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WO2005079307A2 - Utilisation d'outils derives de il-14 comme therapeutique pour les tumeurs malignes et les maladies auto-immunes et animaux transgeniques correspondant - Google Patents

Utilisation d'outils derives de il-14 comme therapeutique pour les tumeurs malignes et les maladies auto-immunes et animaux transgeniques correspondant Download PDF

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WO2005079307A2
WO2005079307A2 PCT/US2005/004420 US2005004420W WO2005079307A2 WO 2005079307 A2 WO2005079307 A2 WO 2005079307A2 US 2005004420 W US2005004420 W US 2005004420W WO 2005079307 A2 WO2005079307 A2 WO 2005079307A2
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mice
transgenic mice
lymphocytes
cells
transgenic
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WO2005079307A3 (fr
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Julian L. Ambrus, Jr.
Richard Ford
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The Research Foundation Of State University Of New York
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/544—IL-14
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0381—Animal model for diseases of the hematopoietic system

Definitions

  • the present invention relates to the field of using IL-14 related compositions to affect immunologically related responses and diseases.
  • IL-14 formerly known as High Molecular Weight B Cell Growth Factor (HMW-BCGF)
  • HMW-BCGF High Molecular Weight B Cell Growth Factor
  • IL-14 cytokine with a molecular weight of about 55kDa with 498 amino acids.
  • the IL-14 cDNA was originally identified by expression cloning using a monoclonal antibody and polyclonal antisera recognizing HMW-BCGF. Recombinant IL-14 proteins reproduce many of the activities of native HMW-BCGF (12). When IL-14 was identified in 1984 it was observed to preferentially induced the proliferation of large-elutriated B-lymphocytes (7). Large-elutriated B-lymphocytes were shown to consist primarily of CD77+ germinal center B-lymphocytes.
  • IL-14 was shown to preferentially expand B-lymphocytes grown for several weeks in vitro and sorted IgDiow B-lymphocytes- from human tonsils, which contain both Bl lymphocytes and normal memory B2 lymphocytes (8).
  • IL- 14 was shown to expand B-lymphocytes from patients with chronic lymphocytic leukemia (9), which are derived from Bl lymphocytes (10).
  • a monoclonal antibody recognizing a putative IL-14 receptor was identified, and shown to be expressed exclusively on activated B-lymphocytes and B-lymphocyte derived tumors (9, 11-13).
  • This receptor has not yet been molecularly characterized, and several receptors may exist for various IL-14 derived proteins.
  • a purification scheme for IL-14 was developed in 1985 (14). Sufficient material was not available for attempts at amino acid sequencing by Edman degradation, however, until 1992.
  • the IL-14 cDNA was identified by expression cloning using a monoclonal antibody and a rabbit polyclonal antisera (15).
  • Several unresolved problems working with IL-14 exist despite the identification of this cDNA, such as the instability of the recombinant protein, a property similar to the native IL-14.
  • the initially predicted open reading frame did not include many peptide sequences deduced from sequencing the native IL-14, and the activity of the recombinant protein was variable in B-lymphocyte proliferation assays, probably because of instability of the protein(s), but also for additional factors.
  • the identification of sequencing errors in the originally published IL-14 cDNA it was realized that peptides sequenced from the native protein were encoded by predicted open reading frames on both the positive and negative strands of the IL- 14 gene (36).
  • the potential role of IL-14 in many immunological processes and diseases has remained poorly defined.
  • SLE Systemic Lupus Erythromatoses is an autoimmune disease in which cellular and humoral reactivity to various self-tissues results in damage to potentially every organ in the body. Both genetic and environmental factors participate in disease pathogenesis (16-19). Animal studies have demonstrated that several distinct genetic alterations can result in phenotypes resembling certain aspects of human SLE. None of the genetic alterations identified so far have reproduced the variability of disease manifestations over time in individual SLE patients or the variability in disease manifestations in genetically- related SLE patients (20-24). Interestingly, expression of various genes enhancing B cell survival, including bcl-2 and TALL-1, has resulted in SLE-like diseases (24, 25). Thus, there is a need for methods to address autoimmune diseases such as SLE.
