WO2015001047A1 - Method of providing anti-human cytokine antibodies for pharmaceutical use - Google Patents

Method of providing anti-human cytokine antibodies for pharmaceutical use Download PDF

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Publication number
WO2015001047A1
WO2015001047A1 PCT/EP2014/064246 EP2014064246W WO2015001047A1 WO 2015001047 A1 WO2015001047 A1 WO 2015001047A1 EP 2014064246 W EP2014064246 W EP 2014064246W WO 2015001047 A1 WO2015001047 A1 WO 2015001047A1
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cytokine
human
antibody
cells
antibodies
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PCT/EP2014/064246
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French (fr)
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Syeda F. Y. HAQUE
Adrian HAYDAY
Martin Woodward
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Immunoqure Ag
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    • 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

Definitions

  • Inappropriate responses of the immune system may cause stressful symptoms to the involved organism.
  • Exaggerated immune answers to foreign substances or physical states which usually do not have a significant effect on the health of an animal or human may lead to allergies with symptoms ranging from mild reactions, such as skin irritations to life- threatening situations such as an anaphylactic shock or various types of vasculitis.
  • Immune answers to endogenous antigens may cause autoimmune disorders such as systemic lupus erythematosus, idiopathic autoimmune hemolytic anemia, pernicious anemia, type 1 diabetes mellitus, blistering skin diseases, psoriasis and different kinds of arthritis.
  • ID intradermal cytokine injections
  • M Measurements - ear thickness
  • S Sacrifice of the animals
  • ID - cytokine injections tested antibodies 26B9, 19D11, Ref.A and the control IgG were injected at day 0 (IP).
  • the present invention generally relates to a method of isolating and providing anti-huma cytokine antibodies and cytokine-binding fragments thereof for pharmaceutical use, in particular recombinant human anti-human cytokine antibodies and cytokine-binding fragments thereof. More specifically, the present invention, relates to a method of validating an isolated antibody or antigen-binding fragment thereof for therapeutic use in an in vivo, non-human animal assay also referred to herein as "HuCytoMab"-Assay. More specifically, the preset invention relates to a method of providing an anti-human cytokine antibody or cytokine-binding fragment thereof for pharmaceutical use, the method being characterized by
  • an. assay could be developed, wherein a candidate anti-human cytokine antibody can be tested and validated regarding its possible neutralizing activity and thus suitability to be further developed as a drug for immunotherapy.
  • pharmaceutical, use and “immunotherapy” are used as common in the art and interchangeably herein, and refer to both, therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow dow n (lessen) an undesired physiological change or disorder, in particular for the treatment of inflammatory, autoinflammatory, immune and autoimmune disorders.
  • Preferred diseases include, for example, RA, IBD, including Crohn's disease and ulcerative colitis, ANCA-associated vasculitis, lupus, MS, Sjogren's syndrome, Graves' disease, IDDM, pernicious anemia, thyroiditis, and glomerulonephritis, and APS-1. Still more preferred are RA, IBD, lupus, and MS, and more preferred RA and IBD, and most preferred RA.
  • NSIP Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient acantholytic dermatosis, cirrhosis such as primary biliary cirrhosis and pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac or Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia such as mixed cryoglobulinemia, amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease such as autoimmune inner ear disease (AIED), autoimmune hearing loss, polychondritis such as refractory or relapsed or relapsing polychond
  • the sample for isolating the antibody of interest comprises or consists of peripheral blood mononuclear cells (PBMC) and serum for the detection of possible antibody reactivities.
  • PBMC peripheral blood mononuclear cells
  • the sample derived from the subject may either be directly used for, e.g., testing seroreactivity against one or more of the desired antigen(s) or may be further processed, for example enriched for B lymphocytes.
  • the sample comprises or is derived from B cells that produce the antibody of interest, most preferably memory B-cells.
  • the candidate antibody or cytokine-binding fragment is provided by a method characterized by isolating B cells from short term, oligoclonal cultures of activated B cells that secrete antibodies of IgG isotype comprising the following steps in the sequence:
  • step (ii) the culture conditions in step (b) and/or step (d) do not comprise a cytokine
  • step (v) in step (d) the transferred new mentioned selected cells are exposed to the second polyclonal activator for about eight to fourteen days; and or
  • cDNA is preferably prepared from, single B cells sorted from, the short term, oligoclonal cultures of activated B cells secreting the antibody of interest in order to isolate and produce the monoclonal antibody of the present invention. Accordingly, the method of the present invention typically comprises the steps of:
  • step (ii) obtaining cDNA from the mRNA of step (i);
  • RT-PCR of single sorted cells is preferably employed for obtaining the immunoglobulin gene repertoire for said antibody.
  • a method of obtaining human antibodies using inter alia single cell RT-PCR is described for example in the international application WO2008/110372, the disclosure content of which is incorporated herein by reference, in particular the Supplementary Methods section and Example 2.
  • Tumor Necrosis Factor Ligand Superfamily Member 13 Tumor Necrosis Factor Ligand Superfamily Member 14 Tumor Necrosis Factor Ligand Superfamily Member 15
  • the method of the present invention further comprises the step of admixing the isolated and validated candidate antibody or cytokine-binding fragment thereof with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier is available in Remington's Pharmaceutical. Sciences (Mack Publishing Company, N.J. 1991) and in Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472.
  • Preferred forms for administration include forms suitable for parenteral, administration, e.g. by injection or infusion, for example by bolus injection or continuous infusion.
  • the product may take the form, of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain agents commonly used in pharmaceutical formulations, such as suspending, preservative, stabilizing and/or dispersing agents.
  • the antibody molecule may be in dry form, for reeonstit utio before use with. an. appropriate sterile liquid.
  • the compositions can. be administered directly to the subject. It is preferred that the compositions are adapted for administration to huma subjects.
  • the present invention also relates to a method of preparing a human anti-human cytokine antibody or cytokine binding fragment thereof for pharmaceutical use or as target for therapeutic intervention in the treatment of any one of the above-identified autoimmune and/or inflammatory disorders and diseases, comprising the steps of any of the above- described methods of the present invention, optionally wherein the human anti-huma cytokine antibody or cytokine binding fragment thereof is detectably labeled or attached to a functional domain or drug, preferably wherein the detectable label, is selected from, the group consisting of an. enzyme, radioisotope, a fluorophore and a heavy metal
  • ear thickness measurements of the animals were taken with a Mitutoyo digital micrometer during the IL-32 administration by daily measurements prior to IL-32 injection. Furthermore, body weight has been monitored during the treatment, however, no significant weight changes have been observed in any of the animal groups due to the treatment applied; see Fig. 3.
  • H&E hematoxylin and eosin; see Harris, H.F., J. Appl. Microscopy III (1900), 777-781 and Mallory, F.B.: Pathological technique. Philadelphia, Saunders, (1938)
  • histology stainings of the ears are performed.
  • the experimental time frame is designed as indicated above for the ear inflammation assay, with injections of the anti-IL-32 antibody, respective the control groups obtaining either PBS or human IgGs of IL-32 non-related binding specificity as indicated above. Reduction of the ankle swelling is used as a readout of the therapeutic effect of the antibodies.
  • Fig. 13 shows an exemplary experimental set up of the Cyto Ankle assay (Fig. 13 A, B) and the dose dependency of IL-32 in inducing inflammation in the CytoAnkle assay (Fig. 13 C, D).
  • the anti-IL-32 inflammatory effect of the 2C2 antibody could be confirmed in the CytoAnkle assay (Fig. 14 C-E).
  • Table 5 Group allocation of animals to the different cytokines tested, n - number of animals in the group, ng/20 ⁇ - amount of cytokine injected per ear.
  • ear thickness measurements of the animals were taken with a Mitutoyo digital micrometer during the cytokine administration by 2 measurements per ear prior to cytokine injection at Day 0 and at alternate days at Day 1 , Day 3, Day 5 (indicated by letter M in Fig. 4A) and alternatively or in addition at Day 6 after sacrifice of the animal.
  • IFNA14 was the most potent especially at Day 5 in this experiment.
  • IFNA2a and IFNA4 induced similar levels of ear thickening.
  • IFNA2b was most similar to both IFNA4 and IFNA 14 induced swelling.
  • IFNA2b induced swelling more than the IFNA2a isoform in this experiment; see Figs. 4-6 and the experimental results summary in the table in Fig. 7.
  • the results of this experiment show the applicability of the ear inflammation assay for tests of the therapeutic applicability of the antibodies of the present invention. Since the exemplary anti-IFN-a antibodies 19D11, 26B9, 31B4, 5D1 and 13B11 did not show any apparent cross- reaction with at least murine IFN-a subtypes 2, 4 and 14, they are tested in the HuCytoMab- Assay of the present invention in respect of their neutralization properties towards human IFN-a used for induction of inflammation.
  • the IFNA neutralizing potential of the antibodies reduce the induced ear inflammation phenotype and/or prevent such an induction which is examined by comparison of ear swelling (thickness) observed in animals obtaining the anti-IFN- ⁇ antibody treatment and the control groups obtaining either PBS or human IgGs of a binding specificity directed towards other molecules than human IFNA subtypes (of IFN-a non-related binding specificity).
  • body weight is monitored during the treatment, to observe any possible weight changes due to the inflammation induction or its respective reduction due to the treatment applied.
  • H&E hematoxylin and eosin; see supra
  • histology stainings of the ears are performed. This assay is used preferably as a surrogate model psoriasis.
  • IM11A11/P-EP filed on July 03, 2014, the disclosure content which is incorporated herein by reference, in particular Examples 1 to 8, Table 1 and Figure 1 disclosing the nucleotide and amino acid sequences of the variable and constant regions (VH, VL, CH, CL) regions of IgG4, lambda, IL-20 specific 20A10 antibody and of IgGl, lambda, IL-20 specific 2A11, 7D1 and 6E11 antibodies subject of this EP application as well as their functional characterization including sources of IL-20 molecules.
  • VH, VL, CH, CL variable and constant regions
  • Ear inflammation phenotype was induced in 8 weeks old C57BL/6J (WT; from Charles River) mice by intradermal injection of human cytokine IL-20 or PBS control into each ear given on alternate days at Day 1, Day 3, Day 6 and Day 8 (20 ⁇ /ear, lOOOng/ear, 2000 ng/mouse/day) using a 30-gauge needle.
  • Treatment with the exemplary anti-IL-20 2A11, 7D1 and 20A10 antibodies were tested on these animals in respect of their neutralizing potential to reduce the induced ear inflammation phenotype.
  • Two IP injections of 2A11, 7D1 and 20A10 or control human IgG [200 ⁇ g,] were administered to the animals at day 0 and day 6. The mice were sacrificed at day 10.
  • ear thickness measurements of the animals were taken with a Mitutoyo digital micrometer during the IL-20 administration by daily measurements prior to IL-20 injection. Furthermore, body weight has been monitored during the treatment, however, no significant weight changes have been observed in any of the animal groups due to the treatment applied. In addition, after sacrifice of the animals H&E (hematoxylin and eosin; see Harris, H.F., J. Appl. Microscopy III (1900), 777-781 and Mallory, F.B.: Pathological technique. Philadelphia, Saunders, (1938)) histology stainings of the ears are performed.
  • H&E hematoxylin and eosin; see Harris, H.F., J. Appl. Microscopy III (1900), 777-781 and Mallory, F.B.: Pathological technique. Philadelphia, Saunders, (1938)

Abstract

Provided is a novel method of isolating and providing anti-human cytokine antibodies for pharmaceutical use, in particular recombinant human anti-human cytokine antibodies. More specifically, a method of validating an isolated anti-human cytokine antibody or cytokine- binding fragment thereof for therapeutic use in an in vivo, non-human animal assay is described.

Description

Method of providing anti-human cytokine antibodies for pharmaceutical use Field of the invention
The present invention relates to a method of isolating and providing anti-human cytokine antibodies and cytokine-binding fragments thereof for pharmaceutical use, in particular recombinant human anti-human cytokine antibodies and cytokine-binding fragments thereof. More specifically, the present invention relates to a method of validating an isolated anti- human cytokine antibody or cytokine-binding fragment thereof for therapeutic use in an in vivo, non-human animal assay also referred to herein as "HuCytoMab"-Assay.
