thawed
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PCT/GB2000/003040
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English (en)
Inventor
Kenneth Crook
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Celltech R&D Limited
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Publication date
Application filed by Celltech R&D Limited filed Critical Celltech R&D Limited
Priority to EP00949803A priority Critical patent/EP1198582A1/fr
Priority to AU63066/00A priority patent/AU6306600A/en
Publication of WO2001011069A1 publication Critical patent/WO2001011069A1/fr

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    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • This invention relates to lipid complexes, to a process for their preparation and to their use in medicine.
  • FCS foetal calf serum
  • the method involves treating a lipid complex to a freeze/thaw regime prior to contact with cells. Transfection of cells by complexes treated in this way is maintained in the presence of serum, but is completely ablated when untreated complexes are used under identical conditions.
  • the levels of transfection by treated complexes is markedly enhanced in the presence of serum over levels in the absence of serum.
  • a process for transfecting a cell by a lipid complex which comprises 1 ) subjecting the lipid complex to one or more freeze/thaw cycles and subsequently 2) bringing the freeze/thawed lipid complex into contact with the cell in the presence of serum
  • each freeze/thaw cycle will generally involve freezing the hpid complex at a low temperature, for example from around -5°C, e g around -20°C, to around -210°C and then thawing at an elevated temperature, for example from around 10°C e g from around ambient temperature such as around 20°C to around 60°C
  • a low temperature for example from around -5°C, e g around -20°C
  • an elevated temperature for example from around 10°C e g from around ambient temperature such as around 20°C to around 60°C
  • the precise temperature used at each stage of a cycle is not critical and may be varied over a wide range but may need to be selected depending on the nature of the lipid complex and in particular the nature of any material complexed with it
  • a complex contains a material which is subject to degradation and/or denaturation at low and/or high temperatures, for example a protein
  • prolonged exposure to extremes of temperature should be avoided where possible
  • freeze/thaw cycle More than one freeze/thaw cycle may be employed, for example two, three, four or more cycles Each cycle may immediately follow the preceding one and may employ the same or different freezing and thawing conditions Alternatively a freeze/thawed complex may be stored for a period of time before being subjected to one or more further freeze/thaw cycles Similarly, in the second part of the process according to the invention freeze/thawed hpid complex may be brought into contact with a cell directly after a freeze/thaw cycle or, where desired, after a period of time during which the freeze/thawed complex has been stored under appropriate conditions, for example at a low temperature such as around 4°C, to maintain high transfection activity
  • the pid complex for use in the invention may in general comprise one or more negatively charged (anionic), neutral or positively charged (cationic) lipids complexed with one or more extraneous materials
  • the complex may in particular be in the form of a hposome
  • lipids Numerous lipids have been described in the literature and/or are commercially available which are capable of forming complexes with extraneous materials and it will be appreciated that the precise nature of the lipid is not crucial to the process of the invention
  • suitable lipids include those described in International Patent Specifications Nos WO 88/04924, WO 90/09782, WO 91/05545, WO 91/05546, WO 93/19738, WO 94/20073, WO 94/22429, WO 95/21931 , WO 96/10038, WO 96/17823, WO 96/18273, WO 96/25508, WO 96/26179, WO 96/41606, WO 97/18185, WO 97/25339, WO 97/30170 and WO 97/31934
  • Targeted lipid complexes especially targeted liposomes in which the lipid is non-covalently associated or covalently linked to a targeting molecule which is capable of directing the lipid to a selected target such as a cell are particularly suitable for use in the process of the invention
  • the targeting molecule may be a peptide, including a glycopeptide, a polypeptide, protein, including a glycoprotein or phosphoprotein, a carbohydrate, glycohpid, oligonucleotide, polynucleotide or other organic molecule, e g a vitamin, which can specifically bind to a receptor, ligand, antigen or other natural or synthetic molecule
  • Antibodies and antigen-binding fragments and derivatives thereof and antibody mimetic molecules produced by combinatorial or other synthetic means form one particular class of suitable targeting molecules
  • Antibodies and antigen-binding fragments thereof e g Fab, Fab', F v and single chain F fragments are especially useful
  • interferons for example interferons ⁇ , ⁇ and ⁇ , tumour necrosis factors ⁇ and ⁇
  • interleukins for example interleukins 1 to 15, chemokines, for example MIP-1 ⁇ , MIP-1 ⁇ and RANTES
  • growth factors for example PDGF, VEGF, EGF, TGF ⁇ , TBF ⁇ , GM-CSF, G-CSF, M-CSF, FGF, IGF, bombesins, thrombopoietin, erythropoietin, oncostatin and endothelin 1
  • peptide hormones for example LH, FSH, TRH, TSH, ACTH, CRH, PRH, MRH, MSH, glucagon and prolactin, transfer ⁇ n, lactofernn, angiotensin, histamine, insulin, lectins, apolipoproteins, for example apolipoprotein E, kmins, and vitamins, for example LH,
  • adhesion moelcules and their binding partners or binding fragments thereof may be used in the invention as targeting molecules
  • Particular examples include VLA-4, VMAC-1 , fibronectin, LFA-1 , MAC- 1.
  • Suitable targeting molecules include monosaccha ⁇ des and oligosacchandes such as galactose, lactose and mannose
