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WO2012110596A1 - Fusion protein for the treatment of immunologic or allergic reactions - Google Patents

Fusion protein for the treatment of immunologic or allergic reactions Download PDF

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Publication number
WO2012110596A1
WO2012110596A1 PCT/EP2012/052681 EP2012052681W WO2012110596A1 WO 2012110596 A1 WO2012110596 A1 WO 2012110596A1 EP 2012052681 W EP2012052681 W EP 2012052681W WO 2012110596 A1 WO2012110596 A1 WO 2012110596A1
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fusion protein
patient
dcs
antigen
toxin
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PCT/EP2012/052681
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French (fr)
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Gerold Schuler
Andreas Baur
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Friedrich-Alexander-Universität Erlangen-Nürnberg
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Publication of WO2012110596A1 publication Critical patent/WO2012110596A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A61K2039/55527—Interleukins
    • A61K2039/55533—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55544—Bacterial toxins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin

Definitions

  • the present invention relates to the field of human medicine, namely the treatment of undesirable immunological or allergic reactions.
  • Dendritic cells are considered the organizers of the immune system. In principle, they can mediate two reactions. On the one hand, they organize the immune defense for the control and elimination of microbes and foreign structures/cells, such as cancer cells. On the other hand, DCs prevent this very immune defense from attacking endogenous cells and structures. The latter is also referred to as induction or maintenance of tolerance or briefly tolerization. If tolerance is broken, the result is graft rejections, allergic reactions or autoimmune diseases with the known chronic complaints, for example, of rheumatoid arthritis. Hardly any therapies or therapy regimens for the induction of tolerance, for example, towards particular cell structures or antigens, have been developed to date.
  • DAB 389 IL-2 Denileukin Difitox
  • Ontak SEQ ID NO : l
  • DAB 389 IL-2 is a medicament approved in the U.S.A. that has been employed for the treatment of cutaneous T-cell lymphomas (CTCL) for more than 10 years (i.v. application). It is supplied by the Japanese pharmaceutical company Eisai or Cephalon. It is a recombinant fusion protein produced in E. coli and consisting of a fusion between fragments A and B of diphtheria toxin as well as human interleukin 2 (IL-2). In addition to its application for CTCL, the medicament was employed in experimental approaches for the depletion of regulatory T cells in humans.
  • CCL cutaneous T-cell lymphomas
  • DCs dendritic cells
  • the underlying molecular mechanism could be elucidated. It involves the upregulation of particular tolerogenic genes. This effect can be used therapeutically, for example, in transplantation medicine to avoid graft rejections, or for desensitization by means of adendritic cell (DC) vaccine. Further, the medicament can be applied therapeutically for immune suppression.
  • DC dendritic cell
  • a bacterial toxin domain comprising a bacterial toxin that lacks the cellular receptor binding sequence of the toxin
  • a cellular binding domain comprising a cellular binding protein that is different from the native binding sequence of the toxin and that targets the fusion protein to mammalian cells
  • DC dendritic cells
  • the bacterial toxin domain comprises fragments A and B of diphtheria toxin
  • the cellular binding domain comprises human interleukin 2 (IL-2) or a fragment thereof;
  • DCs dendritic cells
  • a fusion protein as defined in (1) to (3) above for use in the treatment of immunological diseases, including autoimmune diseases, that are induced and/or maintained by a DC-T cell interaction that causes detrimental T cell proliferation/ activation and immunopathology;
  • a method for treating immunological diseases including autoimmune diseases that are induced and/or maintained by a DC-T cell interaction that causes detrimental T cell proliferation/activation and immunopathology in a patient, which method comprises administering the patient a suitable amount of the fusion protein as defined in (1) to (3) above; (7) a method for generating tolerogenic antigen-loaded DCs ex vivo, which comprised loading DCs with the fusion protein as defined in (1) to (3) above;
  • Fig. 1 Expression of surface molecule on day 10 mBMDC after ONTAK (48 h) plus LPS.
  • ONTAK was present during DC-T cell co-culture and inhibits the proliferation of murine T cells in allo-MLR.
  • Fig. 3 ONTAK was present during DC cell maturation (48 h) and inhibits DC mediated T cell proliferation of murine T cells in allo-MLR.
  • ONTAK reduced paralyses symptoms associated with EAE.
  • ONTAK (1 g/animal) was injected i.p. at day -1, 1, 3, EAE was induced at day 0. At day 30 a second EAE was induced.
  • Fig. 5 EAE was induced on day 0, at peak of disease (day 15) mice were divided in two groups stage (4/4/3), one group was injected with ONTAK five times i .p. from day 15 to day 19, the other group was left untreated.
  • DD-treatment induces increased expression of Stat3, ⁇ -catenin and CIITA in PBMC and skin tissue.
  • Protein expression was assessed in patient ' s PBMC by FACS analysis and in tissue by the robot-automated multi-epitope ligand cartography (MELC) technique (Schubert, W. et al., Nat. Biotechnol . 24, 1270- 1278 (2006)).
  • MELC robot-automated multi-epitope ligand cartography
  • £bl maDC were prepared from healthy donors and treated with DD (0, 10 and lOOng/ml) for 24 h and subsequently stained for intracellular Stat3 by FACS. Results for one representative donor are shown, (c) Tissue sections of healthy skin obtained from patient #19 (see results for patient #21 in Fig. 8) on day 0 and day 4 were stained side-by-side using MELC robots and the indicated antibodies.
  • White arrows depict epidermal Langerhans cells expressing CIITA. Dark arrows indicate co-localization of ⁇ -catenin and CDla, respective ⁇ -catenin expression in Langerhans cells.
  • Fig. 7 Sampling and processing scheme for DC mRNA array analysis, (a ⁇ DC and monocytes were obtained from patients # 19 and #21 before and after (dark arrows) two administrations of DD (black arrows). The blue lines describe the mDCl cell count (in %) with respect to original levels at the time of sampling. (b) ImDC were generated from monocytes of a healthy donor and matured by a standard maturation cocktail (TNFa, IL-lb, IL-6 and PGE2 as described in Methods) as indicated. Subsequently the cells were incubated/not incubated with lOOng/ml for 48 h before they were harvested for array analysis.
  • TNFa standard maturation cocktail
  • DD-treatment induces increased expression of ⁇ -catenin and CIITA in Langerhans cells and keratinocytes.
  • Tissue sections of healthy skin obtained from patient #21 on day 0 and day 4 were stained side-by-side using MELC robots and the indicated antibodies.
  • White arrows depict Langerhans cells expressing CIITA.
  • dark arrows depict co-localization of ⁇ -catenin and CDla, respective ⁇ -catenin expression in Langerhans cells.