Background of the invention
Inappropriate responses of the immune system may cause stressful symptoms to the involved organism. Exaggerated immune answers to foreign substances or physical states which usually do not have a significant effect on the health of an animal or human may lead to allergies with symptoms ranging from mild reactions, such as skin irritations to life- threatening situations such as an anaphylactic shock or various types of vasculitis. Immune answers to endogenous antigens may cause autoimmune disorders such as systemic lupus erythematosus, idiopathic autoimmune hemolytic anemia, pernicious anemia, type 1 diabetes mellitus, blistering skin diseases, psoriasis and different kinds of arthritis.
Immune responses occur in a coordinated manner, involving several cell types and requiring communication by signaling molecules such as cytokines between the cell types involved. This communication may be influenced or inhibited by, e.g., interception of the signals or block of the respective receptors.
Cytokines are secreted soluble proteins, peptides and glycoproteins acting as humoral regulators at nano- to picomolar concentrations behaving like classical hormones in that they act at a systemic level and which, either under normal or pathological conditions, modulate the functional activities of individual cells and tissues. Cytokines differ from hormones in that they are not produced by specialized cells organized in specialized glands, i.e. there is not a single organ or cellular source for these mediators as they are expressed by virtually all cells involved in innate and adaptive immunity such as epithelial cells, macrophages, dendritic cells (DC), natural killer (NK) cells and especially by T cells, prominent among which are T helper (Th) lymphocytes. Depending on their respective functions, cytokines may be classified into three functional categories: regulating innate immune responses, regulating adaptive immune responses and stimulating hematopoiesis. Due to their pleiotropic activities within said three categories, e.g., concerning cell activation, proliferation, differentiation, recruitment, or other physiological responses, e.g., secretion of proteins characteristic for inflammation by target cells, disturbances of the cell signaling mediated by aberrantly regulated cytokine production have been found as a cause of many disorders associated with defective immune response, for example, inflammation and cancer.
Accordingly, immunotherapy, i.e. use of anti-cytokine antibodies has raised great interest in the therapy of substantially every disease which can be targeted to an undesired cytokine expression and/or activity. However, while methods of providing human and human-like antibodies, respectively, have emerged the screening of biologically and thus therapeutically active antibodies may be cumbersome because of the necessity of using and establishing appropriate in vivo models such as transgenic animals which may not always be available. In addition, the generation of transgenic animal models may be costly and restricted in view desired phenotype and therapeutic target.
Accordingly, it would be desirable to provide a simple and cost sensitive method of validating antibodies with respect to their biological activity and in order to select appropriate antibody drag candidates for immunotherapy. This problem, has bee solved by the embodiments characterized in the claims and following below as illustrated in the Examples and Figures.
Summary of the invention
The present invention relates to a method of isolating and providing anti-human cytokine antibodies and cytokine-binding fragments thereof for pharmaceutical use, in particular recombinant human anti-human cytokine antibodies and cytokine-binding fragments thereof. More specifically, the present invention relates to a method of validating an isolated antibody or antigen-binding fragment thereof for therapeutic use in an in vivo, non-human animal assay also referred to herein as "HuCytoMab"-Assay.
In the method and assay of the present invention, a candidate antibody, i.e. anti-human cytokine antibody or cytokine-binding fragment thereof is subjected to a non-human mammal in which an inflammatory phenotype is induced such as swelling of/at ear, knee, joint or ankle by local administration of a human cytokine, wherein a reduction of the induced inflammatory phenotype, i.e. typically swelling, compared to a control non- human mammal having received a mock antibody indicates that the candidate anti-human cytokine antibody or cytokine- binding fragment thereof has neutralizing activity and thus is suitable for the pharmaceutical use. In this context, unless otherwise stated the term "animal" and "non-human mammal" are used interchangeably herein. Preferably, the candidate human anti-human cytokine antibodies originated in memory B cells obtained from patients suffering from a disease which is caused by or involves activation of the immune system such as an autoimmune and inflammatory disorder.
Accordingly, in one aspect the present invention provides high affinity neutralizing monoclonal antibodies to cytokines derived from the naturally matured humoral immune response of a predetermined subject, which are considered to be safe and effective therapeutics for disorders in which those cytokines are involved.
The present invention further relates to the anti-human cytokine antibody, cytokine-binding fragment thereof as well as equivalent cytokine binding/neutralizing compounds which have be isolated and selected in accordance with the HuCytoMab-Assay of the present invention for pharmaceutical use, in particular for the treatment of inflammatory disorders.
Definitions and Embodiments
Unless otherwise stated, a term and an embodiment as used herein is given the definition as provided and used in international application WO2013/098419 Al and WO2013/098420 Al . Supplementary, a common term as used herein is given the definition as provided in the Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 0 19 850673 2.
Several documents are cited throughout the text of this specification. The contents of all cited references (including literature references, issued patents, published patent applications as cited throughout this application and manufacturer's specifications, instructions, etc.) are hereby expressly incorporated by reference; however, there is no admission that any document cited is indeed prior art as to the present invention. Description of the Figures
Fig. 1: HuCytoMab- Assay for anti- human IL-32 antibodies. Ear thickness measurements calculated as fold change relative to day 0 measurements, then normalized to relevant PBS controls, for each cohort. Mean +/- SEM, N stated on figure. P values obtained by 2-way ANOVA testing, ns (not significant) = P > 0.05; * = P < 0.05; ** = P < 0.01; *** = PO.001, **** = p<0.0001. IP= intraperitoneal antibody injection, ID= intradermal ear injection.
Fig. 2: HuCytoMab- Assay for anti-human IL-32 antibodies. Ear thickness measurements shown as absolute values (mm) for each cohort. Mean +/- SEM, N stated on figure. P values obtained by ANOVA testing. IP= intraperitoneal antibody injection, ID= intradermal ear injection. P value indications as in Fig. 1.
Fig. 3: HuCytoMab- Assay for anti- human IL-32 antibodies, here weight monitoring. No significant weight changes were observed in either of the animals tested in the experiment.
Fig. 4: HuCytoMab-Assay for anti-human IFN-alpha (IFNA) antibodies. Ear inflammation assay-test of the proinflammatory effect of human IFN subtypes in mice. A: Exemplary 6-day experimental timeline. B: Ear thickness measurements calculated as fold change relative to day 0 measurements than normalized to relevant PBS controls, for each cohort. Bl: Overview of the effect of all normalized measurements. B2: Effects of the IFNa2a and IFNa2b injections. B3: Effects of the IFNa4 and IFNal4 injections. All four human IFNA subtypes tested were able to significantly induce ear swelling following ID. All ears were markedly thicker than PBS treated ears. IFNal4 was the most potent proinflammatory agent. Mean +/- SEM, 11-3 or ID = intradermal cytokine injections, M = Measurements - ear thickness and animal weight, S = Sacrifice of the animals; short arrows - cytokine injections; long arrows - exemplary days of anti-IFN-a antibody injections.
Fig. 5: HuCytoMab-Assay for anti-human IFN-alpha (IFNA) antibodies. Ear inflammation assay-test of the proinflammatory effect of human IFN subtypes in mice. Ear thickness measurements are shown as absolute values (mm) for each cohort. A: Overview of the effect of all measurements. B: Effects of the IFNa2a and IFNa2b injections. C: Effects of the IFNa4 and IFNal4 injections. All indications as in Fig. 4.
Fig. 6: HuCytoMab-Assay for anti-human IFN-alpha (IFNA) antibodies. Ear inflammation assay-test of the proinflammatory effect of human IFN subtypes in mice. Ear thickness measurements are shown fold change from Day 0 for each cohort. The thickness on Day 0 has been set as 1. A: Overview of the effect of all measurements. B: Effects of the IFNa2a and IFNa2b injections. C: Effects of the IFNa4 and IFNal4 injections. All indications as in Fig. 4.
Fig. 7: HuCytoMab- Assay for anti-human IFN-alpha (IFNA) antibodies. Summary of the ear inflammation assay in respect of human IFN-a subtype capabilities for induction of ear inflammation. P values obtained by 2-way ANOVA testing, ns (not significant) = P > 0.05; * = P < 0.05; ** = P < 0.01; *** = PO.001, **** = p<0.0001. All ears were markedly thicker than PBS treated ears; this was significant in all groups after the 2nd ID, from Day 3 until the end of the experiment.
Fig. 8: Ear inflammation assay CytoEar IFNA14. Testing the effect of different IFNA blocking antibodies of the present invention following hIFNA14 induced inflammation. To induce inflammation 20 μΐ IFNal4 was injected per ear at a concentration of 25 μg/ml. Measurement of thickness was also performed before giving the injection, and with two measurements per ear. A: Exemplary 10-day experimental timeline. B: Overview of the experimental treatment of the experimental animal groups A to I. Ref. A - IFN A- specific reference antibody. CytoEar ear thickness measurements calculated as fold change relative to day 0 measurements than normalized to relevant PBS controls, for each cohort. C: Overview of the effect of all normalized measurements. D: Effects of 26B9 treatment following IFNal4 injections. E: Effects of 19D11 treatment following IFNal4 injections. F: Effect of the treatment with reference anti-IFN-a antibody Ref. A following IFNal4 injections. Treatment with antibodies 26B9 and 19D1 1 (significant reduction of ear thickness at days 7, 9, 10, respective at days 4, 7-10 for 19D11) of the present invention leads to pronounced reduction of the ear thickness resulting from IFNal4 injections compared to the control treatment with IgG (of IFN-a non-related binding specificity) and of treatment with Ref. A. Mean +/- SEM, ID = intradermal cytokine injections, M = Measurements - ear thickness, S = Sacrifice of the animals; ID - cytokine injections; tested antibodies 26B9, 19D11, Ref.A and the control IgG were injected at day 0 (IP).
Fig. 9: Ear inflammation assay CytoEar IFNA5. Testing the effect of different IFNA blocking antibodies of the present invention following hIFNA5 induced inflammation. To induce inflammation 20 μΐ IFNa5 was injected per ear at a concentration of 25 μg/ml. Measurement of thickness was also performed before giving the injection, and with two measurements per ear. A: Exemplary 10-day experimental timeline. B: Overview of the experimental treatment of the experimental animal groups A to I. Ref. A - reference IFNA- specific antibody. CytoEar ear thickness measurements calculated as fold change relative to day 0 measurements than normalized to relevant PBS controls, for each cohort. C: Overview of the effect of all normalized measurements. D: Effects of 26B9 treatment following IFNa5 injections. E: Effects of 19D11 treatment following IFNa5 injections. F: Effect of the treatment with reference anti-IFN-a antibody Ref. A following IFNa5 injections. Treatment with antibodies 26B9 and 19D1 1 of the present invention leads to a reduction of the ear thickness resulting from IFNa5 injections (significant reduction for 26B9 at days 4, 6, 7, 8 and 9: for 19B11 at days 7-9). See description of Fig. 26 for further details. Tested antibodies 26B9, 19D11, Ref.A and the control IgG were injected at day 0 (IP).
Fig. 10: Ear inflammation assay CytoEar IFNw. Testing the effect of different IFNA blocking antibodies of the present invention following hlFNw (IFNco) induced inflammation To induce inflammation 20 μΐ IFNw was injected per ear at a concentration of 6.25 μg/ml, 125 ng/ear. Measurement of thickness was also performed before giving the injection, and with two measurements per ear. A: Exemplary 10-day experimental timeline. B: Overview of the experimental treatment of the experimental animal groups A to I. Ref. A - reference IFNA- specific antibody. CytoEar ear thickness measurements calculated as fold change relative to day 0 measurements than normalized to relevant PBS controls, for each cohort. C: Overview of the effect of all normalized measurements. D: Effects of 26B9 treatment following IFNw injections. E: Effects of 19D11 treatment following IFNw injections. F: Effect of the treatment with reference anti-IFN-a antibody Ref. A following IFNw injections. Treatment with antibody 26B9 of the present invention leads to a significant reduction of the ear thickness resulting from IFNw injections at experimental Day 9. Treatment with 19D11 or Ref.A leads to apparently none or very slight reduction of the ear thickness (not significant (ns) compared to control IgG injections at all days). See description of Fig. 26 for further details. Tested antibodies 26B9, 19D11, RefA and the control IgG were injected at day 0 (IP).