  • the extraneous material which forms part of the pid complex may in general be any organic compound ranging in size from a low molecular weight molecule through to a macromolecule Alternatively the material may be a complex, for example complexed metal and other ions One of more different materials may be present
  • the extraneous compound or complex may be in particular a bioactive substance
  • Each bioactive substance may be for example a pharmacologically active agent, including an endosomolytic agent, a diagnostic agent or any agent able to modify the genotype and/or phenotype of a cell
  • pharmacologically active agent including an endosomolytic agent, a diagnostic agent or any agent able to modify the genotype and/or phenotype of a cell
  • Such substances include bioactive proteins, peptides, polysaccha ⁇ des, nucleic acids including synthetic polynucleotides, oligonucleotides and derivatives thereof, lipids, glyco pids, poproteins, lipopolysaccha ⁇ des and viral, bacterial, protozoal, cellular or tissue fractions
  • the bioactive substance possesses a net negative charge and is thus polyanionic
  • Particular polyanionic bioactive substances include nucleic acids, for example single, double or triple stranded, circular or supercoiled DNA or RNA, and hybrids e g chimeroplasts, and derivatives thereof Where desired the DNA may be part of a structure such as a plasmid
  • lipid complex substances which may be present as part of the lipid complex include condensing agents, especially for example polycationic compounds such as polylysine, where the extraneous material is a polyanion such as a nucleic acid
  • Suitable cells include cells associated with the immune system such as lymphocytes e g cytotoxic T-lymphocytes, tumour infiltrating lymphocytes, natural killer cells, neutrophils, basophils or T-helper cells, dendritic cells, B-cells, haematopoietic stem cells, macrophages, monocytes or NK cells
  • lymphocytes e g cytotoxic T-lymphocytes, tumour infiltrating lymphocytes, natural killer cells, neutrophils, basophils or T-helper cells, dendritic cells, B-cells, haematopoietic stem cells, macrophages, monocytes or NK cells
  • the freeze/thawed complex may be added to cells removed from a host and in the presence of serum To achieve appropriate levels of transfection the complex and cells may need to be left in contact for a period of time at an appropriate temperature e g around 37°C Once transfected, the cells are reintroduced into the host using standard techniques
  • freeze/thawed complex may be administered to a host using any convenient approach, for example as described in more detail below. Once administered transfection of host cells can occur according to the second part of the process according to the invention.
  • the freeze/thawed lipid complexes obtained according to the first part of the process of the invention may be put to any use depending on the nature of the extraneous compounds and/or complexes associated with them.
  • the extraneous material is a pharmaceutical agent
  • the complex is of use in medicine and the invention includes a method of treatment of a human or animal subject, the method comprising administering to the subject an effective amount of a freeze/thawed lipid complex.
  • the exact amount of complex to be used will depend on the age and condition of the patient, the nature of the disease or disorder and the route of administration, but may be determined using conventional means, for example by extrapolation of animal experiment derived data.
  • the number of transfected effector cells required may be established by ex vivo transfection and re-introduction into an animal model of a range of effector cell numbers.
  • the quantity required for in vivo use may be established in animals using a range of lipid complex concentrations.
  • the freeze/thawed lipid complex may be useful in the treatment of a number of diseases or disorders.
  • diseases or disorders may include those described under the general headings of infectious diseases, e.g. HIV infection; inflammatory disease/autoimmunity e.g. rheumatoid arthritis, osteoarthritis, inflammatory bowel disease; cancer; allergic/atopic diseases e.g. asthma, eczema; congenital e.g. cystic fibrosis, sickle cell anaemia; dermatologic, e.g. psoriasis; neurologic, e.g. multiple sclerosis; transplants e.g. organ transplant rejection, graft-versus-host disease; metabolic/idiopathic disease e.g. diabetes.
  • infectious diseases e.g. HIV infection
  • inflammatory disease/autoimmunity e.g. rheumatoid arthritis, osteoarthritis, inflammatory bowel disease
  • cancer allergic/atopic diseases e.g. asthma,
  • the freeze/thawed lipid complex may be formulated with other materials such as one or more other lipids or other pharmaceutically acceptable carriers, excipients or diluents before use in the second part of the process of the invention and the invention includes such compositions and their uses
  • the compositions may take any other form suitable for oral, buccal, parenteral, nasal, topical or rectal administration, or a form suitable for administration by inhalation or insufflation
  • compositions may take the form of, for example, liquid preparations such as solutions, syrups or suspensions
  • liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents, emulsifying agents, non-aqueous vehicles and preservatives
  • pharmaceutically acceptable additives such as suspending agents, emulsifying agents, non-aqueous vehicles and preservatives
  • the preparations may also contain buffer salts, flavouring, colouring and sweetening agents as appropriate
  • the freeze/thawed lipid complex may be formulated for parenteral administration by injection, including bolus injection or infusion or particle mediated injection