  • the bacterial toxin domain comprises a toxin selected from diphtheria toxin and exotoxins of Pseudomonas and Cholera
  • the toxin domain comprises is fragments A and B of diphtheria toxin (e.g. a protein having the sequence of amino acid residues 1-387 of SEQ ID NO : l) or domains II and III of exotoxins A of Pseudomonas and Cholera (e.g. proteins having the sequences of SEQ ID NOs: 2 and 3, respectively).
  • the cellular binding domain is selected from heterologous proteins/molecules (heterologous relative to the protein of the toxin domain) that target the toxin domain to immature DCs, mature DCs or both, and that are of non-bacterial (i .e. mammalian or even human) origin and are not toxic to the DCs including receptor binding ligands, as for example interleukin2 (IL-2), GM-CSF or fragments thereof, and single-chain antibodies directed against dendritic cell surface receptors as for example DEC205 or DC-SIGN .
  • receptor binding ligands as for example interleukin2 (IL-2), GM-CSF or fragments thereof, and single-chain antibodies directed against dendritic cell surface receptors as for example DEC205 or DC-SIGN .
  • the non-toxicity is achieved by utilizing mammalian or even human receptor binding ligands.
  • the cellular binding domain is IL-2, hIL-2 or a fragment thereof.
  • the toxin domain can be directly or through a linker domain fused to the cellular binding domain, while such linker includes single amino acid residues or an arbitrary oligopeptide.
  • the toxin domain can be fused at its C- or N-terminus to the cellular binding domain. It is however preferred that the toxin domain is linked at its N-terminus to the cellular binding domain.
  • the fusion protein for use as a medicament to tolerize DCs in a patient has a bacterial toxin domain that comprises fragments A and B of diphtheria toxin, and a cellular binding domain that comprises human IL-2 or a fragment thereof.
  • the bacterial toxin domain consists of fragments A and B of diphtheria toxin, preferably has the sequence of amino acid residues 1-387 of SEQ ID NO : l, and/or that the cellular binding domain consists of the sequence of human IL-2, preferably has the sequence of amino acid residues 389 to 521 of SEQ ID NO : l; and/or that the toxin domain is directly fused at its N-terminus to the cellular binding domain.
  • the particularly preferred fusion protein to be utilized according to aspect (3) of the invention is the one of SEQ ID NO: l (Ontak).
  • the medicament is suitable to tolerize all or most dendritic cells in the patient.
  • patient within the present invention not only includes human patients but also other mammals, human patients are, however, preferred.
  • the medicament is suitable to be applied to the patient within a defined timeframe, such as a few days, which is determined by time of treatment (days), concentration of fusion protein (per kg bodyweight) and thorough numeric assessment of peripheral DC subpopulations.
  • the medicament is suitable for tolerization to nominal antigen, including defined peptide epitopes, as well as alloantigens, including transplanted organ and graft-versus-host-disease (GVHD).
  • the medicament is suitable for tolerization in vivo in the setting of organ transplantation requires treatment of donor, preferably also the recipient, with the fusion protein prior to transplantation, and optionally, after transplantation the recipient can (again) be treated with the fusion protein.
  • the medicament is suitable for tolerization for hematopoietic transplants/hematopoietic stem cell transplantation.
  • the medicament is suitable for antigen-specific tolerization in vivo, which is achievable by combining all possible vaccination procedures, including subcutaneous peptide injection or DC vaccination, with the fusion protein treatment, and is guided and controlled by antigen-specific peptide profiling (immunomonitoring) before and after fusion protein treatment and vaccination.
  • the method for tolerizing DC in a patient of aspect (4) of the invention is suitable for tolerization regimen set forth in connection with aspects (1) to (3) above.
  • the fusion protein as defined above is for use in the treatment for immunological diseases that are induced and/or maintained by a DC-T cell interaction that causes detrimental T cell proliferation/activation and immunopathology.
  • autoimmune diseases include multiple sclerosis, insulin dependent diabetes mellitus, thyroid diseases such as Hashimoto's thyroiditis and Grave's disease, acute rheumatic fever, rheumatoid arthritis and the like.
  • the method for treating immunological diseases of aspect (6) of the invention can preferably be used for treating the autoimmune diseases mentioned hereinbefore.
  • Aspect (7) of the invention provides for a method for generating tolerogenic antigen-loaded DCs ex vivo, which comprised loading DCs with the fusion protein as defined above.
  • the DCs to be loaded can be mature and immature DCs. After loading the DCs can be frozen according to well-known procedures without loss of viability.
  • the tolerogenic antigen-loaded DCs of aspect (8) can be freshly prepared, be in the frozen state or be re-thawed.
  • such tolerogenic antigen-loaded DCs freshly prepared or re-thawed
  • the tolerogenic antigen-loaded DCs of aspect (8) are suitable for identifying molecules and mechanisms mediating the tolerogenic effect. Further, according to aspect (11) of the invention the tolerogenic antigen-loaded DCs of aspect (8) can be used for co-culturing with T cells ex vivo to generate regulatory/tolerogenic T cells for adoptive transfer of tolerogenic T cell populations to induce antigen-specific tolerance in the patient. According to the invention said regulatory T cells can also be further expanded with or without cloning before adoptive transfer. Furthermore, said regulatory T cells are suitable to clone TCR and indentify molecules mediating the tolerogenic effect(s) .
  • Dendritic cells have been employed therapeutically for years, especially for the experimental immune therapy of cancers.
  • progenitor cells i.e., so-called monocytes
  • monocytes i.e., so-called monocytes
  • Ontak has been employed for treating CTCL for many years. This is based on the idea that the fusion protein will bind to the IL-2 receptor of tumor cells with its interleukin-2 portion, become internalized and induce cell death (apoptosis) of the tumor cell with its toxin portion. In this way, the number of tumor cells is supposed to be reduced. Actually, a significant reduction of tumor cells can be observed in CTCL upon treatment with Ontak. Due to this supposed mechanism, Ontak was also employed for the depletion of regulatory T cells in humans. Regulatory T cells have a particularly high density of IL-2-receptor and therefore appear to be an ideal target population for Ontak. Actually, several studies have reported that Ontak reduces the number or circulating T reg s significantly and enduringly.
  • Ontak was administered to melanoma patients (stage four of the disease) intravenously on three consecutive days before vaccination with dendritic cells. Before, during and after the Ontak treatment, blood and tissue samples were taken from the patients and processed and analyzed under immunological aspects. It was found that:
  • Ontak-treated dendritic cells show a similar gene regulation pattern in transcriptome analysis as in patients.
  • Ontak-treated dendritic cells show a significant upregulation of Stat3 and CIITA.
  • the tolerogen of aspect (1) to (3) as defined above is applied to the patient within a defined timeframe, such as a few days, which is determined by time of treatment (days), concentration of the fusion protein, notably Ontak (per kg bodyweight) and thorough numeric assessment of peripheral DC subpopulations. (e.g. by the DC enumeration kit from Miltenyi).