Fig. 11: Effect of 19A1 blocking antibody following hIL-32y induced inflammation, comparison to 2C2 in CytoEar assay. To induce inflammation hIL-32y was injected at a concentration of 6.25 μg/ml, 125 ng/ear. A: Exemplary 10-day experimental timeline. B: Overview of the experimental treatment of the experimental animal groups A to F. C-F: Mice cohorts (C57/BL6, 8 weeks) were IP injected with stated amounts of 2C2 or 19A1 antibodies (or IgG control) at experiment initial day, while 125 ng hrIL-32y cytokine in 20 μΐ of PBS (or PBS control) was intradermally injected into mice ears every 48-72 hours. Ear thickness measurements were taken with a Mitutoyo digital micrometer. CytoEar ear thickness measurements calculated as fold change relative to initial day measurements, then normalised to relevant PBS controls, for each cohort. Mean +/- SEM, N stated on figure. P values obtained by ANOVA testing. IP= intraperitoneal antibody injection, ID= intradermal ear injection, NT= non-treated control.
Fig. 12: Effect of different doses of 19A1 antibody following hIL-32y induced inflammation in CytoEar assay. To induce inflammation hIL-32y was injected at a concentration of 6.25 μg/ml, 125 ng/ear. A: Exemplary 10-day experimental timeline. B: Overview of the experimental treatment of the experimental animal groups A to K. C-F: Mice cohorts (C57/BL6, 8 weeks) were IP injected with stated amounts of 2C2 or 19A1 antibodies (or IgG control) at experiment initial day, while 125 ng hrIL-32y cytokine in 20ul of PBS (or PBS control) was intradermally injected into mice ears every 48-72 hours. Ear thickness measurements were taken with a Mitutoyo digital micrometer. CytoEar ear thickness measurements calculated as fold change relative to initial day measurements, then normalised to relevant PBS controls, for each cohort. Mean +/- SEM, N stated on figure. P values obtained by ANOVA testing. IP= intraperitoneal antibody injection, ID intradermal ear injection, NT= non-treated control.
Fig. 13: Dose dependency of IL-32 in inducing inflammation in CytoAnkle assay. To induce inflammation mice obtained intraarticular ankle injections of 10 μΐ hIL-32y in the right ankle, whereas the left ankle was injected with PBS. A: Exemplary 13- day experimental timeline. B: Overview of the experimental treatment of the experimental animal cages Al to D2. C-D: Mice cohort (C57/BL6, 8 weeks) were IA injected with stated amounts of hrIL-32y cytokine in lOul of PBS (or PBS control) into mice ankles every 48-72 hours. Axial ankle thickness measurements were taken with a Mitutoyo digital micrometer. CytoAnkle thickness measurements calculated as fold change relative to initial day measurements, then normalised to relevant PBS controls, for each cohort. Mean +/- SEM, N stated on figure. P values obtained by ANOVA testing. IA= intraarticular ankle injection.
Fig. 14: Effect of 2C2 antibody following hIL-32y induced inflammation in CytoAnkle assay. CytoAnkle test: +/- IL-32y +/- 2C2. A: Exemplary 10-day experimental timeline. B: Overview of the experimental treatment of the experimental animal cages Al to D2. C-D: Mice cohort (C57/BL6, 8 weeks) were IP injected with 200 μg of 2C2 antibodies (or IgG control) at experiment initial day, while 500 ng of hrIL-32y cytokine in 10 μΐ of PBS (or PBS control) was IA injected into mice ankles every 48-72 hours. Axial ankle thickness measurements were taken with a Mitutoyo digital micrometer. CytoAnkle thickness measurements calculated as fold change relative to initial day measurements, then normalised to relevant PBS controls, for each cohort. Mean +/- SEM, N stated on figure. P values obtained by ANOVA testing. IP= intraperitoneal antibody injection, IA= intraarticular ankle injection.
Fig. 15: Ear inflammation assay CytoEar IL-20. Testing the effect of different IL-20 blocking antibodies of the present invention following hIL-20 induced inflammation. To induce inflammation IL-20 was injected at a concentration of 1000 ng in a volume of 20 μΐ which gave the optimal experimental results after titrating different concentrations of 125 ng, 250 ng, 500 ng and 1000 ng. A: Exemplary 10-day experimental timeline. B: Overview of the experimental treatment of the experimental animal groups A to M. CytoEar ear thickness measurements calculated as fold change relative to day 0 measurements than normalized to relevant PBS controls, for each cohort. C: Overview of the effect of all normalized measurements. D: Overview of the effect of all normalized measurements. E: Overview of the effect of all normalized measurements showing IL-20 results only for the sake of clarity. Treatment with antibodies 20A10 and 7D1 (significant reduction of ear thickness at days 6 to 10, respective at days 8 to 10 for 2A11) of the present invention leads to pronounced reduction of the ear thickness resulting from IL-20 injections compared to the control treatment with IgG (of IL-20 non-related binding specificity). Mean +/- SEM, ID = intradermal cytokine injections, M = Measurements - ear thickness, S = Sacrifice of the animals; ID - cytokine injections; tested antibodies 2A11, 20A10, 7D1, and the control IgG were injected at day 0 and 6 (IP). IP= intraperitoneal antibody injection, ID= intradermal ear injection.
Disclosure of the invention
As mentioned hereinbefore, the present invention generally relates to a method of isolating and providing anti-huma cytokine antibodies and cytokine-binding fragments thereof for pharmaceutical use, in particular recombinant human anti-human cytokine antibodies and cytokine-binding fragments thereof. More specifically, the present invention, relates to a method of validating an isolated antibody or antigen-binding fragment thereof for therapeutic use in an in vivo, non-human animal assay also referred to herein as "HuCytoMab"-Assay. More specifically, the preset invention relates to a method of providing an anti-human cytokine antibody or cytokine-binding fragment thereof for pharmaceutical use, the method being characterized by
(a) subjecting a candidate anti-human cytokine antibody or cytokine-binding fragment thereof to a non-human mammal in which
(i) a local inflammatory phenotype;
(ii) is induced by a predetermined human cytokine;
wherein
(b) a reduction of the induced inflammatory phenotype compared to a control, indicates that the candidate anti-human, cytokine antibody or cytokine-binding fragment thereof is suitable for the pharmaceutical use.
The present invention is based on the observation that inflammation induced by injecting a human cytokine to a local area, such, as ear, knee, or ankle of a laboratory animal, such as mice induced a local, inflammatory phenotype, in particular swelling which could be prevented or ameliorated when, co-injecting a candidate anti-human cytokine antibody which is capable of neutralizing the cytokine. Fortunately, the local injection of human antibody does seem to have side effects such, as a mouse anti-human response which, would substantially affect the outcome of the assay. As illustrated in the appended Examples and shown in the Figures, the assay of the present invention has been validated for four different cytokines and corresponding anti-cytokine antibodies, i.e. with respect to interleukin-32 (IL- 20), alpha- interferon (IFNA, IFN-o) including different subtypes, omega- interferon (IFNW, IFN-ω) and interleukin-20 (IL-20) as well, as at different locations of the animal, body (ear and ankle, i.e. CytoEar and CytoAnkle assay), which demonstrates the general applicability of the HuCytoMab- Assay.
Thus, in accordance with the present invention an. assay could be developed, wherein a candidate anti-human cytokine antibody can be tested and validated regarding its possible neutralizing activity and thus suitability to be further developed as a drug for immunotherapy. If not stated otherwise, the terms "pharmaceutical, use" and "immunotherapy" are used as common in the art and interchangeably herein, and refer to both, therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow dow n (lessen) an undesired physiological change or disorder, in particular for the treatment of inflammatory, autoinflammatory, immune and autoimmune disorders. The term "pharmaceutical, use" further comprises the galenic formulation of the candidate antibodies or antigen binding fragments thereof into drugs, solutions or mixtures intended for to be used for diagnosis, cure, mitigation, treatment, or prevention of disease of either man or other animals. In. this connection, the terms "drug," "medicine," or "medicament" are used interchangeably herei and shall include but are not limited to all (A) articles, medicines and preparations for internal, or external use, and any substance or mixture of substances intended to be used for diagnosis, cure, mitigation, treatment, or prevention of disease of either man. or other an.im.als; and (B) articles, medicines and preparations (other than food) intended to affect the structure or any function of the body of man or other animals; and (C) articles intended for use as a component of any article specified in clause (A) and (B). The term "drug," "medicine," or "medicament" shall, include the complete formula of the preparation intended for use in either man or other animals containing one or more "agents," "compounds", "substances" or "(chemical) compositions" as and in some other context also other pharmaceutically inactive e cipients as fillers, disintegrants, lubricants, glidants, binders or ensuring easy transport, disintegration, disaggregation, dissolution and biological availability of the "drug," "medicine," or "medicament" at an intended target location within the body of man or other animals, e.g., at the skin, in the stomach or the intestine. The terms "agent," "compound" or "substance" are used interchangeably herein and shall include, in a more particular context, but are not limited to all pharmacologically active agents, i.e. agents that induce a desired biological or pharmacological effect or are investigated or tested for the capability of inducing such a possible pharmacological effect by the methods of the present invention. In one embodiment of the present invent ion, the term " pharmaceutical use" or "immunotherapy" also includes the diagnostics use of the anti-huma cytokine antibody and cytokine binding fragment of the present invention in vivo, for example for use in in vivo imaging and the like.
Besides the easy set-up of the so called HuCytoMab assay of the present invention, it has the advantage to address questions of disease pathogenesis and to validate therapeutic agents for human target proteins which have no animal homobgue or which target domain is not conserved in a corresponding animal homologue.
Thus, besides they broad utility of the HuCytoMab- Assay of the present invention towards in principle any human cytokine, in particular pro inflammatory cytokines, it is possible to identify and validate anti-human cytokine antibodies and equivalent drugs which either target both the human cytokine as well as its non-human mammalian, homologue or is specific to either the human cytokine or its non-human mammalian homologue. Accordingly, the HuCytoMab- Assay of the present invention provides an important tool for the development of potential anti-human cytokine antibody and equivalent human cytokine-binding drag candidates from a possible lead compound to the final pharmaceutical product.
Cytokines are small glycoprotein messengers which where variously identified in the absence of a unified classification system by numeric order of discovery, a given functional, activity, a kinetic or functional, role in inflammatory responses, a primary cell of origin, or structural homologues shared with related molecules; see, e.g., Mclnnes in Kelley's Textbook of Rheumatology, Elsevier Health Sciences 9th Edition Vol. 1 by Firestein et al. (2012), 3rd part, chapter 23, 367-377. The cytokines in accordance with the present invention are described but not limited to the cytokines described in. Mclnnes (2012) 3rd part, chapter 23, 367-377) depicted in Tables 23-1 to 23-8, the disclosure content of which is incorporated herei by reference, and in the Cytokine listing; see the table infra. In a preferred embodiment of the assay of the present invention, the cytokine is an interferon or interleukin, most preferably an interfero or interleukin as described in the appended Examples. As illustrated in the Examples, in a preferred embodiment of the present invention the non- human, mammal used in the method of the present invention is a laboratory animal such as mice. However, other laboratory animals may be used as well, in particular rodents such as rats and gerbiis. In some embodiments, the use of higher non-human mammals may be desired, for example cats, dogs, cows or primates, for example depending on the human cytokine and corresponding anti-human cytokine antibody to be investigated and the cytokine background of a give non-human mammal with respect to the presence of an endogenous homologue of the huma cytokine. In one embodiment of the method of the present invention, the candidate antibody or cvtokine-bmding fragment does not cross-react with the non-huma mammalian homologue of the human cytokine and/or the non-human mammal lacks a homologue of the human cytokine. In a preferred embodiment of the method of the present invention the non-human mammal in the 1 1 uCyto ab- Assay is a mouse and/or the subject mammal as the source for the candidate antibody is a human.
The local inflammatory phenotype is preferably induced at/in a local, site of the non-human- mammal which is represents a typical part of the huma and non-human mammal body, which is affected by the corresponding inflammatory or autoimmune disorder, for example, knee or ankle in case of rheumatoid arthritis and skin, e.g., of the ear in case of psoriasis. Furthermore, the local, site of the non-human, mammal, is preferably characterized in exhibiting the most distinct phenotype as response to an inflammatory stimulus. Accordingly, the local inflammatory phenotype, such as swelling of ear, knee, joint or ankle can. preferably be easily monitored, preferably by eye or by simple measurements, such as measurements or perception of changes of thickness, length, color or weight of body parts, organs or the whole animal.
