WO2000074724A2 - Conjugates comprising cytokines and nucleic acids for treating proliferating cells - Google Patents

Conjugates comprising cytokines and nucleic acids for treating proliferating cells Download PDF

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Publication number
WO2000074724A2
WO2000074724A2 PCT/GB2000/002014 GB0002014W WO0074724A2 WO 2000074724 A2 WO2000074724 A2 WO 2000074724A2 GB 0002014 W GB0002014 W GB 0002014W WO 0074724 A2 WO0074724 A2 WO 0074724A2
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WIPO (PCT)
Prior art keywords
product
gene
growth factor
cells
cytokine
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PCT/GB2000/002014
Other languages
French (fr)
Other versions
WO2000074724A3 (en
Inventor
Christopher Ralph Franks
Ruggero Della Bitta
Norman James Maitland
David Jonathan Knight
Original Assignee
Bioinnovation Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority claimed from GBGB9912807.6A external-priority patent/GB9912807D0/en
Application filed by Bioinnovation Limited filed Critical Bioinnovation Limited
Priority to JP2001501258A priority Critical patent/JP2003501402A/en
Priority to AU50886/00A priority patent/AU5088600A/en
Priority to EP00935338A priority patent/EP1185304A2/en
Priority to MXPA01012500A priority patent/MXPA01012500A/en
Publication of WO2000074724A2 publication Critical patent/WO2000074724A2/en
Publication of WO2000074724A3 publication Critical patent/WO2000074724A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2013—IL-2
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    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/642—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
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Definitions

  • the present invention relates to novel products which use a prohferatively active moiety as a vector to target nucleic acid material to proliferating cells and to induce proliferation of the target cells It also relates to products which use a moiety having a high affinity receptor binding activity as vectors for delivering genetic material to selected cells More particularly, the invention relates to novel compositions of matter for use in delivering genetic materials to cells, in gene therapy
  • cytokine Interleuk ⁇ n-2 is also known as T-cell Growth Factor Cytokines and growth factors are both peptide hormones
  • cytokines regulate the functional status of their target cells (I e they can stimulate or suppress both quantitatively and qualitatively), whilst growth factors are more focused on promotion, regulation and maintenance of proliferation and differentation, and the survival of their target cell lineages
  • cytokines and growth factors recognise specific membrane receptors on their target cells, which are unique for that particular cytokine or growth factor Each receptor, m turn, can express a dynamic avidity towards its specific cytokine or growth factor, based on physiological and/or pathological conditions These receptors can be categorised as low, medium, or high affinity Most important of all, high affinity receptors only recognise, capture, and internalise their related cytokine or growth factor These receptors and their gands are discussed in more detail below with reference to cytokines
  • Cytokines are a group of molecules, other than antibodies, which are produced by lymphocytes and are involved in signalling between cells of the immune system, for the purpose of stimulating or suppressing cell function Cytokine activity is often mediated by specific receptors expressed on target cells Cytokines are glycosylated or non-glycosylated polypeptides and can be secreted by both T-cells and B-cells, though T- cells are assumed to be the major source in cell-mediated responses Complications in the study of cytokines have arisen from the fact that in vivo no cytokine ever operates in isolation This is illustrated by the observation that many cytokine actions are synergistic Important cytokines include interleukins (ILs), tumour necrosis factors (TNFs) and interferons (IFNs) In addition, various colony stimulating factors (CSFs) are secreted by myeloervthroid cells Receptors for numerous cvtokines have now been cloned, and their structures (a
  • a domain is a sequence or segment of a protein which forms a discrete structural unit, able to capture and/or convert specific signals
  • the domains of interest are the extra-cellular regions (those located at the surface of a given target cell-lineage)
  • CKR-SF or haematopoietic receptor super family
  • these sites have low (e g 1 - l OnM) or high (e g at least lpM and more usually 10-lOOpM) affinity to a given ligand (cytokine or growth factor)
  • additional sub-units have been identified which are required for high affinity receptor expression
  • These sub-units also referred to as affinity convenors or convertor chains
  • affinity convenors or convertor chains are often expressed on the cell surface after a given activatory or inhibitory stimulus is applied through a receptor ligand This results
  • T-cells must change from a resting to an activated state
  • T-cells stimulated by foreign antigens enter a program of cellular activation leading to de novo synthesis of IL-2
  • Resting T-cells do not express high affinity receptors but these are rapidly expressed after activation
  • Interaction of IL-2 with its induced cellular receptors triggers cellular proliferation culminating m the emergence of effector T-cells that are required for the full expression of immune responses
  • IL-2/IL-2r complex in many of the diseases described herein, this physiological tuning is disrupted (primarily by neoplastic transformation, secondary to viruses), or is automamtamed (autoimmune reactions/diseases, transplant rejection), leading to systemic multi-organ failure
  • High affinity receptors therefore include those w ith an affinity constant of 10 l0 M or less, and, more particularly those with an affinity constant of 10 ' "M or less
  • Representative high affinity receptors include those w ith affinir. constants of between 10 " and 10 12 M
  • IL-2 mterleuk ⁇ n-2
  • three forms of receptor for mterleuk ⁇ n-2 (IL-2) can be distinguished on the basis of their affinitv for IL-2 with IL-2 binding affinities of 10 ' "M (high affinity), 10 '9 M (intermediate affinity) and 10 "8 M (low affinity) (Refs 1 -4)
  • IL-2 receptors are well described in the prior arts (Refs 5 & 6)
  • TNF- ⁇ has been described as having two isoform receptors with high affinity on the target cells for TNF These target cells are macrophages and osteoclasts (Ref 7) M-CSF (macrophage colony stimulating factor) has a high affinity receptor on macrophages and osteoclasts
  • the high affinity receptor is a 150 Kda glycoprotem (Ref 8)
  • IFN- ⁇ has a 90 KDa glycoprotem as a high affinity receptor
  • a different receptor present on activated lymphocytes, macrophages, endothelial cells and fibroblasts has been recognised as the high affinity receptor of IFN- ⁇ and IFN- ⁇ (Ref 9)
  • FGF fibroblastic growth factor
  • a high affinity receptor which is a 140 KDa glycoprotem on mesodermic and neuroectodermic lineage cells, such as activated fibroblasts, macrophages, endothelial cells, chondrocytes, astrocytes, glioma cells, hepatocytes, epithelial cells, neurones, ovarian cells, pituitary cells, and keratmocytes
  • the pharmacological properties of FGF are primarily related to angiogenesis, ovarian steroidogenesis, ostoblast activation, and nerve growth (during the foetal phase) (Ref 10).
  • GnRH-III (GIp-H ⁇ s-T ⁇ -Ser-H ⁇ s-Asp-T ⁇ -Lys-Pro-Gly-NH 2 ) from the sea lamprey, Petromyzon martnus, has been found to suppress growth of breast, prostate and endomet ⁇ al cancer cells but not to have endocrine activity at the concentrations effective against growth of cancer cells (Refs 25-28)
  • Epidermal growth factor is a 53 ammo acid peptide which efficiently stimulates cell growth via a receptor mediated mechanism It is a classical example of a tyrosme kmase/SH2 domain receptor, in which extracellular EGF binding induces receptor dime ⁇ sation, autophosphorylation and binding of downstream signalling molecules to the activated receptor via their SH2 domains (Ref 29) Since the receptors for EGF are present m multiple potentially heterodime ⁇ c forms on the surfaces of a large number of cell types, this receptor type is widely applicable
  • IGF insulin-like growth factor
  • IGF has a high affinity receptor on eterotetrame ⁇ c complex present in different tissues and in mammary adenocarcinoma (Ref 1 1 )
  • TGF ⁇ Transforming Growth Factor ⁇
  • IGF ⁇ Transforming Growth Factor ⁇
  • TGF ⁇ is a non-glycosylated homodyme ⁇ c protein secreted by fibroblasts. epithelial cells, platelets, astrocytes, monocytes. bone cells, and g oblastoma cells The physiological target cells are primarily fibroblasts, osteoblasts, neutrophils. hematopoietic progenitors.
  • T B lymphocytes and a range of tumor cells
  • the cytokine interacts with a high affinity receptor, expressed by the target cells, in response to parac ⁇ ne microenvironmentai stimulation, located on the cell surface of the above cells
  • a high affinity receptor expressed by the target cells, in response to parac ⁇ ne microenvironmentai stimulation, located on the cell surface of the above cells
  • type 1 or type 2 receptors 55 and 80 Kda
  • GM-CSF granulocyte/ macrophage colony stimulating factor
  • SCF stem cell factor
  • G-CSF granulocyte colony stimulating factor
  • EPO erythropoietin
  • IL-6 ( ⁇ nterleuk ⁇ n-6) has an ⁇ -/ ⁇ - high affinity receptor
  • the alpha chain binds IL-6 with low affinity and exists in a soluble form
  • the beta chain is a 130 KDa protein which simultaneously binds IL-6/IL-6r, becoming a trime ⁇ c complex which initiates target cell stimulation IL-6 high affinity receptor induction, following specific stimuli, is primarily positioned on activated cells such as T/B lymphocytes, fibroblasts, myeloid precursors, neurones, keratmocytes, and hepatocytes
  • multiple myeloma cells produce IL-6, and express IL-6 receptors working as an autoc ⁇ ne cancer growth factor, inducing at the same time osteoclastogenesis (bone lytic lesions) IL-6 from stromal cells can also be involved in bone metastatic lesions through different tumour histotypes
  • Gonadotropin-releasing hormone (GnRH, Glp-H ⁇ s-T ⁇ -Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2 ) is the central regulator of the hypothalamic-pituitary-gonadal axis GnRH analogues are used to treat sex-hormone dependent cancers of the breast, prostate and ovaries GnRH receptor RNA is expressed in human pituitary, breast, ovary, prostate and endolinguisticim As described by Palyi et al (Ref 24) certain cancer cell lines have been found by previous workers to express high- and low- affinity binding sites for GnRH GnRH is therefore a peptide hormone for which there are high affinity receptors
  • WO 92/20364 describes hybrid molecules containing a first portion which is a molecule capable of decreasing cell viability (especially a cytotoxin) and a second portion which is a molecule capable of specifically binding to a cytokine receptor (especially all or a binding portion of a cytokine)
  • the second portion targets the first portion to the cytokine receptor and is exemplified as IL-2
  • the IL-2 portion preferably lacks IL-2 activity because the molecules will then prevent proliferation of the target cells
  • GnRH gonadotropin-releasing hormone
  • the conjugated polymer protects the peptide against proteolysis and enhances its antiproliferative effect, possibly as a result of enhanced binding of the peptide conjugates with external domains of the receptor and adjacent membrane structures and/or internal isation of receptor- conjugate complexes
  • Gene therapy is the transfer of genetic material or gene function modulators to target cells of a patient for the pu ⁇ ose of preventing or altering a disease state, and may be for the treatment of non-genetic as well as genetic disorders Gene transfer may be ex ⁇ vo or in vivo Ex vivo techniques usually involve the genetic alterations of cells, mostly by use of viral vectors, prior to
  • liposomes especially retrovirus and adenovirus vectors
  • receptor-mediated endocytosis for example using DNA linked to a targeting molecule such as polylysine
  • GDEPT gene-directed enzyme-prodrug therapy
  • viral vectors are seen as potentially undesirable because of doubts raised about their safety, immunity to them and the relative difficulty of large scale culture of viral vectors Effective alternatives to viral vectors are therefore being actively sought
  • the invention of International patent application No PCT/GB98/03509 is based in one aspect on an insight that a medicament which contains an active promoter of proliferation, for example an active IL-2, can beneficially be used to deliver pharmacologically desirable species to cells whose proliferation is not desired
  • some medicaments of that invention control or inhibit proliferation using a molecule which contains an active promoter of proliferation
  • Preferred embodiments are based on an appreciation that, by using the high affinity of receptor super families, it is possible to drive drugs or genetic material, for example, into specific cell lineages which are predominantly responsible for many clinical events
  • these prohferatively active vectors are not only active as transporters of genes but also promote the rearrangement of DNA, in addition to opening DNA chains in target cells
  • Thev are therefore ideal for integrating genes both in vitro and in vivo
  • No other system available has a comparable bi-modal activity, and without the associated risks of viruses (used currently as vectors)
  • PCT/GB98/03509 also describes and claims products comprising a biologically active agent linked to a moiety which is a peptide hormone, which has a high affinity receptor, or is a molecule functionally equivalent to the peptide hormone in relation to the high affinity receptor
  • the present application relates in particular to novel products of the types described and claimed in PCT/GB98/03509 More particularly the invention provides products or compositions of matter comprising a prohferatively active moiety linked to genetic or nucleic acid material which is associated with protective material There is considerable evidence that transported genes are more active in dividing cells and the prohferatively active moiety therefore potentiates the cells whose proliferation is caused towards the genetic or nucleic acid material
  • the invention is concerned with products which potentially use prohferatively active moieties both as vectors for protected nucleic acid material and as mitogens to stimulate proliferation of the target cells
  • the protective material serves to protect the genetic or nucleic acid material from degradation and may by way of example comprise any known protective material Specifically, the genetic or nucleic acid material may be protected b> encapsulation in a micelle, especially a hposome, or by being complexed, for example with a protective protein such as, e g , polylysine
  • the invention includes products in which genetic or nucleic acid material is protected by an associated material the invention is not restricted to products in which the associated material is protective or solely protective in function It includes also products in which genetic or nucleic acid material is otherwise associated with cationic DNA-binding material
  • the invention provides products or compositions of matter comprising a prohferativeh active moietv linked to a nucleotide which is associated with cationic DNA-binding material
  • nucleic acid or genetic material is linked to a prohferatively active moiety Unlike prior art chimeric proteins containing solely the receptor-bmding domain of IL-2, therefore, these products induce cellular proliferation, enabling anti-prohferative drugs to be highly effective, even at ultra-low doses in the case of prohferatively active moieties with high affinity receptors
  • the invention therefore enables low systemic toxicity to be achieved
  • An additional benefit at least in the case of IL-2 is that IL-2 induces expression of the high affinity IL-2 receptor when the relevant antigen is present
  • IL-2 and protected antisense DNA/RNA designed to block a retrovirus gene obtains the following effects
  • the rep cative stimulus given by the IL-2 stimulates also replication of the viral genome, resulting in stronger inhibitory activity by the antisense DNA/RNA,
  • the product is a combination of existing moieties (or of moieties functionally equivalent thereto), each of which retains its function and, optionally, its entire structure (except at any covalent linkage site to the other moiety)
  • the product can be administered at exceedingly low dosages, so that little or no systemic toxicity results
  • the growth factor/cytokine stimulates the target svstem and the nucleic acid moiety induces a therapeutic effect
  • Fig 1 An overview of the invention is shown in Fig 1 , which for convenience illustrates the invention with reference to a product in which the prohferatively active moiety is an IL-2