  • Regulatory/tolerogenic T cells and tolerogenic DC can be isolated from Ontak treated patients (from their blood, bone marrow or organs / biopsies) and used as above (e.g. in vitro for expansion before further use for treatment of patients or for the identification of functional molecules and mechanisms).
  • Ontak is suitable for the systemic (for example, grafted organ) and antigen-specific (for example, peptide allergen) tolerization treatment in humans.
  • the antigen specificity can be achieved by any kind of vaccination (for example, subcutaneous injection of peptides, vaccination with dendritic cells).
  • vaccination for example, subcutaneous injection of peptides, vaccination with dendritic cells.
  • treatment with Ontak before and optionally after the administration of the antigen is required.
  • the success of the Ontak treatment and time of vaccination/grafting is chosen after the dendritic cells have been determined numerically in the peripheral blood.
  • the patient to be treated is treated with Ontak i.v. (5-12 pg/kg of body weight) on three consecutive days, and the drop of their dendritic cells in the peripheral blood is determined by FACS analysis. On the fourth day, the vaccination with specific antigens or the grafting of the donor organ is effected. Thereafter, the Ontak treatment is continued for another three days.
  • the success of the tolerization treatment can be determined by means of ex-vivo Elispot, MLPC assay and KLH stimulation test.
  • Table 1 shows the relative increase of RNA in differently treated dendritic cells (DCs) (e.g., LPS) or dendritic cells isolated from patients who were under Ontak treatment ⁇ in vivo Ontak treated DC). It is specifically shown that coreceptors are being upregulated only in dendritic cells treated with LPS or cocktail (which are maturing stimuli), but not in dendritic cells from patients under Ontak treatment.
  • DCs dendritic cells
  • LPS dendritic cells
  • Table 2 shows that only dendritic cells from patients under Ontak treatment upregulate tolerogenic factors (Cheng F. et al., Immunity. l9(3) :425-36 (2003); Manicassamy S. et al., Science. 329(5993) : 849-53 (2010), and Foligne B. et al., PLoS One. 2007,
  • Table 3 shows that the antigen-presenting HLA genes in dendritic cells from patients under Ontak treatment are significantly upregulated. In accordance with this, the responsible transcription factor CIITA is also upregulated. This means that said dendritic cells present antigens in the absence of coreceptors (Table 1), which essentially corresponds to the functionality of a tolerogenic dendritic cell .
  • Table 4 shows a number of genes from the network of the NK (natural killer) cell- mediated cytotoxicity that are upregulated in dendritic cells of Ontak-treated patients to a strikingly significant extent. All in all, this gene regulation leads to inhibition of the NK cell-mediated cytotoxicity.
  • TRAIL TNFSF10
  • the high upregulation of TRAIL leads to the cell death of CD4+ and CD8+ immune cells (see Lichtner M . et al ., AIDS Res Hum Retroviruses. 20(2) : 175-82 (2004), and Vidalain P. O. et al ., J Virol. 74(l) : 556-9 (2000)).
  • Tables 5-8 show a comparison between dendritic cells obtained from patients under Ontak treatment and dendritic cells treated with Ontak in vitro. A large number of parallels can be observed although both cell populations are not directly comparable due to their genesis and stimulation. Thus, for example, the dendritic cells generated in vitro additionally obtain a maturing cocktail.
  • mice were treated with Ontak before and during the experimental induction of EAE (experimental autoimmune encephalitis) which is a model disease in mice to demonstrate the antigen-specific (here called MOG peptide) induction of autoimmune disease induced/caused through antigen presentation by dendritic cells.
  • MOG peptide antigen-specific
  • Example 3 Upregulation of tolerogenic effectors and antigen presentation in Dendritic Cells by Ontak
  • Table 9 shows fold change of mRNA levels of selected genes in DC after exposure to DD in vivo and in vitro and of controls.
  • Monocytes; mDCl were obtained by FACS sorting before (day 0) and during (day 2) DD-treatment (see details in Fig. 7a). Numbers in these rows represent fold mRNA change (mean values of two patients) after two DD infusions (12mg/kg) (day 2).
  • MaDC+DD prepared from healthy donors and treated with lOOng/ml DD for 48h (details in Fig. 7b). Numbers represent fold mRNA change (mean values of triplicates) with respect to an untreated control .
  • ImDC+MC; imDC+LPS existing data sets of maturing DC stimulated by a conventional maturation cocktail (MC, see Methods) (12 h exposure) or LPS (12 h). Numbers represent fold mRNA changes with respect to an untreated control.
  • ⁇ -catenin and NOD2 factors also implicated in DC tolerance (Manicassamy, S. et al., Science 329, 849-853 (2010); Magalhaes, J.G. et al., J. Immunol . 181, 7925-7935 (2008)), were up-regulated in mDCl .
  • ⁇ -catenin was upreguiated in Langerhans cells and upper layer keratinocytes (stratum spinosum and granulosum; Fig. 6c, dark arrows and Fig. 8).

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Abstract

The present invention relates to the field of human medicine, namely the treatment of undesirable immunological or allergic reactions.

Description

Fusion Protein for the Treatment of Immunologic or Allergic Reactions Summary of the Invention
The present invention relates to the field of human medicine, namely the treatment of undesirable immunological or allergic reactions.
Background of the Invention
Dendritic cells (DC) are considered the organizers of the immune system. In principle, they can mediate two reactions. On the one hand, they organize the immune defense for the control and elimination of microbes and foreign structures/cells, such as cancer cells. On the other hand, DCs prevent this very immune defense from attacking endogenous cells and structures. The latter is also referred to as induction or maintenance of tolerance or briefly tolerization. If tolerance is broken, the result is graft rejections, allergic reactions or autoimmune diseases with the known chronic complaints, for example, of rheumatoid arthritis. Hardly any therapies or therapy regimens for the induction of tolerance, for example, towards particular cell structures or antigens, have been developed to date. In the field of allergology, the method of desensitization, for example, to pollen antigens, has been known. In this method, doses of the disease-inducing substances/antigens are repeatedly injected into the skin of the patient according to a particular time schedule under clinical supervision. With certain antigens, this results in an induction of tolerance. This method has been approved only for a few antigens with varying degree of success.
In transplantation medicine, there has been no method for "habituating" the recipient's immune system to the new organ or for inducing tolerance towards the new organ. Therefore, graft recipients must be treated with immune suppressants for life. This treatment is expensive on the one hand and is a burden on the patient on the other hand due to the increased infection risk and the side effects, which are in part significant.