  • B-Lvmphocyte Derived Malignancies Leukemia and lymphomas of lymphoid origin continue to increase in incidence in the 20 ⁇ and 21st century (26). In the case of the high-grade B cell lymphomas, such as
  • the present invention provides tools derived from the IL-14 gene.
  • the tools include the plus and minus strands of the IL-14 gene, proteins translated from each ORF, a protein obtained by splicing of the two ORF transcripts (FL IL-14), as well as other molecules that may mimic the effects of the IL-14 proteins, polypeptides or peptides, or nucleotide sequences.
  • the tools may also comprise polynucleotide having sequences of the IL-14 gene or portions thereof. Such polynucleotides may comprise antisense DNA or RNA molecules or siRNAs.
  • the present invention also provides the use of IL-14 derived proteins to be used as adjuvants to enhance immunological memory after vaccination.
  • IL-14 peptides may also be used as therapeutic agents for malignancy such as, but not limited to, lymphomas.
  • Transgenic IL-14 mice are also provided.
  • Figure 1 is a graphical representation of the human and murine IL-14 genes, which consist of 11 exons and 10 introns (17.9 KB in human and 12.4 KB in mouse).
  • the 210#2 transcript is identical in sequence to the plus strand of the IL-14 gene and utilizes 9 exons.
  • the 210#1 transcript is identical in sequence to the minus strand of the IL- 14 gene and utilizes only part of the 11th exon, starting at the very 3' end.
  • the transcripts for 210#1 and 210#2 have very little overlap.
  • "P" and "P/TATA” indicate predicted promoter and franscriptional start sites.
  • Figure 2 is a sequence alignment demonstrating that the 210#1 sequences in human and mouse are very different.
  • Figure 3 is a representation of a western blot and shows that the proteins 210#1 and 210#2 are made in vivo.
  • Namalva cells were stimulated with PHA for 48 hours, lysed and the resulting proteins separated by sucrose density centrifugation. Proteins were separated by SDS-PAGE, transferred to PVDF paper, and analyzed by Western blots using antisera specific for 210#1 or 210#2 as labeled.
  • Figure 4 is a representation of a gel demonstrating expression of IL-14 transcripts by PMBC after stimulation with various activation agents.
  • Human PBMC treated in vitro for 24 hours lane 1 - media alone, 2 - PHA (lOug/ml), 3 - cycloheximide (15ug/ml), 4 - PHA + cycloheximide, 5 - ConA (20ug/ml), 6 - PWM (20ug/ml) were evaluated by RT - PCR using primers spanning only 210#1, 210#2, the entire IL-14 mRNA (FL-LL-14), or actin, as indicated. Lane 7 is a positive and Lane 8 a negative control for the PCR reaction. 210#1 was found to be constitutively expressed, and 210#2 and FL-IL-14 produced only after mitogen stimulation.
  • FIG. 5 is a representation of gels demonstrating expression of 210#2 and 210# 1 in splenocytes from C57BL/6 mice.
  • S molecular weight markers
  • 1 and 5 is mRNA freshly isolated from spleens of two different mice
  • 2 and 5 is mRNA from the lymphocytes of the same mice cultured for 24 hours in media alone
  • +C is the positive and -C the negative control for the PCR reaction.
  • Splenic lymphocytes were obtained from normal 20-week-old C57BL/6 mice.
  • FIG. 6 is a graphical representation of results representing proliferation of human
  • Human B-lymphocytes were purified from tonsils and placed in culture with media (RPMI / 5%FBS), recombinant CD40L (gp39, lO ⁇ g/ml), and a wide dose range (lpg/ml - lOOng/ml) of recombinant IL-4, 210#1 or 210#2.
  • Proliferation was determined by 3H-thymidine incorporation after 120 hours of culture at 370C. Data shown are the optimal proliferation for each cytokine throughout the dose range studied, and the mean for quadruplicate wells.
  • FIG 7 is a graphical representation of results representing human IL-14 induction of proliferation of murine B-Lymphocytes.