  • the IL-2 is linked to a protected (in this case polylysine complexed) expression vector (in this case a plasmid), suitably by attaching to the IL-2 a linker having a functional group reactive with the polylysine as well as a functional group reactive with the IL-2, and then reacting the linker with the polylysine before or after the latter is combined with the plasmid (expression vector)
  • a protected expression vector in this case plasmid
  • the resultant product is administered in vivo or in vitro and the IL-2 (prohferatively active moiety) serves as a vector to direct the product to cells presenting IL-2 receptors, especially high affinity receptors
  • the product is internalised, after which the IL-2-polylysme link is cleaved (e g hydrolysed), allowing the IL-2 to promote division and the expression vector to migrate to the nucleus
  • IL-2-polylysme link is cleaved (e g hydrolysed), allowing the IL-2 to promote division and the expression vector to migrate to the nucleus
  • the prohferatively active moiety which is not internalised by its receptor, in which case the link with the remainder of the product is normally cleaved extracellularly and the expression vector (or other nucleotide) is internalised
  • Figure 1 is an overview of the invention, illustrating the structure and function of products of the invention with reference to an IL-2-Express ⁇ on Vector construct
  • Figures 2, 3 and 4 are diagrammatic illustrations (linearised circles) of plasmid constructs of the invention
  • Figure 5 is an SDS-PAGE gel run using the product of a reaction between Aldesleukm and LC-SPDP,
  • Figure 6 illustrates a 96 well plate as used in a growth assay
  • Figure 7 is a data chart of cell densities resulting from the incubation of HT-2 cells with modified Aldesleukm
  • Figure 8 is a data chart showing growth responses of HT-2 cells to various IL-2 compositions
  • Figures 9a, b and c are data charts showing the results of a toxicity assay in which serial dilutions of 25k PEI were added to HT-2 cells cultured at 1 0, 0 5 and 0 2ng ml RhIL-2 and cell viability assayed for three days
  • the material associated w ith the genetic or nucleic acid material is protective in one class of products It may form a micelle, especially a hposome
  • the posome or micelle mav encapsulate the nucleic acid material or it may be positively charged and hold the nucleic acid on its surface (a so-called lipoplex)
  • Liposomes are formed by phospholipids and similar amphipathic lipids, and are commercially available Cholesterol is frequently included in hposome formulations
  • Cholesterol is frequently included in hposome formulations
  • An alternative class of encapsulating composition comprises artificial viral envelopes (see Ref 39)
  • One class of protective mate ⁇ als therefore, comprises complexmg materials and includes not only cationic liposomes but also other cationic materials, especially polymers
  • suitable polymers there may be mentioned polylvsine (especially poly-D-lysine), polylysine derivatives (eg phospholipid derivatives of, in particular, poly-L-lysme) and polyethyleneimine (PEI)
  • PKI polyethyleneimine
  • suitable complexmg agents are dend ⁇ mers, especially polyamidoamine dend ⁇ mers (which are cationic)
  • the associated material is not protective, or exclusively protective, in all the products of the invention
  • a cationic DNA-bmdmg moiety forms a bridge between the prohferatively active product and the nucleic acid material (nucleotide)
  • Suitable DNA-binding moieties are cationic polymers such as polylysine or PEI Additionally there may be cationic or other protective material
  • nucleic acid or genetic material is not critical to the invention, which is concerned primarily with methods for delivery, l e transfer of a nucleotide into the target cell and especially into its nucleus
  • the nucleic acid material generally comprises either a therapeutic gene or an antisense nucleotide (ohgo- or poly- nucleotide)
  • Antisense nucleotides are single strand nucleotides containing sequences complementary to target mRNA or DNA in order to block the production of disease-causing proteins Antisense therapy is often considered to be a form of gene therapy because it is modulation of gene function for therapeutic pu ⁇ oses
  • Therapeutic nucleotides are suitably phosphorothioate ohgodeoxynucleotides (ODNs), as these are nuclease-resistant
  • ODNs phosphorothioate ohgodeoxynucleotides
  • antisense nucleotides may conveniently be protein nucleic acids (PNAs)
  • Antisense nucleotides are useful for the treatment of viral diseases and cancer (see Ref 25) and the invention includes the use of antisense products for such treatments as well as the treatment of neurodegenerative and cerebrovascular disorders
  • the products of the invention may be materials comprising an expression vector for expression of a therapeutic gene
  • the expression vector will contain a gene encoding a protein operably linked to a control sequence
  • the control sequence will include a promoter and preferably an enhancer derived from, for example, immunoglobulin genes, SV40, cytomegalovirus (CMV), and a polyadenvlation sequence
  • the expression vector comprises a plasmid construct (recombinant plasmid)
  • the expression vectors are suitably in the form of recombinant plasmids
  • a bacterial expression cassette permits large scale preparation of the construct in a prokaryotic system
  • Illustrative plasmids are shown in Figures 2, 3, and 4 in linearised form
  • Figure 2 shows a plasmid comprising a therapeutic gene, which in this case is a cytotoxic gene, represented by a gene-directed enzvme-prodrug therapy gene shown to be a thymidine kinase gene
  • the therapeutic gene is operably linked to a control sequence, shown to be a promoter and specifically a CMV promoter
  • the plasmid will also contain a poly A/ termination site, a bacterial origin and an antibiotic resistance gene
  • Figure 3 shows a plasmid similar to that of Figure 3 but additionally containing an episomal maintenance sequence, which will maintain the therapeutic gene outside of the cell chromosome as an episome This will enhance its expression and prevent cellular silencing of the therapeutic gene by insertion into heterochromatm
  • the construct of Figure 4 contains two therapeutic genes which again are represented as cytotoxic genes
  • the construct optionally contains an IRES (internal ⁇ bosome entry site) to ensure that the two genes are expressed simultaneously from the same promoter
  • IRES internal ⁇ bosome entry site
  • an indicator gene e g the EGFP gene which causes expression of a green marker protein
  • a therapeutic gene e g he ⁇ es simplex virus (HSV) thymidine kinase gene which will activate gancyclovir when expressed
  • Cells which have been treated for transduction with the construct can be monitored for successful gene transduction by detecting for green cells
  • the killing drug gancyclovir in this case
  • the process is suitably monitored by time lapse photomicroscopy, to provide information about the efficiency of cell kill as well as about the percentage of cells in the population transduced with the genes and killed
  • the gene may be any gene having a therapeutic function and, by wav of non-limiting example, may be a therapeutic gene now known to those skilled in the art of gene therapy
  • Representative classes of genes include immunogenes (e g cytokine gene therapy DNA-based cancer vaccines), cytotoxic genes and especially genes for enzy e-prodrug therapy (e g genes encoding viral thymidine kinase, bacterial or other cvtosme deammase, cytochrome P-450 or bacterial nitroreductase) and defect correction genes (e g tumour suppressor genes and especially p53)
  • the invention also includes products in which the nucleic acid material has a non-specific and/or non- antisense effect, for example products which inhibit viral infection by interference with abso ⁇ tion, penetration or uncoating of viruses
  • the prohferatively active moiety is usually a cytokine or growth factor
  • the cytokine may be an interleukin for example a TNF, for example an M-CSF, an IFN, for example an FGF, an IGF, a TGF, for example a GM-CSF, an SCF, a G-GSF, or an EPO
  • the cytokine is preferably a human cytokine
  • the growth factor may be a haematopoietic or lymphopoietic growth factor They are a family of glycoprotem hormones which regulate survival, proliferation, and differentiation of progenitor cells, in addition to impacting on some functional activities of mature lymphohaematological cells
  • Suitable growth factors include Erythropoietin (Epo),
  • SCF Stetem cell factor
  • Multi-CSF also known as lnterleuk ⁇ n-3
  • M-CSF Multi-CSF
  • E-CSF (or lnterleuk ⁇ n-5)
  • IGF- 1 Insulin- 1 (Insuhn- ke growth factor)
  • PDGF Platinum-derived growth factor
  • TGF beta2 Transforming growth factor -beta2
  • the prohferatively active or mitogenic moiety is internalised by target cells in a preferred class of products but in another class of products is not internalised
  • the mitogenic moiety may be a growth factor such as an EGF or FGF which is Tyr k ⁇ nase/SH2 mediated
  • the nucleic acid material is preferably cleavable from the mitogen and/or associated with a delivery material, e g a hposome or DNA binding material
  • FGF products include their use as antiangiogenic factors in solid cancers, and to block hyperactivation of fibroblasts in scleroderma
  • FGF-2 and FGF-7 both of which have high affinity receptors, have been implicated in prostate cancers
  • the present invention provides products comprising a domain functional to bind to an EGF, FGF-2 or FGF- 7 receptor (especially a high affinity receptor) to promote proliferation and a nucleotide, e g genetic material
  • the domain or moiety having EGF, FGF-2 or FGF-7 receptor binding function serves as a vector for directing the second domain or moiety to cells having, as the case may be, EGF receptors, FGF-2 receptors or FGF-7 receptors and especially EGF high affinity receptors, FGF-2 high affinity receptors or FGF-7 high affinity receptors
  • the products having FGF-2 or FGF-7 receptor binding function are useful for targeting anti-cancer drugs to breast, stomach, oesophageal and prostate tumour cells
  • the products having EGF receptor binding function are useful for targeting anti-cancer drugs to most tumour types but particularly those of breast, stomach, ovarian, bladder and prostatic origin
  • the two domains or moieties are suitably linked by a physiologically cleavable link which will be cleaved in the receptor-bound product
  • the linkage between the two domains or moieties may be covalent but is not covalent in some of the products
  • One class of products comprises molecules having a multi-part bridging group as described above
  • Cytokines or growth factors may be native or a mutein representing the native molecule modified by one or more amino acid alterations (deletions, additions or substitutions)
  • Such muteins usable in the present invention, possess the biological activity of the native protein, in the sense of having both functional affinity for the receptor (and in one class of embodiments functional affinity for the high affinity receptor) and in many cases a capability of forming, with the receptor, a product internalised by the cell presenting the receptor
  • the cytokines and growth factors are preferably recombinant molecules but may be produced by cultivating cytokine or growth factor producing cell lines, for example peripheral blood lymphocytes
  • the products comprise a nucleotide associated with protective and or DNA- binding material linked to a molecule which is functional to have a high affinity with a cytokine or growth factor high affinity receptor, and to form a complex with such a receptor which is, in one class of products, internalised by the cell presenting the receptor
  • the molecule may be a native or mutein cytokine.
  • the molecule may be a native or mutein growth factor, or a fragment thereof
  • IL-2 is a lymphokine which is produced by normal peripheral blood lvmphocvtes, and induces proliferation of antigen or mitogen stimulated T-cells after exposure to plant lectins antigens, or other stimuli IL-2 was first described bv Morgan, D A .
  • T-cell growth factor because of its ability to induce proliferation of stimulated T lymphocytes it is now recognised that, in addition to its growth factor properties, it modulates a variety of functions of immune system cells in vitro and in vivo, and has been renamed interleukm-2 (IL-2)
  • Interleukm-2 may be made by cultivating human peripheral blood lymphocytes (PBL), as described, for example, in US Patent No 4,401 ,756
  • PBL peripheral blood lymphocytes
  • the IL-2 may be recombinant Taniguchi, T et al , Nature (1983), 302 305-310 and Devos, R , Nucleic Acids Research (1983), 1 1 4307-4323 have reported cloning the human IL-2 gene and expressing it in micro-organisms
  • IL-2 mutein desala [ -IL-2 er j 25 (Aldesleukm) is available commercially from Chiron B V of Amsterdam, Netherlands under the trade mark Proleukin®
  • a bridging group is directly linked to the two of them by covalent bonding
  • a bridging group is directly covalently bonded between the prohferatively active moiety and the associated material
  • the bridging group may comprise a known heterobifunctional or homobifunctional linker, or it may be formed bv the interbonding of two heterobifunctional linkers
  • a peptide linker is used, for example between the proliferativelv active material and the envelope of a hposome (see for example Ref 19)
  • the prohferatively active moiety is usuallv peptidic and is in a preferred class of products covalently bonded to the nucleic acid or the associated material
  • the peptidic moietv mav be covalentlv bonded to the nucleic acid or associated material via a bridging moietv which is bonded to the peptide through a p ⁇ mar. amine residue thereof and to the other material through a functional group thereof
  • a polylysine or PEI complexmg moiety mav be linked through one of its a ine groups to the bridging group
  • a nucleic acid may be linked through one of its hydroxy groups to the bridging groups
  • the two active domains of the molecule are in a preferred class of molecules bonded to each other via a bridging moiety which comprises a first heterobifunctional cross-linker residue bonded to the prohferatively active moiety and to a second heterobifunctional cross-linker which in turn is bonded to the nucleic acid or associated material
  • the invention also includes in another aspect a method of treating by prophylaxis or therapy a disease or disorder involving cells bearing a high affinity receptor for, in particular, a cytokine or growth factor, comprising administering to a patient an effective amount of a product comprising an agent which is biologically active when in said cells and is linked to said cytokine or growth factor, such products and preparations containing them form a further aspect of the invention
  • a product of the invention for use as a pharmaceutical, especially in internalising the biologically active agent into a cell having a high affinity receptor for the prohferatively active agent, cytokine or growth factor of the product
  • Another aspect of the invention resides in the use of a product of the invention for the manufacture of a medicament for internalising the biologically active agent into a cell having a high affinity receptor for the pro ferativelv active agent, cytokine or growth factor of the product and optionally for stimulating lymphocyte proliferation
  • the products of the invention preferably act only on cells presenting a high affinity receptor for the cytokine or growth factor, which are typically lvmphocytes or other cells involved in the immune response
  • the action of a product of the invention on its target cells depends on the function of the active agent
  • the molecular ratio of the active agent nucleic acid material in the products of the invention is not critical Thus the invention includes ratios of 1 1 or less but in some embodiments the ratio is greater than 1 1 , e g 1 1000 or more, l e a plurality of active agent molecules/atoms may be bound to each prohferatively active moietv
  • inventive products will now be described in more detail, by way of example, with reference to illustrative products and product classes
  • IL-2/ant ⁇ sense fusion products are useful for introducing specific antisense sequences (ohgonucleotides) into lymphocytes bearing the IL-2 receptor
  • an antisense compound for a given tract of the HIV genome e g genome coding for an envelope protein, or for the enzyme transcriptase
  • a pharmacological strategy such as IV administration, for example
  • IL-2/ antisense products may also be used to introduce antisense compounds or antioncogenes into the T-cell lineage affected by neoplastic transformation where gene mutation, or oncogene hyperexpression is known
  • the IL-2/act ⁇ ve compound fusion products are useful for diseases for which the lymphocyte is mainly involved in tissue damage, and the resultant development of a given disease entity
  • Interleuk ⁇ n-2 high-affinity receptor-directed lmmunosuppressive therapy acts pharmacologically, but only on recently activated lymphocytes (particularly T- cells), which bear this structure on the cellular membrane
  • the activation signal is absent from the surface of resting T-cells and all other non-lymphoid tissues.