As further illustrated in the Examples, the huma cytokine may be administered to the animal, here by reference to mice, in a concentration/dose of about 1 n.g to 1 mg, preferably 0, 1 ,ug to 10 ,ug, more preferably 0,5 to 5 ag and typically 1 ,ug total/mouse/day. The dose administered to the mouse may be split up into 2 or more doses. Typically, two doses are used in the amount of about 250, 500 or 1000 ng; see also the Examples. Typically, the cytokine is administered in a volume of about 5 to 100 μΐ, preferably 10 or 20 μΐ per injection. As a solvent for administering the human cytokine in principle any pharmaceut ically or physiologically acceptable career may be used, preferably phosphate-buffered saline (PBS) as illustrated in the Examples. Administration, of the huma cytokine will, be effected preferably every other day. However, depending on the laboratory animal and the local side for inducing the inflammation different intervals and frequency of administration may be used.
The candidate antibody or cytokine-binding fragment is subjected prior, concomitantly and/or after induction of the local inflammatory phenotype, preferably in the same solvent and volume as the human cytokine. The usual dose of the candidate antibody or cytokine-binding fragment may range from 0.1 μg to 1 mg, preferably from. 1 μg to 500 ,ug and most preferably from, about 50 μg to 250 ,ug; see also the Examples. In. one embodiment, the candidate antibody or cytokine-binding fragment is injected in combination with the human cytokine or prior to its application. A.s a control, instead of the candidate antibody for example either the solvent alone or an. unrelated human antibody or IgG fraction, may be used.
Administration, of the human cytokine into the local site for inducing the inflammatory phenotype ca effected by means well known, in the art, e.g., intradermal injection, or injection into the knee joint or ankle, i.e. intraarticularly or periarticular administration. Typically, the candidate antibody or cytokine-binding fragment is administered in the manner as the human, cytokine.
I order to determine the inflammatory phenotype, for example, when, using the ears for the local side for inducing inflammation ear thickness is measured at the beginning of the assay before giving the injection, typically with, two measurements per ear, on the day or days in between, the day or days for injection and on the last day of the assay, before or after sacrifice of the animal. A. reductio n of the i nduced inflammatory phenotype compared to a control, fo r example reduced or suppressed swelling of the ear compared to the swelling of the ear of a corresponding non-huma mammal which did not receive the candidate anti-human, cytokine antibody o a swelling comparable to the swelling in control, animals obtaining PBS infusions instead of the cytokine indicates that the candidate anti-human, cytokine antibody or cytokine- binding fragment thereof is suitable for pharmaceutical use.
In case of other local inflammation sides, e.g., knee joint or ankle the inflammation may be induced and the respective phenotype may be measured by other means known in. the art; see, e.g., injection of human 11.-32 in murine knee joints, measurement of murine joint inflammation and histology described in Joosten et al., Proc. Natl. Acad. Sci. USA. 103 (2006), 3298-3303, the disclosure content of which, is incorporated herein by reference. In Joosten et al. (2006), the local injection of human IL-32 has been shown to induce severe joint inflammation. However, though the corresponding experiments were used as evidence that IL-32 appears to be a pro -inflammatory cytokine in rheumatoid arthritis (RA), the use of human IL-32 as therapeutic target in the treatment of an inflammatory disorder such as RA and the assessment of candidate compounds capable of neutralizing the biological activity of IL-32 has not been contemplated.
Hence, a further advantageous aspect of the method of the present invention is that the HuCytoMab-Assay in most instances not only is capable of determining the neutralizing activity of given candidate compound, in particular anti-human cytokine antibody but also allows a reasonable prediction of its therapeutic utility and tolerability in human since the principle of exogenously inducing a local inflammatory phenotype in animal models for addressing the questions of disease development and validation of therapeutic targets is widely used in the art, for example collagen- induced arthritis (CIA) as an animal model of rheumatoid arthritis (RA); see also Hod rick et al, J. Clin. Investigation 119 (2009), 2317- 2329 for ! L-23 induced psoriasis-like inflammation in mice and references cited therei or Watanabe et al, J. Investigative Dermatology 129 (2009), 650-656 for IL-17F in psoriasis induced in mice. However, also in these publications, the use of 11.-23 and I.L-17F, respectively, as therapeutic target in the treatment of an inflammatory disorder such as psoriasis and the assessment of candidate compounds capable of neutralizing the biological activity of human IL-23 and IL-17F has not been contemplated.
Thus, the HuCytoMab-Assay of the present invention provides for the first time a surrogate model for testing anti-human cytokine antibodies and like cytokine neutralizing compounds in the treatment of inflammatory diseases such as RA and psoriasis.
In summary, the HuCytoMab- Assay and method of the present invention is a valuable tool in determining the biological activity, in particular neutralizing activity of anti-human cytokine antibodies and equivalent cytokine antagonizing compounds as well as their specificity towards the human cytokine and their utility in the immunotherapy of inflammatory disorders. In this context, most embodiments are illustrated by way of reference to a human recombinant anti-human cytokine antibody. However, if not stated otherwise or evident, the description of these embodiments though preferred are used to illustrate the present invention and are not intended to exclude equivalent huma cytokine binding and/or neutralizing compounds from the scope of the appended claims in any way. In principle the candidate antibody or equivalent human cytokine binding agent could be provided by any possible source including the classical hybndoma approach as well as combinatorial libraries for producing synthetic antibodies and antibody like cytokine binding molecules. Thus, antibody engineering has become a well-developed discipline, encompassing discovery methods, production strategies, and modification techniques for natural and non-natural human antibody repertoires and their mining with non-combinatorial and combinatorial strategies. This also includes the production and selectio of huma antibodies (mAbs) from naive, immune, transgenic and synthetic human antibody repertoires using methods based on hybridoma technology, clonal expansion of peripheral B cells, single- cell PCR, phage display, yeast display, mammalian cell display and in silico design; see for review of the characteristics of natural and non-natural human antibody repertoires and their mining with non-combinatorial and combinatorial strategies, e.g., Beerli and Rader in Mining human antibody repertoires, mAbs 2 (2010); 365-378 and Campbell, et ah, British Journal of Pharmacology 162 (2011), 1470-1484; the disclosure content of which is incorporated herein by reference.
However, as mentioned before, in a preferred embodiment of the method of the present invention, the candidate anti huma cytokine antibody or cytokine binding fragment thereof is derived from, a human antibody provided by a method comprising isolating a monoclonal antibody or antigen-binding fragment thereof, wherein, a B cell expressing the monoclonal antibody is isolated from a sample obtained from, a subject mammal. In the past decades several technologies have been developed to isolate monoclonal antibodies and to produce humanized or fully human antibodies; see, e.g., references cited in international application WO 2007/068758 also granted as European patent EP 1 974 020 Bl, in particular in sections [0002] to [0027], the disclosure content of which is incorporated herein by reference. Typically, the isolation of antibodies, for example monoclonal antibodies from B cells relies on cloning and expression of the immunoglobulin genes. This can be done by using phage display libraries of scramble VH and VL genes from B cells, or by isolation of paired VH and VL genes from single B cells using single cell PCR or from immortalized B cell clones.
In. accordance with the present invention the use of candidate anti human, cytokine antibodies which, originated from B cells of a patients suffering from an. autoimmune and/or inflammatory disease turned out to be in. particular valuable, i.e. in particular for providing human antibodies. Accordingly, in one embodiment of the present invention, the subject mammal, as the source of the antibody is suffering from, an autoimmune and/or inflammatory disease.
As used herein, an "autoimmune disorder" can be an organ-specific disease (i.e., the immune response is specifically directed against an organ system such as the endocrine system, the hematopoietic system, the skin, the cardiopulmonary system, the gastrointestinal and liver systems, the renal system, the thyroid, the ears, the neuromuscular system, the central nervous system, etc.) or a systemic disease that can affect multiple organ systems (for example, systemic lupus erythematosus (SLE), rheumatoid arthritis, polymyositis, autoimmune polyendocrinopathy syndrome type 1 (APS-l)/autoimmune polyendocrinopathy-candidiasis- ectodermal dystrophy (APECED) etc. Preferred such diseases include autoimmune rheumatologic disorders (such as, for example, rheumatoid arthritis, Sjogren's syndrome, scleroderma, lupus such as SLE and lupus nephritis, polymyositis/dermatomyositis, cryoglobulinemia, anti-phospholipid antibody syndrome, and psoriatic arthritis), autoimmune gastrointestinal and liver disorders (such as, for example, inflammatory bowel diseases (e.g., ulcerative colitis and Crohn's disease), autoimmune gastritis and pernicious anemia, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, and celiac disease), vasculitis (such as, for example, ANCA-negative vasculitis and ANCA-associated vasculitis, including Churg-Strauss vasculitis, Wegener's granulomatosis, and microscopic polyangiitis), autoimmune neurological disorders (such as, for example, multiple sclerosis (MS), opsoclonus myoclonus syndrome, myasthenia gravis, neuromyelitis optica, Parkinson's disease, Alzheimer's disease, and autoimmune polyneuropathies), renal disorders (such as, for example, glomerulonephritis, Goodpasture's syndrome, and Berger's disease), autoimmune dermatologic disorders (such as, for example, psoriasis, atopic dermatitis, urticaria, pemphigus group diseases, bullous pemphigoid diseases, and cutaneous lupus erythematosus), hematologic disorders (such as, for example, thrombocytopenic purpura, thrombotic thrombocytopenic purpura, post-transfusion purpura, and autoimmune hemolytic anemia), atherosclerosis, uveitis, autoimmune hearing diseases (such as, for example, inner ear disease and hearing loss), Behcet's disease, Raynaud's syndrome, organ transplant, and autoimmune endocrine disorders (such as, for example, diabetic-related autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM), Addison's disease, autoimmune thyroid disease (e.g., Graves' disease and thyroiditis)) and diseases affecting the generation of autoimmunity such as autoimmune polyendocrinopathy syndrome type 1 (APS-l)/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) Myasthenia Gravis (MG/Thymoma. Preferred diseases include, for example, RA, IBD, including Crohn's disease and ulcerative colitis, ANCA-associated vasculitis, lupus, MS, Sjogren's syndrome, Graves' disease, IDDM, pernicious anemia, thyroiditis, and glomerulonephritis, and APS-1. Still more preferred are RA, IBD, lupus, and MS, and more preferred RA and IBD, and most preferred RA.