  • Splenic B-lymphocytes were purified from Balb/C mice using anti-CD 19 magnetic beads and Percoll density centrifugation, as described (38). Cells were plated at 105 cells/well in 96 well plates with RPMI / 5% FBS, 0.01% 2-merca ⁇ toethanol and l ⁇ g/ml LPS. Cytokines were added as shown. IL-14 was produced in CHO cells and consisted of a roughly equal mixture of 210#1 and 210#2. Proliferation was determined on day 5 by incorporation of 3H- thymidine, as previously described (15).
  • FIG. 8 is a graphical representation of results demonstrating that purified B- lymphocytes stimulated with recombinant IL-14 and CD40L remain viable in vitro for greater than 20 days.
  • Human tonsiUar B cells were placed in culture with RPMI/5% FCS and either IL-14 (210#1 + 210#2; 100 ng ml), CD40L (g ⁇ 39; a soluble recombinant product provided by Bristol-Myers/Squibb; used at 10 ⁇ g/ml), or IL-14 plus CD40L. Viable cells were counted every 4 days using trypan blue exclusion. Data shown are from a single experiment and representative of 4 done.
  • Figure 9 is a graphical representation of a gel demonstrating that human B- Lymphocytes stimulated with recombinant IL-14 express BCL-2.
  • 20 x 106 purified human tonsiUar B-lymphocytes were activated with Sac for 48 hr. and then cultured in either media alone (RPMI/5% FCS; Media) or media plus recombinant IL-14 (100 ng/ml) for 24 hr.
  • Cellular proteins were then analyzed by Western blot using a monoclonal antibody recognizing human bcl- 2 (kindly provided by S. Korsmeyer), as described (48).
  • FIG. 10 is a graphical representation of results representing expression of IL-14 (210#2) in B and T cells.
  • B and T cells were eliminated from freshly isolated human tonsils with anti-CD19 and anti-CD3 Mab respectively. The remaining cells were then sorted into CD3-CD77-HJ2+ and CD3-CD77-HJ2- populations.
  • RT-PCR for 210#2 was performed on each population of cells as described in Figure 3.
  • RT-PCR was also performed for IL-2, IL-4, IL-6, IL-10, interferon- ⁇ , and TNF- ⁇ . None of these mRNA were identified.
  • FIG 11 is a graphical representation of results representing expression of LL-14 (210#2) in TonsiUar T lymphocytes.
  • TonsiUar T lymphocytes were selected by magnetic beads coated with anti-CD4.
  • CD4 positive T lymphocytes were separated into CD57+ (Leu7; germinal center) and CD57 - (Leu7-; non-germinal center) populations using magnetic beads coated with anti-Leu 7.
  • RNA was isolated and RT-PCR performed as described in Figure 3.
  • Germinal center T cells express IL-14, but not IL-4 or IL-10. T cells outside the germinal center produce IL-4 and IL-14.
  • Figure 12 is a graphical representation of a gel demonstrating expression of 210# 1 and 210#2 in Purified Follicular Dendritic Cells.
  • Purified Human FDC were obtained from Greg Burton. TonsiUar B (CD19+, CD77 +/-) and T (CD4+, CD57+) lymphocytes were isolated by cell sorting. RT-PCR was performed as described above for 210#1, 210#2 and actin. Both 210#1 and 210#2 mRNA are expressed in human FDC.
  • Figure 13 is a graphical representation of a gel demonstrating that expression of
  • Figure 14 is a graphical representation of a gel demonstrating expression of 210#2 and 210#1 in murine models of SLE.
  • Figure 15 is a graphical representation of a gel demonstrating northern blot analysis of 210#2 expression ins the spleens of autoimmune mice.
  • Figure 16 is a graphical representation of results demonstrating anti-TNP responses in IL-14 transgenic mice immunized with TNPOVA.
  • mice Groups of 3 - control, 3 - 210#1 transgenic and 3 - 210#2 transgenic mice were immunized with lOO ⁇ g TNP-OVA in incomplete Freund's adjuvant on day 0 and 14.
  • FIG. 18 is a graphical representation of results demonstrating 210#1 transgenic mice produce enhanced IgM Responses to NP-Ficoll compared to littermate controls.
  • the experiment was performed exactly the same as the experiment shown in Figure 15, except NP-Ficoll rather than TNP-OVA was used as the immunogen.