  • lymphocytes Since the receptor is only transiently expressed during the brief pro ferative phase, when lymphocytes respond to antigen stimuli (autologous-antigen in the case of autoimmune diseases, and heterologous- antigens in the case of transplantation), it is possible to achieve selective in vivo immunosuppression, directed solely towards activated lymphocytes (ohgoclonal immunosuppression) This pharmacological action is totally different from the general immunosuppression action exerted by conventional lmmunosuppressive drugs
  • autoimmune diseases transplant rejection HIV- infection
  • lymphoprohferative diseases diseases which can benefit from this approach include autoimmune diseases transplant rejection, HIV- infection, and lymphoprohferative diseases
  • Autoimmune Diseases are a wide variety of disorders with a common pathogenic pathway immune attack on target organs due to abnormal recognition of tissue antigens, and/or cellular antigens, by the immune system, particularly by T-lymphocytes ( 17)
  • the clinical disorders under this heading and their target organs include the following:
  • Idiopathic thrombocytopenic pu ⁇ ura (ITP) > platelet 14
  • Autoimmune neutropenia > neutrophil
  • T-cells The evidence to support the role of T-cells in the pathogenesis of specific disease and progression of targeted tissue damage is substantial In most of the diseases listed above, CD4 cells (a cytotoxic subset of T-cells) are the dominant T-cell phenotype in the target tissues T-cells express several activation markers Experimentally, there is evidence that autoimmune diseases improve when T-cell targeted intervention occurs, as in thoracic duct drainage, total lymphoid irradiation, and administration of Cyclospo ⁇ n
  • autoimmune diseases are treated by attempting to reduce the function of the immune system using lmmunosuppressive and anti-inflammatory drugs This therapeutic strategy is conducted m a non-specific way, resulting at times in latrogenic toxicity and a failure to control the overall disease process
  • Lymphocytes responsible for the acute phase of a given autoimmune attack, all bear the high-affinity IL-2 receptor on the membrane They are antigen-activated, or cytokine-activated, lymphocytes with a high avidity for IL-2
  • IL-2 as the vector of pharmacologically active nucleic acids to exert immunosuppression
  • the IL- 2/ ⁇ mmunosuppressant nucleic acid products selectively bind to. and interact with, only those cells bearing the high-affinity receptor of IL-2 This means that immune cells, responsible for tissue damage and disease progression, are inactivated selectively, potentially curing patients with chronic diseases, or reducing their relapse rate
  • Recombinant proteins for example recombinant IL-2 and other recombinant cytokines and growth factors, usually have low lmmunogenicity and good tissue distribution After parenteral administration, every tissue compartment in the body is exposed, including all lymphocytes (circulating lymphocytes, lymphocytes in the tissues, and lymphocytes in the lymph nodes)
  • the acute or chronic rejection of a transplanted organ is related to heterologous antigens (antigen specific to the donor transplanted organ(s)) presenting to host T-cells Following antigen presentation and recognition, immune cells enter into a pro ferative phase, during which the high-affinity receptor for IL-2 is expressed This leads to activation of the cytotoxic process, and to damage and subsequent failure of the transplanted organ
  • IL-2 as a vector to target lmmunosuppressive nucleic acids achieves longevity of transplanted organs without the associated toxicity of conventional lmmunosuppressive therapy (acute, delayed, and long term)
  • Allogeneic bone marrow transplantation is used to treat and cure leukemias (both lvmphoid and mveloid). thalassemia and solid tumours
  • the products of the invention have the potential to reduce the incidence of Graft-Versus-Host-Disease (GVHD) which is the reaction of the donor immune svstem against tissue antigens of the host, without compromising the global immune-svstem (of graft origin)
  • GVHD Graft-Versus-Host-Disease
  • the administration of low-dose lL-2/ant ⁇ sense nucleotide specific to HIV-genome fraction exerts in vivo an immunostimulatory effect on HIV-negative lymphocytes bearing the high-affinity receptor
  • the antisense nucleotide is introduced into the cytoplasm of HIV-infected lymphocytes (CD4 cells), leading to a selective destruction of infected cells, without impacting on the normal reactive lymphocytes which are stimulated
  • IL-2 cytotoxic gene construct it is possible to selectively kill the neoplastic lymphoid lineage expressing the high-affinity IL-2 receptor, without inducing any critical systemic toxicity on non-lymphoid compartments
  • Products containing growth factors or cytokines other than IL-2 may be used in the therapies described below
  • TNF- ⁇ has as its target cells macrophages and osteoclasts TNF- ⁇ /blocking compound products may be used to insert into macrophages a blocking nucleotide (which blocks cellular function and/or kills the cell)
  • a blocking nucleotide which blocks cellular function and/or kills the cell
  • Such products potentially provide an important tool in some pathological conditions, e g advanced solid cancers where macrophage hyperstimulation and activation is responsible for cachexia and tumour progression, monocyto-macrophage neoplasms (e g histiocytosis), transplant rejection and GVHD, autoimmunit , and neurological degenerative diseases (the TNF receptor in its extracellular domain is similar to nerve Growth Factor receptor)
  • M-CSF macrophage colony stimulating factor
  • TNF- ⁇ blocking nucleotide products may be used to form products containing blocking nucleotides. useful for treating the same classes of conditions as TNF- ⁇ blocking nucleotide products
  • M-CSF is also responsible for microghal proliferation in the CNS
  • Other potential applications are therefore in some degenerative diseases of the CNS. such as Alzheimer's svndrome. and in bone diseases
  • the IFN/nucleic acid products may be used to modify the function of activated lymphocytes, macrophages, endothelial cells and fibroblasts, or to incapacitate them in different pathological conditions, such as in HIV-infection ( AIDS), and fibroblast-related diseases such as scleroderma
  • AIDS HIV-infection
  • FGF products include their use as antiangiogenic factors in solid cancers, and to block hyperactivation of fibroblasts in scleroderma
  • the invention enables the preparation of FGF products capable of acting as an antagonist in relation to the cell types listed earlier in this specification as having FGF high affinity receptor when activated
  • IGF products may be used to treat breast cancer Due to the presence of the high affinity receptor in CNS (neurog a), it could also be used in some degenerative neurological disorders
  • TGF ⁇ products have applications similar to those of FGF and IGF constructs
  • GM-CSF constructs may be used to selectively kill myeloid blasts responsible for myeloid leukemias
  • G- CSF products have similar pharmacological activity to the GM-CSF products, but bind to a different high- affinity receptor present only in multipotent stem cells in the bone marrow
  • Epo fusion products may be used for diseases such as polycythemia and erythroleukemia, for example
  • Epo/gene sequence fusion products which the DNA fraction is the normal gene for haemoglobin beta- chain, may be used for introducing the normal gene into the erythroid lineage in patients affected by beta- Thalassemia
  • the abnormal gene coding for a non-functional haemoglobin beta- chain
  • the insertion of the normal gene, through the Erythropoietm vector, selectively into the bone marrow erythroblastic lineage, represents true in vivo gene-therapy, to potentially cure patients with this disease
  • the same consideration applies to another genetic haemoglobin disease sickle cell anaemia
  • IL-6 constructs may be used to block cells, having IL-6 high affinity receptors, which are involved in multiple myeloma, osteoclastic hyperactivation (metastasis to the bone), cancer-related bone lesions and osteoporosis
  • Epo/SS-wild gene is Sickle Cell Anemia (or Sickle Cell Disease)
  • Lysosomal storage diseases include most of the lipid storage disorders, the mucopolysaccha ⁇ doses and glycoprotem storage diseases which are characterised by mono-enzymatic defects (beta-galactosidase, beta-glucocerebrosidase deficiency in Gaucher's disease, alpha-fucosidase deficiency in Fucosidosis, ceramidase deficiency in Farber's disease and hexosammidase-A deficiency in Tay-Sachs syndrome) All these serious congenital conditions may have their onset in infantile, juvenile and adult age
  • M-CSF/wiId gene coding for a functional enzyme selected on the basis of the specific deficiency, once integrated into macrophages and transc ⁇ pted into protein, will compete with the nonfunctional protein and repair the defect in vivo
  • the invention includes products comprising a moiety having M-CSF, SCF or GM-CSF function linked to a functional acid sphingomyehnase gene
  • ADA Adenosine Deammase
  • SCID SCID
  • T-cell dysfunction a reduction in, and abnormal function of, both T and B lymphocytes
  • ADA deficiency can result in slowly progressive immune dysfunction, which presents at birth but also in adolescents and adults
  • ADA ADA-gene abnormality
  • ADA autosomal recessive inherited SCID
  • the gene for ADA is located on chromosome 20q More than 25 single base changes within the coding region, as well as several deletion and splicing mutations leading to loss of enzymatic activity have been identified ADA catalyzes the irreversible deamination of adenosine to inosine, and of 2-deoxyadenosme to 2-deoxymosine
  • Allogeneic bone marrow transplantation is currently the treatment of choice for ADA-deficient SCID, but an HLA-identical donor is available only for a minority of patients
  • An alternative therapy is the injection of PEG-ADA (daily enzyme replacement)
  • ADA gene has been sequenced and the structure of the enzyme has been determined.
  • Several patients have been treated with ADA-cDNA ex-vivo transduced autologous T-lymphocytes using retroviral vectors (Refs 26-36) Results have been poor and the major barrier to effective gene therapy remains the low efficiency of stem cell transduction with retroviral vectors
  • Recombinant IL-2 has been used at low doses m SCID and ADA-SCID patients (Ref 37) Therapy with low dose rIL-2 has resulted in a marked clinical improvement as well as improved T-cell function Futhermore IL-2 is normally used to expand T-cells in vitro from ADA-SCID patients before ADA-cDNA transfection with retroviral vectors.
  • Lymphocytes and lymphoblasts from ADA-SCID patients and ADA-SCID animal model are responsive to low dose IL-2 stimulation, both in vitro and in vivo
  • IL-2 low dose IL-2 stimulation
  • the invention therefore includes products comprising a first domain which comprises an IL-2 sequence functional to be recognised by high affinity IL-2 receptors and to promote proliferation linked to a second domain which comprises a gene for functional ADA
  • the gene is usually associated with protective material as described above, e g polylysine
  • the invention is a product comprising a functional IL-2 linked to an expression vector comprising a gene for functional ADA Preparation
  • Polynucleotides containing a desired gene can be prepared by any suitable method including, for example, cloning and restriction of appropriate sequences as discussed supra, or by direct chemical synthesis by methods such as the phosphotnester method of Narang et al Meth Enzymol 68 90-99 (1979), the phosphodiester method of Brown et al , Meth Enzymol 68 109- 151 (1979), the diethylphosphoramidite method of Beaucage et al , Tetra Lett , 22 1859-1862 (1981), the solid phase phosphoramidite tnester method described by Beaucage and Caruthers ( 1981 ), Tetrahedron Letts , 22(20) 1859-1862, e g , using an automated synthesizer, e g , as described in Needham-VanDevanter et al ( 1984) Nucleic Acids Res , 12 6159-6168 and, the solid support method of U S Patent No 4,458,066 Chemical synthesis
  • Nucleic acids may be modified by site-directed mutagenesis. As is well known in the art Native and other nucleic acids can be amplified by in vitro methods Amplification methods include the polymerase chain reaction (PCR), the hgase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (SSR) A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well-known to persons of skill
  • mice or cationic materials for example cationic polymers, are commercially available and the use of liposomes in gene therapy is reviewed by Lasic and Templeton (Lasic D D, Templeton D S (1996) Advanced Drug Reviews 20 221 -226)
  • the prohferatively active moiety and one of the nucleic acid material and protective/DNA-bindmg material are suitably interlinked using a multifunctional (e g bifunctiona!) linker which reacts with respective functional groups on the two components
  • a multifunctional linker which reacts with respective functional groups on the two components
  • the two constituent parts are linked by an intracellularly cleavable link
  • the link is intracellularly stable
  • the link may comprise a bridging group comprising the inter-bonded residues of two heterobifunctional cross-linkers, as described in our US provisional patent application filed on the same day as this application under the title "Novel Constructs"
  • the potential benefit of this technique is that it helps avoid the preparation of dimers, by separately preparing (I) an active moiety-linker construct and (n) a nucleic acid- hnker construct or protective material- or DNA-b dmg material- linker construct, and then reacting together constructs (I) and (n) to join their respective linkers together
  • the linkers are obviously chosen to be reactive to each other, as well as to the material forming the residue of the construct (I) or (n)
  • linkers are multifunctional, and especially bifunctional compounds capable of reacting with a polypeptide and a nucleotide
  • acid-cleavable linker reagents for interlinking two polypeptides
  • Such acid-cleavable linker reagents based on orthoester, acetal and ketal functionalities, have been described previously (Ref 18), and are bifunctional compounds whose hydrolytic rate constants increase as the pH decreases
  • the crosshnkers react with the proteins via heterobifunctional groups (e g maleimide or N-hydroxysuccinimide ester) or homobifunctional groups (e g bis-maleimide or bis-succinimidyl)
  • cross-linking agents which may be used are 1 Disuccinimidyl suberate This is a homo-bifunctional cross-linking reagent containing the N hydroxy succmimide (“NHS”) ester reactive group, which is reactive towards amino groups
  • NHS N hydroxy succmimide
  • Products in which the prohferatively active moiety (especially cytokine or growth factor) and nucleotide are acid-cleavably linked benefit from the potential advantage that the product is cleaved in the endosome to release the nucleotide in free form
  • Heterobifunctional linkers suitable for conjugating cytokines and growth factors to biologically active agents are obtainable from Pierce & Warner (UK) Limited. 44 Upper Northgate Street, Chester CH I 4EF, UK, or Pierce Chemical Company, PO Box 1 17, Rockford, IL 61 105, USA, whose literature provides further information
  • the IL-2 and other cytokines or growth products may be administered parenterally, in contemplated amounts of from 10 000 - 1 000 000 International Units, and suitably by intravenous, intramuscular or subcutaneous injection e g (less than 1 ⁇ g to 0 1 mg of recombinant protein), to give very low plasma concentrations
  • the plasma IL-2 concentrations may be close to the dissociation constant
  • the products of the invention may be formulated as human or veterinary pharmaceutical formulations in practice comprising a pharmaceutically acceptable diluent carrier or excipient
  • the formulations may be in the form of solutions or suspensions
  • the formulations are suitable for parental (e g lv or sc) administration but, oral formulations are not excluded
  • LC-SPDP Succinimidyl 6-[3'(2-py ⁇ dyld ⁇ th ⁇ o)-prop ⁇ onam ⁇ do]hexanoate
  • the sample is applied to a BioRad Micro Bio-Spin Desalting Column (pre-equi brated in lOOmM sodium acetate, l OOmM sodium chloride, pH4 5), spin and the filtrate used for the subsequent DTT treatment
  • Coniugation Reaction The desalted LC-SPDP-activated Aldesleukm and the desalted PMPI-activated genetic material are mixed in equal proportions and incubated at room temperature for 24 hours The conjugation reaction is then analysed by SDS-PAGE, along with samples from the activation reactions, to assess the conjugation efficiency Conjugated product can then be purified by size-exclusion chromatography
  • Example 2 The cell density in the initial cultures from Example 2 was assessed using a haemocytometer. and the viable cells assessed by Trypan blue exclusion. These were counted in triplets, as were all samples throughout the assay period. The averaged cell densities are shown in following Table 1 :
  • pEGFP-l was transfected into HT-2 cells using Transfectam (Promega).
  • the numbers of cells that expressed EGFP is shown in Table 2.
  • the highest transfection efficiency (approximately 0.08%) was observed with a cationic lipid : DNA ratio of 4: 1 using either Dfx20 or Dfx50.
  • the number of cells that were transfected was considerably higher when cultured at 1.Ong/ml RhIL-2 compared with 0.2ng/ml RhlL- 2. This may reflect the fact that these cells are proliferating more quickly and as a consequence of nuclear breakdown during the cell cycle it is easier for the plasmids to enter the nucleus and express their genes. Support for this theory comes from the observation that many of the EGFP expressing cells were found in 50 pairs which suggests that they may have recently divided These results indicate that IL-2 and other prohferatively active moieties potentiate cells towards genetic or nucleic acid material.