Specific examples of other autoimmune disorders as defined herein, which in some cases encompass those listed above, include, but are not limited to, arthritis (acute and chronic, rheumatoid arthritis including juvenile-onset rheumatoid arthritis and stages such as rheumatoid synovitis, gout or gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, menopausal arthritis, estrogen-depletion arthritis, and ankylosing spondylitis/rheumatoid spondylitis), autoimmune lymphoproliferative disease, inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails, atopy including atopic diseases such as hay fever and Job's syndrome, atopic dermatitis, allergic and toxic contact dermatitis (acute and chronic), exfoliative dermatitis, allergic dermatitis, hives, dermatitis herpetiformis, nummular dermatitis, seborrheic dermatitis, non-specific dermatitis, x-linked hyper IgM syndrome, allergic intraocular inflammatory diseases, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, cold urticarial, including chronic autoimmune urticaria, myositis, polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including local and systemic forms of scleroderma), multiple sclerosis (MS) such as spino-optical MS, primary progressive MS (PPMS), and relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, ataxic sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease (IBD) (for example, Crohn's disease, autoimmune-mediated gastrointestinal diseases, gastrointestinal inflammation, colitis such as ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, and transmural colitis, and autoimmune inflammatory bowel disease), bowel inflammation, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndrome, including adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, an autoimmune hematological disorder, graft-versus-host disease, angioedema such as hereditary angioedema, cranial nerve damage as in meningitis, herpes gestationis, pemphigoid gestationis, pruritis scroti, autoimmune premature ovarian failure, sudden hearing loss due to an autoimmune condition, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, various forms of asthma, Chronic Obstructive Pulmonary Disease (COPD), encephalitis such as Rasmussen's encephalitis and limbic and/or brainstem encephalitis, uveitis, such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis, glomerulonephritis (GN) with and without nephrotic syndrome such as chronic or acute glomerulonephritis such as primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (MPGN), including Type I and Type II, and rapidly progressive GN (RPGN), proliferative nephritis, autoimmune polyglandular endocrine failure, balanitis including balanitis circumscripta plasmacellularis, balanoposthitis, erythema annulare centrifugum, erythema dyschromicum perstans, eythema multiform, granuloma annulare, lichen nitidus, lichen sclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus, lichen planus, epidermolytic hyperkeratosis, premalignant keratosis, pyoderma gangrenosum, allergic conditions and responses, food allergies, drug allergies, insect allergies, rare allergic disorders such as mastocytosis, allergic reaction, eczema including allergic or atopic eczema, asteatotic eczema, dyshidrotic eczema, and vesicular palmoplantar eczema, asthma such as asthma bronchiale, bronchial asthma, and auto-immune asthma, conditions involving infiltration of T cells and chronic inflammatory responses, immune reactions against foreign antigens such as fetal A-B-0 blood groups during pregnancy, chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte adhesion deficiency, lupus, including lupus nephritis, lupus cerebritis, pediatric lupus, nonrenal lupus, extra-renal lupus discoid lupus erythematosus including all its clinical forms, SLE, such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and lupus erythematosus disseminatus, juvenile onset (Type I) diabetes mellitus, including pediatric IDDM, adult onset diabetes mellitus (Type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, diabetic retinopathy, diabetic nephropathy, diabetic colitis, diabetic large-artery disorder, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis including lymphomatoid granulomatosis, agranulocytosis, vasculitides (including large-vessel vasculitis such as polymyalgia rheumatica and giant-cell (Takayasu's) arteritis, medium- vessel vasculitis such as Kawasaki's disease and polyarteritis nodosa/periarteritis nodosa, immuno vasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis such as fibrinoid necrotizing vasculitis and systemic necrotizing vasculitis, A CA-negative vasculitis, and ANCA-associated vasculitis such as Churg-Strauss syndrome (CSS), Wegener's granulomatosis, and microscopic polyangiitis), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia perniciosa), Addison's disease, pure red cell anemia or aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia(s), cytopenias such as pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, Alzheimer's disease, Parkinson's disease, multiple organ injury syndrome such as those secondary to septicemia, trauma or hemorrhage, antigen-antibody complex- mediated diseases, anti-glomerular basement membrane disease, anti-phospho lipid antibody syndrome, motoneuritis, allergic neuritis, Beliefs disease/syndrome, Castleman's syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens- Johnson syndrome, pemphigoid or pemphigus such as pemphigoid bullous, cicatricial (mucous membrane) pemphigoid, skin pemphigoid, pemphigus vulgaris, paraneoplastic pemphigus, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, and pemphigus erythematosus, epidermolysis bullosa acquisita, ocular inflammation, preferably allergic ocular inflammation such as allergic conjunctivis, linear IgA bullous disease, autoimmune-induced conjunctival inflammation, autoimmune polyendocrinopathies, Reiter's disease or syndrome, thermal injury due to an autoimmune condition, preeclampsia, an immune complex disorder such as immune complex nephritis, antibody-mediated nephritis, neuroinf ammatory disorders, polyneuropathies, chronic neuropathy such as IgM polyneuropathies or IgM-mediated neuropathy, thrombocytopenia (as developed by myocardial infarction patients, for example), including thrombotic thrombocytopenic purpura (TTP), post-transfusion purpura (PTP), heparin- induced thrombocytopenia, and autoimmune or immune-mediated thrombocytopenia including, for example, idiopathic thrombocytopenic purpura (ITP) including chronic or acute ITP, scleritis such as idiopathic cerato-scleritis, episcleritis, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases including thyroiditis such as autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave's disease, Grave's eye disease (ophthalmopathy or thyroid-associated ophthalmopathy), polyglandular syndromes such as autoimmune polyglandular syndromes, for example, type I (or polyglandular endocrinopathy syndromes), paraneoplastic syndromes, including neurologic paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis such as allergic encephalomyelitis or encephalomyelitis allergica and experimental allergic encephalomyelitis (EAE), myasthenia gravis such as thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant-cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, pneumonitis such as lymphoid interstitial pneumonitis (LIP), bronchiolitis obliterans (non-transplant) vs. NSIP, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient acantholytic dermatosis, cirrhosis such as primary biliary cirrhosis and pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac or Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia such as mixed cryoglobulinemia, amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease such as autoimmune inner ear disease (AIED), autoimmune hearing loss, polychondritis such as refractory or relapsed or relapsing polychondritis, pulmonary alveolar proteinosis, keratitis such as Cogan's syndrome/nonsyphilitic interstitial keratitis, Bell's palsy, Sweet's disease/syndrome, rosacea autoimmune, zoster-associated pain, amyloidosis, a non-cancerous lymphocytosis, a primary lymphocytosis, which includes monoclonal B cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and channelopathies of the CNS, autism, inflammatory myopathy, focal or segmental or focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, Dressler's syndrome, alopecia areata, alopecia totalis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), male and female autoimmune infertility, e.g., due to anti-spermatozoan antibodies, mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, parasitic diseases such as leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, fibrosing mediastinitis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, flariasis, cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), or Fuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, SCID, acquired immune deficiency syndrome (AIDS), echovirus infection, sepsis (systemic inflammatory response syndrome (SIRS)), endotoxemia, pancreatitis, thyroxicosis, parvovirus infection, rubella virus infection, post-vaccination syndromes, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, giant-cell polymyalgia, chronic hypersensitivity pneumonitis, conjunctivitis, such as vernal catarrh, keratoconjunctivitis sicca, and epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, transplant organ reperfusion, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway/pulmonary disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders (cerebral vascular insufficiency) such as arteriosclerotic encephalopathy and arteriosclerotic retinopathy, aspermiogenese, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica, enteritis allergica, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearing loss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leucopenia, mononucleosis infectiosa, traverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica (sympathetic ophthalmitis), neonatal ophthalmitis, optic neuritis, orchitis granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma gangrenosum, Quervain's thyreoiditis, acquired spenic atrophy, non-malignant thymoma, lympho follicular thymitis, vitiligo, toxic-shock syndrome, food poisoning, conditions involving infiltration of T cells, leukocyte-adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome, antigen-antibody complex -mediated diseases, antiglomerular basement membrane disease, autoimmune polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, autoimmune polyglandular syndromes, including polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), cardiomyopathy such as dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, allergic sinusitis, an eosinophil-related disorder such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or granulomas containing eosinophils, anaphylaxis, spondyloarthropathies, seronegative spondyloarthritides, poly endocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome, angiectasis, autoimmune disorders associated with collagen disease, rheumatism such as chronic arthrorheumatism, lymphadenitis, reduction in blood pressure response, vascular dysfunction, tissue injury, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and disease accompanying vascularization, allergic hypersensitivity disorders, glomerulonephritides, reperfusion injury, ischemic re-perfusion disorder, reperfusion injury of myocardial or other tissues, lymphomatous tracheobronchitis, inflammatory dermatoses, dermatoses with acute inflammatory components, multiple organ failure, bullous diseases, renal cortical necrosis, acute purulent meningitis or other central nervous system inflammatory disorders, ocular and orbital inflammatory disorders, granulocyte transfusion-associated syndromes, cytokine -induced toxicity, narcolepsy, acute serious inflammation, chronic intractable inflammation, pyelitis, endarterial hyperplasia, peptic ulcer, valvulitis, and endometriosis.
With respect to the use of subject mammals, in particular human patients suffering from, an autoimmune and/or inflammatory disorder the candidate antibody is preferably isolated from mammals, in particular humans, which are affected with an impaired central and/or peripheral tolerance or loss of self-tolerance which may be due to or associated with a disrupted or deregulated genesis of self-tolerance, preferably caused by a monogenic autoimmune disorder. Examples of mammals which provide a particularly suitable source for autoantibodies in accordance with the present invention are mammals, e.g., humans having a disorder associated with a mutation in the AIRE (Autoimmune Regulator) gene such as Autoimmune polyendocrinopathy syndrome type 1 (APS 1 ) and autoimmune polyendocrinopathy-candidiasis-ectodermal. dystrophy (APECED), respectively, (Peterson et al, Nat. Rev. Immunol. 8 (2008), 948-957), Autoimmune polyendocrinopathy syndrome type 2 (APS2) (Baker et al, J. Clin. Endocrinol. Metab. 95 (2010), E263-E270) and immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX) (Powell et al, J. Pediatr. 100 (1982), 731-737; Ochs et al, Immunol. Rev. 203 (2005), 156-164). Preferably, the subject mammal from which the candidate antibodies are isolated are displaying seroreactivity against the predetermined human cytokine. For further details concerning APECED/APS1 patients and the screening of their auto-immunosome; see the description of applicant's co-pending international application WO2013/098419 Al, the disclosure content of which is incorporated herein by reference in its entirety, and the Examples described therein, in particular the Material and Methods section on pages 112-117; Example 1 on pages 117-118 and Example 7 on page 128 and the following Tables 1 to 14; and Example 17 on pages 168-171, the disclosure content of which is incorporated herein by reference.
In this context it is noted that though as mentioned in principle the generation of human antibodies has been reported for some antigen classes such as amyloid-beta and viral antigens, the provision of isolated and recombinant human anti-cytokine antibodies which matured in the human body does not seem to have been reported yet. Therefore, in accordance with the present invention human candidate anti-human cytokine antibodies are preferably cloned by a novel and proprietary method of isolating human antibodies, which is disclosed in applicant's co-pending international application WO2013/098420 Al, the disclosure content of which is incorporated herein by reference in its entirety.
Briefly, the sample for isolating the antibody of interest comprises or consists of peripheral blood mononuclear cells (PBMC) and serum for the detection of possible antibody reactivities. The sample derived from the subject may either be directly used for, e.g., testing seroreactivity against one or more of the desired antigen(s) or may be further processed, for example enriched for B lymphocytes. In particular, it is preferred that the sample comprises or is derived from B cells that produce the antibody of interest, most preferably memory B-cells. The memory B cells are cultured under conditions allowing only a definite life span of the B cells, typically no more than 1 to 2 weeks until singling out the cells from B cell cultures which are reactive against the desired antigen subsequently followed by RT-PCR of single sorted cells for obtaining the immunoglobulin gene repertoire; see for detailed description Examples 1 and 2 on pages 118 to 120 of WO2013/098419 Al and in particular Examples 1 to 4 on pages 27 to 31 as well as Figures 1 and 6 of WO2013/098420 Al, the disclosure content of which is incorporated herein by reference. Naturally, the present invention extends to the immortalized human B memory lymphocyte and B cell, respectively, that produces the antibody having the distinct and unique characteristics as defined herein above and below
Indeed, with respect to the use of patients suffering from an autoimmune and/or inflammatory disorder such as A.PECED patients it turned out that the method disclose in applicant's copending international, application WO2013/098419 Al and WO2013/098420 Al is particular suitable for isolating anti-human cytokine antibodies. Therefore, in one further preferred embodiment of the method of the present invention the candidate antibody or cytokine-binding fragment is provided by a method characterized by isolating B cells from short term, oligoclonal cultures of activated B cells that secrete antibodies of IgG isotype comprising the following steps in the sequence:
(a) selecting B cells that express antibodies against a human cytokine of interest from one or more biological, samples on the basis of the expression of at least one cell-surface membrane marker and/or antigen binding, and preferably wherein the cells are depleted from IgM and IgD isotypes;
(b) stimulating the selected cells with, a first polyclonal B cell activator under cell culture conditions;
(c) separating the cells from said activator;
(d) activating the stimulated cells with a second polyclonal B cell activator under cell culture conditions;
(e) screening the activated cells that express IgG isotype antibodies of interest and preferably;
(e') single-cell harvesting of oligoclonal cultures producing the antibody of interest;
(f) sequencing and/or cloning the cDNA of at least the variable light and heavy chai regions and optionally constant region of the antibody of interest.