  • FIG. 19 is a graphical representation of results demonstrating 210#2 transgenic mice produce enhanced IgG Responses to NP-OVA compared to littermate controls. The experiment was performed exactly the same as the experiment shown in Figure 15, except NP-OVA rather than TNP-OVA was used as the immunogen. There was no difference in the IgM anti-NP response between the 210#2 transgenic mice and the littermate controls.
  • FIG. 20 is a graphical representation of results demonstrating 210#2 transgenic mice produce enhanced IgM Responses to NP-Ficoll compared to littermate control. The experiment was performed exactly the same as the experiment shown in Figure 15, except NP-Ficoll rather than TNP-OVA was used as the immunogen. There were no difference in the IgG anti-NP responses between the 210#2 transgenic mice and the littermate controls (data not shown).
  • FIG. 21 is a graphical representation of results demonstrating IL-14 transgenic mice immunized with NP-Ficoll show enhanced IgM anti-NP memory responses.
  • Figure 22 is a graphical representation of results demonstrating IL-14 transgenic mice immunized with NP-Ficoll show enhanced IgG anti-NP memory responses. This is the same experiment as in Figure 21, but the IgG anti-NP response is shown.
  • Figure 23 is a graphical representation of results demonstrating IL-14 transgenic mice immunized with NP-OVA show normal IgM anti-NP responses. This is the same experiment as Figure 21, but NP-OVA vaccination was used. The IgM anti-NP response is shown.
  • Figure 24 is a graphical representation of results demonstrating IL-14 transgenic mice immunized with NP-OVA show enhanced IgG anti-NP memory responses.
  • Figure 25 is a graphical representation of results demonstrating Ig levels and autoantibodies in IL-14 transgenic and littermate control mice at 20 weeks of age.
  • Figure 26 Panel A is a graphical representation of results demonstrating immunoglobulin levels at 30 weeks of age and autoantibodies at 4 and 16 months of age in IL-14 transgenic and littermate control mice. Sera were obtained from normal littermate controls (CI, C2, C3), 210#1 transgenic (210#1- 1, 2, 3) or 210#2 transgenic (210#2 - 1,2,3) mice and evaluated by ELISA for IgM , IgG , and ANA or anti-chromatin IgG.
  • FIG. 27 is a representation of renal histology results from of IL-14 transgenic mice. Spleens were harvested at 30 weeks of age. Giemsa staining and staining with PE- conjugated anti-IgG and Texas red conjugated anti-IgM was performed by standard techniques.
  • Sections shown are representative of 3 kidneys studies from each group - littermate controls, 210#1 transgenic mice and 210#2 transgenic mice.
  • Figure 28 is a representation of histology results from EL- 14 transgenic mice demonstrating infiltration of salivary glands with lymphocytes by 17 months of age.
  • Figure 29 is a representation of histology results from IL-14 transgenic mice demonstrating lymphomas in lymph and spleen.
  • Figure 30 is a graphical representation of results demonstrating tumors in IL-14 210#2 x Myc transgenic mice at 3 months of age.
  • Figure 31 is a representation of a gel demonstrating siRNA based on 210#2 specifically blocks expression of 210#2.
  • the MS - lymphoma B cell line was grown in media (control) or in media with siRNA based on a random sequence (D7), GADPH or IL-14/ 210#2. Cells were harvested at 24 hours and mRNA expression of 210#2 and actin determined by RT-PCR.
  • Figure 32 is a graphical representation of results demonstrating siRNA based on 210#2 blocks the proliferation of a B cell lymphoma cell line in vitro. Incorporation of , 3H-thymidine incorporation was determined over the last 18 hours of a 72 hour culture period.
  • Figure 33 is a graphical representation of results demonstrating IL-14 enhances IgG anti-tetanus toxoid responses in mice.
  • mice Groups of two BALB/c mice were immunized i.p. with TT 1 mg on day 0 and 0.1 mg on day 14. TT was given in an emulsion with incomplete Freund's adjuvant. In addition to TT, mice were given either recombinant IL- 14 (500 ng in culture supernatant of CHO cells transfected with SFFV.neo-IL-14; contains 210#1, 210#2 and FL-IL-14), culture supernatant of CHO cells transfected with a non- cytokine cDNA (media), or with no additive (data not shown; not significantly different from "media”) every 3 days intraperitoneally.