Abstract

A product comprising a proliferatively active moiety linked to genetic or nucleic acid material which is associated with protective material. The proliferatively active moiety is typically a cytokine or growth factor. The protective material may form a micelle, especially a liposome, which may encapsulate the nucleic acid material or be positively charged and hold the nucleic acid on its surface (a so-called lipoplex). One class of protective materials, therefore, comprises complexing materials and includes not only cationic liposomes but also other cationic materials, especially polymers. As suitable polymers there may be mentioned polylysine (especially poly-D-lysine), polylysine derivatives (e.g. phospholipid derivatives of, in particular, poly-L-lysine) and polyethyleneimine (PEI). Other suitable complexing agents are dendrimers, especially polyamidoamine dendrimers (which are cationic). The genetic or nucleic acid material may be a cytotoxic gene, a defect correction gene or an immunogene. Suitable cytotoxic genes are for expressing an enzyme to convert a prodrug into a toxic drug.

Description

GENE THERAPY PRODUCTS
FIELD OF THE INVENTION
The present invention relates to novel products which use a prohferatively active moiety as a vector to target nucleic acid material to proliferating cells and to induce proliferation of the target cells It also relates to products which use a moiety having a high affinity receptor binding activity as vectors for delivering genetic material to selected cells More particularly, the invention relates to novel compositions of matter for use in delivering genetic materials to cells, in gene therapy
BACKGROUND OF THE INVENTION
Proliferation, differentiation and functional activity of most cells, particularly haematopoietic cells and cells of the immune svstem are regulated by proteins called cytokines and growth factors Separation of the two groups is difficult, due to several overlapping mechanisms and effects on target cells For example, the cytokine Interleukιn-2 is also known as T-cell Growth Factor Cytokines and growth factors are both peptide hormones
More specifically, cytokines regulate the functional status of their target cells (I e they can stimulate or suppress both quantitatively and qualitatively), whilst growth factors are more focused on promotion, regulation and maintenance of proliferation and differentation, and the survival of their target cell lineages
Both cytokines and growth factors recognise specific membrane receptors on their target cells, which are unique for that particular cytokine or growth factor Each receptor, m turn, can express a dynamic avidity towards its specific cytokine or growth factor, based on physiological and/or pathological conditions These receptors can be categorised as low, medium, or high affinity Most important of all, high affinity receptors only recognise, capture, and internalise their related cytokine or growth factor These receptors and their gands are discussed in more detail below with reference to cytokines
Cytokines are a group of molecules, other than antibodies, which are produced by lymphocytes and are involved in signalling between cells of the immune system, for the purpose of stimulating or suppressing cell function Cytokine activity is often mediated by specific receptors expressed on target cells Cytokines are glycosylated or non-glycosylated polypeptides and can be secreted by both T-cells and B-cells, though T- cells are assumed to be the major source in cell-mediated responses Complications in the study of cytokines have arisen from the fact that in vivo no cytokine ever operates in isolation This is illustrated by the observation that many cytokine actions are synergistic Important cytokines include interleukins (ILs), tumour necrosis factors (TNFs) and interferons (IFNs) In addition, various colony stimulating factors (CSFs) are secreted by myeloervthroid cells Receptors for numerous cvtokines have now been cloned, and their structures (amino acid sequences) analysed As a result, it is possible to group many of these into super families, based on common homology regions in their primary structures For the purpose of this invention, the main super families recognised are cytokine receptor super family (CKR-SF) sometimes called haemopoietic receptor super family, and the interferon receptor super family (IFNR-SF), also termed cytokine receptor super family Type II (Ref 1 )
The term "super family" should be used only to describe proteins with amino acid sequence homology of
50% or less Proteins, with ammo acid sequence homology of greater than 50% are designated by the term
"family"
Many cytokine and growth factor receptors have combinations of different domains or repeats A domain is a sequence or segment of a protein which forms a discrete structural unit, able to capture and/or convert specific signals For the purpose of this invention, the domains of interest are the extra-cellular regions (those located at the surface of a given target cell-lineage) Studies focussing on receptor binding have revealed the existence of more than one binding affinity for several members of the CKR-SF (or haematopoietic receptor super family) Typically, these sites have low (e g 1 - l OnM) or high (e g at least lpM and more usually 10-lOOpM) affinity to a given ligand (cytokine or growth factor) For most of these receptor complexes, additional sub-units have been identified which are required for high affinity receptor expression These sub-units (also referred to as affinity convenors or convertor chains) are often expressed on the cell surface after a given activatory or inhibitory stimulus is applied through a receptor ligand This results in an amplification of effects, but only in those cells bearing the high affinity receptor and not in resting cells (which usually bear the low-active receptor complexes), and is the physiological basis of any paracπne stimulation/inhibition, in the absence of any involvement of distπct/regional/systemic networks
Thus to mediate immune responses T-cells must change from a resting to an activated state T-cells stimulated by foreign antigens enter a program of cellular activation leading to de novo synthesis of IL-2 Resting T-cells do not express high affinity receptors but these are rapidly expressed after activation Interaction of IL-2 with its induced cellular receptors triggers cellular proliferation culminating m the emergence of effector T-cells that are required for the full expression of immune responses Taking the example of IL-2/IL-2r complex, in many of the diseases described herein, this physiological tuning is disrupted (primarily by neoplastic transformation, secondary to viruses), or is automamtamed (autoimmune reactions/diseases, transplant rejection), leading to systemic multi-organ failure
Further information about cytokines and their receptors may be found in Callard R E, Gearing A J H, The Cytokine-Factsbook. Academic Press - Harcourt Brace & Company, Publishers 1994, 18-25
High affinity receptors therefore include those w ith an affinity constant of 10 l0M or less, and, more particularly those with an affinity constant of 10'"M or less Representative high affinity receptors include those w ith affinir. constants of between 10 " and 10 12M For example three forms of receptor for mterleukιn-2 (IL-2) can be distinguished on the basis of their affinitv for IL-2 with IL-2 binding affinities of 10'"M (high affinity), 10'9M (intermediate affinity) and 10"8M (low affinity) (Refs 1 -4) IL-2 receptors are well described in the prior arts (Refs 5 & 6)
TNF-α has been described as having two isoform receptors with high affinity on the target cells for TNF These target cells are macrophages and osteoclasts (Ref 7) M-CSF (macrophage colony stimulating factor) has a high affinity receptor on macrophages and osteoclasts The high affinity receptor is a 150 Kda glycoprotem (Ref 8)
High affinity receptors have been described also for IFNs (interferons) IFN-γ has a 90 KDa glycoprotem as a high affinity receptor A different receptor present on activated lymphocytes, macrophages, endothelial cells and fibroblasts has been recognised as the high affinity receptor of IFN-α and IFN-β (Ref 9)
In the case of FGF (fibroblastic growth factor), there is a high affinity receptor which is a 140 KDa glycoprotem on mesodermic and neuroectodermic lineage cells, such as activated fibroblasts, macrophages, endothelial cells, chondrocytes, astrocytes, glioma cells, hepatocytes, epithelial cells, neurones, ovarian cells, pituitary cells, and keratmocytes The pharmacological properties of FGF are primarily related to angiogenesis, ovarian steroidogenesis, ostoblast activation, and nerve growth (during the foetal phase) (Ref 10).
A variant GnRH-III (GIp-Hιs-Tφ-Ser-Hιs-Asp-Tφ-Lys-Pro-Gly-NH2) from the sea lamprey, Petromyzon martnus, has been found to suppress growth of breast, prostate and endometπal cancer cells but not to have endocrine activity at the concentrations effective against growth of cancer cells (Refs 25-28)
Epidermal growth factor (EGF) is a 53 ammo acid peptide which efficiently stimulates cell growth via a receptor mediated mechanism It is a classical example of a tyrosme kmase/SH2 domain receptor, in which extracellular EGF binding induces receptor dimeπsation, autophosphorylation and binding of downstream signalling molecules to the activated receptor via their SH2 domains (Ref 29) Since the receptors for EGF are present m multiple potentially heterodimeπc forms on the surfaces of a large number of cell types, this receptor type is widely applicable
IGF (insulin-like growth factor) has a high affinity receptor on eterotetrameπc complex present in different tissues and in mammary adenocarcinoma (Ref 1 1 )
Transforming Growth Factor β (TGFβ) is similar to IGF TGFβ is a non-glycosylated homodymeπc protein secreted by fibroblasts. epithelial cells, platelets, astrocytes, monocytes. bone cells, and g oblastoma cells The physiological target cells are primarily fibroblasts, osteoblasts, neutrophils. hematopoietic progenitors. T B lymphocytes, and a range of tumor cells The cytokine interacts with a high affinity receptor, expressed by the target cells, in response to paracπne microenvironmentai stimulation, located on the cell surface of the above cells These are type 1 or type 2 receptors (55 and 80 Kda), and are able to bind to TGFβ l, 2, and 3
GM-CSF (granulocyte/ macrophage colony stimulating factor) and SCF (stem cell factor) possess a dimeπc high affinity receptor in multipotent cells in the bone marrow (Ref 12) G-CSF (granulocyte colony stimulating factor) also has a high affinity receptor present, but only in multipotent cells m the bone marrow
EPO (erythropoietin) has a multimeπc high affinity receptor present on erythroid precursors in the bone marrow
IL-6 (ιnterleukιn-6) has an α-/β- high affinity receptor The alpha chain binds IL-6 with low affinity and exists in a soluble form The beta chain is a 130 KDa protein which simultaneously binds IL-6/IL-6r, becoming a trimeπc complex which initiates target cell stimulation IL-6 high affinity receptor induction, following specific stimuli, is primarily positioned on activated cells such as T/B lymphocytes, fibroblasts, myeloid precursors, neurones, keratmocytes, and hepatocytes In addition, multiple myeloma cells produce IL-6, and express IL-6 receptors working as an autocπne cancer growth factor, inducing at the same time osteoclastogenesis (bone lytic lesions) IL-6 from stromal cells can also be involved in bone metastatic lesions through different tumour histotypes
Gonadotropin-releasing hormone (GnRH, Glp-Hιs-Tφ-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) is the central regulator of the hypothalamic-pituitary-gonadal axis GnRH analogues are used to treat sex-hormone dependent cancers of the breast, prostate and ovaries GnRH receptor RNA is expressed in human pituitary, breast, ovary, prostate and endometruim As described by Palyi et al (Ref 24) certain cancer cell lines have been found by previous workers to express high- and low- affinity binding sites for GnRH GnRH is therefore a peptide hormone for which there are high affinity receptors
WO 92/20364 describes hybrid molecules containing a first portion which is a molecule capable of decreasing cell viability (especially a cytotoxin) and a second portion which is a molecule capable of specifically binding to a cytokine receptor (especially all or a binding portion of a cytokine) The second portion targets the first portion to the cytokine receptor and is exemplified as IL-2 The IL-2 portion preferably lacks IL-2 activity because the molecules will then prevent proliferation of the target cells
Palyi et al (Ref 19) describe gonadotropin-releasing hormone (GnRH) analogue conjugates comprising peptidic analogues of human GnRH linked through lysyl side chains and a spacer (Gly-Phe-Leu-Gly) to poly(N-vιnylpyrrolιdιne-co-maleιc acid) The conjugated polymer protects the peptide against proteolysis and enhances its antiproliferative effect, possibly as a result of enhanced binding of the peptide conjugates with external domains of the receptor and adjacent membrane structures and/or internal isation of receptor- conjugate complexes Gene therapy is the transfer of genetic material or gene function modulators to target cells of a patient for the puφose of preventing or altering a disease state, and may be for the treatment of non-genetic as well as genetic disorders Gene transfer may be ex \ιvo or in vivo Ex vivo techniques usually involve the genetic alterations of cells, mostly by use of viral vectors, prior to implanting these into the tissues of the living body In vivo gene therapy means direct introduction of genetic material into the body, and usually suffers poor efficiency because of poor access to target tissues Proposed vectors for gene therapy include viruses
(especially retrovirus and adenovirus vectors), liposomes and receptor-mediated endocytosis, for example using DNA linked to a targeting molecule such as polylysine
A variety of gene therapy strategies are known for cancer, including haemopoietic gene transfer, immunogene therapy, delivery of toxic genes, e g gene-directed enzyme-prodrug therapy (GDEPT), and correction of genetic defects GDEPT exploits the differences in gene expression between cancer cells and their normal counteφarts to increase the specificity of cell destruction A foreign gene is introduced which encodes an enzyme capable of converting a harmless prodrug into a cvtotoxic compound The system is designed so that significant transcription of the enzyme gene occurs oniv in tumour cells
For further information on gene therapy see "Textbook of Gene Therapy", K K Ja , Hogrefe & Huber Publishers, 1998 (Ref 24)
The most common candidate vectors for gene therapy are viruses However, viral vectors are seen as potentially undesirable because of doubts raised about their safety, immunity to them and the relative difficulty of large scale culture of viral vectors Effective alternatives to viral vectors are therefore being actively sought
The invention of International patent application No PCT/GB98/03509, the content of which is included herein by reference, is based in one aspect on an insight that a medicament which contains an active promoter of proliferation, for example an active IL-2, can beneficially be used to deliver pharmacologically desirable species to cells whose proliferation is not desired For example some medicaments of that invention control or inhibit proliferation using a molecule which contains an active promoter of proliferation Preferred embodiments are based on an appreciation that, by using the high affinity of receptor super families, it is possible to drive drugs or genetic material, for example, into specific cell lineages which are predominantly responsible for many clinical events
International patent application No PCT/GB98/03509, therefore, describes a class of products which comprise a prohferatively active compound, especially a cytokine or growth factor, linked to a pharmacologically active compound for example a conventional drug or a gene The prohferatively active moiety (or cvtokine or growth factor in the preferred aspect) retains its functional activity, which can come into play once the product targets its receptor The prohferatively active moietv binds to the receptor and is then internalised by the cell so that each active domain of the product (the pro ferativelv active moietv and the biologically active agent) can perform its respective function It is contemplated that the two domains of the product will separate intracellularly in commercially viable products, but this is not essential and the invention is not restricted to products which are intracellularly cleavable In preferred embodiments, the events which follow binding of the prohferatively active moiety to the receptor typically include internal isation of the product (typicallv a fusion compound) into the cytosol (by the endosome pathway), endosome acidification (by the proton H+ pump mechanism), and a separation of functional domains into the receptor domain, the prohferativelv active domain, and the active agent domain Since the receptor domain and the prohferatively active domain (normally a cytokine or growth factor domain) retain their functional integrity, the prohferatively active domain (cytokine or growth factor) will trigger cell activation/division through DNA interaction (G2-M phase enrichment)
As the International application teaches, therefore, these prohferatively active vectors are not only active as transporters of genes but also promote the rearrangement of DNA, in addition to opening DNA chains in target cells Thev are therefore ideal for integrating genes both in vitro and in vivo No other system available has a comparable bi-modal activity, and without the associated risks of viruses (used currently as vectors)
PCT/GB98/03509 also describes and claims products comprising a biologically active agent linked to a moiety which is a peptide hormone, which has a high affinity receptor, or is a molecule functionally equivalent to the peptide hormone in relation to the high affinity receptor
SUMMARY OF THE INVENTION