The term "oligoclonal culture" refers to a culture of cells producing the antibody of interest derived from one or a few cells that have been activated. Preferably, the oligoclonal culture is derived from one single B cell, which may also be referred to as "B cell clone". As mentioned above, and unless stated otherwise, the terms "oligoclonal" and "clone" do not imply or refer to immortalized cells. As illustrated in the Examples, the biological sample is preferably derived from peripheral blood mononuclear cells of a patient whose serum has been screened for the presence of auto-antibodies against the cytokine of interest.
As further illustrated in. the Examples of international application WO2013/098420 Al the first polyclonal. B cell, activator is preferably Epstein-Barr virus (EBV) and/or the second polyclonal B cell activator is preferably a CpG-based oligonucleotide. Further preferred embodiments disclosed in international application WO2013/098420 Al are also preferably used in context with HuCytoMab-Assay of the present invention. Thus, in one embodiment the above-mentioned method of present invention is characterized in that
(i) the population of cells in step (a) are memory B cells and the cell surface marker is CD22;
(ii) the culture conditions in step (b) and/or step (d) do not comprise a cytokine;
(hi) in step (b) the selected cells are stimulated for about three to five hours;
(iv) in step (c) the activator is removed by diluting of or washing out;
(v) in step (d) the transferred new mentioned selected cells are exposed to the second polyclonal activator for about eight to fourteen days; and or
(vi) in step (d) and/or (e) the cells are cultured under oligoclonal. conditions with about ten. cells per well, in eight to fourteen days short term, cultures.
For details of those embodiments, see international, applicatio WO2013/098420 Al .
As illustrated in Figures 1 and 6 of international application WO2013/098420 A l , cDNA is preferably prepared from, single B cells sorted from, the short term, oligoclonal cultures of activated B cells secreting the antibody of interest in order to isolate and produce the monoclonal antibody of the present invention. Accordingly, the method of the present invention typically comprises the steps of:
(i) obtaining mR A from bulk or single B cells secreting the candidate antibody;
(ii) obtaining cDNA from the mRNA of step (i);
(iii) using a primer extension reaction to amplify from said cDNA the gene repertoire corresponding to the heavy chains (HC) and the light chains (LC) and optionally constant domain of the candidate antibody;
(iv) using said repertoire to express the candidate antibody or an cytokine-binding fragment thereof in a host cell, preferably animal cell;
(v) identifying the antibody sample presumably responsible for the reactivity of the parental B cell culture; and
(vi) isolating the monoclonal candidate antibody or a cytokine-binding fragment thereof;; optionally wherein the DNA is manipulated between steps (iii) and (iv) to introduce restriction sites, to change codon usage, introduce coding sequences for functional domains or peptide linkers; and/or to add or optimize transcription and/or translation regulatory sequences. RT-PCR of single sorted cells is preferably employed for obtaining the immunoglobulin gene repertoire for said antibody. A method of obtaining human antibodies using inter alia single cell RT-PCR is described for example in the international application WO2008/110372, the disclosure content of which is incorporated herein by reference, in particular the Supplementary Methods section and Example 2. As used herein, the terms "cDNA" and "mRNA" encompass all forms of nucleic acid, including but not limited to genomic DNA, cDNA, and mRNA. Cloning and heterologous expression of the antibody or antibody fragment can be performed using conventional techniques of molecular biology and recombinant DNA, which are within the skill of the art (Wrammert et al., Nature 453 (2008), 667-671 and Meijer et al, 2006 J. Mol. Bio. 358 (2006), 764-772). Such techniques are explained fully in the literature, for example in Sambrook, 1989 Molecular Cloning; A Laboratory Manual, Second Edition. For retrieval of VH/VL sequences and expression the method of Tiller et al., in J. Immunol. Methods 329 (2008), 112-124 can be used. Any appropriate host cell, for expressing the recombinant human antibody may be used, e.g., a yeast, a plant cell or an animal cell. Preferably, mammalian host cells such CHO cells and HE . cells are used; see also, e.g., European patent EP 1 974 020 B 1 in sections [0164] to [0171] the disclosure content of which is incorporated herein by reference.
In one embodiment the constant region of the antibody of the present invention or part thereof, in particular the CFI2 and/or CF13 domain but optionally also the CHI domain is heterologous to the variable region of the native human monoclonal, antibody isolated in accordance with the method of the present invention. In this context, the heterologous constant region(s) are preferably of human origin, in case of therapeutic applications of the antibody of the present invention but could also be of for example rodent origin in case of animal studies.
Regarding the huma cytokine used in the HuCytoMab-Assay of the present invention, in principle any human cytokine and related protein, may be used; see also Table 1 below.
Cytokine
Chemokines Interferons beta-Thromboglobulin Interferon Type I
Chemokines, C Interferon Type I, Recombinant:
Chemokines, CC: Interferon Alfa -2 a
Chemokine CCL1
Interferon Alfa-2b
Chemokine CCL3
Chemokine CCL4 Interferon AIfa-2e
Chemokine CCL5 In terferon-alpha:
Chemokine CCL11 Interferon Al a- ■1
Chemokine CCLl 7 Interferon Alfa- ■2a
Chemokine CCL19 Interferon Aifa- ■2b
Chemokine CCL20 Interferon Alfa- ■2c
Chemokine CCL21
Chemokine CCL22 Interferon Alfa-4
Chemokine CCL24 Interferon Alfa- ■5
Chemokine CCL27 Interferon Alta- ■6
Monocyte Chemoattractant Proteins: Interferon Alfa- ■7
Chemokine CCL2 Interferon Alfa- ■8
Chemokine CCL7 Interferon Alfa- ■10
Chemokine CCL8 Interferon Alfa- ■13
Chemokines, CXC:
Chemokine CXCLI Interferon Alfa- ■14 Chemokine CXCL2 Interferon Alfa- ■16 Chemokine CXCL5 Interferon Alfa- ■17 Chemokine CXCL6 Interferon Alfa- ■21 Chemokine CXCL9 Interferon-beta
Chemokine CXCL10 Interfero -kappa
Chemokine CXCL I I In terferon-omega
Chemokine CXCLI 2
Chemokine CXCL13 Interferon-gumma
Interleukin-8 In terferon -gam m a, Recombin ant Platelet Factor 4
Chemokines, CX3C: Growth Differentiation Factor 15
Chemokine CX3CL1
Chemokines, XCL:
Chemokine XCLl
Chemokine XCL2
Macrophage Inflammatory Proteins:
Chemokine CCL3
Figure imgf000028_0001
Chemokine CC.L4
Chemokine CCLl 9 Oncostatin M
Chemokine CCL20
Chemokine CXCL2
ontin Cytokine
Intei eukins Lymphokines
Interleukin 1 Receptor Antagonist Interferon-gamnta
Protein Interleukin-!
Interleukin- 1 : Leukocyte Migration-Inhibitory
Interleukin- 1 alpha Factors
Interleukin- 1 beta Lymphotoxin-alpha
Interleukin -2 Macrophage-A ctivating Factors:
Interleukin-3 Interferon-gamma
Interl.eukin-4 Macrophage Migration-Inhibitory
Interleukin- 5 Factors
Interleukin- 6 Neuroleukin
Interleukin-7 Suppressor Factors, Immunologic
Interleukin-8 Transfer Factor
Interleukin-9
Interleukin- 10
Interleukin- 11
Interleukin- 12:
Interleukin- 1 2 Subunitp35
Interleukin- 12 Subunit p40 Monokines
Interleukin- 13 Interleukin- 1
Interleukin- 15 In terleukin-1 alpha
Interleukin- 16 In terleakin- 1 beta
Interleukin- 1 7
Interleukin- 18
Interl.eukin-23:
Interleukin- 12 Subunit p40
Interleukin-23 Subunit p 19
Interleuki -26
Interleukin- 27 Transforming Growth Factor beta
Interleukin- 28 Transforming Growth Factor betal
Interleukin-29 Transforming Growth Factor beta!
Interleukin- 30 Transforming Growth Factor betaS
Interleukin-31
Interleukin- 32
Interleukin- 34
Interleukin- 35
Interleukin- 37 Cytokines
Tumor Necrosis Factor
Antigens, CD70
B-Cell Activating Factor
4-1 BB Ligand
CD30 Ltgand
CD40 Ligand
Ectodysplasins
Fas Ligand Protei
Lympho toxin-alpha
Lymphotoxin alpha 1, beta.2 Heterotrimer
Lymphotoxin-beta
OX40 Ligand
RANK Ligand
TNF-Related Apoptosis-Inducing Ligand
Tumor Necrosis Factor-alpha
Tumor Necrosis Factor Ligand Superfamily Member 13 Tumor Necrosis Factor Ligand Superfamily Member 14 Tumor Necrosis Factor Ligand Superfamily Member 15
Table 1: List of cytokines for use in the HuCytoMab- Assay of the present invention.
Preferably, the human cytokine is a pro-inflammatory cytokine capable of inducing an inflammatory response at the local side when exogenously administered to non-human, mammal, preferably mice. In one embodiment, the huma cytokine is selected from the group consisting of leukotrienes, lymphokines, interleukins, interferons, chemokines and members of the TNF family. in one embodiment, the present invention does not include the use of candidate human anti- interleukin- 17 antibodies isolated from A PEC ED patients as disclosed in applicant's copending international application WO2013/098419 Al, the disclosure content of which is incorporated herein by reference, particularly as described in Example 13 and Figures 40 and 41, wherein in a method of the present invention the local inflammatory phenotype is induced in the ear of mice. The isolated antibodies of the present invention may of course not be applied as such to a patient, but usually have to be pharmaceutically formulated to ensure, e.g., their stability, acceptability and bioavailability in the patient. Therefore, in one embodiment, the method of the present invention further comprises the step of admixing the isolated and validated candidate antibody or cytokine-binding fragment thereof with a pharmaceutically acceptable carrier. A thorough discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical. Sciences (Mack Publishing Company, N.J. 1991) and in Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472. Preferred forms for administration include forms suitable for parenteral, administration, e.g. by injection or infusion, for example by bolus injection or continuous infusion. Where the product is for injection, or infusion, it may take the form, of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain agents commonly used in pharmaceutical formulations, such as suspending, preservative, stabilizing and/or dispersing agents. Alternatively, the antibody molecule may be in dry form, for reeonstit utio before use with. an. appropriate sterile liquid. Once formulated, the compositions can. be administered directly to the subject. It is preferred that the compositions are adapted for administration to huma subjects. The pharmaceutical compositions may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intraperitoneal, intrathecal, intraventricular, transdermal, transcutaneous, topical, subcutaneous, intranasal, enteral, sublingual, intra va inal or rectal, routes.
The antibodies of the present invention or fragments thereof may be directly used as a therapeutic agent. However, in one embodiment the huma anti-human cytokine antibody or cytokine binding fragment thereof which is provided by the present invention, is detectably labeled or attached to a drug, for example wherein the detectable label, is selected from, the group consisting of an enzyme, a radioisotope, a fluorophore and a heavy metal. Labeled human, anti-human cytokine antibody or cytokine binding fragment of the present invention, may be used to detect specific targets in vivo r i vitro including "immunochemistry/immunolabelling" like assays in. vitro. In vivo they may be used in a manner similar to nuclear medicine imaging techniques to detect tissues, cells, or other material expressing the antigen of interest. Labels, their use in diagnostics and their coupling to the human cytokine binding molecules of the present inventio are known to the person skilled in. the art. In this context, in one embodiment the term pharmaceutical use includes diagnostic use in vivo, in particular in. vivo imaging in human. Hence, the present invention also relates to a method of preparing a human anti-human cytokine antibody or cytokine binding fragment thereof for pharmaceutical use or as target for therapeutic intervention in the treatment of any one of the above-identified autoimmune and/or inflammatory disorders and diseases, comprising the steps of any of the above- described methods of the present invention, optionally wherein the human anti-huma cytokine antibody or cytokine binding fragment thereof is detectably labeled or attached to a functional domain or drug, preferably wherein the detectable label, is selected from, the group consisting of an. enzyme, radioisotope, a fluorophore and a heavy metal
EXAMPLES
The Examples which follow and corresponding Figures further illustrate the invention, but should not be construed to limit the scope of the invention in any way. Detailed descriptions of conventional methods, such as those employed herein can be found in the cited literature; see also "The Merck Manual of Diagnosis and Therapy" Seventeenth Ed. ed. by Beers and Berkow (Merck & Co., Inc., 2003).
