WO1998006431A2 - Procede de traitement des diabetes - Google Patents

Procede de traitement des diabetes Download PDF

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WO1998006431A2
WO1998006431A2 PCT/US1997/014448 US9714448W WO9806431A2 WO 1998006431 A2 WO1998006431 A2 WO 1998006431A2 US 9714448 W US9714448 W US 9714448W WO 9806431 A2 WO9806431 A2 WO 9806431A2
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interferon
cells
insulin
cell
diabetes
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PCT/US1997/014448
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WO1998006431A3 (fr
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Kurt B. Osther
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Osther Kurt B
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Priority to EP97938359A priority patent/EP0859633A2/fr
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Publication of WO1998006431A3 publication Critical patent/WO1998006431A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/249—Interferons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention is in the field of the prevention and treatment of diabetes mellitus.
  • Insulin dependent diabetes also known as Type I or juvenile diabetes
  • Type 1 diabetes is a disease characterized by reduced or absent pancreatic secretion of insulin.
  • Type 1 diabetes is associated with the progressive loss of pancreatic ⁇ cells and with several autoimmune disorders (W. Gepts and P.M. LeCompte in The Diabetic Pancreas (B.W. Volk and E.R.
  • HIT T-15 represses insulin II gene transcription in the rat (Stein and Ziff, Mol. Cell. Biol.
  • MHC major histocompatibility complex
  • cytokines e.g., interferon- ⁇
  • cytokines can be induced in certain epithelial cells as well, and among these epithelial cells are pancreatic ⁇ cells (A.R. Collins, Adv. Exp. Med. Biol. 276:497, 1990).
  • Interferon- ⁇ can induce NK cell activity (G. Semenzato et al, Blood 68:293, 1986; P. Conn et al., J. Immunopharmacol. 10:907, 1988; M. Sarzotti et al., Nat. Immun. Cell Growth Regul. 8:66, 1989; A. Verhagen et al., Nat Immun. Cell Growth Regul. 9:325, 1990; B.-L.
  • the instant invention teaches methods and compositions for effective treatment of type I diabetes by intervening with the interferon- ⁇ activity in diabetics.
  • This type of treatment for diabetes is relevant in human diabetics for several reasons. It is known that ⁇ cells of type I diabetes expresses interferon- ⁇ rather than interferon beta. Further, it is believed that the islets of human diabetics type I show histologically obvious changes before lymphocytes producing interferon- ⁇ , appear (A.K. Foulis et al, Lancet 11:1423, 1987). Thus, islet cell damage is induced by endogenously produced in:erferon- ⁇ production, and this production becomes the preferentially expressed transcript overwhelming and inhibiting insulin production.
  • the instant invention provides for a method for treating type I diabetes comprising intervention of interferon alpha activity.
  • the instant invention encompasses methods for treating early type I diabetes comprising administering an effective inhibitory amount of an antisense oligonucleotide directed to the mRNA encoding for interferon- ⁇ . Effective inhibition encompasses the complete or partial inhibition of transcription of the interferon alpha gene, or complete or partial inhibition of translation of the interferon alpha mRNA transcript.
  • the method of the instant invention also encompasses administering an effective inhibitory amount of anti-interferon- ⁇ antibodies.
  • anti-interferon- ⁇ antibodies Such antibodies, by binding specifically to interferon- ⁇ would be effective in inhibiting the inflammatory effects of the cytokine.
  • Effective inhibition by the specific binding of anti-interferon alpha antibodies can also be accomplished by active binding fragments of such antibodies (such as Fab, Fab 2 , and single chain antibodies) where said antibodies are either monoclonal or polyclonal.
  • the instant invention also encompasses a method for treating diabetes comprising administering an effective therapeutic amount of anti-interferon- ⁇ antibodies in combination with antisense oligonucleotide directed to the mRNA sequence encoding interferon- ⁇ .
  • the instant invention also encompasses intervention in the regulatory pathway which controls interferon production or other related cellular regulatory protein activity (i.e. enzymes), and responses to interferon alpha.
  • the method of the instant invention embodies inhibition of anti-viral cellular response enzyme activity by specific binding.
  • targeted anti-viral cellular enzymes include 2', 5' oligoadenylate synthetase (2 ⁇ 5' A synthetase), the La antigen (p47,6), and protein kinase p68 (PKR).
  • Inhibitory specific binding may be accomplished by specific monoclonal or polyclonal antibody and binding fragments thereof.
  • the inhibition is accomplished by dsRNA molecules which are competitively and selectively bound to the enzyme molecule.
  • Such inhibitory dsRNA molecules can contain one or more substituted nucleic acids whereby the overall molecule is stabilized to resist degredation.
  • a further embodiment of the instant invention provides for the generation of pancreatic ⁇ -cells, where such cells are suitable for implantation in mammals, that are still insulin production competent but altered such that they are either non-responsive to interferon alpha or interferon alpha inducing signals, or are unable to functionally produce active interferon alpha
  • Such pancreatic ⁇ -cells of the instant invention can be altered by gene- knockout or other molecular biology technique such that interferon alpha genes or interferon alpha production related signal genes or nucleic acid sequences are rendered inoperative.