  • IL- 14 500 ng in culture supernatant of CHO cells transfected with SFFV.neo-IL-14; contains 210#1, 210#2 and FL-IL-14
  • FIG. 34A-C show the development of lymphomas in c-myc transgenic mice and in c-myc x 210#2 EL- 12 transgenic mice.
  • Figure 35 shows the results of DNA gene chip analysis for chromosome 12 and 15 on tumors from myc x IL-14 transgenic mice.
  • Figure 36 A-D shows the results of long term vaccination studies that support a role for 11-14 in supporting memory B cell responses to T-dependent and T-independent antigens.
  • Figure 37A -B shows the IgG subclass levels, as well as IgM, IgA and IgE levels in 9-month-old IL-14 transgenic mice and littermate controls.
  • Figure 38 shows a histological section showing thromboses in the splenic vessels of IL-14 transgenic mice. Such thrombosis was not observed in the littermate controls.
  • Figures 39A-B show anti-cardiolipin antibodies in IL-14 transgenic mice compared to littermate controls at 6, 9 and 12 months.
  • Figures 40 A-D is a representation of regulation of IL- 14 levels In vitro by alloantigen and in vivo following renal transplantation.
  • the present invention comprises a method of increasing B cell ⁇ memory by administration of peptides produced from the EL- 14 gene, such as the 210#2 protein, the 210#1 protein, or combinations thereof.
  • the present invention comprises the administration of 210#2 or 210#1 proteins or combinations thereof as adjuvants.
  • the present invention provides a method for inhibiting IL- 14 expression in individuals with autoimmune diseases such as SLE.
  • the method comprises administering antisense DNA or RNA or siRNA that inhibits EL- 14 production.
  • the present invention comprises a method for inhibiting IL-14 expression in individuals with germinal center derived malignancies, such as lymphocyte derived lymphomas, comprising the steps of administering antisense DNA or RNA or siRNA that inhibits EL- 14 production.
  • the present invention provides animal models in the form of transgenic mice expressing the 210#2 or 210#1 proteins.
  • splenic lymphocytes were obtained from normal 20 week old C57BL/6 mice. Some lymphocytes were immediately harvested for mRNA while others were cultured for 24 hours in RPMI/5% FCS with or without PHA. After 24 hours all the cells were harvested for mRNA.
  • RT-PCR was performed to evaluate the transcripts encoding 210#2 and actin as labeled. The results demonstrate that the transcript encoding 210#1 is constitutively expressed in human peripheral blood mononuclear cells and murine splenocytes, while the transcript encoding 210#2 is expressed only after stimulation with PHA.
  • the production of RNA transcripts that are translated into proteins from both strands of a gene has been previously described (37), although this phenomenon has only rarely been appreciated in mammalian genetics.
  • EXAMPLE 2 Recombinant human 210#1 and 210#2 in E.Coli was produced using methods known to those skilled in the art. Unexpectedly, the smaller 210#1 protein showed greater activity in inducing human B-rymphocyte proliferation in vitro than the 210#2 protein ( Figure 6). Recombinant human IL-14 proteins induced proliferation of murine B- lymphocytes in vitro as well ( Figure 7), consistent with the ability of many human cytokines to have similar activities on both human and murine cells.
  • EXAMPLE 3 Different functional activities may be directed by 210# 1 , 210#2 or a spliced product of 210#1 and 210#2, FL-IL-14 that has been documented in vivo (data not shown). Both the 210#1 and 210#2 proteins are unstable in purified form. The 210#2 protein is recognized by both the monoclonal antibody and polyclonal antisera that were used to identify the IL-14 cDNA. Recombinant IL-14 is shown to maintain the viability of a small population of B-lymphocytes in vitro when used in combination with recombinant CD40L (gp39) ( Figure 8). This maybe related to the ability of IL-14 to enhance BCL-2 expression in B lymphocytes.
  • FIG. 9 demonstrates that recombinant IL-14 induces expression of BCL-2 in normal human B-lymphocytes.
  • B-lymphocytes surviving selection in the germinal center, which are memory cells, transiently express BCL-2 (40, 41). Prolonged expression of BCL-2 leads to prolonged survival of B-lymphocytes (42).