  • Formulations for injection may be presented in unit dosage form, e g in glass ampoule or multi dose containers, e g glass vials or a device containing a compressed gas such as helium for particle mediated administration
  • the compositions for bolus injection or infusion may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising, preserving and/or dispersing agents
  • the complex may also be formulated as a depot preparation
  • Such long acting formulations may be administered by implantation or by intramuscular injection
  • the complex may be conveniently delivered in the form of an aerosol spray presentation for pressurised packs or a nebu ser, with the use of suitable propellant, e g dichlorodifluoromethane, t ⁇ chlorofluoromethane, dichlorotetrafluoro- ethane, carbon dioxide or other suitable gas or mixture of gases
  • suitable propellant e g dichlorodifluoromethane, t ⁇ chlorofluoromethane, dichlorotetrafluoro- ethane, carbon dioxide or other suitable gas or mixture of gases
  • the hpid complex may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient
  • the pack or dispensing device may be accompanied by instructions for administration
  • the quantity of lipid complex required for any particular application will to a large extent depend on the nature of the extraneous material being delivered. Another important factor will include whether the particle is intended for ex vivo or in vivo use. If the latter, the route of administration and particular formulation chosen as well as factors such as the age and condition of the subject will govern the quantity of complex used. In general however up to around 50 mg of complex can be used for every kilogram of body weight.
  • PBMCs human PBMCs or Jurkats
  • FCS foetal calf serum
  • Transfection complexes were prepared as follows: DNA (pCMV ⁇ gal, 4 ⁇ g/10 6 cells) was mixed with a cationic condensing peptide at a wt:wt ratio of 2:1 (peptide: DNA).
  • Liposomes were prepared by rehydration of a lipid film consisting of dioleoylphosphatidylethanolamine (DOPE): cholesteroholeic acid in a molar ratio of 40:40:20.
  • DOPE dioleoylphosphatidylethanolamine
  • Antibodies either recognising CD3 (OKT 3) or an isotype matched control (lgG2a) not recognising a T cell-expressed ligand were coupled to the external surface of the liposome using conventional coupling methods.
  • liposomes were mixed with the condensed DNA particle at various wt:wt ratios e.g. 8:1 (lipid.DNA). These liposome complexes were then subjected to four cycles of freezing in liquid nitrogen and then thawing in a water bath heated to 60°C. The complexes were then added to the cells and incubated overnight at 37°C. Cells were then prepared for assay of reporter gene expression by a ⁇ gal colorimetric assay or ⁇ gal ELISA kit (see below).
  • Non-freeze/thawed complexes were added to cells directly after mixing the liposomes with the DNA:peptide condensate. For transfections in the absence of serum, complexes were incubated with cells for three hours in medium with no serum. After this time the medium was removed and replaced with fresh medium containing 10% FCS. This was followed by overnight incubation and reporter gene assay as above.
  • lysis buffer serum, sample buffer, wash buffer (x10), anti- ⁇ -galactosidase antibody coated 96-well microwell plate, digoxi ⁇ conjugated anti- ⁇ - galactosidase antibody (anti- ⁇ -Gal DIG), peroxidase conjugated anti- digoxin antibody (anti- ⁇ -DIG-POD), ⁇ -galactosidase standard at 1024 pg/ml.
  • Lyophilised reagents and wash buffer were reconstituted in de-ionised water as per the manufacturer's instructions. Lysis buffer was diluted 3 volumes to 10 volumes of phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the TMB substrate reagent A+B pack (supplied by Pierce & Warriner Ltd., Chester, UK) was used in place of the kit substrate; equal volumes of reagent A and B were mixed just prior to use. Eight doubling dilutions of the ⁇ - galactosidase standard were prepared in lysis buffer.
  • the anti- ⁇ -Gal-DIG reagent was diluted: 20 ⁇ l per 10ml sample buffer and the anti-DIG-POD diluted: 40 ⁇ l per 10 ml sample buffer.
  • microplate was blocked by incubation for 1 h with 300 ⁇ l per well of 20%) new-born bovine serum, then washed twice with wash buffer and 10O ⁇ l sample buffer added per well.
  • the first ELISA stage involved adding 100 ⁇ l of sample or standard per well in triplicate and incubating for 1.5h at room temperature with orbital agitation at 300 rpm. The wells were washed 4 times with 400 ⁇ l wash buffer per well.
  • the second ELISA stage required 200 ⁇ l of diluted anti- ⁇ -Gal-DIG per well and incubation for 1 h as above followed by the same wash regimen.
  • the third ELISA stage required 200 ⁇ l of diluted anti-DIG-POD per well and incubation for 1 h and wash as above.
  • the final stage required the addition of 200 ⁇ l per well TMB substrate reagent and incubating for 30 min at room temperature, with agitation at 300 rpm. Colour intensity in the wells was measured at 630nm using a plate reader with a reference wavelength set to 490nm.
  • Figure 1 shows the results of one experiment using freeze/thawed and untreated lipids on transfection levels in Jurkat cells, a human T cell line.
  • the ratios in the figure refer to the relative proportion of lipid: DNA.
  • the results show that freeze/thawing has little effect on transfection levels in the absence of serum. However, when serum is included, transfection by the non-freeze/thawed complexes, as expected, drops to background levels, while transfection with the freeze/thawed complexes is maintained.
  • Figure 2 shows the results of a further experiment using T cells prepared from human peripheral blood and various lipid:DNA ratios. Once again serum completely ablates transfection by untreated complexes. Unexpectedly, however, not only was transfection by freeze/thawed complexes maintained in the presence of serum, but it was markedly enhanced compared to levels obtained in the absence of serum.