  • pancreatic ⁇ -cells are suitable for implantation into human tissue. It is further preferred that such pancreatic ⁇ -cells are from a human, or from a transgenic mammal (such as a pig) where the histocompatibility markers of the cell have been genetically altered to be adapted to human
  • the instant invention provides for methods for treating type I diabetes comp ⁇ sing interfe ⁇ ng with either or both the endogenous interferon- ⁇ production by pancreatic ⁇ -cells and the inflammatory activity of interferon alpha protein
  • pancreatic ⁇ cells being subjected to an exogenously or endogenously de ⁇ ved agent which induces them to produce mterferon-a
  • This production of interferon- ⁇ has a number of consequences of which two are very important
  • the first consequence is that the insulin production by the pancreatic ⁇ cells is overridden by the activated interferon- ⁇ production, having the effect that insulin levels become lowered in the bloodstream and that blood sugar levels get out of control
  • the second consequence is that the ⁇ cells become susceptible to va ⁇ ous cytotoxic effects due to the general cell inhibiting effects of interferon- ⁇ The ⁇ cells thus become more "fragile" and subsequently die out as a consequence of their constant state of inhibition
  • the instant disclosure teaches a number of inventive methods and means for the treatment of insulin dependent diabetes
  • the invention relates to a method for the treatment and/or prophylaxis of insulin dependent diabetes, the method comp ⁇ sing interfe ⁇ ng with the effects of interferon- ⁇ and its role in the destruction of insulin producing cells
  • One method of interfe ⁇ ng with the effects of interferon- ⁇ is to effect the contacting between interferon- ⁇ and a substance (e.g. a monoclonal or polyclonal antibody or an antibody fragment raised against interferon- ⁇ ) which binds to interferon- ⁇ in such a way that the cell inhibiting effects of this cytokine are blocked or inhibited.
  • a substance e.g. a monoclonal or polyclonal antibody or an antibody fragment raised against interferon- ⁇
  • Such a block or inhibition should of course be specific, i.e. the effect must be ascribable to a specific binding between the substance and interferon- ⁇ and not merely ascribable to non-specific interference of the substance on a wide variety of binding phenomena.
  • Another method encompassed by the instant invention is to interfere with the production of interferon- ⁇ . This can be accomplished by direct interference with the genetic material encoding interferon- ⁇ or by interfering with the transcription products of this genetic material using the antisense technique (Gustafsson et al, Immunol. Rev. 141: 59, 1994; all references described are incorporated by reference in their entirety).
  • antisense fragments both DNA and RNA fragments may be used, but also PNA fragments (peptide nucleic acids; Nielsen P. E. et al, Science 254: 1497-1500, 1991) are also candidates for use in such therapy, as these molecules have been demonstrated to exhibit superior and very dynamic hybridization profiles.
  • An additional method would be to inactivate the interferon- ⁇ gene by a gene knock-out technique described in EP-A-546073 (GenPharm International, Inc; incorporated by reference).
  • a successful inhibition of interferon- ⁇ activity may also be accomplished by the effective alteration of the active residues involved in effecting binding.
  • interferon expression is one aspect of a generalized pro-inflammatory immune mechanism where both gene expression and induced cell regulatory proteins (i.e. enzymes) are involved.
  • Interferon directed expression of certain cell regulatory proteins (i.e. enzymes) and the inherent dependency of these proteins on double-stranded RNA molecules (viral or synthetic origin) for induction and regulation of their activity are prime targets for the method of intervention of the instant invention.
  • Most important as targets for intervention, of the genes expressed during the viral defense response, are the 2 ',5' oligoadenylate synthetase (2',5' A synthetase), the protein kinase p68 (PKR), and the La antigen (p47,6) genes and related transcripts.
  • the dsRNA dependent 2', 5' A synthetase exists in five isoforms localized in the cytoplasm (p42, p46), ribosomes (plOO), and cell membranes (p69/71).
  • the 2',5' A synthetase link ATP molecules together by 2',5' di-ester bonds yielding 2', 5' oligomers of different chain lengths which are specific activators of the RNA degrading latent ribonuclease (RNase L).
  • RNase L RNA degrading latent ribonuclease
  • the PKR enzyme is expressed in most mammalian cells and is associated with ribosomes, and also found within the nucleolus.
  • the PKR gene has been recently cloned. Two domains of the protein bind dsRNA with high-affinity leading to auto-phosphorylation whereby the enzyme becomes activated.
  • the activation of PKR by synthetic, viral or cellular dsRNA phosphorylates the eukaryotic protein synthesis initiation factor (eIF-2), which thereby becomes activated.
  • eIF-2 eukaryotic protein synthesis initiation factor
  • the effect of this process is to produce an anti-viral state via inhibition of cellular protein synthesis and thus inhibition of viral protein synthesis.
  • Over expression of PKR has a significant anti-proliferative effect and may eventually lead to cell death.
  • RNA molecules have been found to play a significant role in the regulation of PKR (i.e. Epstein-Barr virus, EBER-1 adenovirus VA.RNA). Viral small RNAs are capable of inhibiting PKR activity by competitive interference with dsRNA binding to the enzyme.
  • the 46.7 kDa cellular La antigen has been shown to have a DNA-RNA and dsRNA unwinding activity resulting in single-stranded RNA which cannot activate PKR. Therefore, viruses with La antigen will escape the degrading effects of the PKR enzymes and replicate uninhibited, and there is increased risk of incorporation of viral genome into host cell genome and/or persistent generation of virus particles in host cell cytoplasm. This may lead to perpetual viral antigen presentation and processed viral neoantigen presentation to immune competent T- lymphocytes.
  • a transgenic mouse model has been developed to initiate an autoimmune response model for diabetes, capable of destroying ⁇ cells when the ⁇ cells are made capable of co- expressing interferon- ⁇ , thus giving a syndrome comparable to diabetes type 1.
  • transgenic mice that carry a transgene containing the regulatory region of the human insulin gene, and a cDNA that encodes a hybrid human interferon- ⁇ , because ordinary interferon- ⁇ is not active in mice (S.A. Chen et al, J. Interferon Res. 8:597, 1988; L. Martin et al, J. Immunol. 150:1234, 1993; O. Bohoslawec et al, J. Interferon Res. 6:207, 1986). Furthermore, the polyadenylate addition signal from hepatitis surface antigen was also transfe ⁇ ed. This general form of a transgene can efficiently drive expression of a heterologous gene in the ⁇ cells (N. Sarvetnick et al, Cell 52:773, 1988).
  • mice were called Iia founder mice, which were then backcrossed to outbred albino mice (CDI) and to inbred mice (C57BL/6).
  • CDI outbred albino mice
  • C57BL/6 inbred mice
  • the Iia x CDI transgenic mice became diabetic at a rate of more than 50%, with a median time to onset of the type 1 diabetes of 4 to 6 weeks.