  • IL-14 we purified follicular dendritic cells and germinal center T- lymphocytes from human tonsils using the monoclonal antibody HJ2 that recognizes follicular dendritic cells (47) and CD3 / CD57 that recognize germinal center T- lymphocytes.
  • RT-PCR performed on the HJ2 positive cells revealed mRNA for IL-14 (210#2), but not IL-2, IL-4, IL-6, TNF- ⁇ , or IFN- ⁇ (Figure 10).
  • the germinal center T cells contained IL-14 (210#2) mRNA, but not IL-4 or IL-10 mRNA.
  • Non-germinal center T cells produced mRNA for IL-4, as well as some IL-14.
  • we obtained purified normal human follicular dendritic cells and demonstrated that they expressed mRNA for both 210#1 and 210#2 without any stimulation (Figure 12).
  • EXAMPLE 5 We evaluated the expression of 210#1 and 210#2 encoding transcripts in the peripheral blood mononuclear cells of several patients with SLE. Shown in Figure 13 are the studies evaluating one patient with active SLE and nephritis, one patient with inactive SLE and predominantly skin and joint disease and one normal control (Figure 13). Normal donors express 210#1 but not 210#2 constitutively. ( Figure 4). Patients with SLE express both 210#1 and 210#2 constitutively. In these two patients disease activity did not influence the expression of 210#2.
  • This band contains the normal 210#2 mRNA plus retained intron 5, which also has the normal intron 5 sequence.
  • the expression of 210#1 in murine lupus is decreased compared to normal C57BL/6 mice, in contrast to human SLE where its expression is similar to the expression in normal donors.
  • the increased expression of 210#2 observed in (NZB x NZW)F1 spleens comes from NZW and is at a similar level as spleens from C57BL/6 mice after PHA stimulation (Figure 15).
  • EXAMPLE 6 For the evaluation of the effects of IL-14 in vivo, and its potential roles in SLE and lymphoma, we chose to make IL-14 transgenic mice using pE ⁇ SV.
  • the vector pE ⁇ SV leads to production of IL-14 in the B- > T- lymphocyte compartment (25, 52), making it available to the cells normally responsive to it. It also reproduces the aberrant overproduction of IL-14 that occurs in SLE and lymphoma (13, 51).
  • the IL-14 transgenic mice were produced with pE ⁇ SV, using standard procedures. Several founder lines were established of transgenic mice expressing 210#1 or 210#2.
  • transgenic vectors have been shown to contain the transgenic vectors by Southern blot analysis, to express the transgenic vectors by RT-PCR, and to produce 210#1 or 210#2 protein that can be detected in the sera of the mice (data not shown). Because the zygotes for injection are produced from C3H oocytes fertilized with C57BL/6 sperm, the resulting transgenic mice are on a mixed C3H x C57BL/6 background. Backcrosses are being done to produce transgenic mice on a pure C57BL/6 background. The preliminary data were generated after between 3-6 backcrosses on to C57BL/6.
  • EXAMPLE 7 To determine whether IL-14 influences memory responses in vivo, we immunized groups of 3 - control, 3 - 210#1 transgenic and 3 - 210#2 transgenic mice with lOO ⁇ g TNP-OVA in incomplete Freund's adjuvant on day 0 and 14. We determined TNP - specific IgM and IgG in the sera of these mice by ELISA on day 0, 14, 28 and 77. Figure 16 demonstrates that both groups of IL-14 transgenic mice produced TNP-specific IgG antibody more rapidly and at higher levels than the littermate control animals. The TNP- 16 specific IgM remained elevated in the 210#2, but not the 210#1 or control mice at day 77.
  • mice were immumzed i.p. with TT 1 mg on day 0 and 0.1 mg on day 14.
  • TT was given in an emulsion with incomplete Freund's adjuvant.
  • mice were given either recombinant IL-14 (500 ng in culture supernatant of CHO cells transfected with SFFV.neo-IL-14; contains 210#1, 210#2 and FL-IL-14), culture supernatant of CHO cells transfected with a non-cytokine cDNA (media), or with no additive (data not shown; not significantly different from "media”) every 3 days intraperitoneally.