Example 1: Providing candidate anti-human cytokine antibody
As mentioned in. the preceding description, i principle any anti-cytokine antibody may be isolated, produced and assessed, respectively, in the HuCytoMab- Assay of the present invention. For example, human-like anti-cytokine antibodies Adalimumab (Humira®; Abbott) and Golirnumab (Simponi®; Johnson. & Johnson.) against TNFalpha, Canakinumab (Ilaris®; Novartis) against 11 - l b or Ustekinumab (Stelara®; Johnson & Johnson) against IL-12 and IL- 23 or Sifalimumab (Medimmune), an. anti-INFa antibody may be used as a control.
However, as mentioned in the preceding description, rather than, human-like or humanized antibodies preferably patient-derived human, anti-human cytokine antibodies are used as candidate antibody which, can be isolated as described in the international, applications WO2013/098419 Al and WO2013/098420 Al as briefly illustrated for anti-human IL-32 antibodies. Patients selection, peripheral blood mononuclear cells (PBMC) isolation from APECED/APS1 Patients memory, B cell culture and antibody isolation were carried out as described in the international applications WO2013/098419 Al and WO2013/098420 Al with the difference that specificity of the antibodies isolated and analyzed was directed towards IL- 32 isotypes as defined hereinabove and below instead of IL-17 and IL-22, which were specifically used in the mentioned PCT applications; see Examples sections therein, in particular Examples 1 and 2 on pages 117 to 120 and Example 17 on pages 168-171 of WO2013/098419 Al and Examples 1 to 4 on pages 27 to 31 of WO2013/098420 Al, the disclosure content of which is incorporated herein by reference.
The molecular cloning of human antibodies of the present invention and subsequent antibody production and purification were performed as described in the international application WO2013/098419 Al ; see the Examples section of the application and in particular Examples 1 to 3 on pages 117-120 therein, the disclosure content of which is incorporated herein by reference.
For a more detailed description of exemplary anti-IL-32 antibodies see applicant's copending international application PCT/EP2014/064163 "Human anti-IL-32 antibodies" (Attorney's docket no. IM11A07/P-WO) filed on July 03, 2014, the disclosure content which is incorporated herein by reference, in particular Examples 1 to 6, Table 1 and Figure 1 disclosing the nucleotide and amino acid sequences of the variable and constant regions (VH, VL, CH, CL) regions of IgG3, lambda, IL-32 specific 2C2 antibody and of IgGl , lambda, IL-32 specific 14B3, 19A1 and 26A6 antibodies of the invention subject of this application as well as their functional characterization including source of IL-32 cytokine.
Example 2: HuCytoMab- Assay for providing human anti-human IL-32 antibodies for pharmaceutical use
Ear inflammation assay
Ear inflammation phenotype was induced in 8 weeks old C57BL/6J (WT; from Charles River) mice by intradermal injection of human cytokine IL-32y, or IgG control in 20μ1 of PBS (or PBS control) into each ear given on alternate days at Day 1, Day 4, Day 6 and Day 8 (20μl/ear, 125ng/ear, 250ng/mouse/day) using a 30-gauge needle. Treatment with the exemplary anti-IL-32 2C2 antibody was tested on these animals in respect of its neutralizing potential to reduce the induced ear inflammation phenotype. Only one IP injection of 2C2 or control human IgG [200μg, 100μg or 50μg/IP] was administered to the animals at Day 0, prior to induction of ear inflammation. The mice were sacrificed at day 11.
To test a potential therapeutic effect of the antibodies of the present invention ear thickness measurements of the animals were taken with a Mitutoyo digital micrometer during the IL-32 administration by daily measurements prior to IL-32 injection. Furthermore, body weight has been monitored during the treatment, however, no significant weight changes have been observed in any of the animal groups due to the treatment applied; see Fig. 3. In addition, after sacrifice of the animals H&E (hematoxylin and eosin; see Harris, H.F., J. Appl. Microscopy III (1900), 777-781 and Mallory, F.B.: Pathological technique. Philadelphia, Saunders, (1938)) histology stainings of the ears are performed.
Combination of two independent experiments shows that the induction of ear swelling with intradermal injection of human IL-32y is reduced in the presence of 2C2 neutralizing antibody; see Figs. 1 and 2. This is significant at Day 9 for the lower antibody doses of 100 μg, respective 50 μg/IP; see Fig. 1C, D and Fig. 2C, D. For the highest dosage of 200 μg/IP, significant reduction of the ear swelling can be even observed starting with day 5; see Fig. IB and Fig. 2B. The level of ear swelling following the continuous intradermal injection of PBS control is not affected by the presence of IgG or 2C2; see Fig. 2A-D.
Exemplary anti-IL-32 antibody 2C2 demonstrates dose dependent effects and is able to neutralize the injected IL-32y in the HuCytoMab-Assay. Furthermore, as shown in Figs. 11 and 12 antibody 19A1 effectively neutralizes hIL-32y induced inflammation in comparison to antibody 2C2 in the CytoEar assay.Accordingly, the data presented herein indicates that the anti-human IL-32 antibody is effective against IL-32y in cytokine induced ear inflammation experiments, demonstrating the therapeutic value of the IL-32 specific binding molecule.
Cy to Ankle assay:
Despite the fact that no mouse homologue of IL-32 has been identified yet, there are hints, as for example the induced ear swelling phenotype as described supra, that at least some members of the IL-32 pathway are present in the mice as well. Furthermore, Joosten et al. (2006) have injected human IL-32 into the knee joints of mice, which led to the induction of joint swelling, and used such experiments as a model for RA. Such experiments have been also performed in connection with the present invention, however, the animals have demonstrated no measurable swelling after IL-32 injections, which might be due to the difficulty of measuring knee thickness through intervening muscle tissue. Due to this fact the present invention established a novel assay to test the effects of IL-32 in mice and in particular of the exemplary anti-human IL-32 antibodies, as described below.
In this assay mice cohorts (C57/BL6, 7-8weeks) are intraarticular (IA) injected with 62.5- 250ng cytokine, e.g., an IL-32 isotype such as IL32y or IL-32a or mixtures of several IL-32 isotypes in lOul of PBS (or PBS control) into ankles every 48-72 hours. Axial ankle thickness measurements are than taken with a Mitutoyo digital micrometer. Animals are weighed each day and respective IL-32 isotype or isotypes are administered while the mice are anaesthetized with isofluorane. The experimental time frame is designed as indicated above for the ear inflammation assay, with injections of the anti-IL-32 antibody, respective the control groups obtaining either PBS or human IgGs of IL-32 non-related binding specificity as indicated above. Reduction of the ankle swelling is used as a readout of the therapeutic effect of the antibodies.
In addition the weight of the anti-IL32 antibody treated and of the control animals is monitored during the treatment, and after sacrifice of the animals H&E (hematoxylin and eosin) histology stainings of the ears are performed.
Fig. 13 shows an exemplary experimental set up of the Cyto Ankle assay (Fig. 13 A, B) and the dose dependency of IL-32 in inducing inflammation in the CytoAnkle assay (Fig. 13 C, D). As further shown in Fig 14, the anti-IL-32 inflammatory effect of the 2C2 antibody could be confirmed in the CytoAnkle assay (Fig. 14 C-E).
Example 3: HuCytoMab- Assay for providing human anti-human IFN-alpha antibodies for pharmaceutical use
As another example, human anti-human IFN-alpha (IFNA) antibodies have been isolated and proved in the HuCytoMab- Assay of the present invention to be suitable in immunotherapy of cytokine mediated disorders. For a detailed description of exemplary anti-IFN-alpha antibodies and IFNA subtypes and molecules see applicant's co-pending international application PCT/EP2014/064167 "Human anti-IFN-alpha antibodies" (Attorney's docket no. IM11A06/P-WO) filed on July 03, 2014, the disclosure content which is incorporated herein by reference, in particular Examples 1 to 9, Table 1 and Figure 1 disclosing the nucleotide and amino acid sequences of the variable and constant regions (VH, VL, CH, CL) regions of IgGl, kappa, IFNA, e.g. IFNA2, -4 and-14 recognizing antibodies 5D1, 13B11, 19D11 , 25C3, 26B9 and 31B4 subject of this EP application as well as their functional characterization including sources of IFNA subtype molecules.
Ear inflammation assay
First, the effect of different IFN-a subtypes was tested in vivo for the induction of ear inflammation in mice., in particular, the proinflammatory activity of human subtypes IFNA2a, IFNA2b, IFNA4 and IFNA14. Ear inflammation phenotype was induced in 8 weeks old C57BL/6J (WT; from Charles River) mice by intradermal injection of human IFNA2a, IFNA2b, IFNA4 and IFNA14 in 20 μΐ of PBS, or PBS control into each ear given on alternate days at Day 0, Day 2 and Day 4 (20 μΐ/ear, 500 ng/ear, 1 μg total/mouse/day) using a 30-gauge needle. The mice were sacrificed at day 6; see Table 5 below and Fig. 4A for the experimental timeline.
Figure imgf000036_0001
Table 5: Group allocation of animals to the different cytokines tested, n - number of animals in the group, ng/20 μΐ - amount of cytokine injected per ear.
To test the proinflammatory effect of the injected IFNA subtypes ear thickness measurements of the animals were taken with a Mitutoyo digital micrometer during the cytokine administration by 2 measurements per ear prior to cytokine injection at Day 0 and at alternate days at Day 1 , Day 3, Day 5 (indicated by letter M in Fig. 4A) and alternatively or in addition at Day 6 after sacrifice of the animal.
Furthermore, body weight is monitored during the treatment, to observe any possible weight changes due to the inflammation induction or its respective reduction due to the treatment applied. In addition, after sacrifice of the animals H&E ((hematoxylin and eosin; see Harris, H.F., J. Appl. Microscopy III (1900), 777-781 and Mallory, F.B.: Pathological technique. Philadelphia, Saunders, 1938.) histology stainings of the ears are performed.
All four human IFNA subtypes tested were able to significantly induce ear swelling following cytokine injection; see Figs. 4-6 and results of the experiment summarized in the table in Fig. 7. All ears were markedly thicker than PBS treated ears, this was significant in all groups after the 2nd intradermal injection, from Day 3 until the end of the experiment. IFNA14 was the most potent especially at Day 5 in this experiment. IFNA2a and IFNA4 induced similar levels of ear thickening. IFNA2b was most similar to both IFNA4 and IFNA 14 induced swelling. IFNA2b induced swelling more than the IFNA2a isoform in this experiment; see Figs. 4-6 and the experimental results summary in the table in Fig. 7. The results of this experiment show the applicability of the ear inflammation assay for tests of the therapeutic applicability of the antibodies of the present invention. Since the exemplary anti-IFN-a antibodies 19D11, 26B9, 31B4, 5D1 and 13B11 did not show any apparent cross- reaction with at least murine IFN-a subtypes 2, 4 and 14, they are tested in the HuCytoMab- Assay of the present invention in respect of their neutralization properties towards human IFN-a used for induction of inflammation. Apparent binding affinity of exemplary anti-IFN- α antibody 25C3 towards murine INFA2 and the affinity of exemplary anti-IFN-a antibodies 5D1 and 19D11 towards murine IFNA1 is taken into consideration when designing the in vivo Ear neutralization experiments described herein. The tests are performed with the exemplary anti-IFN-α antibodies by injection of the antibodies at different time points during the above drafted experimental timeline (see also Fig. 4A) for testing induction of inflammation by human IFN-a. For example, for testing the preventive and/or therapeutic effect one or more of the exemplary antibodies are injected together with or separately to the IFNA subtype or subtypes at Day 0 of the experiment. In addition or alternatively, one or more of the antibodies are injected on alternate days with the IFNA subtype or subtypes. For example, if the IFNA subtype or subtypes are injected as indicated above at Days 0, 2 and 4 (short arrows in Fig. 4A), the antibodies are injected on the alternate Days 1 , 3 and/or 5 (long arrows in Fig. 4A).
The IFNA neutralizing potential of the antibodies reduce the induced ear inflammation phenotype and/or prevent such an induction which is examined by comparison of ear swelling (thickness) observed in animals obtaining the anti-IFN-α antibody treatment and the control groups obtaining either PBS or human IgGs of a binding specificity directed towards other molecules than human IFNA subtypes (of IFN-a non-related binding specificity).