  • the incidence in Iia x C57BL/6 mice was less than 5%.
  • One major consequence of the co-expression of interferon- ⁇ transgene in ⁇ cells was to induce development of hypoinsulinemic diabetes.
  • NOD non-obese diabetic mice
  • spontaneously diabetic BB rats are another model system by which early intervention to prevent the effects of interferon, as a means of preventing the development of diabetes can be tested where there is a demonstrated genetic predisposition to develop diabetes.
  • interferon- ⁇ The antibody used for the detection of interferon- ⁇ in these islets was raised by immunizing sheep with lymphoblast interferon (Hu INF- ⁇ Ly Namalwa: Wellferon from Wellcome Research Laboratories, Beckenham, UK) an antigen.
  • the hyper-expression of the major histocompatibility complex class I (MHC Class I) appeared to be related to the presence of interferon- ⁇ in the islets.
  • MHC Class I major histocompatibility complex class I
  • 93% contained interferon- ⁇ , compared to only 0.4% of those islets showing no hyper-expression.
  • Interferon- ⁇ is known to enhance MHC class I expression by pancreatic endocrine cells in vitro (Pujol-Borell R. et al., Clin. Exp. Immunol. (1986) 65, 128).
  • Part of this invention is the identification of the species of interferon- ⁇ that are produced by the ⁇ cells, because the anti-interferon- ⁇ species specific antibody(ies) that preferably should be administered into the patients could be targeted specifically by tailor- making the antibody specificity to the certain species of interferon- ⁇ produced.
  • the interferon- ⁇ family consists of 24 or more genes or pseudogenes. Two families of interferon- ⁇ are distinguishable (type I and II) consisting of > 1 of bovine type I. Mature type I interferons ⁇ are 166 amino acids long (one is 165 amino acids long), type II is 172 amino acids long.
  • non-allelic genes or pseudo-genes for human interferon- ⁇ have been identified. Eighteen of these including at least 4 pseudo-genes are for IFN ⁇ (type I), and 6 of the genes of which at least 5 pseudo-genes are for IFN ⁇ (type II). Amino acid sequence for human interferon ⁇ D (IFN ⁇ type I) and for human interferon all (IFNa(Il) (IFN ⁇ ) is given below and is from Swissprot.
  • IFN ⁇ amino acid sequence for human IFN ⁇ (type II) also called IFN ⁇ (Capon DJ. et al. Mol. Cell. Biol. (1985), 5, 768): Accession code: Swissprot P05000 (SEQ ID NO:2)
  • the insulin producing ⁇ cells are in a constant activated interferon- ⁇ producing state. It is remarkable that this active state is not an on - off mechanism as in other interferon- ⁇ producing cells and, furthermore, it is also remarkable that the repressor proteins produced when interferon- ⁇ is induced in cells which normally would participate in shutting off the mRNA for interferon within hours, does not elicit the same repressor function on the production of interferon- ⁇ product in ⁇ cells. There could be at least one or more hypothetical reasons for this phenomenon that the interferon- ⁇ mRNA in the ⁇ cells is not shut off by repressor proteins.
  • interferon- ⁇ is a type such as for instance type II (bovine type), induced by virus.
  • type II bovine type
  • a spontaneous IFN production in the absence of any added inducers can occur in cultures of macrophages, of lymphoblastoid cells and of leukemic and normal peripheral leukocytes (Northrop R. and Deinhardt F., J. Nat. Cancer Inst. (1967) 39, 685; Pickering L. et al., Proc. Natl. Acad. Sci.
  • IFN production resulting in demonstrable virus resistance can be rendered permissible by treatment of anti-IFN ⁇ serum or of IFN ⁇ / ⁇ serum (Jarvis A. and Colby C, Cell (1978) 14, 355; Gresser I. et al., J. Virol. (1985) 53, 221). Therefore antibodies to IFN ⁇ , to subtypes of IFN ⁇ , or to IFN ⁇ / ⁇ serum can interrupt a spontaneous IFN production or a induced IFN production that will not be interrupted even if the inducer(s) have disappeared. Hence, it is conceivable that IFN production in insulin producing ⁇ cells can be interrupted by antibodies. It is therefore important to establish what subtype of IFN ⁇ (and, may be IFN ⁇ ) the insulin producing ⁇ cells are producing.
  • IFN ⁇ and/or IFN ⁇ such as colony stimulating factor 1 (CSF-1) (She ⁇ , C. et al., Cell (1985) 41, 665); Moore R. et al., Science (1984) 223, 178).
  • CSF-1 colony stimulating factor 1
  • PDGF platelet-derived growth factor
  • Mu IFN ⁇ mRNA in confluent monolayers of murine BALB/c 3T3 cells (Zullo J. et al., Cell (1985) 43, 793).
  • Interleukin 1 (IL-1) (and tumor necrosis factor (TNF) are regulatory cytokines with pleiotropic activities which in human diploid fibroblasts can induce the synthesis Hu IFN ⁇ .
  • Interleukin 2 vital for T- cell function can induce IFN in bone marrow cells, and IFN ⁇ can sometimes act as an inducer of IFN ⁇ .
  • IFN ⁇ Part of the antiviral activity induced in mouse L-929 cells by IFN ⁇ can be neutralized by monoclonal antibody to Mu IFN ⁇ (Hughes T. and Byron S., J. Biol. Regul. Ho eostat. Agents (1987) 1 , 29).
  • mRNA is synthesized as a large precursor molecule in the nucleus. The mature mRNA moves to the cytoplasm for translation. For many eukaryotes mRNA is the functional half-life in the cytoplasm can be relatively long, up to 24 hours. Some mRNAs however, have much shorter half-lives, of 30 minutes or lower. Rapid degradation of mRNA in the absence of further transcription is one way of quickly terminating the synthesis of a protein (Caput D. et al., Proc. Natl. Acad. Sci. USA (1986) 83, 1670; Shaw G. and Kamen R., Cell (1986) 46, 659).