On the other hand, Denileukin Difitox (DAB389IL-2; Ontak; SEQ ID NO : l) is a medicament approved in the U.S.A. that has been employed for the treatment of cutaneous T-cell lymphomas (CTCL) for more than 10 years (i.v. application). It is supplied by the Japanese pharmaceutical company Eisai or Cephalon. It is a recombinant fusion protein produced in E. coli and consisting of a fusion between fragments A and B of diphtheria toxin as well as human interleukin 2 (IL-2). In addition to its application for CTCL, the medicament was employed in experimental approaches for the depletion of regulatory T cells in humans.
Summary of the Invention
It was now found that the i.v. application of Ontak results in a tolerization of dendritic cells (DCs). The underlying molecular mechanism could be elucidated. It involves the upregulation of particular tolerogenic genes. This effect can be used therapeutically, for example, in transplantation medicine to avoid graft rejections, or for desensitization by means of adendritic cell (DC) vaccine. Further, the medicament can be applied therapeutically for immune suppression. The invention thus provides
(1) a fusion protein comprising
(a) a bacterial toxin domain comprising a bacterial toxin that lacks the cellular receptor binding sequence of the toxin, and
(b) a cellular binding domain comprising a cellular binding protein that is different from the native binding sequence of the toxin and that targets the fusion protein to mammalian cells
for use as a medicament to tolerize dendritic cells (DC) in a patient;
(2) a preferred embodiment of (1) above, wherein the bacterial toxin domain comprises fragments A and B of diphtheria toxin, and the cellular binding domain comprises human interleukin 2 (IL-2) or a fragment thereof;
(3) a further preferred embodiment of (1) above, wherein the fusion protein has the sequence of SEQ ID NO : l (Ontak);
(4) a method for tolerizing dendritic cells (DCs) in a patient, which comprises administering the patient a suitable amount of the fusion protein as defined in (1) to (3) above;
(5) a fusion protein as defined in (1) to (3) above for use in the treatment of immunological diseases, including autoimmune diseases, that are induced and/or maintained by a DC-T cell interaction that causes detrimental T cell proliferation/ activation and immunopathology;
(6) a method for treating immunological diseases, including autoimmune diseases that are induced and/or maintained by a DC-T cell interaction that causes detrimental T cell proliferation/activation and immunopathology in a patient, which method comprises administering the patient a suitable amount of the fusion protein as defined in (1) to (3) above; (7) a method for generating tolerogenic antigen-loaded DCs ex vivo, which comprised loading DCs with the fusion protein as defined in (1) to (3) above;
(8) a tolerogenic antigen-loaded DCs obtainable by the method of (7) above;
(9) a tolerogenic antigen-loaded DCs of (8) above for use in inducing antigen- specific T cell tolerance in a patient in vivo (also referred to as tolerogenic vaccination);
(10) a method for inducing antigen-specific T cell tolerance in a patient in vivo (tolerogenic vaccination), which comprises administering the patient a suitable amount of the antigen-loaded DCs of (8) above;
(11) the use of the tolerogenic antigen-loaded DCs of (8) above for identifying molecules and mechanisms mediating the tolerogenic effect; and
(12) the use of the tolerogenic antigen-loaded DCs of (8) above for co-culturing with T cells ex vivo to generate regulatory/tolerogenic T cells for adoptive transfer of tolerogenic T cell populations to induce antigen-specific tolerance in the patient.
Short Description of the Figures
Fig. 1 : Expression of surface molecule on day 10 mBMDC after ONTAK (48 h) plus LPS.
Fiq.2 : ONTAK was present during DC-T cell co-culture and inhibits the proliferation of murine T cells in allo-MLR.
Fig. 3 : ONTAK was present during DC cell maturation (48 h) and inhibits DC mediated T cell proliferation of murine T cells in allo-MLR.
Fig. 4: ONTAK reduced paralyses symptoms associated with EAE. ONTAK (1 g/animal) was injected i.p. at day -1, 1, 3, EAE was induced at day 0. At day 30 a second EAE was induced.
Fig. 5 : EAE was induced on day 0, at peak of disease (day 15) mice were divided in two groups stage (4/4/3), one group was injected with ONTAK five times i .p. from day 15 to day 19, the other group was left untreated.
Fig. 6: DD-treatment induces increased expression of Stat3, β-catenin and CIITA in PBMC and skin tissue. Protein expression was assessed in patient 's PBMC by FACS analysis and in tissue by the robot-automated multi-epitope ligand cartography (MELC) technique (Schubert, W. et al., Nat. Biotechnol . 24, 1270- 1278 (2006)). (al PBMC from patients # 19 and #21 (3xl2mg/kg) were stained for intracellular β-catenin and Stat3 on day 0 and day 4 after DD-treatment and mDCl were analyzed by FACS. £bl maDC were prepared from healthy donors and treated with DD (0, 10 and lOOng/ml) for 24 h and subsequently stained for intracellular Stat3 by FACS. Results for one representative donor are shown, (c) Tissue sections of healthy skin obtained from patient #19 (see results for patient #21 in Fig. 8) on day 0 and day 4 were stained side-by-side using MELC robots and the indicated antibodies. White arrows depict epidermal Langerhans cells expressing CIITA. Dark arrows indicate co-localization of β-catenin and CDla, respective β-catenin expression in Langerhans cells.
Fig. 7 : Sampling and processing scheme for DC mRNA array analysis, (a} DC and monocytes were obtained from patients # 19 and #21 before and after (dark arrows) two administrations of DD (black arrows). The blue lines describe the mDCl cell count (in %) with respect to original levels at the time of sampling. (b) ImDC were generated from monocytes of a healthy donor and matured by a standard maturation cocktail (TNFa, IL-lb, IL-6 and PGE2 as described in Methods) as indicated. Subsequently the cells were incubated/not incubated with lOOng/ml for 48 h before they were harvested for array analysis.
Fig. 8 : DD-treatment induces increased expression of β-catenin and CIITA in Langerhans cells and keratinocytes. Tissue sections of healthy skin obtained from patient #21 on day 0 and day 4 were stained side-by-side using MELC robots and the indicated antibodies. White arrows depict Langerhans cells expressing CIITA. dark arrows depict co-localization of β-catenin and CDla, respective β-catenin expression in Langerhans cells.
Detailed Description of the Invention
In the fusion protein of aspect (1) of the invention it is preferred that the bacterial toxin domain comprises a toxin selected from diphtheria toxin and exotoxins of Pseudomonas and Cholera, preferably the toxin domain comprises is fragments A and B of diphtheria toxin (e.g. a protein having the sequence of amino acid residues 1-387 of SEQ ID NO : l) or domains II and III of exotoxins A of Pseudomonas and Cholera (e.g. proteins having the sequences of SEQ ID NOs: 2 and 3, respectively). Further it is preferred that the cellular binding domain is selected from heterologous proteins/molecules (heterologous relative to the protein of the toxin domain) that target the toxin domain to immature DCs, mature DCs or both, and that are of non-bacterial (i .e. mammalian or even human) origin and are not toxic to the DCs including receptor binding ligands, as for example interleukin2 (IL-2), GM-CSF or fragments thereof, and single-chain antibodies directed against dendritic cell surface receptors as for example DEC205 or DC-SIGN . The non-toxicity is achieved by utilizing mammalian or even human receptor binding ligands. Particularly preferred is that the cellular binding domain is IL-2, hIL-2 or a fragment thereof.