The present application relates in particular to novel products of the types described and claimed in PCT/GB98/03509 More particularly the invention provides products or compositions of matter comprising a prohferatively active moiety linked to genetic or nucleic acid material which is associated with protective material There is considerable evidence that transported genes are more active in dividing cells and the prohferatively active moiety therefore potentiates the cells whose proliferation is caused towards the genetic or nucleic acid material In other words the invention is concerned with products which potentially use prohferatively active moieties both as vectors for protected nucleic acid material and as mitogens to stimulate proliferation of the target cells
The protective material serves to protect the genetic or nucleic acid material from degradation and may by way of example comprise any known protective material Specifically, the genetic or nucleic acid material may be protected b> encapsulation in a micelle, especially a hposome, or by being complexed, for example with a protective protein such as, e g , polylysine
Whilst the invention includes products in which genetic or nucleic acid material is protected by an associated material the invention is not restricted to products in which the associated material is protective or solely protective in function It includes also products in which genetic or nucleic acid material is otherwise associated with cationic DNA-binding material
Accordingly the invention provides products or compositions of matter comprising a prohferativeh active moietv linked to a nucleotide which is associated with cationic DNA-binding material
It is a feature of the above aspects of the invention that the nucleic acid or genetic material is linked to a prohferatively active moiety Unlike prior art chimeric proteins containing solely the receptor-bmding domain of IL-2, therefore, these products induce cellular proliferation, enabling anti-prohferative drugs to be highly effective, even at ultra-low doses in the case of prohferatively active moieties with high affinity receptors The invention therefore enables low systemic toxicity to be achieved An additional benefit at least in the case of IL-2 is that IL-2 induces expression of the high affinity IL-2 receptor when the relevant antigen is present
For example a construct of IL-2 and protected antisense DNA/RNA designed to block a retrovirus gene obtains the following effects
a in infected cells, the rep cative stimulus given by the IL-2 stimulates also replication of the viral genome, resulting in stronger inhibitory activity by the antisense DNA/RNA,
b immunostimulation of uninfected lymphocytes, with the potential benefit of increased immune surveillance
Amongst the features of preferred embodiments are
• the product is a combination of existing moieties (or of moieties functionally equivalent thereto), each of which retains its function and, optionally, its entire structure (except at any covalent linkage site to the other moiety)
• the product can be administered at exceedingly low dosages, so that little or no systemic toxicity results
• the growth factor/cytokine stimulates the target svstem and the nucleic acid moiety induces a therapeutic effect
• biodistπbution is predictable and good
• targeting is good • immunogenicity is low
An overview of the invention is shown in Fig 1 , which for convenience illustrates the invention with reference to a product in which the prohferatively active moiety is an IL-2 As shown in Fig 1 , the IL-2 is linked to a protected (in this case polylysine complexed) expression vector (in this case a plasmid), suitably by attaching to the IL-2 a linker having a functional group reactive with the polylysine as well as a functional group reactive with the IL-2, and then reacting the linker with the polylysine before or after the latter is combined with the plasmid (expression vector)
The resultant product is administered in vivo or in vitro and the IL-2 (prohferatively active moiety) serves as a vector to direct the product to cells presenting IL-2 receptors, especially high affinity receptors The product is internalised, after which the IL-2-polylysme link is cleaved (e g hydrolysed), allowing the IL-2 to promote division and the expression vector to migrate to the nucleus Less preferred products of the invention have a prohferatively active moiety which is not internalised by its receptor, in which case the link with the remainder of the product is normally cleaved extracellularly and the expression vector (or other nucleotide) is internalised
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is an overview of the invention, illustrating the structure and function of products of the invention with reference to an IL-2-Expressιon Vector construct,
Figures 2, 3 and 4 are diagrammatic illustrations (linearised circles) of plasmid constructs of the invention,
Figure 5 is an SDS-PAGE gel run using the product of a reaction between Aldesleukm and LC-SPDP,
Figure 6 illustrates a 96 well plate as used in a growth assay,
Figure 7 is a data chart of cell densities resulting from the incubation of HT-2 cells with modified Aldesleukm, and
Figure 8 is a data chart showing growth responses of HT-2 cells to various IL-2 compositions
Figures 9a, b and c are data charts showing the results of a toxicity assay in which serial dilutions of 25k PEI were added to HT-2 cells cultured at 1 0, 0 5 and 0 2ng ml RhIL-2 and cell viability assayed for three days
DESCRIPTION OF PREFERRED EMBODIMENTS
The products of the invention will now be described in more detail taking each component in turn The Associated Material
The material associated w ith the genetic or nucleic acid material is protective in one class of products It may form a micelle, especially a hposome
The posome or micelle mav encapsulate the nucleic acid material or it may be positively charged and hold the nucleic acid on its surface (a so-called lipoplex) Liposomes are formed by phospholipids and similar amphipathic lipids, and are commercially available Cholesterol is frequently included in hposome formulations A review of cationic liposomes as vectors for gene transfer may be found in Ref 38 An alternative class of encapsulating composition comprises artificial viral envelopes (see Ref 39)
One class of protective mateπals, therefore, comprises complexmg materials and includes not only cationic liposomes but also other cationic materials, especially polymers As suitable polymers there may be mentioned polylvsine (especially poly-D-lysine), polylysine derivatives (eg phospholipid derivatives of, in particular, poly-L-lysme) and polyethyleneimine (PEI) Other suitable complexmg agents are dendπmers, especially polyamidoamine dendπmers (which are cationic)
The associated material is not protective, or exclusively protective, in all the products of the invention For example, in a class of product falling within the invention a cationic DNA-bmdmg moiety forms a bridge between the prohferatively active product and the nucleic acid material (nucleotide) Suitable DNA-binding moieties are cationic polymers such as polylysine or PEI Additionally there may be cationic or other protective material
The Nucleic Acid Material
The specific identity or function of the nucleic acid or genetic material is not critical to the invention, which is concerned primarily with methods for delivery, l e transfer of a nucleotide into the target cell and especially into its nucleus
The nucleic acid material generally comprises either a therapeutic gene or an antisense nucleotide (ohgo- or poly- nucleotide) Antisense nucleotides are single strand nucleotides containing sequences complementary to target mRNA or DNA in order to block the production of disease-causing proteins Antisense therapy is often considered to be a form of gene therapy because it is modulation of gene function for therapeutic puφoses Therapeutic nucleotides are suitably phosphorothioate ohgodeoxynucleotides (ODNs), as these are nuclease-resistant Alternatively, antisense nucleotides may conveniently be protein nucleic acids (PNAs) Antisense nucleotides are useful for the treatment of viral diseases and cancer (see Ref 25) and the invention includes the use of antisense products for such treatments as well as the treatment of neurodegenerative and cerebrovascular disorders (Ref 24)
The products of the invention may be materials comprising an expression vector for expression of a therapeutic gene The expression vector will contain a gene encoding a protein operably linked to a control sequence The control sequence will include a promoter and preferably an enhancer derived from, for example, immunoglobulin genes, SV40, cytomegalovirus (CMV), and a polyadenvlation sequence In one class of products, the expression vector comprises a plasmid construct (recombinant plasmid)
The expression vectors are suitably in the form of recombinant plasmids The use of a bacterial expression cassette permits large scale preparation of the construct in a prokaryotic system Illustrative plasmids are shown in Figures 2, 3, and 4 in linearised form Thus, Figure 2 shows a plasmid comprising a therapeutic gene, which in this case is a cytotoxic gene, represented by a gene-directed enzvme-prodrug therapy gene shown to be a thymidine kinase gene The therapeutic gene is operably linked to a control sequence, shown to be a promoter and specifically a CMV promoter The plasmid will also contain a poly A/ termination site, a bacterial origin and an antibiotic resistance gene
Figure 3 shows a plasmid similar to that of Figure 3 but additionally containing an episomal maintenance sequence, which will maintain the therapeutic gene outside of the cell chromosome as an episome This will enhance its expression and prevent cellular silencing of the therapeutic gene by insertion into heterochromatm
The construct of Figure 4 contains two therapeutic genes which again are represented as cytotoxic genes The construct optionally contains an IRES (internal πbosome entry site) to ensure that the two genes are expressed simultaneously from the same promoter The two genes may be repeat copies of the same gene or may have different functions
For experimental puφoses, it is useful to make a construct having an indicator gene (e g the EGFP gene which causes expression of a green marker protein) and a therapeutic gene (e g heφes simplex virus (HSV) thymidine kinase gene which will activate gancyclovir when expressed) Cells which have been treated for transduction with the construct can be monitored for successful gene transduction by detecting for green cells The killing drug (gancyclovir in this case) can then be added and should target the green cells by virtue of the TK gene The process is suitably monitored by time lapse photomicroscopy, to provide information about the efficiency of cell kill as well as about the percentage of cells in the population transduced with the genes and killed
The gene may be any gene having a therapeutic function and, by wav of non-limiting example, may be a therapeutic gene now known to those skilled in the art of gene therapy Representative classes of genes include immunogenes (e g cytokine gene therapy DNA-based cancer vaccines), cytotoxic genes and especially genes for enzy e-prodrug therapy (e g genes encoding viral thymidine kinase, bacterial or other cvtosme deammase, cytochrome P-450 or bacterial nitroreductase) and defect correction genes (e g tumour suppressor genes and especially p53)
The invention also includes products in which the nucleic acid material has a non-specific and/or non- antisense effect, for example products which inhibit viral infection by interference with absoφtion, penetration or uncoating of viruses
The Prohferatively Active Moietv
Since the invention is concerned primarily with a delivery technique for nucleic acids and not with the treatment of a specific disease, the products of the invention are not restricted as to the precise identity or function of the prohferatively active moiety However, the prohferatively active moiety is usually a cytokine or growth factor The cytokine may be an interleukin for example a TNF, for example an M-CSF, an IFN, for example an FGF, an IGF, a TGF, for example a GM-CSF, an SCF, a G-GSF, or an EPO
The cytokine is preferably a human cytokine
The growth factor may be a haematopoietic or lymphopoietic growth factor They are a family of glycoprotem hormones which regulate survival, proliferation, and differentiation of progenitor cells, in addition to impacting on some functional activities of mature lymphohaematological cells
Suitable growth factors include Erythropoietin (Epo),
GM-CSF,
G-CSF,
SCF (Stem cell factor),
Multi-CSF (also known as lnterleukιn-3), M-CSF
E-CSF (or lnterleukιn-5),
IGF- 1 (Insuhn- ke growth factor),
PDGF (Platelet-derived growth factor),
TGF beta2 (Transforming growth factor -beta2)
The prohferatively active or mitogenic moiety is internalised by target cells in a preferred class of products but in another class of products is not internalised For example, the mitogenic moiety may be a growth factor such as an EGF or FGF which is Tyr kιnase/SH2 mediated In the case of non-intemahsed mitogens. the nucleic acid material is preferably cleavable from the mitogen and/or associated with a delivery material, e g a hposome or DNA binding material
Applications of FGF products include their use as antiangiogenic factors in solid cancers, and to block hyperactivation of fibroblasts in scleroderma In particular, FGF-2 and FGF-7, both of which have high affinity receptors, have been implicated in prostate cancers
The present invention provides products comprising a domain functional to bind to an EGF, FGF-2 or FGF- 7 receptor (especially a high affinity receptor) to promote proliferation and a nucleotide, e g genetic material The domain or moiety having EGF, FGF-2 or FGF-7 receptor binding function serves as a vector for directing the second domain or moiety to cells having, as the case may be, EGF receptors, FGF-2 receptors or FGF-7 receptors and especially EGF high affinity receptors, FGF-2 high affinity receptors or FGF-7 high affinity receptors The products having FGF-2 or FGF-7 receptor binding function are useful for targeting anti-cancer drugs to breast, stomach, oesophageal and prostate tumour cells
The products having EGF receptor binding function are useful for targeting anti-cancer drugs to most tumour types but particularly those of breast, stomach, ovarian, bladder and prostatic origin
The two domains or moieties are suitably linked by a physiologically cleavable link which will be cleaved in the receptor-bound product The linkage between the two domains or moieties may be covalent but is not covalent in some of the products One class of products comprises molecules having a multi-part bridging group as described above
Cytokines or growth factors (or pro ferative agents) may be native or a mutein representing the native molecule modified by one or more amino acid alterations (deletions, additions or substitutions) Such muteins, usable in the present invention, possess the biological activity of the native protein, in the sense of having both functional affinity for the receptor (and in one class of embodiments functional affinity for the high affinity receptor) and in many cases a capability of forming, with the receptor, a product internalised by the cell presenting the receptor
The cytokines and growth factors are preferably recombinant molecules but may be produced by cultivating cytokine or growth factor producing cell lines, for example peripheral blood lymphocytes
In one class of embodiments, the products comprise a nucleotide associated with protective and or DNA- binding material linked to a molecule which is functional to have a high affinity with a cytokine or growth factor high affinity receptor, and to form a complex with such a receptor which is, in one class of products, internalised by the cell presenting the receptor In a particular class of products, the molecule may be a native or mutein cytokine. or a fragment thereof In another class of products, the molecule may be a native or mutein growth factor, or a fragment thereof Particularly preferred is the cytokine ιnterleukιn-2 (IL-2) IL-2 is a lymphokine which is produced by normal peripheral blood lvmphocvtes, and induces proliferation of antigen or mitogen stimulated T-cells after exposure to plant lectins antigens, or other stimuli IL-2 was first described bv Morgan, D A . at el , Science (1976), j_93 1007-1008 Then called T-cell growth factor because of its ability to induce proliferation of stimulated T lymphocytes it is now recognised that, in addition to its growth factor properties, it modulates a variety of functions of immune system cells in vitro and in vivo, and has been renamed interleukm-2 (IL-2)
Interleukm-2 may be made by cultivating human peripheral blood lymphocytes (PBL), as described, for example, in US Patent No 4,401 ,756 As a preferred alternative, the IL-2 may be recombinant Taniguchi, T et al , Nature (1983), 302 305-310 and Devos, R , Nucleic Acids Research (1983), 1 1 4307-4323 have reported cloning the human IL-2 gene and expressing it in micro-organisms
US Patent No 4,518,584 describes and claims muteins of IL-2 in which the cvsteine normally occurring at position 125 of the wild-type or native molecule has been replaced with a neutral amino acid, such as seπne or alanme An oxidation-resistant mutein of IL-2 which is biologically active may be prepared wherein each methionine residue of the protein from which the mutein is derived is replaced with a conservative amino acid such as alanine, the methionine resιdue(s) is/are susceptible to chloramine T or peroxide oxidation These IL-2 muteins possess the biological activity of native IL-2 US Patents Nos 4,530,787 and 4,569,790 disclose and claim methods for purifying recombinant native IL-2 and muteins thereof, as well as purified forms of IL-2 The aforesaid US patents are included herein by reference
The IL-2 mutein desala[ -IL-2 erj25 (Aldesleukm) is available commercially from Chiron B V of Amsterdam, Netherlands under the trade mark Proleukin®
The Linkage between the Prohferatively Active Moiety and the Nucleic Acid
The nature of the linkage between the prohferatively active moiety and the nucleic acid material is not critical to the invention In some instances a bridging group is directly linked to the two of them by covalent bonding In some other cases, a bridging group is directly covalently bonded between the prohferatively active moiety and the associated material The bridging group may comprise a known heterobifunctional or homobifunctional linker, or it may be formed bv the interbonding of two heterobifunctional linkers In some cases, a peptide linker is used, for example between the proliferativelv active material and the envelope of a hposome (see for example Ref 19)