  • Superinduction can enhance the translation or the transcription or the combination of both. Stabilization of cytoplasmic IFN and mRNA probably contributes to the enhanced and prolonged synthesis of Hu IFN ⁇ in polyrlrC - induced human fibroblasts that have been treated with cycloheximide and Actinomycin D in a superinduction scheme; in cells not exposed to these metabolic inhibitors, active degradation of IFN mRNA starts a few hours after induction after induction by polyrlrC, whereas in superinduced cells IFN synthesis goes on for several more hours (Raj N. and Pitha P., Proc. Natl. Acad. Sci. USA (1983) 77, 4918).
  • RNA virus infection Other mechanisms than the formation of dsRNA during interferon synthesis induced by an RNA virus infection is conceivable.
  • an interaction between a virus surface component and the mononuclear cell membrane is sufficient to induce an IFN ⁇ synthesis.
  • the glycoproteins of Sendai virus are capable of inducing acid stable IFN ⁇ type I in human leukocytes, human lymphoblastoid cells (Namalwa), in murine spleen cells, but are unable to do so in mouse L fibroblasts (Ito Y. et al., Virology (1978) 88, 128).
  • IFN induction can or should be explained by a universal inducer which would be a common denominator of the many agents, viral and others, shown to stimulate IFN ⁇ / ⁇ synthesis.
  • IFN inducers activate several other genes at the same time.
  • polyrlrC induces, in addition to Hu IFN ⁇ , the synthesis of at least 13 and maybe as many as 23 other proteins, which have not been further characterized (Raj N. and Pitha P., Proc. Natl. Acad. Sci. USA (1980) 77, 4918; Content J. et al., Proc. Natl. Acad. Sci. USA (1982) 79, 2768).
  • Some of these co-induced genes are probably linked, but unrelated to the IFN gene or chromosome 9, Gross G. et al., In: The Biology of the Interferon System (eds. E. DeMaeyer, G. Gallasso, and H.
  • Gene expression in eukaryotic cells is controlled at several levels. To allow for transcription, the gene must be in an active state, which requires changes in the chromatin structure to permit access of RNA polymerase. Acquisition of the "active" state is the first requirement for gene activation and has in fact been shown to occur in IFN genes upon induction. The second step of gene activation consists of initiation of transcription and is controlled by the promoter region of the structural gene.
  • the leftward boundary of the promoter is always upstream of the TATA box, a ubiquitous sequence of 7 or 8 bp that lies usually about 20 to 30 bp upstream from the starting point of transc ⁇ ption (Wagner R., Nature (1964) 204, 49).
  • Functional IFN mRNA cannot be extracted from unmduced cells. Transc ⁇ ption starts early after induction, the actual time being a function of the inducer-cell system studied. When human lymphoblastoid Namalwa cells are treated with the Sendai virus, IFN ⁇ and IFN ⁇ are produced (Havell E. et al., J. Gen. Virol. (1977) 38, 51).
  • Detectable levels of mRNA are present 3 hours after the onset of induction, reach a maximum at 9 hours and then decline (Shuttleworth J. et al., Eur. J. Biochem. (1983) 133, 399).
  • the cause of the shutoff of IFN synthesis is unknown; it occurs in cell cultures and in the animal and is usually followed by a penod of hypo-responsiveness to renewed induction This does not seem to be the case with regard to the insulin producing ⁇ cells.
  • Crude IFN preparations often contain one or several substances that can decrease IFN production (Friedman R., J Immunol. (1966) 96, 872). These substances will be effective inhibitors of IFN ⁇ , as used in the present invention. It is possible to isolate and characte ⁇ ze these substances, using known techniques, in order todemonstrate that these substances are able to a ⁇ est the synthesis of IFN in insulin producing ⁇ cells.
  • Demethylation of cytosine at CpG sites plays a role in the control of eukaryotic gene expression (Felsenfeld G and McGhee J., Nature (1982), 296, 602)
  • a demethylating agent such as 5-azacyt ⁇ d ⁇ ne
  • NDV induced IFN ⁇ production Tovcy M. et al., In: The Biology of the Interferon System, E. DeMaeyer and H. Schellekens, eds.) Elsevier, Amsterdam, 1983, pp. 45-50).
  • the rate of transc ⁇ ption of the IFN genes can also be influenced by IFN itself, a phenomenon known as "p ⁇ ming" (Stewart II, W. et al., J. Virol. (1971) 7, 792).
  • This loop-back mechanism may also be the reason why insulin producing ⁇ cells continue synthesis of IFN Substances can be found m crude interferon shown to repress interferon production may thus play a role in interrupting this function
  • antibodies to IFN may be capable of preventing this loop-back-priming phenomenon.
  • priming of interferon by interferon because transcription of other genes than IFN genes, clustered near the IFN gene are also affected by priming (Nir U. et al., J. Biol. Chem. 1985), 260, 14242).
  • mRNA stability is one of the factors intervening in the control of protein synthesis, and rapid degradation of mRNA in the absence of further transcription is one way of quickly terminating the synthesis of a protein. Normally, this appears to be the case for IFN, since IFN mRNAs in general are rapidly degrading mRNAs. In this respect they are comparable to mRNAs of other cytokines and lymphokines and of some proto-oncogenes.
  • the HuIFN ⁇ l gene promoter region contains the necessary information: when the 675 bp upstream of the IFN ⁇ l coding region are joined to the transcription unit of the rabbit globin gene, and mouse cells are then transformed by this hybrid gene. If, on the other hand, the globin promoter is fused to the IFN ⁇ l coding sequence, correct transcription no longer depends on viral induction, but instead, has become constitutive. It has been revealed that the region required for inducible transcription is located between positions- 1 17 to -74. This region contains a purine-rich stretch of 42 bp. located immediately downstream of position - 117, which is highly conserved in all HuIFN ⁇ genes (Benjamin R.