According to the invention the toxin domain can be directly or through a linker domain fused to the cellular binding domain, while such linker includes single amino acid residues or an arbitrary oligopeptide. According to the invention the toxin domain can be fused at its C- or N-terminus to the cellular binding domain. It is however preferred that the toxin domain is linked at its N-terminus to the cellular binding domain.
According to preferred aspect (2) of the invention the fusion protein for use as a medicament to tolerize DCs in a patient has a bacterial toxin domain that comprises fragments A and B of diphtheria toxin, and a cellular binding domain that comprises human IL-2 or a fragment thereof. Here it is preferred that the bacterial toxin domain consists of fragments A and B of diphtheria toxin, preferably has the sequence of amino acid residues 1-387 of SEQ ID NO : l, and/or that the cellular binding domain consists of the sequence of human IL-2, preferably has the sequence of amino acid residues 389 to 521 of SEQ ID NO : l; and/or that the toxin domain is directly fused at its N-terminus to the cellular binding domain.
The particularly preferred fusion protein to be utilized according to aspect (3) of the invention is the one of SEQ ID NO: l (Ontak).
In the fusion protein for use as a medicament to tolerize DCs in a patient according aspects (1) to (3) of the invention the medicament is suitable to tolerize all or most dendritic cells in the patient. The term "patient" within the present invention not only includes human patients but also other mammals, human patients are, however, preferred.
Further the medicament is suitable to be applied to the patient within a defined timeframe, such as a few days, which is determined by time of treatment (days), concentration of fusion protein (per kg bodyweight) and thorough numeric assessment of peripheral DC subpopulations.
Even further the medicament is suitable for tolerization to nominal antigen, including defined peptide epitopes, as well as alloantigens, including transplanted organ and graft-versus-host-disease (GVHD). Also the medicament is suitable for tolerization in vivo in the setting of organ transplantation requires treatment of donor, preferably also the recipient, with the fusion protein prior to transplantation, and optionally, after transplantation the recipient can (again) be treated with the fusion protein. Moreover the medicament is suitable for tolerization for hematopoietic transplants/hematopoietic stem cell transplantation. Finally, the medicament is suitable for antigen-specific tolerization in vivo, which is achievable by combining all possible vaccination procedures, including subcutaneous peptide injection or DC vaccination, with the fusion protein treatment, and is guided and controlled by antigen-specific peptide profiling (immunomonitoring) before and after fusion protein treatment and vaccination. The method for tolerizing DC in a patient of aspect (4) of the invention is suitable for tolerization regimen set forth in connection with aspects (1) to (3) above. According to aspect (5) of the invention the fusion protein as defined above is for use in the treatment for immunological diseases that are induced and/or maintained by a DC-T cell interaction that causes detrimental T cell proliferation/activation and immunopathology. Particular immunological diseases that can be treated are autoimmune diseases include multiple sclerosis, insulin dependent diabetes mellitus, thyroid diseases such as Hashimoto's thyroiditis and Grave's disease, acute rheumatic fever, rheumatoid arthritis and the like. Similarly the method for treating immunological diseases of aspect (6) of the invention can preferably be used for treating the autoimmune diseases mentioned hereinbefore.
Aspect (7) of the invention provides for a method for generating tolerogenic antigen-loaded DCs ex vivo, which comprised loading DCs with the fusion protein as defined above. The DCs to be loaded can be mature and immature DCs. After loading the DCs can be frozen according to well-known procedures without loss of viability.
Similarly the tolerogenic antigen-loaded DCs of aspect (8) can be freshly prepared, be in the frozen state or be re-thawed. According to aspects (9) and (10) of the invention such tolerogenic antigen-loaded DCs (freshly prepared or re-thawed) can be utilized for inducing antigen-specific T cell tolerance (also referred to as tolerogenic vaccination) in a patient in vivo.
According to aspect (11) of the invention the tolerogenic antigen-loaded DCs of aspect (8) are suitable for identifying molecules and mechanisms mediating the tolerogenic effect. Further, according to aspect (11) of the invention the tolerogenic antigen-loaded DCs of aspect (8) can be used for co-culturing with T cells ex vivo to generate regulatory/tolerogenic T cells for adoptive transfer of tolerogenic T cell populations to induce antigen-specific tolerance in the patient. According to the invention said regulatory T cells can also be further expanded with or without cloning before adoptive transfer. Furthermore, said regulatory T cells are suitable to clone TCR and indentify molecules mediating the tolerogenic effect(s) .
The invention is hereinafter further explained in connection with aspect (3) of the invention (Ontak). This explanation is, however, not to be construed as limiting the invention.
Dendritic cells have been employed therapeutically for years, especially for the experimental immune therapy of cancers. In principle, progenitor cells, i.e., so- called monocytes, are withdrawn from the patient to be treated, matured to dendritic cells in vitro by means of cytokines, loaded with tumor antigens and subsequently administered intravenously or subcutaneously. To date, this method has been applied in numerous clinical studies, showing the safety and feasibility of this method, among others. Within the scope of these DC immune therapies, the only object was to induce antigen-specific immune responses, i.e., to provoke defense reactions against particular target structures. An experimental immune therapy for tolerance induction has not yet been performed, at least not within the scope of clinical studies. One reason for this is the fact that there is no method recognized for humans for preparing tolerogenic dendritic cells.
In the U.S.A., Ontak has been employed for treating CTCL for many years. This is based on the idea that the fusion protein will bind to the IL-2 receptor of tumor cells with its interleukin-2 portion, become internalized and induce cell death (apoptosis) of the tumor cell with its toxin portion. In this way, the number of tumor cells is supposed to be reduced. Actually, a significant reduction of tumor cells can be observed in CTCL upon treatment with Ontak. Due to this supposed mechanism, Ontak was also employed for the depletion of regulatory T cells in humans. Regulatory T cells have a particularly high density of IL-2-receptor and therefore appear to be an ideal target population for Ontak. Actually, several studies have reported that Ontak reduces the number or circulating Tregs significantly and enduringly.