Furthermore, or alternatively body weight is monitored during the treatment, to observe any possible weight changes due to the inflammation induction or its respective reduction due to the treatment applied. In addition, after sacrifice of the animals H&E (hematoxylin and eosin; see supra) histology stainings of the ears are performed. This assay is used preferably as a surrogate model psoriasis.
In a second experimental round, the above indicated assay has been used with some modifications to test neutralization properties of exemplary antibodies 26B9 and 19D1 1 towards inflammation induced in mice ears by injections of human IFNA14 (Fig. 8), IFNA5 (Fig. 9) and IFNW (Fig. 10). As may be seen from the time schemes shown in Figs. 8A, 9A and 10A the experimental time line has been prolonged here to 10 days with tested antibodies and controls injected at experimental day 0 (IP), intradermal cytokine injections at days 1 , 3, 6 and 8 and a sacrifice of the test animals at day 10. Group allocations of animals to the different cytokines tested and the respective concentrations and amounts of the cytokines, respective antibodies tested are indicated in tables in panels B of the respective figures. Both antibodies, 26B9 and 19D11 have shown pronounced preventive and/or therapeutic potential due to a significant reduction of the ear thickness on several experimental days after IFNA14 (Figs. 8 D and E) and IFNA5 (Figs. 9 D and E) induced ear inflammation in comparison to IgG controls. Treatment with the reference IFN-a specific antibody (Ref. A in Figs. 8, 9, in particular Figs. 8F and 9F) led also to a significant reduction of ear swelling after IFNA14 treatment on several experimental days, however with a slightly lower reduction at day 10 compared with antibodies 26B9 and 19D11 of the present invention (compare curves F for 26B9 in Fig. 8D and G for 19D11 in Fig. 8E with curve H for Ref. A in Fig. 8F and with curve E in each Fig. for the IgG-control). Furthermore, exemplary antibody 26B9 has shown a significant reduction of IFNW induced ear swelling at experimental day 9 (Fig. 10D), wherein injections of antibody 19D11 and Ref. A did not show any significant reduction of the ear swelling compared to non-specific IgG treatment. Accordingly, antibodies provided with the present invention have a high potential for use in prevention and/or treatment of diseases associated with enhanced IFNA and/or IFNW activity.
Cy to Ankle assay:
In this assay mice cohorts (C57/BL6, 7-8weeks) are intraarticular (IA) injected with 62.5- 1000 ng cytokine, e.g., at least one IFNA subtype such as IFNA2a, IFNA2b, IFNA4 or IFNA14 or mixtures of several IFNA subtypes in 10 μΐ of PBS (or PBS control) into ankles every 48-72 hours. Axial ankle thickness measurements are than taken with a Mitutoyo digital micrometer. Animals are weighed each day and respective IFNA subtype or subtypes are administered while the mice are anaesthetized with isofluorane. The experimental time frame is designed as indicated above for the ear inflammation assay, with injections of the anti-IFN- α antibody respective the control groups obtaining either PBS or human IgGs of IFN-a non- related binding specificity as indicated above. Reduction of the ankle swelling is used as readout of the therapeutic effect of the antibodies of the present invention. This assay is used preferably as a surrogate model for arthritis, e.g., rheumatoid arthritis. Therapeutic effect of the exemplary anti-IFNA antibodies is further confirmed by preliminary data obtained from monitoring the weight of candidate antibody treated animals showing nearly no or at least greatly reduced weight loss after inflammation induction by the cytokine.
Example 4: HuCytoMab-Assay for providing human anti-human IL-20 antibodies for pharmaceutical use
As another example, human anti-human 11-20 antibodies have been isolated and proved in the HuCytoMab-Assay of the present invention to be suitable in immunotherapy of cytokine mediated disorders. For a detailed description of exemplary anti-IL-20 antibodies see applicant's co-pending European patent application EP 14 175 585.0 "Human-derived anti- human IL-20 antibodies" (Attorney's docket no. IM11A11/P-EP) filed on July 03, 2014, the disclosure content which is incorporated herein by reference, in particular Examples 1 to 8, Table 1 and Figure 1 disclosing the nucleotide and amino acid sequences of the variable and constant regions (VH, VL, CH, CL) regions of IgG4, lambda, IL-20 specific 20A10 antibody and of IgGl, lambda, IL-20 specific 2A11, 7D1 and 6E11 antibodies subject of this EP application as well as their functional characterization including sources of IL-20 molecules.
When testing anti-Il-20 antibodies in concern of their neutralizing activity towards human IL- 20 in an animal disease model, for example mice, it has to be ensured that human IL-20 induces diseased phenotypes in mice and that no cross-reaction occurs between the tested IL- 20 antibodies and the murine IL-20 homologues. Since no adequate model system for IL-20 was available in the prior art, the present Example describes and provides such a system to test IL-20 neutralizing antibodies that do not cross react with mouse IL-20.
Ear inflammation assay
Ear inflammation phenotype was induced in 8 weeks old C57BL/6J (WT; from Charles River) mice by intradermal injection of human cytokine IL-20 or PBS control into each ear given on alternate days at Day 1, Day 3, Day 6 and Day 8 (20 μΐ/ear, lOOOng/ear, 2000 ng/mouse/day) using a 30-gauge needle. Treatment with the exemplary anti-IL-20 2A11, 7D1 and 20A10 antibodies were tested on these animals in respect of their neutralizing potential to reduce the induced ear inflammation phenotype. Two IP injections of 2A11, 7D1 and 20A10 or control human IgG [200 μg,] were administered to the animals at day 0 and day 6. The mice were sacrificed at day 10. To test a potential therapeutic effect of the antibodies of the present invention ear thickness measurements of the animals were taken with a Mitutoyo digital micrometer during the IL-20 administration by daily measurements prior to IL-20 injection. Furthermore, body weight has been monitored during the treatment, however, no significant weight changes have been observed in any of the animal groups due to the treatment applied. In addition, after sacrifice of the animals H&E (hematoxylin and eosin; see Harris, H.F., J. Appl. Microscopy III (1900), 777-781 and Mallory, F.B.: Pathological technique. Philadelphia, Saunders, (1938)) histology stainings of the ears are performed.
These experiments show that the induction of ear swelling with intradermal injection of human IL-20 is reduced in the presence of 2 Al l, 7D1 and 20A10 neutralizing antibody; see Fig. 15. This is significant from Day 6 onwards to varying degrees of statistical significance. Exemplary anti-IL-20 antibodies 2A11, 7D1 and 20A10 are able to neutralize the injected IL- 20 in a mouse model. Accordingly, the data presented herein indicates that the anti-IL-20 antibodies are effective against IL-20 in cytokine induced ear inflammation experiments, demonstrating their therapeutic value.

Claims

Claims
1. A method of providing an anti-human cytokine antibody or cytokine-binding fragment thereof for pharmaceutical use, the method being characterized by
(a) subjecting a candidate anti- human cytokine antibody or cytokine-binding fragment thereof to a non-human mammal in which
(i) a local inflammatory phenotype; preferably swelling of ear, knee, joint or ankle
(ii) is induced by a predetermined human cytokine, preferably by intradermal, intraarticularly or periarticular injection of the human cytokine;
wherein
(b) a reduction of the induced inflammatory phenotype compared to a control indicates that the candidate anti-human cytokine antibody or cytokine-binding fragment thereof is suitable for the pharmaceutical use.
2. The method of claim 1, wherein the candidate antibody or cytokine-binding fragment does not cross-react with the non-human mammalian homologue of the human cytokine and/or the non-human mammal lacks a homologue of the human cytokine.
3. The method of claim 1 or 2, wherein candidate antibody or cytokine-binding fragment is subjected prior, concomitantly and/or after induction of the inflammatory phenotype.
4. The method of any one of claims 1 to 3, wherein the candidate antibody or cytokine- binding fragment is provided by a method comprising isolating a monoclonal antibody or antigen-binding fragment thereof, wherein a B cell expressing the monoclonal antibody is isolated from a sample obtained from a subject mammal.
5. The method of claim 4, wherein the subject mammal is suffering from an autoimmune and/or inflammatory disease.
6. The method of claim. 4 and 5, wherein the subject mammal is affected with a disorder related to an impaired central and/or peripheral, tolerance or loss of self-tolerance, preferably wherein, the disorder is autoimmune pol.yen.docrinopathy-candidiasi.s- ectodermal dystrophy (APECED).
7. The method of any one of claims 1 to 6, wherein the non-human mammal, is a mouse and or the subject mammal is a human.
8. The method of any one of claims 1 to 7, wherein the candidate antibody or cytokine- binding fragment is provided by method characterized by isolating B cells from short term oligoclonal cultures of activated B cells that secrete antibodies of IgG isotype comprising the following steps in the sequence:
(a) selecting B cells that express antibodies against a human cytokine of interest from one or more biological samples on the basis of the expression of at least one cell- surface membrane marker and/or antigen binding, and preferably wherein the cells are depleted from IgM and IgD isotypes;
(b) stimulating the selected cells with a first polyclonal B cell activator under cell culture conditions;
(c) separating the cells from said activator;
(d) activating the stimulated cells with a second polyclonal B cell activator under cell culture conditions;
(e) screening the activated cells that express IgG isotype antibodies of interest and preferably;
(e') single-cell harvesting of oligoclonal cultures producing the antibody of interest;
(f) sequencing and/or cloning the cDNA of at least the variable light and heavy chain regions and optionally constant region of the antibody of interest.
9. The method of any one of claims 4 to 8, wherein the biological sample is derived from subject mammal sera screened for the presence of auto-antibodies against the human cytokine.
10. The method of claim. 8 or 9, wherein, the first polyclonal B cell, activator is Epstein-Barr vims (EBV) and/or the second polyclonal B cell activator is a CpG-based oligonucleotide.
11. The method of any one of claims 8 to 10, wherein
(i) the population of cells in step (a) are memory B cells and the cell surface marker is CD22;
(ii) the culture conditions in step (b) and/or step (d) do not comprise a cytokine;
(iii) in. step (b) the selected cells are stimulated for about three to five hours; (iv) in step (c) the activator is removed by diluting of or washing out;
(v) in step (d) the transferred new mentioned selected ceils are exposed to the second polyclonal activator for about eight to fourteen days; and/or
(vi) in step (d) and/or (e) the cells are cultured under oligoclonal. conditions with about ten cells per well in eight to fourteen days short term cultures.
The method of any one of claims 1 to 11, wherein the candidate antibody or cytokine- binding fragment is provided by a step comprising
(i) obtaining mR A from bulk or single B cells secreting the candidate antibody;
(ii) obtaining cDNA from the mRNA of step (i);
(iii) using a primer extension reaction to amplify from said cDNA the gene repertoire corresponding to the heavy chains (HC) and the light chains (LC) and optionally constant domain of the candidate antibody;
(iv) using said repertoire to express the candidate antibody or an cytokine- binding fragment thereof in a host cell, preferably animal cell;
(v) identifying the antibody clone presumably responsible for the reactivity of the parental B cell culture; and
(vi) isolating the monoclonal candidate antibody or a cytokine-binding fragment thereof;
optionally wherein the DNA is manipulated between steps (iii) and (iv) to introduce restriction sites, to change codon usage, introduce coding sequences for functional domains or peptide linkers; and/or to add or optimize transcription and/or translation regulatory sequences.
The method of any one of claims 1 to 12, wherein the human cytokine is selected from the group consisting of leukotrienes, lymphokines, interleukins, interferons and chemokines.
The method of any one of claims 1 to 13 further comprising the step of admixing the isolated candidate antibody or cytokine-binding fragment thereof with a pharmaceutically acceptable carrier.
A method for preparing a human anti-human cytokine antibody or cytokine binding fragment thereof for pharmaceutical use or as a target for therapeutic intervention in the treatment of an autoimmune and/or inflammatory disorder, comprising the steps of the method of any one of claims 1 to 14, optionally wherein the human anti-human cytokine antibody or cytokine binding fragment thereof is detectably labeled or attached to a functional domain or drug, preferably wherein the detectable label is selected from the group consisting of an enzyme, radioisotope, a fluorophore and a heavy metal.
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