  • the instant invention also encompasses a method of administering anti-interferon- ⁇ antibody(ies) that can be tolerated by the humans, produced either as monoclonal antibodies (as for instance mouse/human chimeric .antibodies), as other chimeric combinations of antibodies, or as monoclonal antibodies produced by human hybridoma cells.
  • the invention encompasses the use of polyclonal antibodies produced in animals (e.g., pigs) or from transgenic animals (e.g., transgenic pigs) immunized with one or several species of human interferon- ⁇ .
  • Antibodies to other cytokines that may be useful in the treatment of diabetes are also considered, among those, antibodies to tumor necrosis factor ⁇ , tumor necrosis factor ⁇ , etc. It is possible to develop a strain of pigs which produces human immune globulins either together with the porcine immune globulins, which could be separated by affinity chromatography, or from a strain of pigs which produces human immune globulins exclusively. Previous work has achieved impressive clinical effect from H1V-1 hyperimmunized pigs that also have been immunized with a protein complex, named gp48 which is derived from cellular proteins (cell membrane associated) found in CD4+ cells such as H9 cell lines.
  • gp48 protein complex
  • the gp48 protein complex was first found to be present in HIV-1 lysate as described by Kurt Osther (Kurt B. Osther, U.S. Patent No. 5,286,852 incorporated by reference in the entirety). Later on the gp48 protein complex was identified in H9 cells and harvested from these cells, purified to be used as a separate immunogen composition in pigs that were immunized with an antigen or antigens such as for instance tumor necrosis factor ⁇ in order to produce porcine immunoglobulins that at the same time would neutralize tumor necrosis factor ⁇ in humans and at the same time be tolerated well by the recipient due to the anti gp48 which among others contain antibodies against MHC class I and MHC class II. The anti gp48 antibodies will thus prevent the human from identifying the porcine immunoglobulin as xenogenic or foreign.
  • Pigs immunized with human interferon- ⁇ specie(s) and with the gp48 protein complex, thereby rendering and thus produced porcine immune globulins capable of escaping the human immune surveillance system could then be useable in neutralizing circulating interferon- ⁇ specie(s) with tolerance identical to other porcine hyperimmune globulins such as a porcine anti TNF- ⁇ immune globulins. It is anticipated that other species of animals may be escaping in the same manner as pigs provided these animals are co-immunized with gp48 protein complex or with antibodies to other T cell antigens such as gp39.
  • the ligand for CD40 which delivers signals to B cells can synergize with those provided by other B cell surface receptors to induce B cells proliferation and antibody class switching as well as modulating the cytokine production and cell adhesion.
  • the ligand for CD40 has recently been found to be a cell surface protein of ⁇ 39kDa expressed by activated T cells, called gp39 (HoUenbaugh D. et al., The EMBO Journal (1992) 11, 4313).
  • the gp39 is a type II membrane protein with homology to TNF.
  • the gp39 protein has been constructed and characterized in a soluble recombinant form.
  • the results from HoUenbaugh et al.'s work has indicated that B cells require a second signal besides gp39-CD40 to drive proliferation and soluble gp39 alone in a non-membrane bound form is able to provide co-stimulatory signals to B cells.
  • Noelle et al. has shown that monoclonal antibodies to the 39 kDa membrane protein or gp39 that is selectively expressed by T helper cells (CD4+ cells) inhibited CD40-Ig binding and also inhibited the activation of B cells by T helper cells (CD4+ cells) (Noelle R. et al., Proc. Natl.
  • porcine antibodies produced by co- immunizing pigs with gp39 together with the gp48 protein complex which, besides cloaking the MHC class I and class II may be capable of producing antibodies inhibiting the activation of B cells with regard to both antibody production and cytokine production, because blocking the binding gp39 with an antibody from binding with CD40 inhibits T-helper (CD4+) cell dependent B cell activation as shown by Noelle et al.
  • Porcine anti-interferon- ⁇ antibodies may in this way be constructed to be tolerated by humans with juvenile diabetes.
  • the mechanism with diabetes is also encompassing other cytokines such as tumor necrosis factor ⁇ in which case administration of anti TNF- ⁇ antibodies such as those that we produce in pigs, co-immunized with gp48 complex, and, possibly, also gp39 ligand.
  • An antisense molecule capable of basepairing with DNA and/or RNA is introduced to the interior of the target cells. Inside the cells, the antisense molecule blocks synthesis of the target protein by interfering with the DNA or RNA sequence needed for synthesis of the protein in question.
  • the antisense molecule can be a native nucleic acid (DNA or RNA) or a modified nucleic acid.
  • modified antisense molecules one can use a phosphorothioate, a methylphosphonate, a PNA (Peptide Nucleic Acid), or any of the modifications commonly practiced ( see Crooke R.M. "In vitro toxicology and pharmacokinetics of antisense oligonucleotides, " 1991 , Anti-Cancer Drug Design, 6, 609-646.)
  • the antisense molecule can be introduced to the inte ⁇ or of the target cells by unspecific methods, such as pmocytosis (Stein CA. et al "Dynamics of the Intemalization of Phosphodiester Oligodeoxynucleotides in HL60 Cells," 1993, Biochemistry, 32, 4855- 4861).
  • the antisense molecules can also be introduced to the inte ⁇ or of the target cells by specific methods; the antisense molecule is conjugated to a molecule (a earner molecule).
  • the con j ugate interacts specifically with receptors/molecules on the surface of the target cells (e.g., membrane receptors) and is thus presented to the inte ⁇ or of the cells by endocytose.
  • the antisense can block synthesis of interferon ⁇ or interferon- ⁇ receptor on DNA level or RNA level (Crooke S.T. "Progress Toward Oligonucleotide Therapeutics- Pharmacodynamic Properties," 1993, FASEB Journal, 7, 533-539).
  • the antisense can block transcription by * Formation of triple-stranded structures
  • the antisense can block transcription by * Binding to the translation initiation codon
  • RNA-DNA duplex which is a specific substrate for Rnase H * Interfering with capping at the 5 -end or polyadenylation at the 3 -end
  • Pancreatic cells are isolated and grown in vitro. These cells are then subjected to gene knockout experiment to destroy their interferon- ⁇ production capability.