Within the scope of a clinical study relating to the experimental immune therapy of malignant melanoma, Ontak was administered to melanoma patients (stage four of the disease) intravenously on three consecutive days before vaccination with dendritic cells. Before, during and after the Ontak treatment, blood and tissue samples were taken from the patients and processed and analyzed under immunological aspects. It was found that:
(I) a significant drop of the most important lymphocyte population and of dendritic cells could be observed in blood immediately after intravenous Ontak administration. Despite their high density of IL-2 receptor, regulatory T cells are not more strongly affected than other populations. After the end of the treatment, the cell population recovers relatively quickly. Depending on the dose applied, dendritic cells show a delayed recovery.
(II) Patients who obtained Ontak before being vaccinated show no improvement of the course of their disease.
(III) Patients who obtained Ontak before being vaccinated show no tumor antigen- specific immune response (MLPC assay).
(IV) Patients who obtained Ontak before being vaccinated show no immune response to vaccinated control peptides (ex vivo Elispot).
(V) PBMCs from patients having obtained Ontak before being vaccinated do not show IFNgamma upon KLH stimulation (as would be expected), but an IL-4 secretion pattern.
(VI) Patients having obtained Ontak before being vaccinated may lose previously existing antigen-specific immune responses (1 case).
(VII) In transcriptome analysis, dendritic cells from Ontak-treated patients show (a) upregulation of "tolerogenic" genes (Stat3, beta-catenine, NOD2), (b) upregulation of CIITA and HLA genes in the absence of immunostimulatory coreceptors (which are needed for an immune defense reaction), and (c) upregulation of genes that block the NK cell-mediated immune response.
In addition to the results obtained by processing the patient material, in vitro data were obtained with Ontak, which confirmed its tolerance-inducing effects on dendritic cells. Thus, we were able to show that:
(i) Ontak-treated dendritic cells will block the expansion/proliferation of interacting T cells.
(ii) Ontak-treated dendritic cells will enhance the suppressive (tolerogenic) effect of
TregS.
(iii) Ontak-treated dendritic cells show a similar gene regulation pattern in transcriptome analysis as in patients. (iv) Ontak-treated dendritic cells show a significant upregulation of Stat3 and CIITA.
The numerous results obtained from patients and in vitro experiments yield a clear picture. The treatment of dendritic cells with Ontak induces a tolerogenic phenotype of these cells and at the same time blocks essential mechanisms of immune defense.
The tolerogen of aspect (1) to (3) as defined above is applied to the patient within a defined timeframe, such as a few days, which is determined by time of treatment (days), concentration of the fusion protein, notably Ontak (per kg bodyweight) and thorough numeric assessment of peripheral DC subpopulations. (e.g. by the DC enumeration kit from Miltenyi).
Regulatory/tolerogenic T cells and tolerogenic DC can be isolated from Ontak treated patients (from their blood, bone marrow or organs / biopsies) and used as above (e.g. in vitro for expansion before further use for treatment of patients or for the identification of functional molecules and mechanisms).
Ontak is suitable for the systemic (for example, grafted organ) and antigen-specific (for example, peptide allergen) tolerization treatment in humans. The antigen specificity can be achieved by any kind of vaccination (for example, subcutaneous injection of peptides, vaccination with dendritic cells). For both systemic and antigen-specific tolerization, treatment with Ontak before and optionally after the administration of the antigen is required. The success of the Ontak treatment and time of vaccination/grafting is chosen after the dendritic cells have been determined numerically in the peripheral blood.
The invention is further described in connection with the following Examples, which are, however, not to be construed as limiting the invention
Examples
Example 1 : Tolerization treatment
The patient to be treated is treated with Ontak i.v. (5-12 pg/kg of body weight) on three consecutive days, and the drop of their dendritic cells in the peripheral blood is determined by FACS analysis. On the fourth day, the vaccination with specific antigens or the grafting of the donor organ is effected. Thereafter, the Ontak treatment is continued for another three days. The success of the tolerization treatment can be determined by means of ex-vivo Elispot, MLPC assay and KLH stimulation test. Coreceptor expression
Gene name Cocktail-treated LPS-treated DCs In vivo ONTAK- DCs 12h 12h treated DCs
CD80 11.65 8.1 N/A
CD86 2.84 N/A N/A
CD83 4.38 N/A N/A
CD40 N/A 4.1 N/A
Like all Tables (see below), Table 1 shows the relative increase of RNA in differently treated dendritic cells (DCs) (e.g., LPS) or dendritic cells isolated from patients who were under Ontak treatment {in vivo Ontak treated DC). It is specifically shown that coreceptors are being upregulated only in dendritic cells treated with LPS or cocktail (which are maturing stimuli), but not in dendritic cells from patients under Ontak treatment.
Table 2 : Tolerogenic / Th2 response markers
Gene name Cocktail-treated LPS-treated DCs In vivo ONTAK- DCs 12h 12h treated DCs
CTNN B1 2.63 N/A 5.46
STAT3 N/A N/A 5.68
NOD2 N/A N/A 6.94
Table 2 shows that only dendritic cells from patients under Ontak treatment upregulate tolerogenic factors (Cheng F. et al., Immunity. l9(3) :425-36 (2003); Manicassamy S. et al., Science. 329(5993) : 849-53 (2010), and Foligne B. et al., PLoS One. 2007,
21;2(3) : 313 (2007)).
Table 3 : Maior histocompatibility complex markers
Gene name Cocktail-treated LPS-treated DCs In vivo ONTAK- DCs 12h 12h treated DCs
CIITA 0.31 N/A 5.4
HLA-DMB 0.28 0.38 6.14
HLA-A N/A N/A 4.02
HLA-B N/A N/A 4.52
HLA-C N/A N/A 4.25
HLA-E N/A N/A 4.29
HLA- DMA N/A N/A 4.3
Table 3 shows that the antigen-presenting HLA genes in dendritic cells from patients under Ontak treatment are significantly upregulated. In accordance with this, the responsible transcription factor CIITA is also upregulated. This means that said dendritic cells present antigens in the absence of coreceptors (Table 1), which essentially corresponds to the functionality of a tolerogenic dendritic cell .