The prohferatively active moiety is usuallv peptidic and is in a preferred class of products covalently bonded to the nucleic acid or the associated material The peptidic moietv mav be covalentlv bonded to the nucleic acid or associated material via a bridging moietv which is bonded to the peptide through a pπmar. amine residue thereof and to the other material through a functional group thereof For example, a polylysine or PEI complexmg moiety mav be linked through one of its a ine groups to the bridging group, and a nucleic acid may be linked through one of its hydroxy groups to the bridging groups
The two active domains of the molecule (the prohferatively active moiety and the nucleic acid or genetic material) are in a preferred class of molecules bonded to each other via a bridging moiety which comprises a first heterobifunctional cross-linker residue bonded to the prohferatively active moiety and to a second heterobifunctional cross-linker which in turn is bonded to the nucleic acid or associated material
Other Features
The invention also includes in another aspect a method of treating by prophylaxis or therapy a disease or disorder involving cells bearing a high affinity receptor for, in particular, a cytokine or growth factor, comprising administering to a patient an effective amount of a product comprising an agent which is biologically active when in said cells and is linked to said cytokine or growth factor, such products and preparations containing them form a further aspect of the invention
Additionally included in the invention is a product of the invention for use as a pharmaceutical, especially in internalising the biologically active agent into a cell having a high affinity receptor for the prohferatively active agent, cytokine or growth factor of the product
Another aspect of the invention resides in the use of a product of the invention for the manufacture of a medicament for internalising the biologically active agent into a cell having a high affinity receptor for the pro ferativelv active agent, cytokine or growth factor of the product and optionally for stimulating lymphocyte proliferation
The invention will now be illustrated by way of example with reference to certain specific cytokines, growth factors, biologically active agents and diseases Of course, the invention is not limited to these specific features
The products of the invention preferably act only on cells presenting a high affinity receptor for the cytokine or growth factor, which are typically lvmphocytes or other cells involved in the immune response The action of a product of the invention on its target cells depends on the function of the active agent
The molecular ratio of the active agent nucleic acid material in the products of the invention is not critical Thus the invention includes ratios of 1 1 or less but in some embodiments the ratio is greater than 1 1 , e g 1 1000 or more, l e a plurality of active agent molecules/atoms may be bound to each prohferatively active moietv The inventive products will now be described in more detail, by way of example, with reference to illustrative products and product classes
Interleukιn-2/antιsense product
IL-2/antιsense fusion products are useful for introducing specific antisense sequences (ohgonucleotides) into lymphocytes bearing the IL-2 receptor
Using cytokines (e g IL-2) which internalise into target cells, an antisense compound for a given tract of the HIV genome (e g genome coding for an envelope protein, or for the enzyme transcriptase) may be introduced specifically into the lymphocytes of a given patient, using a pharmacological strategy, such as IV administration, for example
IL-2/ antisense products may also be used to introduce antisense compounds or antioncogenes into the T-cell lineage affected by neoplastic transformation where gene mutation, or oncogene hyperexpression is known
Treatable Diseases
The IL-2/actιve compound fusion products are useful for diseases for which the lymphocyte is mainly involved in tissue damage, and the resultant development of a given disease entity
In essence, Interleukιn-2 high-affinity receptor-directed lmmunosuppressive therapy acts pharmacologically, but only on recently activated lymphocytes (particularly T- cells), which bear this structure on the cellular membrane The activation signal is absent from the surface of resting T-cells and all other non-lymphoid tissues As such, very low doses of cytokine/nucleic acids can be targeted
Since the receptor is only transiently expressed during the brief pro ferative phase, when lymphocytes respond to antigen stimuli (autologous-antigen in the case of autoimmune diseases, and heterologous- antigens in the case of transplantation), it is possible to achieve selective in vivo immunosuppression, directed solely towards activated lymphocytes (ohgoclonal immunosuppression) This pharmacological action is totally different from the general immunosuppression action exerted by conventional lmmunosuppressive drugs
Diseases which can benefit from this approach include autoimmune diseases transplant rejection, HIV- infection, and lymphoprohferative diseases
Autoimmune Diseases Autoimmune diseases are a wide variety of disorders with a common pathogenic pathway immune attack on target organs due to abnormal recognition of tissue antigens, and/or cellular antigens, by the immune system, particularly by T-lymphocytes ( 17)
This immune attack is implemented by a network of T-cell-mediated cytotoxicity, humoral autoimmune antibodies produced by B-lymphocytes, complement activation and consumption, and finally by tissue damage The central role of the abnormal activation of the T-lymphocytes lineage in all autoimmune diseases is well recognised
The clinical disorders under this heading and their target organs include the following
1 Autoimmune diabetes melhtus (Type I diabetes) — > endocrine pancreas
2 Autoimmune thyroiditis (Hashimoto and others) — > thyroid
3 Autoimmune hepatitis (chronic active hepatitis) — > liver 4 Rheumatoid arthritis — > synovial/joints/viscera
5 Autoimmune Nephritis (glomerulonephπtis) — > kidney
6 Uveitis (Behcet's syndrome) — > eye
7 Multiple sclerosis — > CNS/PNS
8. Sjogren syndrome — > saliva glands 9 Scleroderma — > skin/viscera
10 Dermatopo myositis — > skin/muscle/viscera
1 1 Systemic Lupus Erythematosus(SLE) — > viscera/skin/hematopoieses/ mucose
12 Autoimmune hemolytic anaemia — > erythrocyte
13 Idiopathic thrombocytopenic puφura (ITP) — > platelet 14 Autoimmune neutropenia — > neutrophil
15 Vascu tis — > vessels
16 Crohn's disease — > bowel
17 Ulcerative colitis — > bowel
18 Coeliac disease — > bowel 19 Psoriasis — > skin/joints/viscera
20 Sarcoidosis — > lung/viscera/skin
21 Atopic syndromes
In the majority of these pathological manifestations, there is a pathogenic lymphocyte-mediated reaction, and cytotoxicity
The evidence to support the role of T-cells in the pathogenesis of specific disease and progression of targeted tissue damage is substantial In most of the diseases listed above, CD4 cells (a cytotoxic subset of T-cells) are the dominant T-cell phenotype in the target tissues T-cells express several activation markers Experimentally, there is evidence that autoimmune diseases improve when T-cell targeted intervention occurs, as in thoracic duct drainage, total lymphoid irradiation, and administration of Cyclospoπn
Furthermore active autoimmune disease is generally less severe in AIDS patients who have CD4 cytopenia
Most autoimmune diseases are treated by attempting to reduce the function of the immune system using lmmunosuppressive and anti-inflammatory drugs This therapeutic strategy is conducted m a non-specific way, resulting at times in latrogenic toxicity and a failure to control the overall disease process
Lymphocytes, responsible for the acute phase of a given autoimmune attack, all bear the high-affinity IL-2 receptor on the membrane They are antigen-activated, or cytokine-activated, lymphocytes with a high avidity for IL-2
The parenteral administration of very-low doses of IL-2/ lmmunosuppressive nucleic acids enables the use of IL-2 as the vector of pharmacologically active nucleic acids to exert immunosuppression The IL- 2/ιmmunosuppressant nucleic acid products selectively bind to. and interact with, only those cells bearing the high-affinity receptor of IL-2 This means that immune cells, responsible for tissue damage and disease progression, are inactivated selectively, potentially curing patients with chronic diseases, or reducing their relapse rate
Recombinant proteins, for example recombinant IL-2 and other recombinant cytokines and growth factors, usually have low lmmunogenicity and good tissue distribution After parenteral administration, every tissue compartment in the body is exposed, including all lymphocytes (circulating lymphocytes, lymphocytes in the tissues, and lymphocytes in the lymph nodes)
Transplantation
The acute or chronic rejection of a transplanted organ is related to heterologous antigens (antigen specific to the donor transplanted organ(s)) presenting to host T-cells Following antigen presentation and recognition, immune cells enter into a pro ferative phase, during which the high-affinity receptor for IL-2 is expressed This leads to activation of the cytotoxic process, and to damage and subsequent failure of the transplanted organ
The use of IL-2 as a vector to target lmmunosuppressive nucleic acids achieves longevity of transplanted organs without the associated toxicity of conventional lmmunosuppressive therapy (acute, delayed, and long term)
Allogeneic bone marrow transplantation (ABMT) is used to treat and cure leukemias (both lvmphoid and mveloid). thalassemia and solid tumours The products of the invention have the potential to reduce the incidence of Graft-Versus-Host-Disease (GVHD) which is the reaction of the donor immune svstem against tissue antigens of the host, without compromising the global immune-svstem (of graft origin) As a result, there could be a reduction in mortality rate due to GVHD (currently in excess of 40%), a reduction in infectious complications, and a positive anti-tumour effect on minimal residual disease (the so-called Graft-
Versus-Leukaemia effect)
HIV-Infection
The administration of low-dose lL-2/antιsense nucleotide specific to HIV-genome fraction exerts in vivo an immunostimulatory effect on HIV-negative lymphocytes bearing the high-affinity receptor The antisense nucleotide is introduced into the cytoplasm of HIV-infected lymphocytes (CD4 cells), leading to a selective destruction of infected cells, without impacting on the normal reactive lymphocytes which are stimulated
Lvmphoprohferative diseases (Lvmphoblastic leukaemia and lvmphomas)
Using an IL-2 cytotoxic gene construct, it is possible to selectively kill the neoplastic lymphoid lineage expressing the high-affinity IL-2 receptor, without inducing any critical systemic toxicity on non-lymphoid compartments
Products containing growth factors or cytokines other than IL-2 may be used in the therapies described below
TNF-α has as its target cells macrophages and osteoclasts TNF-α/blocking compound products may be used to insert into macrophages a blocking nucleotide (which blocks cellular function and/or kills the cell) Such products potentially provide an important tool in some pathological conditions, e g advanced solid cancers where macrophage hyperstimulation and activation is responsible for cachexia and tumour progression, monocyto-macrophage neoplasms (e g histiocytosis), transplant rejection and GVHD, autoimmunit , and neurological degenerative diseases (the TNF receptor in its extracellular domain is similar to nerve Growth Factor receptor)
M-CSF (macrophage colony stimulating factor) may be used to form products containing blocking nucleotides. useful for treating the same classes of conditions as TNF-α blocking nucleotide products
M-CSF is also responsible for microghal proliferation in the CNS Other potential applications are therefore in some degenerative diseases of the CNS. such as Alzheimer's svndrome. and in bone diseases
The IFN/nucleic acid products (IFN-α, -β or -γ) may be used to modify the function of activated lymphocytes, macrophages, endothelial cells and fibroblasts, or to incapacitate them in different pathological conditions, such as in HIV-infection ( AIDS), and fibroblast-related diseases such as scleroderma Potential applications of FGF products include their use as antiangiogenic factors in solid cancers, and to block hyperactivation of fibroblasts in scleroderma The invention enables the preparation of FGF products capable of acting as an antagonist in relation to the cell types listed earlier in this specification as having FGF high affinity receptor when activated
IGF products may be used to treat breast cancer Due to the presence of the high affinity receptor in CNS (neurog a), it could also be used in some degenerative neurological disorders
TGFβ products have applications similar to those of FGF and IGF constructs
GM-CSF constructs may be used to selectively kill myeloid blasts responsible for myeloid leukemias G- CSF products have similar pharmacological activity to the GM-CSF products, but bind to a different high- affinity receptor present only in multipotent stem cells in the bone marrow
Epo fusion products may be used for diseases such as polycythemia and erythroleukemia, for example
Epo/gene sequence fusion products, which the DNA fraction is the normal gene for haemoglobin beta- chain, may be used for introducing the normal gene into the erythroid lineage in patients affected by beta- Thalassemia In this genetic disease, the abnormal gene, coding for a non-functional haemoglobin beta- chain, is present in the erythroblastic progenitors in the bone marrow The insertion of the normal gene, through the Erythropoietm vector, selectively into the bone marrow erythroblastic lineage, represents true in vivo gene-therapy, to potentially cure patients with this disease The same consideration applies to another genetic haemoglobin disease sickle cell anaemia
IL-6 constructs may be used to block cells, having IL-6 high affinity receptors, which are involved in multiple myeloma, osteoclastic hyperactivation (metastasis to the bone), cancer-related bone lesions and osteoporosis
Some specific vector/gene combinations and related disease targets are set out below
1 Epo/functional Hb-beta-gene in Thalassemia
2 Epo/SS-wild gene is Sickle Cell Anemia (or Sickle Cell Disease)
J GM-CSF/ABL_BCR gene in Chronic Myeloid Leukemia (CML) Phyladelphia + (Ph+) and its accellerated phase (Blastic crisis) 4 G-CSF/ABL BCR gene in Chronic Mveloid Leukemia (CML) Phyladelphia + (Ph+) and its accellerated phase (Blastic crisis)
5 SCF/ABL BCR gene in Chronic Myeloid Leukemia (CML) Phyladelphia + (Ph+) and its accellerated phase (Blastic crisis) and AML Ph+ (rare subform of AMLs)
6 IGF-1/P53 tumor-suppressor gene in breast adenocarcinoma
7 IGF- 1 /antisense HER-2Neu in breast adenocarcinoma
8 IL-2 (or) IL-3 (or) GM-CSF/fϊinctional genes in congenital immunodeficiencies (usually these immunodeficiencies are inherited as pohgenic defects severe combined immunodeficiencies, Di George's syndrome, Nezelof s syndrome, Ataxia-teleangiectasia, X-hnked gammaglobulinemia)
9 IL-2/functιonal gene in selective deficiency of T-lymphocvte function such as inherited puπne nucleoside phosphorylase deficiency (PNP syndrome)
10 M-CSF/functional gene in congenital macrophage enzymatic monogenic deficiencies usually present in lysosomal storage diseases Lysosomal storage diseases include most of the lipid storage disorders, the mucopolysacchaπdoses and glycoprotem storage diseases which are characterised by mono-enzymatic defects (beta-galactosidase, beta-glucocerebrosidase deficiency in Gaucher's disease, alpha-fucosidase deficiency in Fucosidosis, ceramidase deficiency in Farber's disease and hexosammidase-A deficiency in Tay-Sachs syndrome) All these serious congenital conditions may have their onset in infantile, juvenile and adult age
M-CSF/wiId gene coding for a functional enzyme selected on the basis of the specific deficiency, once integrated into macrophages and transcπpted into protein, will compete with the nonfunctional protein and repair the defect in vivo
1 1 M-CSF, SCF or GM-CSF/functional gene in acid sphingomye nase deficiency in monocytes/ macrophages in Niemann-Pick disease
The invention includes products comprising a moiety having M-CSF, SCF or GM-CSF function linked to a functional acid sphingomyehnase gene
Adenosine Deammase (ADA) Deficiency ADA deficiency in its most severe form results in the syndrome of Severe Combined Immunodeficiency
Disease (SCID), and presents as a reduction in, and abnormal function of, both T and B lymphocytes Less severe disease is usually associated with T-cell dysfunction and a more variable loss of B-cell function It is now recognised that ADA deficiency can result in slowly progressive immune dysfunction, which presents at birth but also in adolescents and adults
Approximately 20% of all patients with SCID have an ADA-gene mutation Approximately 50% of patients with autosomal recessive inherited SCID have an ADA-gene abnormality, the remainder having other inherited abnormalities The gene for ADA is located on chromosome 20q More than 25 single base changes within the coding region, as well as several deletion and splicing mutations leading to loss of enzymatic activity have been identified ADA catalyzes the irreversible deamination of adenosine to inosine, and of 2-deoxyadenosme to 2-deoxymosine
Allogeneic bone marrow transplantation is currently the treatment of choice for ADA-deficient SCID, but an HLA-identical donor is available only for a minority of patients An alternative therapy is the injection of PEG-ADA (daily enzyme replacement)
The ADA gene has been sequenced and the structure of the enzyme has been determined. Several patients have been treated with ADA-cDNA ex-vivo transduced autologous T-lymphocytes using retroviral vectors (Refs 26-36) Results have been poor and the major barrier to effective gene therapy remains the low efficiency of stem cell transduction with retroviral vectors
Recombinant IL-2 has been used at low doses m SCID and ADA-SCID patients (Ref 37) Therapy with low dose rIL-2 has resulted in a marked clinical improvement as well as improved T-cell function Futhermore IL-2 is normally used to expand T-cells in vitro from ADA-SCID patients before ADA-cDNA transfection with retroviral vectors.