  • This example describes the inactivation of the endogenous interferon- ⁇ gene by homologous recombination in these pancreatic cells.
  • the strategy is to alter the interferon- ⁇ gene sequences with a gene targeting vector derived from this particular gene.
  • This gene knockout vector is constructed to contain most of the genomic sequence of the interferon- ⁇ gene, a gene for neomycin resistance (neor gene) and a disrupted piece of interferon- ⁇ exon.
  • Human interferon- ⁇ gene can be isolated from a genomic phage library derived from the isolated pancreatic ⁇ cells by PCR using portions of interferon- ⁇ nucleotide sequences as primers. The isolated interferon- ⁇ gene is then cloned and propagated in bacteria E. coli for example, to obtain pure interferon- ⁇ gene DNA in quantity for use in the construction of a interferon- ⁇ gene knockout vector.
  • a neo r gene is first inserted into the coding sequence (exon) of the isolated interferon- ⁇ gene.
  • the inserted neor gene will inactive the interferon- ⁇ gene by interrupting its correct gene sequence. It also services as a positive selection marker to promote the growth of cell that have incorporated the targeting vector.
  • the knock-out vector can be engineered to carry a neo r gene in noncoding sequences, fl.anking sequences or introns, and the truncated piece of exons (coding sequences).
  • the knock-out vector Once the knock-out vector is complete, it can be cloned and propagated in bacteria and introduced into the cultured pancreatic B cells by electroporation for homologous recombination to take place.
  • the injected vector will either take the place of the original gene through homologous recombination or fit itself randomly into a chromosome through random insertion or does not become integrated at all.
  • the targeted insertion of vector DNA by homologous recombination can be selected by growing cells in selective media counting neomycin analogue (G418) and ganciclovir.
  • G418 is lethal to cells unless they carry a functional neo r gene and so it eliminates cells in which no integration of vector has occurred.
  • gancicolovir kills any cells that harbor a tk gene from a randomly integrated vector. Virtually the only cells actually have the recombination and knock out, Southern blot analysis with specific probe can be performed as a conformatory test.
  • the cell line should contain a defective interferon- ⁇ gene and is ready for introduction into patients.
  • An additional method of the instant invention would be to inactivate the interferon- ⁇ gene by a gene knock-out technique described in EP-A-546073 (GenPharm International, Inc; incorporated by reference).
  • a successful inhibition of interferon- ⁇ activity may also be accomplished by the effective alteration of the active residues involved in effecting binding.
  • insulin producing cells is herein meant either pancreatic ⁇ cells (preferably of human origin) or cells which have been genetically engineered so as to produce insulin.
  • One example of such genetically engineered cells could be a fibroblast (or any other suitable cell) which have been genetically manipulated by the introduction of the regulatory gene for human insulin, or in other words, introduction of the 5'-flanking region of the insulin gene containing two cis-acting elements, the enhancer and the promoter, which exert their effect on transcription by interacting with various trans-acting factors which normally restrict insulin gene expression to the endocrine pancreas (Walker et al, Nature 306, 557, 1983; Karlsson et al, Proc. Natl. Acad. Sci.
  • an immunologically acceptable cell or cell line is herein meant a cell or cell line which, upon introduction into the intended recipient, will not be rejected by the recipient. It is prefe ⁇ ed that no medication will be necessary in order to accomplish this result (e.g. by using cells from the recipient himself) but it is contemplated that it will be necessary to use cells from donors which do not have a tissue type which is identical with that of the recipient.
  • a certain amount of immunosuppression may be required, but it should not include damaging chemotherapy on the recipient. It would be acceptable if the immunosuppression could be obtained by Cyclosporin, e.g. in combination with hydrocortisone or prednisolone as well as with azathioprine, a combination that has been used in allotransplantation of pancreas islet cell from a donor pig to a recipient pig that was rendered diabetic by a total pancreatectomy (Yamaguchi et al, Transplantation Proceedings 24:1010, 1992). However, the method of using xenogenic islets in man would most probably give rise to rejection unless the human recipient receives heavy chemotherapy.
  • An alternative way of performing successful pancreatic islet xenotransplantation may utilize the methods described by McCurry et al. by producing transgenic pigs that at the fertilization stage (ovum and/or spermatogonia) have received transfer of genes that regulate the human complement system at the level of the alternative complement component C3 pathway (McCurry et al., Nature Med. 1:423, 1995)
  • Another viable method could be to transfer the Cls esterase inhibitor gene in ova and/or spermatogonia in pigs and thereby producing transgenic pigs with a complement inhibition at the complement component C1/C4 level.
  • Cls esterase inhibitor gene in ova and/or spermatogonia in pigs and thereby producing transgenic pigs with a complement inhibition at the complement component C1/C4 level.
  • transformation a cell By the term “transforming a cell” is herein meant the genetic manipulation of a cell which has as an effect that the cell changes its phenotype. Transformations of cells can be performed in a number of ways which are well-known in the art (cf. e.g. Maniatis et al, Molecular Cloning: a laboratory manual. 2nd edition, Cold Spring Harbor Press, 1989). However, care should be taken that methods are not used which have as a result that interferon- ⁇ is expressed by the resulting transformed cell. For instance, transformation of cells capable of producing interferon- ⁇ by introduction of a viral carrier using CAT plasmids (Gorman et al, Mol. Cell. Biol.
  • the ideal cell type to be used as an in vivo as insulin producer is one that will replicate and give nse to a cell population sufficiently large enough to produce part of or all the insulin needed in vivo, initiated by a regulatory region for human insulin gene (or porcine insulin gene) without being susceptible to being significantly depleted due to differentiation into co- expression of interferon- ⁇ .
  • Such cells can be of human ongm, or transgenic porcine cells, or other suitable mammalian cells. If the insulin producing transgenic cell population should decrease, an additional transgenic therapy could be repeated in the same type of cell population or in other cell populations that fulfill the above criteria.