  • EXAMPLE 8 To examine responses to a T-independent antigen, we immunized four mice in each group, 210#1 transgenic mice, 210#2 transgenic mice, littermate controls and C57BL/6 mice, with NP-OVA or NP-Ficoll with incomplete Freund's adjuvant on days 0 and 14. We determined the serum anti-NP responses at various time points. The data for the 210#1 transgenic mice are shown in Figure 17 and 18, while the data for the 210#2 transgenic mice and littermate controls are shown in Figures 19 and 20. The littermate controls and C57BL/6 mice had identical responses, so the responses of the C57BL/6 mice are not shown. The response to NP-OVA was similar to the response to TNP-OVA.
  • the 210#1 and 210#2 transgenic mice showed a statistically significant (P ⁇ .07 and P ⁇ 0.003 respectively) enhanced IgG anti-NP response compared to the littermate controls on days 28 and 70. The difference was not statistically significant on day 14 in either case. In contrast the IgM anti-NP response was statistically significantly different between the 210#1 transgenic mice and control mice only on day 14. There was no statistically significant difference between the 210#2 transgenic mice and the littermate controls.
  • EL-14 not only enhances short term, but also long term specific responses to both T-dependent and T-independent antigens.
  • the IL-14 proteins 210#1 and 210#2 may have different but overlapping roles in this activity.
  • EXAMPLE 10 We determined serum immunoglobulins and anti-nuclear antibodies (ANA) by commercially available ELlSAs at 20 weeks of age ( Figure 25-26).
  • Both the 210#1 and 210#2 transgenic mice demonstrated hypergammaglobulinemia, although 210#1 transgenic mice demonstrated elevations of only IgM and 210#2 transgenic mice demonstrated elevations of IgM and IgG, especially at 30 weeks of age. Both groups of mice developed autoantibodies to chromatin, histone and DNA, deposits of IgG and IgM in their kidneys, and infiltration of their salivary glands with lymphocytes ( Figures 26-8).
  • EXAMPLE 11 We used the IL-14 transgenic in several ways to study the role of IL-14 in B- lymphocyte derived lymphomas. First, we allowed 210#1 transgenic, 210#2 transgenic and littermate control mice to reach 24 months of age.
  • mice in each of these groups none of the littermate controls developed tumors, one of the 210#1 transgenic mice developed a follicular lymphoma ( Figure 29; the other developed diffuse lymphoid hyperplasia), and both of the 210#2 transgenic mice developed a diffuse lymphoma (currently being characterized).
  • 210#1 transgenic mice, 210#2 transgenic mice and littermate controls with Pristane monthly x 3 and followed them for another 3 months. None of these mice have developed tumors .
  • myc transgenic mice also made with pEocSV
  • 210#2 transgenic or myc transgenic mice developed tumors at 3 months of age. However, 10/15 210#2 x myc transgenic mice developed visible tumors (all of the double transgenic mice had tumors at autopsy) , which have been called lymphoblastic lymphoma by the pathologists ( Figure 30). Flow cytometry on the tumors has shown that they are CD19 +, CD5+ and CD21 -, consistent with chronic lymphocytic leukemia or mantle zone lymphoma. Mice transgenic for myc alone develop pre-B cell tumors.
  • EXAMPLE 12 We evaluated the use of siRNA based on IL- 14 / 210#2 on the growth of lymphoma cell lines in vivo.
  • Figure 31 demonstrates that the designed siRNA specifically blocks the expression of 210#2, and
  • Figure 32 demonstrates that this siRNA blocks the proliferation of a Burkitt lymphoma cell line in vivo.
  • IL- 14 is a therapeutic target to for the treatment of particular B eel lymphomas.
  • EXAMPLE 13 This example demonstrates the usefulness of the IL-14 transgenic mice as animals models for the study of lymphomas.
  • the development of tumors was studied in c-myc transgenic mice, IL-14 transgenic mice and IL-14 x c-myc transgenic mice.
  • Figure 34 (A-C) shows the development of tumors in these mice. It was observed that 75% c-myc transgenic mice developed lymphoma at 12 months and 50% IL-14 transgenic mice developed tumors at 18 months while 100% IL-14 x c-myc transgenic mice developed tumors by 4 months.