Lymphocytes and lymphoblasts from ADA-SCID patients and ADA-SCID animal model (ADA-SCID mouse) are responsive to low dose IL-2 stimulation, both in vitro and in vivo In other words, the IL-2 mechanism and its cascade of intracellular events (cytoplasmic internalisation following high affinity receptor binding, internalisation and T-cell proliferation) is maintained in SCID
The invention therefore includes products comprising a first domain which comprises an IL-2 sequence functional to be recognised by high affinity IL-2 receptors and to promote proliferation linked to a second domain which comprises a gene for functional ADA The gene is usually associated with protective material as described above, e g polylysine Also included m the invention is a product comprising a functional IL-2 linked to an expression vector comprising a gene for functional ADA Preparation
Recombinant Polvnucleotides
The skilled person can readily construct a variety of clones containing functional nucleic acids Cloning methodologies to accomplish these ends, and sequencing methods to verify the sequences of nucleic acids, are well known in the art Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found in Sambrook, et al . Molecular Cloning A Laboratory Manual (2nd Ed , Vols 1-3, Cold Spring Harbor Laboratory ( 1989)), Methods in Enzymology, Vol 152 Guide to Molecular Cloning Techniques (Berger and Kimmel (eds ), San Diego Academic Press. Inc ( 1987)), or Current Protocols in Molecular Biology, (Ausubel, et al (eds ), Greene Publishing and Wiley-Interscience, New York ( 1987)
Product information from manufacturers of biological reagents and experimental equipment also provide information useful in known biological methods Such manufacturers include the SIGMA chemical company (Saint Louis, MO), R&D systems (Minneapolis, MN), Pharmacia LKB Biotechnology (Piscataway, NJ), CLONTECH Laboratories, Inc (Palo Alto, CA), Chem Genes Coφ , Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc , GIBCO BRL Life Technologies, Inc (Gaithersberg, MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen, San Diego, CA, and Applied Biosystems (Foster City, CA), as well as many other commercial sources known to one of skill
Polynucleotides containing a desired gene can be prepared by any suitable method including, for example, cloning and restriction of appropriate sequences as discussed supra, or by direct chemical synthesis by methods such as the phosphotnester method of Narang et al Meth Enzymol 68 90-99 (1979), the phosphodiester method of Brown et al , Meth Enzymol 68 109- 151 (1979), the diethylphosphoramidite method of Beaucage et al , Tetra Lett , 22 1859-1862 (1981), the solid phase phosphoramidite tnester method described by Beaucage and Caruthers ( 1981 ), Tetrahedron Letts , 22(20) 1859-1862, e g , using an automated synthesizer, e g , as described in Needham-VanDevanter et al ( 1984) Nucleic Acids Res , 12 6159-6168 and, the solid support method of U S Patent No 4,458,066 Chemical synthesis produces a single stranded o gonucleotide This may be converted into double stranded DNA by hybridization with a complementarv sequence, or by polymerization with a DNA polymerase using the single strand as a template One of skill would recognize that while chemical synthesis of DNA is limited to sequences of about 100 bases, longer sequences may be obtained by the hgation of shorter sequences
Nucleic acids may be modified by site-directed mutagenesis. as is well known in the art Native and other nucleic acids can be amplified by in vitro methods Amplification methods include the polymerase chain reaction (PCR), the hgase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (SSR) A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well-known to persons of skill
Protective Mateπal/DNA-Binding Material
Micelles or cationic materials, for example cationic polymers, are commercially available and the use of liposomes in gene therapy is reviewed by Lasic and Templeton (Lasic D D, Templeton D S (1996) Advanced Drug Reviews 20 221 -226)
Linking
The prohferatively active moiety and one of the nucleic acid material and protective/DNA-bindmg material are suitably interlinked using a multifunctional (e g bifunctiona!) linker which reacts with respective functional groups on the two components In some embodiments the two constituent parts are linked by an intracellularly cleavable link In other embodiments, the link is intracellularly stable
The link may comprise a bridging group comprising the inter-bonded residues of two heterobifunctional cross-linkers, as described in our US provisional patent application filed on the same day as this application under the title "Novel Constructs" The potential benefit of this technique is that it helps avoid the preparation of dimers, by separately preparing (I) an active moiety-linker construct and (n) a nucleic acid- hnker construct or protective material- or DNA-b dmg material- linker construct, and then reacting together constructs (I) and (n) to join their respective linkers together The linkers are obviously chosen to be reactive to each other, as well as to the material forming the residue of the construct (I) or (n)
The preparation of products comprising a polypeptide linked to another moiety is well known, as for example in the case of fusion proteins, and the skilled person will therefore require no elucidation of preparatory techniques In general terms, suitable linkers are multifunctional, and especially bifunctional compounds capable of reacting with a polypeptide and a nucleotide
One exemplary technique involves the use of acid-cleavable reagents for interlinking two polypeptides Such acid-cleavable linker reagents, based on orthoester, acetal and ketal functionalities, have been described previously (Ref 18), and are bifunctional compounds whose hydrolytic rate constants increase as the pH decreases The crosshnkers react with the proteins via heterobifunctional groups (e g maleimide or N-hydroxysuccinimide ester) or homobifunctional groups (e g bis-maleimide or bis-succinimidyl)
Three particular cross-linking agents which may be used are 1 Disuccinimidyl suberate This is a homo-bifunctional cross-linking reagent containing the N hydroxy succmimide ("NHS") ester reactive group, which is reactive towards amino groups The chain of the cross-linking reagent is non-cleavable
2 Ethylene glycobisfsuccinimidyl succinate] This too is a homo-bifunctional cross-linking reagent, containing the NHS ester reactive group which is reactive towards amino groups The chain of the cross- linking reagent is cleavable
3 Succinimidyl 6-[3-(2-pyπdylthιo)-propιonamιdo] hexanoate This is a hetero-bifunctional cross- linking reagent, containing the pyndyldithio and NHS ester reactive groups, which are reactive towards sulfhydryl and amino groups The chain of the cross-linking reagent is cleavable
A table containing more information about these crosshnkers and other suitable candidates appears below
Figure imgf000026_0001
Products in which the prohferatively active moiety (especially cytokine or growth factor) and nucleotide are acid-cleavably linked benefit from the potential advantage that the product is cleaved in the endosome to release the nucleotide in free form
The literature describes a number of techniques for linking substances to proteins, typically by using bifunctional hnker-chelating agents For example, the reader is referred to the textbook "Laboratory Techniques in Biochemistry and Molecular Biology", Volume 19, edited by R H Burdon and P H van Knippenberg, published by Elsevier and to Chapter 3 ("Peptide-Camer Conjugation") of the textbook "Synthetic Polypeptides as Antigens" by M H V Van Regenmortel et al, published by Elsevier (1988)
Heterobifunctional linkers suitable for conjugating cytokines and growth factors to biologically active agents are obtainable from Pierce & Warner (UK) Limited. 44 Upper Northgate Street, Chester CH I 4EF, UK, or Pierce Chemical Company, PO Box 1 17, Rockford, IL 61 105, USA, whose literature provides further information
Administration The IL-2 and other cytokines or growth products may be administered parenterally, in contemplated amounts of from 10 000 - 1 000 000 International Units, and suitably by intravenous, intramuscular or subcutaneous injection e g (less than 1 μg to 0 1 mg of recombinant protein), to give very low plasma concentrations For example, the plasma IL-2 concentrations may be close to the dissociation constant
(KD) of the concentration of IL-2 that saturates 50% of the IL-2 high-affinity receptor isoform
Furthermore, this range of dose is generally without systemic adverse side effects
The products of the invention may be formulated as human or veterinary pharmaceutical formulations in practice comprising a pharmaceutically acceptable diluent carrier or excipient
The formulations may be in the form of solutions or suspensions The formulations are suitable for parental (e g lv or sc) administration but, oral formulations are not excluded
EXAMPLES
Protocols suitable for crosslinking nucleotide material with peptides. exemplified by the IL-2 mutein Aldesleukm, will now be described The specific reaction conditions may of course be varied from those described
Modification of Proteins Using LC-SPDP
LC-SPDP = Succinimidyl 6-[3'(2-pyπdyldιthιo)-propιonamιdo]hexanoate
Materials
A LC-SPDP stock solutions 20mM LC-SPDP in DMSO Note Prepare just before use
B Reaction buffer 20mM sodium phosphate, 150mM sodium chloride, ImM EDTA. pH7 5 C Acetate buffer lOOmM sodium acetate, lOOmM sodium chloride, pH4 5
D BioRad Micro Bio-Spin Desalting Columns
E Dithiothreitol (DTT) 24mg/ml in lOOmM sodium acetate pH4 5, lOOmM sodium chloride
F Aldesleukm stock solution Dissolved at 1 53mg/ml (0 ImM) in reaction buffer
Method
1 To 9μl of Aldesleukm stock solution, add l μl of the 20mM LC-SPDP stock solution
2 Incubate for 30 minutes at room temperature
3 To remove the unconjugated cross-linker, the sample is applied to a BioRad Micro Bio-Spin Desalting Column (pre-equi brated in lOOmM sodium acetate, l OOmM sodium chloride, pH4 5), spin and the filtrate used for the subsequent DTT treatment
4 DTT (dissolved as above in the appropriate buffer) is added to a final concentration of 8mg/ml
5 Incubate at room temperature for 30 minutes 6 To remove the DTT the sample is applied to another BioRad Micro Bio-Spin Desalting Column (pre- equihbrated in conjugation buffer 20mM sodium phosphate pH7 5, 150mM sodium chloride, ImM
EDTA), spin and use the filtrate for the final conjugation reaction (see later)
Modification of Nucleotide Material using PMPI PMPI = N-(p-Maleιmιdophenyl) isocyanate Materials
A PMPI stock solution 50mM in dry DMSO, freshly prepared before use B Reaction Buffer 20mm Tns-HCl pH8 5, l OOmM NaCl C BioRad Micro Bio-Spin desalting columns
Method
1 Add lμl 50mM PMPI in dry DMSO to genetic material Mix thoroughly
2 Incubate at room temperature for 60 minutes 3 Remove the excess unreacted cross-linking reagent using a BioRad Micro Bio-Sp desalting column, pre-equihbrated in conjugation buffer (20mM sodium phosphate pH 7 5, 150mM sodium chloride, ImM EDTA) The filtrate is used for the final conjugation reaction (see below)
Coniugation Reaction The desalted LC-SPDP-activated Aldesleukm and the desalted PMPI-activated genetic material are mixed in equal proportions and incubated at room temperature for 24 hours The conjugation reaction is then analysed by SDS-PAGE, along with samples from the activation reactions, to assess the conjugation efficiency Conjugated product can then be purified by size-exclusion chromatography
Example 1
Production of modified Aldesleukm
Modification of Aldesleukm was performed according to the protocol set out above, except that 7 reactions were run for 0, 10, 20, 30, 40, 50 and 60 minutes at room temperature The reaction mixtures were desalted as per the protocol (using BioRad Micro Bio-spin P6 desalting columns, used according to the manufacturer's protocols) A l μl sample was removed from each filtrate for analysis by SDS-PAGE [A 12% acrylamide gel, run according to the method of Laem (Laemmli UK Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 1970 Aug 15 227 259 680-5)] The gel was then stained using Gelcode Blue (Pierce, used according to manufacturer s protocol)
A gel of a 60 minute run is shown in Figure 5 The picture shows the increase in apparent molecular weight after modification of Aldesleukm by LC-SPDP to illustrate that all of the Aldesleukm is covalently modified Example 2
Growth Assay
The column filtrates from Example 1 were resuspended to approximately 0 20 μg/ml in standard HT-2 culture medium (Iscove's Modified Dulbecco's Medium with added 10% Foetal Bovine serum, 20μM 2- mercaptoethanol) and used in growth assays according to the protocol set out below
The assay samples were set up to contain 20ng/ml modified Aldesleukm species ( 1 set of samples from each reaction product), and plated out onto a 96-well plate. The final row was plated out containing only the recombinant IL-2 carried over from the stock cell culture
(HT-2 cells are standard cells for measuring induction of proliferation of T-lymphocytes, see Watson J Continuous proliferation of muπne antigen-specific helper T lymphocytes in culture. J Exp Med 1979 Dec 1 150:6 1510-9.)
Growth Assay Protocol
A Setting up the Assay.
1 From the same master cell stock, set up 8 x 2ml ahquots of HT-2 cells in sterile containers in Iscove's Modification of Dulbecco's Medium (plus 10% FCS, 20μM β-mercaptoethanol, lOIU/ml recombinant human IL-2, or equivalent), at a cell density of between 20,000 and 30,000 per ml
2 To each, add the IL-2 under test to the desired concentration
3 Plate out the cell suspensions into 96-well plates at 150μl per well as shown in Figure 6 Grow on in an incubator for 4 days, counting the cells each day 4 Pool the remaining cell suspensions from the sterile containers for cell counting to establish a baseline cell density (use n=3 for the counts)
B The Cell Counting
1 Each day, count the cells which appear to be of both relatively normal moφhology (round or nearly round), and exclude trypan blue, using a haemocytometer Note the time at which the cell counts were performed
2 Count three wells for each test condition per day. and average the results Example 3 Cell Viability
The cell density in the initial cultures from Example 2 was assessed using a haemocytometer. and the viable cells assessed by Trypan blue exclusion. These were counted in triplets, as were all samples throughout the assay period. The averaged cell densities are shown in following Table 1 :
Average Cell Densities
Figure imgf000030_0001
The results are presented in a data chart in Figure 7.
Comparative growth responses to various IL-2 compositions comprising Aldesleukin (Proleukin®), native human IL-2 and recombinant human IL-2 are shown in Figure 8. and indicate that any of these IL-2s is a suitable mitogenic vector.