  • An additional method of the invention is to use anti-sense techniques to interrupt interferon- ⁇ expression in humans.
  • this approach one may have to augment natural (in vivo) production of interferon- ⁇ by parenteral injection of exogenous interferon- ⁇ . This technique would be most effective in early stages of diabetes. There may be a possibility that the insulin producing ⁇ cells might regenerate under such a treatment approach.
  • a combination of transgenic transfer of the regulatory region of the human (or porcine) insulin gene to a non-expressing cell type to be re-introduced into a patient during an early onset of diabetes - together with the described antisense and/or with anti -interferon antibody therapy - may be an alternative treatment of diabetes that may approach a cure or at least an improvement of the early onset diabetes type I.
  • the transgenic treatment may be the preferred treatment in order to approach a cure or an improvement of the diabetic condition negating or diminishing the demand for exogenic regulation with insulin and at the same time keep the blood sugar reasonably under control, thereby approaching a better regulated diabetes type I, and complementing remaining endogenous insulin producing cells still functioning.
  • a patient with juvenile diabetes will still have circulating inducer(s) of interferon- ⁇ in cells capable of producing interferon- ⁇ and possibly other cytokines. It is not known whether ⁇ -interferon induction in cells necessarily interferes with a theoretical insulin production. Pancreatic ⁇ cells do not produce ⁇ -interferon. When "healthy" pancreatic islets are transplanted into patients with juvenile diabetes the patients will quickly destroy the insulin production in the cells transplanted. This would be expected if any interferon- ⁇ inducer(s) is "chronically” circulating in these patients.
  • the invention teaches that the ideal cell population for use to produce insulin does not co-produce interferon- ⁇ or possibly even other cytokines and is thus functionally unresponsive, or incapable of producing functional cytokine.
  • the ideal cell population can be selected from among fibroblast cells only producing ⁇ -interferon, or cell types that do not produce interferon at all.
  • a well known cell line that does not produce interferon at all is the monkey kidney cell, Vero cell. This cell type has often been used in bioassays because no auto-induction of interferons occurs.
  • This cell or cells lacking the above described function would be the ideal cell to introduce the regulatory gene for insulin. The concept would then be that the regulatory gene would activate the dormant insulin gene in the cell rende ⁇ ng it capable of producing insulin.
  • the instant invention provides for a cell, capable of expressing insulin, but inhibited in the ability to produce interferon- ⁇ , for use in gene therapy and transplantation into diabetic patients
  • the cell is capable of expressing insulin under the regulation of the human insulin inducer system, and incapable of expressing interferon- ⁇ , because of either knock-out mutation of the IF- ⁇ , or other genetic manipulation of IF- ⁇ expression.
  • the preferred cell for such use is of human o ⁇ gin, but can be of transgenic ongin whereby human Histocompatabhty markers have been transfe ⁇ ed and expressed by other animal cells.
  • the cell of the instant invention can be capable of further differentiation, or preferrably inhibited from further differentiation.
  • the cell of the instant invention can actively divide, but for certain applications it may be better to have cells which are incapable of further cell division.
  • the above described cell such as the Vero cell line was selected, - preferably a human cell that is not capable of producing at least interferon- ⁇ , as the transplanted cell after transfer of the genes necessary for insulin production and expression secretion, it would not contain the gene(s) that are interfe ⁇ ng with the insulin production as for instance cells that can not produce at least interferon- ⁇ .
  • the co ⁇ ect cell line(s) are selected and the genetic expression of insulin is introduced the cell line can then transplanted into the diabetic patient.
  • the order in sequence would be:
  • an animal model suitable for the insulin expressing experiments is the pig.
  • the transfer of the regulatory region of the human or porcine insulin gene (or the entire genes needed to obtain porcine insulin gene expression) is being exemplified using a porcine model where the selected cells are isolated fibroblast cells, capable of producing ⁇ -interferon. and used as target cells for the regulatory region of the human insulin gene.
  • a more ideal approach will be to isolate a porcine cell line that are not producing interferons or may be even not producing cytokines.
  • An alternative method would be to utilize a continuous porcine kidney cell line, PK15 for the construct of porcine or human regulatory region of the insulin gene or the entire genes involved in the insulin production.
  • This cell line or a resembling cell line may be of advantage provided the cell lines selected do not produce interferon- ⁇ (or ⁇ -interferon).
  • This model is advantageous because porcine insulin resembles that of humans to a degree where insulin from pigs have been used in human diabetics for several years. The pigs, treated so that they become diabetic, will be used in the some of the experiments. There are well-known ways of introducing diabetes in pigs. The difference between these pigs and human juvenile diabetes would be the fact that the pigs do not have a true diabetes.
  • the main goal would be to utilize the transgenic techniques or the gene therapy for creating insulin producing cells to demonstrate the transfer the genes necessary to induce an insulin production in cells not used by the organism to produce insulin from said kind of cells.
  • the insulin gene may be dormant in the cell being used for the gene transfer and thereby the gene transfer would be to operably introduce the regulatory region of the insulin gene in order to create the insulin production. What is yet to be learned is whether the insulin gene composition necessary for the cell to excrete insulin otherwise would be present, and/or whether other gene products necessary for an actual production and excretion of insulin from the cell would be possible.
  • the regulatory region of the porcine insulin gene is obtained using the procedure described previously (S.A. Chen et al, J. Interferon Res. 8: 597, 1988; L. Martin et al, J. Immunol. 150: 1234, 1993; O. Bohoslawec et al, J. Interferon Res. 6: 207, 1986).
  • the regulatory region of the porcine insulin gene is expressed in E. coli, B. subtilis or in other such as yeast or from cells containing said regulatory region.
  • Porcine kidney cells are selected and the regulatory region of the human insulin gene is transfe ⁇ ed using the procedures described previously. The methods used have been described by Chen and Martin, who utilized the technology in transgenic mice by standard techniques without transferring the interferon- ⁇ transgene (L. Martin et al., J. Immunol, 150:1234, 1993).
