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  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/20—Interleukins [IL]
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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 1 The column filtrates from Example 1 were resuspended to approximately 0 20 ⁇ g/ml in standard HT-2 culture medium (Iscove's Modified Dulbecco's Medium with added 10% Foetal Bovine serum, 20 ⁇ M 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 mu ⁇ ne antigen-specific helper T lymphocytes in culture. J Exp Med 1979 Dec 1 150:6 1510-9.
• 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.
• 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 inte ⁇ retation 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
• PEI polyethylenimine

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