Example 4
Transfection ofpEGFP-1 into HT-2 cells
pEGFP-l was transfected into HT-2 cells using Transfectam (Promega). The numbers of cells that expressed EGFP is shown in Table 2. The highest transfection efficiency (approximately 0.08%) was observed with a cationic lipid : DNA ratio of 4: 1 using either Dfx20 or Dfx50. The number of cells that were transfected was considerably higher when cultured at 1.Ong/ml RhIL-2 compared with 0.2ng/ml RhlL- 2. This may reflect the fact that these cells are proliferating more quickly and as a consequence of nuclear breakdown during the cell cycle it is easier for the plasmids to enter the nucleus and express their genes. Support for this theory comes from the observation that many of the EGFP expressing cells were found in 50 pairs which suggests that they may have recently divided These results indicate that IL-2 and other prohferatively active moieties potentiate cells towards genetic or nucleic acid material.
Table 2: Number of cells which express EGFP 28 hours after transfection
Figure imgf000031_0001
Example 5
Characterisation of Polyethelinimine (25k)
25k PEI. in contrast to 800k PEI, is more homogeneous in terms of polymer length and has been reported to have fewer toxic effects Since a more homogeneous product would simplify the inteφretation of crosshnked products, 25k PEI was investigated as a possible candidate cationic polymer for this study Initially 25k PEI was run on the column to determine the size profile Comparison with the profile of 800k PEI reveals an equally heterogeneous range of species
A toxicity assay using serial dilutions of 25k PEI added to HT-2 cells cultured at 1 0, 0 5 and 0 2ng/ml RhIL-2 was performed and cell viability assayed for three days (see Figures 9a - c) There was a drop in cell viability at 2μg/ml 25k PEI in cells grown at 1 0 or 0 5ng/ml RhIL-2 whilst those cultured at 0 2ng/ml RhlL- 2 showed a fall in viability at 1 μg/ml 25k PEI 25k PEI is slightly more toxic than 800k PEI for HT-2 cells which is contrast to the published results for other cell lines
Example 6
Transfection of PEGFP.CAT into HT-2 cells using Polyethylenimine
An assay was performed to determine if polyethylenimine (PEI) can form complexes with the reporter plasmid which are capable of transfecting HT-2 cells Various ratios of PEI DNA were used to ensure 1 1 optimal DNA compaction In addition cells were grown at 1 Ong ml RhIL-2 for 24 hours prior to the application of the DNA PEI complexes, before being split into two plates, one at 1 Ong/ml RhIL-2 and the other at 0 2ng/ml RhIL-2 The transfection complexes were then applied to the cells and centrifiiged at 500φm for 5 mm After one hour incubation all the cells were covered with complete media containing 1 Ong/ml RhIL-2 The number of cells expressing EGFP 20 hours after transfection are shown in Table 3
Table 3
Figure imgf000032_0001
The following conclusions may be drawn • The number of cells transfected increased with RhIL-2 concentration
• 25kD PEI is far more efficient at gene delivery than 800kD PEI
• 25kD PEI is 2-3 times more efficient than the commercial transfection reagent Transfectam (comparative data not shown)
• a ratio of 250ng PEI to 500ng DNA was the most efficient • the presence of chloroquinone improved the transfection efficiency
A second assay was performed to investigate the effect of the length of incubation of the 25kD PEI DNA complexes before the addition of complete media (containing serum) The ratio of PEI DNA was further investigated and the effect of chloroquinone more fully examined The number of cells expressing EGFP 18 hours after transfection are shown below in Table 4
Table 4
Figure imgf000032_0002
The following conclusions may be drawn • 200ng of PEI w ith 500ng of DNA gave the highest transfection efficiencv in the presence of chloroquinone • in the absence of chloroquinone the highest transfection efficiency was with lOOng PEI in contrast to the result of the previous assay
• chloroquinone increased transfection efficiency by 3 to 6 times at higher PEI concentrations
• there was no significant increase in transfection efficiency when the incubation time was increased from 1 hour to 2 hours
• 18 hours after transfection was considered most appropriate for examination of gene expression due to the toxic effect of marker gene products
In summary, mild centnftigation (data not shown), the use of chloroquinone and the use of 25kD PEI all helped to improve the delivery of the marker genes to HT-2 cells
As used herein the word "comprises" is not exclusive, l e it indicates that the subject of the verb need not consist only of its object but may include the object of the verb and one or more additional elements Cognate expressions are to be construed accordingly
References
1 Smith. K A (1988) Science 240 1 16-1 176
2 Waldmann. T A (\99\ ) J Biol Chem 266 2681 -2684 3 Waldmann, T A (1989) Annu
Figure imgf000034_0001
Bioc em 58 875-91 1
4 Kuziel, W A and Greene, W C ( 1990) J Invest Dennatol 94 275-325
5 EP 319012 (Du Pont)
6 Waldmann, T A (1993) Immunol Today 14 264-269
7 Cerami and Beuter ( 1988) Immunol Today 9 28 8 Suzu et al (1992) J Biol Chem 267 4345 Sato et al (1992) Cancer Res 52 444
10. Gabbianelli et al ( 1990) J Biol Chem 249 1252
1 1 Zumstein et al (1987) J Biol Chem 262 1252
12 Bussolmo et al (1989) Nature 337 471 13 Signore A et al ( 1987) The Lancet. VOL II, No 8558
14 Signore A et al (1992) Nuclear med Comm 13 713-722
15 Chianelli A et al Nuclear medicine Proceed EAN 'M 1991 143-146
16 Bellan-harel A et al (1989) J Immunologtcal Methods 1 19 127-133 7 Godoman & Gilman The Pharmacological Basis of Therapeutics, Eighth Edition 1990 8 Neville D M et al Jl 989) J Biol Chem 264- 14653-14661 9 Pέlyi I et al, (1999) Proc Natl Acad Sci USA Vol 96, pp2361-2366 0 Palyi I et al, ( 1995) 5th International Congress on Hormones and Cancer ed Labrie J (ESCOM, Leiden, the Netherlands, p87 1 Palyi l et al, ( 1996) Ann Oncol 7, 83 2 Palyi I et al, (1997) Proc Am Assoc Cancer Res 38, 61 3 Vmcze B et al, (1997) Proc Am Assoc Cancer Res 38, 433 4 K K Ja e "Textbook of Gene Therapy", Hografe & Huber Publishers, 1998 5 Wagner R W et al, ( 1997) Molecular Medicine Today 3 31-38 6 Blaese R M et a Treatment of severe combined immunodeficiency disease (SCID) due to adenosine deammase deficiency with CD34+ selected autologous peripheral blood cells transduced with a human ADA gene Amendment to clinical research project. Project 90-C- I95, January 10, 1992 Hum Gene Ther 1993 Aug, 4(4) 521-7 7 Blese R M Development of gene therapy for immunodeficiency adenosine deammase deficiency Pediatr Res 1993 Jan.33(Suppll) S49-53, discussion S53-5 8 Kaptein L C et al Bone marrow gene therapy for adenosine deammase deficiency Immunodeficiency 1993,4( 1 -4) 335-45 Kawamura N et al Elevation of serum IgE level and peripheral eosinophil count during T- lymphocyte-directed gene therapy for ADA deficiency implication of Tc2-hke cells after gene transduction procedure Immunol Lett 1998 Nov 64(1) 49-53/ Onodera M et al Successful peripheral T-Iymphocvte-directed gene transfer for a patient with severe combined immune deficiency caused bv adenosine deammase deficiency Blood 1998 Jan
1 91( 1 ) 30-6 Sakiyama Y Gene therapy for adenosine deammase deficiency Okkatdo Igaku Zasshi 1996 Jan,71( 1 ) 27-32 Kohn D B et al Engraftment of gene-modified umbilical cord blood cells in neonates with adenosine deammase deficiency Nat Med 1995 Oct, 1( 10) 1017-23 Hoogerbrugge P M et al Gene therapy for adenosine deammase deficiency Br Med Bull 1995 Jan, 51(1) 72-81 Blaese R M et al T lymphocyte-directed gene therapy for ADA-SCID initial trial results after 4 years Science 1995,270 475-480 Bordignon C et al Gene therapy in peripheral blood lymphocytes and bone marrow for ADA- lmmunodeficient patients Science 1995,270 470-475 Bordignon C et al Transfer of the ADA gene into bone marrow cells and peripheral blood lymphocytes for the treatment of patients affected by ADA-deficient SCID Hum Gen Ther
1993,4 513-20 Pahwa R et al Recombinant ιnterleukιn-2 therapy in severe combined immunodeficiency disease
Proc Natl Acad Set USA 1989 Jul, 86(13) 5069-73 Gao X et al (1995) Gene Therapy 2 710-722 Philips S ( 1996) Exp Opm Invest Drugs 5 1 101-1 1 15

Claims

1 A product comprising a prohferatively active moiety linked to genetic or nucleic acid material which is associated with protective material
2 A product of claim 1 wherein the protective material comprises a micelle-forming or complex- forming material
3 A product of claim 2, wherein the complex-forming material comprises polylysine
4 A product of claim 2, wherein the micelle-forming material comprises one or more phospholipids
5 A product of any of claims 1 to 4, wherein the genetic material comprises an expression vector containing a gene encoding a protein and operably linked to a control sequence
6 A product of claim 5, wherein said gene is a cytotoxic gene, a defect correction gene or an immunogene
7 A product of claim 6, wherein the cytotoxic gene is for expressing an enzyme to convert a prodrug into a toxic drug
8 A product of claim 7, wherein the enzyme is thymidine kinase, cytosine deammase, cytochrome P- 450 or bacterial nitroreductase
9 A product of any of claims 5 to 8, wherein the control sequence comprises a CMV promoter
10 A product of any of claims 5 to 9, wherein the genetic material contains an episomal maintenance sequence
1 1 A product of any of claims 5 to 10, wherein the genetic material comprises two or more genes, the second and any subsequent genes each being operably linked to an internal πbosomal entry site
12 A product of any of claims 1 to 1 1 , wherein the genetic material comprises a plasmid construct
13 A product of any of claims 1 to 4 wherein the nucleic acid material comprises an anti-sense sequence
14 A product of any of claims 1 to 13 wherein the link between said agent and said moiety is intracellularly cleavable
15 A product of claim 14, wherein the link is cleavable by acid hydrolysis
16 A product of any of claims 1 to 15, wherein target cells of the prohferatively active moiety have high affinity receptors therefor
17 A product of claim 16, wherein the prohferatively active moiety is a cytokine or growth factor or a molecule functionally equivalent thereto
18 A product of claim 17 wherein the moiety is a cytokine or a molecule functionally equivalent to a cytokine
19 A product of claim 18 wherein the cytokine is an IL, a TNF, and M-CSF, an IFN, an FGF, an IGF, a TGF, a GM-CSF, an SCF a G-CSF or an Epo
20 A product of claim 19, wherein the IL is IL-2 or IL-6, the TNF is TNF-α, IFN is IFNα, IFN-β or IFN-γ and the TGF is TGFβ
21 A product of claim 17, wherein the moiety is a growth factor or a molecule functionally equivalent to a growth factor
22 A product of claim 21 , wherein the growth factor is Erythropoietm (Epo),
GM-CSF, G-CSF,
SCF (Stem cell factor),
Multi-CSF (also known as Interleukιn-3),
M-CSF,
E-CSF (or Interleukιn-5), IGF-1 (Insuhn-hke growth factor),
PDGF (Platelet-derived growth factor),
TGF beta2 (Transforming growth factor-beta2)
23 A product of claim 17 wherein the cytokine or growth factor is a human cytokine or growth factor and said molecule is functionally equivalent thereto
24 A product of any of claims 17 to 22 wherein said moiety is a recombinant human cytokine or growth factor, optionally modified by one or more amino acid alterations
25 A product of claim 24 wherein the recombinant human cytokine is recombinant IL-2
26 A product of claim 25, wherein the recombinant IL-2 is desala IL-2 serj25
27 A product comprising a biologically active agent which is provided with a protective material and linked to a cytokine or growth factor or to a molecule functionally equivalent thereto, the biologically active agent being selected from the group consisting of genetic material and antisense nucleotide sequences, and the cytokine or growth factor having target cells capable of presenting a high affinity receptor therefor
28 A product of claim 27, which further includes the feature(s) recited in one or more of claims 2 to
10, 19, 20 or 22 to 26
29 A product comprising first domain which comprises an IL-2 sequence functional to be recognised by high affinitv IL-2 receptors and to promote proliferation linked to a second domain which comprises a biologically active agent selected from the group consisting of antisense nucleotide sequences and genetic material
30 A product of any of claim 29 which further includes the feature(s) recited in one or more of claims 2 to 12.
31 A product comprising a prohferatively active moiety linked to a nucleotide which is associated with cationic DNA-binding material
32 A product of claim 31 wherein the DNA-binding material comprises a polymer, a hposome or a dendπmer
33 A product of claim 32, wherein the polymer comprises polylysine, a polylysine derivative or polyethyleneimine
34 A product of any of claims 3 1 to 33, wherein the DNA-binding material forms a bridge between the active moiety and the nucleotide
35 A product of any of claims 31 to 34 wherein the DNA-binding material forms a complex with the nucleotide
36 A product of any of claims 31 to 35, wherein the nucleotide comprises genetic material as defined in any of claims 5 to 12 or an anti-sense sequence
37 A product of any of claims 31 to 36 which further includes the feature(s) recited in one or more of claims 14 to 26
38 A product comprising a first domain which comprises an IL-2 sequence functional to be recognised by high affinity IL-2 receptors and to promote proliferation linked to a second domain which comprises a gene for functional ADA, the gene optionally being associated with protective material
39 A product comprising a functional IL-2 linked to an expression vector comprising a gene for functional ADA
40 A product of any of claims 1 to 39 for use as a pharmaceutical
41 The use of a product of any of claims 1 to 40 for the manufacture of a medicament for treating by therapy or prophylaxis a disease or disorder involving cells bearing a high affinitv receptor for a prohferatively active moiety
42 The use of claim 41 , wherein the product comprises a prohferatively active moiety having IL-2 function and the disease or disorder is an autoimmune disease, transplant rejection, graft-versus-host- disease, a retroviral disease or a lymphoproliferative disease
43 A pharmaceutical formulation, comprising a product of any of claims 1 to 40 formulated for pharmaceutical use
44 A pharmaceutical composition, comprising a product of any of claims 1 to 40 and a pharmaceutically acceptable diluent, excipient or carrier
45 The use of a product of any of claims 1 to 40 for the manufacture of a medicament for internalising the biologically active agent into a cell having a high affinity receptor for the prohferatively active moiety, cytokine or growth factor of the product and optionally for stimulating lymphocyte proliferation
46 A method of treating by therapy or prophylaxis a disease or disorder involving cells bearing a high affinity receptor for a prohferatively active moiety, comprising administering to a patient an effective amount of a product of any of claims 1 to 40, which product includes a prohferatively active moiety having high affinity for said receptor
47 A product comprising a moiety which is prohferatively active linked to encapsulated or complexed nucleic acid material selected from the group consisting of expression vectors and anti-sense sequences
48. A product comprising a moiety having M-CSF, SCF or GM-CSF function linked to a functional acid sphingomyehnase gene.
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