  • the pigs' pancreas is damaged by toxin so that the islets can not produce insulin, inducing a diabetes condition in the pig.
  • the pig is treated with porcine insulin and the blood sugar is checked.
  • the transgenic cells are re-introduced by infusion of the cells into kidney capsula and into peritoneum.
  • the pig and the diabetic pig is controlled by weekly diminishing the insulin and controlling the blood sugar in order to see when the transgenic production of insulin starts and significantly diminish the need for administration of exogenous insulin.
  • connective tissue is surgically, aseptically removed from the neck region and placed in Minimum Essential Medium (Earle's salt) with 10% fetal bovine serum.
  • the cells are brought to the laboratory where the tissue is cut into cubes under a laminar air flow hood. The cubes are then cut in a petri dish into small cubicles. The cubicles are washed in MEM without fetal bovine serum and treated with 0.25% trypsin in MEM without fetal bovine serum. The trypsin treated cells are then transfe ⁇ ed into a 25 ml tissue culture bottle. Five to 10 ml MEM with 5 to 10% fetal bovine serum is added to the tissue culture bottle.
  • Minimum Essential Medium Earle's salt
  • the cells are brought to the laboratory where the tissue is cut into cubes under a laminar air flow hood. The cubes are then cut in a petri dish into small cubicles. The cubicles are washed in MEM without fetal bovine serum and treated with
  • the cells are cultured in a 37°C incubator. The cells are checked daily. When a confluent cell culture has been obtained the cells are then trypsinized and transfe ⁇ ed to a microinjection microscope. The regulatory region of the porcine or the human insulin gene is transfe ⁇ ed using the process described under example 1.
  • the cells into which the adequate genes are transfe ⁇ ed are cultured in a 20 ml tissue culture bottle. When the cell count is sufficiently big (10 4 to 10 5 cells, or more) are then treated with 0.25% trypsin in MEM without fetal calf serum. When the cells are detached they are treated with MEM with 10% fetal bovine serum and will followingly be washed in sterile PBS at pH 7.2 to 7.4.
  • the pig that shall receive its own gene treated cells has in the meantime been rendered diabetic and exogenous insulin treatment has been initiated.
  • the cells are injected into the peritoneum of the pig and intramuscularly into the neck region of the pig.
  • the pig is monitored for eventual production of endogenous insulin during a follow up time of at least 3 to 6 months.
  • the gene treated cells take over the insulin production the exogenous insulin needed by the pig should accordingly decrease.
  • Lys Tyr Phe Arg Arg lie Thr Leu Tyr Leu Thr Glu Lys Lys Tyr Ser 125 130 135

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Abstract

La présente invention se rapporte à des procédés et à des compositions qui permettent de traiter efficacement les diabètes de type I, lesdits procédés consistant à intervenir au niveau de l'activité de l'interféron alpha.
PCT/US1997/014448 1996-08-16 1997-08-15 Procede de traitement des diabetes WO1998006431A2 (fr)

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
AU2007202840B2 (en) * 2001-01-09 2011-07-28 Baylor Research Institute Methods for treating autoimmune diseases in a subject and in vitro diagnostic assays
US8080638B2 (en) 2005-02-10 2011-12-20 Baylor Research Institute Anti-interferon alpha monoclonal antibodies and methods for use
US8163885B2 (en) 2008-05-07 2012-04-24 Argos Therapeutics, Inc. Humanized antibodies against human interferon-alpha
US20180237751A1 (en) * 2011-10-11 2018-08-23 The Trustees Of Columbia University In The City Of New York Method for generating beta cells

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EP0034307A2 (fr) * 1980-02-16 1981-08-26 Hoechst Aktiengesellschaft Procédé pour la préparation d'interféron de leucocytes humains
WO1993004699A1 (fr) * 1991-08-30 1993-03-18 Genentech, Inc. Procede therapeutique servant a traiter le diabete sucre insulinodependant
EP0588177A2 (fr) * 1992-09-03 1994-03-23 YEDA RESEARCH AND DEVELOPMENT CO., Ltd. Protéine se liant à l'interferon-alpha/beta, sa préparation et les compositions pharmaceutiques la contenant
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EP0034307A2 (fr) * 1980-02-16 1981-08-26 Hoechst Aktiengesellschaft Procédé pour la préparation d'interféron de leucocytes humains
WO1993004699A1 (fr) * 1991-08-30 1993-03-18 Genentech, Inc. Procede therapeutique servant a traiter le diabete sucre insulinodependant
EP0588177A2 (fr) * 1992-09-03 1994-03-23 YEDA RESEARCH AND DEVELOPMENT CO., Ltd. Protéine se liant à l'interferon-alpha/beta, sa préparation et les compositions pharmaceutiques la contenant
WO1996034096A1 (fr) * 1995-04-28 1996-10-31 Abgenix, Inc. Anticorps humains derives de xeno-souris immunisees

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2007202840B2 (en) * 2001-01-09 2011-07-28 Baylor Research Institute Methods for treating autoimmune diseases in a subject and in vitro diagnostic assays
US8080638B2 (en) 2005-02-10 2011-12-20 Baylor Research Institute Anti-interferon alpha monoclonal antibodies and methods for use
US8333965B2 (en) 2005-02-10 2012-12-18 Baylor Research Institute Anti-inteferon alpha monoclonal antibodies and methods for use
US8163885B2 (en) 2008-05-07 2012-04-24 Argos Therapeutics, Inc. Humanized antibodies against human interferon-alpha
US8361463B2 (en) 2008-05-07 2013-01-29 Argos Therapeutics, Inc. Humanized antibodies against human interferon-alpha
US8658771B2 (en) 2008-05-07 2014-02-25 Argos Therapeutics, Inc. Humanized antibodies against human interferon-alpha
US20180237751A1 (en) * 2011-10-11 2018-08-23 The Trustees Of Columbia University In The City Of New York Method for generating beta cells

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