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Abstract

L'invention concerne un procédé assurant la transfection réussie de cellules avec des complexes de lipide en la présence de sérum. Le procédé consiste à soumettre un complexe de lipide à un ou à plusieurs cycles de congélation/décongélation et ensuite à mettre la matière congelée/décongelée en contact avec des cellules soit ex vivo soit in vivo. Le complexe de lipide peut être n'importe quel lipide anionique, cationique ou neutre complexé avec une ou plusieurs matières bioactives telles que des acides nucléiques.
PCT/GB2000/003040 1999-08-06 2000-08-07 Complexes de lipide congeles/decongeles et leur preparation WO2001011069A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP00949803A EP1198582A1 (fr) 1999-08-06 2000-08-07 Complexes de lipide congeles/decongeles et leur preparation
AU63066/00A AU6306600A (en) 1999-08-06 2000-08-07 Freeze/thawed lipid complexes and their preparation

Applications Claiming Priority (2)

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GB9918670.2 1999-08-06
GBGB9918670.2A GB9918670D0 (en) 1999-08-06 1999-08-06 Biological product

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WO (1) WO2001011069A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1791980A2 (fr) * 2004-09-10 2007-06-06 Integral Molecular, Inc. Transfection inverse de reseaux de cellules pour des analyses structurelles et fonctionnelles de proteines
US8828972B2 (en) * 2004-10-19 2014-09-09 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. Formulations containing alkylphosphocholines using novel negative charge carriers

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WO1990009782A1 (fr) 1989-02-24 1990-09-07 Liposome Technology, Inc. Composition liposomique sous forme de gel et son procede de preparation
WO1991005546A1 (fr) 1989-10-20 1991-05-02 Liposome Technology, Inc. Procede et composition de traitement de tumeurs solides
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WO1996041606A2 (fr) 1995-06-08 1996-12-27 Therexsys Limited Compositions pharmaceutiques ameliorees utilisees pour la therapie genique
WO1997018185A1 (fr) 1995-11-14 1997-05-22 Rhone-Poulenc Rorer S.A. Lipopolymamines comme agents de transfection et leurs applications pharmaceutiques
WO1997025339A1 (fr) 1996-01-05 1997-07-17 Commonwealth Scientific And Industrial Research Organisation Composition et procede d'apport d'acides nucleiques
WO1997030170A1 (fr) 1996-02-15 1997-08-21 Boehringer Ingelheim International Gmbh Composition pour la transfection de cellules eucaryotes superieures
WO1997031934A2 (fr) 1996-02-29 1997-09-04 Chemicon Laboratories Gmbh Nouvelles lipopolyamines metabolisables, leur preparation et leur utilisation

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ZELPATI, O ET AL., BIOCHIM. BIOPHYS, ACTA, vol. 1390, 1998, pages 119 - 133

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1791980A2 (fr) * 2004-09-10 2007-06-06 Integral Molecular, Inc. Transfection inverse de reseaux de cellules pour des analyses structurelles et fonctionnelles de proteines
EP1791980A4 (fr) * 2004-09-10 2010-01-06 Integral Molecular Inc Transfection inverse de reseaux de cellules pour des analyses structurelles et fonctionnelles de proteines
US8828972B2 (en) * 2004-10-19 2014-09-09 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. Formulations containing alkylphosphocholines using novel negative charge carriers

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AU6306600A (en) 2001-03-05
GB9918670D0 (en) 1999-10-13

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