Table 4: Natural killer eel 1 mediated cytotoxicity genes
Gene name Cocktail-treated LPS-treated DCs In v/Vo ONTAK- DCs 12h 12h treated DCs
PIK3CD 0.25 N/A 19.12
TNFSF10 N/A N/A 14.99
VAV1 0.39 N/A 9.85
PAK1 0.24 N/A 8.11
NFATC1 N/A N/A 5.64
PTPN6 0.81 N/A 6.89
PRKCB N/A N/A 5.48
PIK3R5 N/A N/A 4.93
HLA-E N/A N/A 4.95
HLA-B N/A N/A 4.52
RAC2 0.27 N/A 4.16
HLA-C N/A N/A 4.25
RAF1 N/A N/A 4.18
HLA-A N/A N/A 4.03
TNFRSF10B N/A N/A 3.77
MAPK3 N/A N/A 3.7
IFNA4 N/A N/A 0.13
GRB2 N/A N/A 0.27
KIR2DL4 N/A N/A 0.16
SOS1 N/A N/A 0.22
PPP3R2 N/A N/A 0.19
KIR2DS4 N/A N/A 0.21
CD244 N/A N/A 0.22
IFNA21 N/A N/A 0.33
KIR3DL1 N/A N/A 0.22
KIR2DL5A N/A N/A 0.26
VAV3 0.28 N/A 0.28
ICAM 1 N/A N/A 0.43
Table 4 shows a number of genes from the network of the NK (natural killer) cell- mediated cytotoxicity that are upregulated in dendritic cells of Ontak-treated patients to a strikingly significant extent. All in all, this gene regulation leads to inhibition of the NK cell-mediated cytotoxicity. In addition, for example, the high upregulation of TRAIL (TNFSF10), as also observed upon infection by HIV or measles virus, leads to the cell death of CD4+ and CD8+ immune cells (see Lichtner M . et al ., AIDS Res Hum Retroviruses. 20(2) : 175-82 (2004), and Vidalain P. O. et al ., J Virol. 74(l) : 556-9 (2000)).
Tables 5-8 show a comparison between dendritic cells obtained from patients under Ontak treatment and dendritic cells treated with Ontak in vitro. A large number of parallels can be observed although both cell populations are not directly comparable due to their genesis and stimulation. Thus, for example, the dendritic cells generated in vitro additionally obtain a maturing cocktail.
Coreceptor expression
Gene name ONTAK in vivo ONTAK in vitro
CD80 N/A N/A
CD86 N/A 0.08
CD83 N/A N/A
CD40 N/A 0.19
Table 6 : Tolerogenic / Th2 response markers
Gene name ONTAK in vivo ONTAK in vitro
CTNN B1 5.46 N/A
STAT3 5.68 57.28
NOD2 6.94 N/A
Table 7 : Maior histocompatibility complex markers
Gene name ONTAK in vivo ONTAK in vitro
CIITA 5.4 10.41
HLA-DMB 6.14 8.82
HLA-A 4.02 0.18
HLA-B 4.52 N/A
HLA-C 4.25 0.18
HLA-E 4.29 0.2
H LA- DMA 4.3 N/A
Table 8 : Natural killer cell mediated cytotoxicity genes
Gene name ONTAK in vivo ONTAK in vitro
PIK3CD 19.12 13.37 TNFSF10 14.99 0.06
VAV1 9.85 N/A
PAK1 8.11 0.15
NFATC1 5.64 N/A
PTPN6 6.89 N/A
PRKCB 5.48 N/A
PIK3R5 4.93 13.73
HLA-E 4.95 0.15
HLA-B 4.52 N/A
RAC2 4.16 9.6
HLA-C 4.25 0.18
RAF1 4.18 N/A
HLA-A 4.03 0.18
TNFRSF10B 3.77 0.14
MAPK3 3.7 N/A
IFNA4 0.13 N/A
GRB2 0.27 N/A
KIR2DL4 0.16 N/A
S0S1 0.22 N/A
PPP3R2 0.19 N/A
KIR2DS4 0.21 9.64
CD244 0.22 N/A
IFNA21 0.33 N/A
KIR3DL1 0.22 0.14
KIR2DL5A 0.26 22.3
VAV3 0.28 N/A
ICAM 1 0.43 N/A
Example 2 : Mice Experiment
Mice were treated with Ontak before and during the experimental induction of EAE (experimental autoimmune encephalitis) which is a model disease in mice to demonstrate the antigen-specific (here called MOG peptide) induction of autoimmune disease induced/caused through antigen presentation by dendritic cells. The results summarized in Figure 4 demonstrate that mice which have been treated with Ontak show a less severe induction of EAE. More importantly, and relevant for the claims in the patent, the EAE challenge was repeated with the same mice (re-vaccination of the same mice with MOG peptide) in the absence of co-treatment with Ontak. Usually a dramatic EAE induction would have been expected in the previously protected mice. However, the symptoms did not worsen indicating that the Ontak treated mice were immunologically protected. This implies that the initial Ontak treatment not only prevented the induction of EAE but induced tolerance to the MOG peptide.
Example 3 : Upregulation of tolerogenic effectors and antigen presentation in Dendritic Cells by Ontak
The tolerogenic conversion of dendritic cells by ONTAK was confirmed by performing mRNA array analyses on mDCl (immature dendritic cells circulating in blood) and monocytes (dendritic cell precursors) sorted from PBMC of two cohort-2 patients (melanoma patients treated with ONTAK) before and during ONTAK-treatment (day 0 and 2). In addition, mature monocyte-derived DC (so- called maDC, generated from precursors in vitro) treated with ONTAK in vitro were analyzed accordingly (see summary in Fig. 7). In our analysis we looked for genes and pathways commonly affected by DD in the different cell populations in vivo (mDCl, monocyte) and in vitro (maDC). In addition, we analyzed factors known to play a role in DC-mediated tolerance. For comparison, existing data sets of maturing DC were analyzed.
In mDCl and monocytes in vivo, 1369 genes were up- and 1292 down-regulated following DD-treatment, whereas in vitro 4978 were up- and 5097 down- regulated (see submissions to Array Express with accession numbers E-MTAB-979 and E-MTAB-977).