  • EXAMPLE 14 This example describes data from running Affymetrix gene chips with mRNA from 11-14 transgenic mice and littermate controls. These data indicate that 1) There are several strongly upregulated genes that are normally upregulated by interferon- ⁇ , although interferon- ⁇ itself is not up-regulated (IgG 2a - 6.23X, cathelicidin antimicrobial peptide - 3.92X, Lstl - 2.35X), suggesting that IL-14 may have interferon-like activity in vivo. 2) Several genes are altered that would encourage B cells to leave the marginal zones of the spleen and lymph nodes (CCR? decreased 2.07X, CCL21b decreased 3.14X, CD21 decreased 3.48X) presumably to go to the germinal centers where memory B cells are formed.
  • CCR? decreased 2.07X
  • CCL21b decreased 3.14X
  • CD21 decreased 3.48X
  • EXAMPLE 15 This example demonstrates that several of the IL-14 transgenic mice, but not the littermate controls had thromboses in their splenic vessels (Figure 37) Because of this observation, we have run anti-cardiolipin antibodies on the IL-14 transgenic mice and littermate controls. We demonstrated statistically significant increases in anti-cardiolipin antibodies in EL-14 transgenic mice compared to littermate controls at 6, 9 and 12 months.
  • Figure 40A-D shows human Normal Lymphocytes stimulated in MLR.
  • Figure 39B shows IL2 mRNA Levels Increase After Con A Stimulation And Are Inhibited by Prograf and Rapamune.
  • Figure 40C shows IL14 mRNA levels increase after Con A stimulation and are inhibited by Prograf and Rapamune.
  • Figure 40D shows in vivo levels of IL14 and IL2 transcripts in PBL of normal individuals compared to PBL collected from patients with stable allograft function at varying times following renal transplantation.
  • HMW-BCGF R human high molecular weight B-cell growth factor receptors
  • Germinal center cells express bcl-2 protein after ' activation by signals which prevent their entry into apoptosis. Eur J Immunol 21:1905. 41. Knox, K. A., and J. Gordon. 1993. Protein Tyrosine Phosphorylation Is Mandatory for

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Abstract

L'invention concerne des outils dérivés du gène IL-14. Lesdits outils comprennent les brins positifs et négatifs du gène IL-14, des protéines translatées à partir de chaque ORF, une protéine obtenue par épissage de deux produits de transcription ORF (FL IL-14) ainsi que d'autres molécules qui peuvent mimer les effets des protéines, polypeptides ou peptides IL-14,, ou de séquences nucléotidiques. Les outils peuvent également comprendre un polynucléotide comprenant des séquences du gène IL-14 ou des parties de celui-ci. Ces polynucléotides peuvent comprend un ADN antisens ou des molécules ARN ou des ARNsi. Les protéines dérivées de IL-14 peuvent être utilisées comme adjuvants afin d'améliorer la mémoire immunologique après une vaccination. En outre, des peptides IL-14 peuvent également être utilisés comme agents thérapeutiques contre les tumeurs maligne, mais ils ne sont pas limités aux lymphomes. L'invention concerne également une souris transgénique IL-14.
PCT/US2005/004420 2004-02-13 2005-02-14 Utilisation d'outils derives de il-14 comme therapeutique pour les tumeurs malignes et les maladies auto-immunes et animaux transgeniques correspondant WO2005079307A2 (fr)

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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
AMBRUS ET AL.: 'Identification of a cDNA for a human high-molecular-weight B-cell growth factor' PROC. NATL. ACAD. SCI. vol. 90, no. 13, July 1993, pages 6330 - 6334, XP003000217 *
FORD ET AL.: 'Identification of B-cell Growth Factors (Interleukin-14; High Molecular Weight-B Cell Growth Factors) in Effusion Fluids From Patients With Agressive B-Cell Lymphomas' BLOOD vol. 86, no. 1, 01 July 1995, pages 283 - 293, XP003000216 *
HOSHINO ET AL.: 'Cutting Edge: II-18-Transgenic mice: In Vivo Evidence of a Broad Role for IL-18 in Modulating Immune Function' J. IMMUNOLOGY vol. 166, June 2001, pages 7017 - 7018, XP002956672 *

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