WO2001011069A1

WO2001011069A1 - Freeze/thawed lipid complexes and their preparation

Info

Publication number: WO2001011069A1
Application number: PCT/GB2000/003040
Authority: WO
Inventors: Kenneth Crook
Original assignee: Celltech R&D Ltd; Celltech Chiroscience Ltd
Current assignee: Celltech R&D Ltd
Priority date: 1999-08-06
Filing date: 2000-08-07
Publication date: 2001-02-15
Anticipated expiration: 2002-02-06
Also published as: GB9918670D0; EP1198582A1; AU6306600A

Abstract

A process is described to enable the successful transfection of cells with lipid complexes in the presence of serum. The process involves subjecting a lipid complex to one or more freeze/thaw cycles and then bringing the freeze/thawed material into contact with cells either ex vivo or in vivo. The lipid complex may be any anionic, cationic or neutral lipid complexed with one or more other bioactive materials such as nucleic acids.

Description

FREEZE /THAWED LIPID COMPLEXES AND THEIR

PREPARATION

This invention relates to lipid complexes, to a process for their preparation and to their use in medicine.

One of the most important barriers to successful in vivo gene therapy, particularly for the use of non-viral vectors such as liposomes, is the presence of serum or other biological fluids. Even in vitro transfections using liposome and other lipid complexes have to be carried out in the absence of serum, as concentrations of foetal calf serum (FCS) as low as 10% will completely ablate gene delivery to cell lines [see for example Zelpati, O er a/ Biochim. Biophys, Acta, 1390. 119-133 (1998)].

Although the mechanisms for this inhibition have not been definitively elucidated, one likely reason is that, since most types of lipid complex (those of a cationic nature) rely on electrostatic interactions to bind to cells, the presence of other polyanionic molecules in serum permits binding of some of these to the complex, thus reducing its interaction with cells. Another possibility is that serum causes some kind of destabilisation and/or molecular reorganisation of the complex, exposing the complexed DNA to nucleases present in serum. Whatever the mechanism, it is clear that any future therapeutic product for in vivo use will need to be able to overcome this inhibition, but to date, few such improvements have been made.

We have now developed a method of treating lipid complexes to alter their transfection ability such that successful transfection can be achieved in the presence of 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. Advantageously, with certain cell types, the levels of transfection by treated complexes is markedly enhanced in the presence of serum over levels in the absence of serum. Thus according to one aspect of the invention we provide 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

In the first part of the process according to the invention 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 In practice 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 Thus for example where 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 Generally the length of time the lipid complex is maintained at each stage of the freeze/thaw cycle may be varied as desired but may depend on the temperature selected and other variables such as the quantity and/or volume of lipid complex Thus for example a large volume of complex will need to be kept at a particular temperature for a longer period of time than a smaller volume to achieve efficient freezing or thawing

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

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 Examples of 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 In these complexes, 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

Other examples include interferons, for example interferons α, β and γ, tumour necrosis factors α and β, interleukins, for example interleukins 1 to 15, chemokines, for example MIP-1 α, MIP-1 β and RANTES, growth factors, for example PDGF, VEGF, EGF, TGFα, TBFβ, 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, transferπn, lactofernn, angiotensin, histamine, insulin, lectins, apolipoproteins, for example apolipoprotein E, kmins, and vitamins, for example folic acid and vitamin B12 Fragments and other synthetic analogues of these molecules may be used, where these retain or have the appropriate selective binding action It will be appreciated that the above list is not exhaustive and may be extended to include other naturally occurring binding molecules, including for example the complementary binding partner, or a binding fragment thereof, of each of those mentioned, for example the PDGF receptor, the VEGF receptor and so on

Similarly, 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. ICAM-1 , ICAM-2, Lewis X, GMP-140, ELAM-1 , S-Lewis X, fibnnogen, GPIIb/llla, CD28, B7, CD40, CD402L, CD4, laminin, VLA-1 , VLA-2, VLA-3 and VLA-6

Other examples of suitable targeting molecules include monosacchaπdes 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 Particular examples of such substances include bioactive proteins, peptides, polysacchaπdes, nucleic acids including synthetic polynucleotides, oligonucleotides and derivatives thereof, lipids, glyco pids, poproteins, lipopolysacchaπdes and viral, bacterial, protozoal, cellular or tissue fractions

Preferably 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

Other 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

Once the freeze/thawed lipid complex has been obtained in the first part of the process according to the invention it may be brought into contact with any cell in the presence of serum either ex vivo or in vivo 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

For ex vivo use, 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

For in vivo use the 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. Where 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. In particular, for ex vivo use 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. Similarly 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. Such 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.

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

For oral administration, the compositions may take the form of, for example, liquid preparations such as solutions, syrups or suspensions Such liquid preparations may be prepared by conventional means with 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

In addition to the formulations described above, the complex may also be formulated as a depot preparation Such long acting formulations may be administered by implantation or by intramuscular injection

For nasal administration or administration by inhalation, 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, tπchlorofluoromethane, 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.

The following Example illustrates the invention:

EXAMPLE

Cells (human PBMCs or Jurkats) were prepared and added to a 24-well plate at a concentration of 1 0⁶ cells/ml/well in RPMI medium supplemented with 10% foetal calf serum (FCS).

Transfection complexes were prepared as follows: DNA (pCMVβgal, 4μg/10⁶ 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. 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. These 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 βgal colorimetric assay or βgal 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.

β-Gal ELISA

The following kit components were used as supplied by the manufacturer,

Roche Dagnostics Limited, Lewes, UK: lysis buffer, sample buffer, wash buffer (x10), anti-β-galactosidase antibody coated 96-well microwell plate, digoxiπ conjugated anti-β- galactosidase antibody (anti-β-Gal DIG), peroxidase conjugated anti- digoxin antibody (anti-β-DIG-POD), β-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). 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 β- galactosidase standard were prepared in lysis buffer. The anti-β-Gal-DIG reagent was diluted: 20μl per 10ml sample buffer and the anti-DIG-POD diluted: 40μl per 10 ml sample buffer.

The microplate was blocked by incubation for 1 h with 300μl per well of 20%) new-born bovine serum, then washed twice with wash buffer and 10Oμl sample buffer added per well.

The first ELISA stage involved adding 100μl 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 400μl wash buffer per well. The second ELISA stage required 200μl of diluted anti-β-Gal-DIG per well and incubation for 1 h as above followed by the same wash regimen. The third ELISA stage required 200μl of diluted anti-DIG-POD per well and incubation for 1 h and wash as above. The final stage required the addition of 200μl 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.

Claims

1. 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 complex into contact with the cell in the process of serum.

2. A process according to Claim 1 in which the lipid complex is in the form of a liposome.

3. A process according to Claim 1 or Claim 2 in which the lipid in the complex is non-covalently associated or covalently linked to a targeting molecule.

4. A process according to Claim 3 wherein the targeting molecule is an antibody or an antigen-binding fragment thereof.

5. A process according to any one of Claims 1 to 4 in which each freeze/thaw cycle is performed at a freezing temperature between

-5°C and -210°Cand a thawing temperature from 10°C to 60°C.