A consistent finding was the increase of Stat3 mRNA, a potent regulator of T cell and DC tolerance (Cheng, F. et al., Immunity. 19, 425-436 (2003); Melillo, J. A. et a/., J. Immunol. 184, 2638-2645 (2010); Herrmann, A. et al., Cancer Res. 70, 7455-7464 (2010); Kortylewski, M . & Yu, H ., Curr. Opin. Immunol. 20, 228-233 (2008); Kortylewski, M . et al., Nat. Med. 11, 1314-1321 (2005)), in all DD- treated DC/monocytes in vivo and in vitro. This effect was not seen in DC treated with a maturation cocktail (cytokine cocktail that matures dendritic cell precursors into dendritic cells), LPS, Candida or TLR7/8 agonist R-848 (Table 9). Furthermore, antigen presenting - (HLA class I and II) and processing genes were strongly increased including the regulating transcription factor CIITA. Importantly, the latter was not seen for co-receptor mRNA, as this would be indicative for a protective immune response and was observed in maturing DC (Table 9). Table 9 :
mDCl Monocytes maDC+DD imDC+MC imDC+
(DD in vivo) (DD in vivo) (48h, in vitro) (12h, in vitro) LPS
(12h, in vitro)
Tolerance/ Th2
Markers
Stat3 5.68 3.80 57.32 nc nc β-Catenin 5.46 nc nc 2.63 nc
NOD2 6.94 nc nc nc nc
MHC complex
CIITA 10.17 16.36 10.42 0.31 nc
HLA-A 4.02 5.67 0.18 nc nc
HLA-B 4.52 4.19 nc nc nc
HLA-C 4.25 6.26 0.18 nc nc
HLA-E 4.29 6.59 0.15 nc nc
H LA-DMA 4.3 nc nc nc nc
HLA-DMB 6.14 nc 16.5 0.28 0.38
HLA-D B4 nc 8.5 nc nc nc
HLA-DRB5 nc 6.9 nc nc nc
HLA-DQB1 nc 5.23 nc nc nc
HLA-DQA2 nc 4.25 14.9 nc nc
HLA-DBP1 5.14 13.12 nc nc nc
HLA-DPA1 nc 11.77 nc nc nc
PSME1 4.81 nc nc nc nc
PSME2 7.32 nc nc nc nc
TAPBP 4.02 9.23 nc nc nc
B2M 3.8 6.02 7.13 nc nc
Coreceptors
CD80 nc nc nc 11.65 5.88
CD86 nc nc nc 2.84 nc
CD83 nc nc nc 4.38 nc
CD40 nc nc 0.08 nc nc
Table 9 shows fold change of mRNA levels of selected genes in DC after exposure to DD in vivo and in vitro and of controls. Monocytes; mDCl : were obtained by FACS sorting before (day 0) and during (day 2) DD-treatment (see details in Fig. 7a). Numbers in these rows represent fold mRNA change (mean values of two patients) after two DD infusions (12mg/kg) (day 2). MaDC+DD : prepared from healthy donors and treated with lOOng/ml DD for 48h (details in Fig. 7b). Numbers represent fold mRNA change (mean values of triplicates) with respect to an untreated control . ImDC+MC; imDC+LPS : existing data sets of maturing DC stimulated by a conventional maturation cocktail (MC, see Methods) (12 h exposure) or LPS (12 h). Numbers represent fold mRNA changes with respect to an untreated control. In addition, β-catenin and NOD2, factors also implicated in DC tolerance (Manicassamy, S. et al., Science 329, 849-853 (2010); Magalhaes, J.G. et al., J. Immunol . 181, 7925-7935 (2008)), were up-regulated in mDCl . Finally, in mDCl and maDC, we found an up- as well as down-regulation of a whole set of genes involved in NK-mediated cytotoxicity, causing a block of NK cell activity according to topological pathway analysis (Draghici, S. et al., Genome Res. 17, 1537-1545 (2007); Tarca, A. L. et al., 25, 75-82 (2009)) (Table 10).
Table 10 :
Gene mDCl Monocytes maDC+DD imDC+MC imDC+LPS
(in vivo) (in vivo) (48h, in vitro) (12h, in vitro) (12h, in vitro)
NK cell-associated cytotoxicity
PIK3CD 19.12 nc 34.9 0.25 nc
TRAIL 15.0 4.42 0.06 nc nc
VAV1 9.85 4.39 nc 0.39 nc
PAK1 8.11 nc 0.15 0.24 nc
NFATC1 5.64 6.67 nc nc nc
PTPN6 6.89 4.02 nc 0.81 nc
PRKCB 5.48 nc nc nc nc
PIK3R5 4.93 nc nc nc nc
RAC2 4.16 nc 9.6 0.27 nc
RAF1 4.18 nc nc nc nc
TNFRSF10B 3.77 nc 0.14 nc nc
MAPK3 3.7 nc nc nc nc
KIR2DS4 0.21 nc nc nc nc
KIR2DL5A 0.26 nc 22.3 nc nc
While efficient antigen presentation by mature DC favors a strong DC-induced T cell response, the combination of the here described effects are expected to be highly tolerogenic (Cheng, F. et al., Immunity. 19, 425-436 (2003); Melillo, J. A. et al., J. Immunol. 184, 2638-2645 (2010); Steinman, R. M . et al., Annu. Rev. Immunol . 21, 685-711 (2003); Pulendran, FJ., et al., Nat. Immunol . 11, 647-655 (2010); Maldonado, R.A. & von Andrian, U. H., Adv. Immunol. 108, 111-165 (2010)) and therefore support our assumption that ONTAK induces tolerogenic DC.
To confirm these results on the protein level, Stat3 and β-catenin were analyzed in mDCl of patient 's PBMC (cohort-2 patients) by FACS. In accordance with the mRNA analysis, we saw an increased expression of both factors in both patients treated with 3xl2μg/kg (Fig. 3a). Importantly, and supporting this finding, DD- treated maDC of healthy individuals revealed a dose-dependent increase of Stat3 (Fig. 3b). Similar as in the mRNA analysis for maDC, however, this effect was not observed for β-catenin (data not shown).
To assess DD-effects in tissue, skin biopsies were analyzed by immunofluorescence on day 0 and day 4, the day the DC-vaccine had been administered. Using the robot-automated multi-epitope ligand cartography (MELC)-technique (Schubert, W. et al., Nat. Biotechnol. 24, 1270-1278 (2006)), we were able to stain day 0 and day 4 biopsies side-by-side at identical conditions and with multiple antibodies. Since the Stat3 antibody was not reactive in tissue, we stained for β-catenin and CIITA. Day 0 epidermal Langerhans cells, identified by anti-CDla, showed no CIITA expression, which, however, strongly increased on day 4 (Fig. 6c, white arrows and second patient in Fig. 8). In addition, β-catenin was upreguiated in Langerhans cells and upper layer keratinocytes (stratum spinosum and granulosum; Fig. 6c, dark arrows and Fig. 8).
Taken together, our in vivo mRNA array results correlated with protein expression in PBMC and tissue and confirmed that DD induced a multilayered tolerogenic change in DC.
Sequence Listing, Free Text
SEQ ID NO : Description
1 fusion protein diptheria toxin AB389lL-2, "Ontak"
2 cholera exotoxin A, domains II and III
3 pseudomonas exotoxin A, domains II, I b and III

Claims

Claims
1. A fusion protein comprising
(a) a bacterial toxin domain comprising a bacterial toxin that lacks the cellular receptor binding sequence of the toxin, and
(b) a cellular binding domain comprising a cellular binding protein that is different from the native binding sequence of the toxin and that targets the fusion protein to mammalian cells
for use as a medicament to tolerize dendritic cells (DCs) in a patient.
2. The fusion protein for use as a medicament to tolerize DCs in a patient of claim 1, wherein
(i) the bacterial toxin domain comprises a toxin selected from diphtheria toxin and exotoxins of Pseudomonas and Cholera, preferably the toxin domain comprises is fragments A and B of diphtheria toxin or domains II and III of exotoxins A of Pseudomonas and Cholera; and/or
(ii) the cellular binding domain is selected from heterologous proteins/molecules that target the toxin domain to immature dendritic cells, mature dendritic cells or both, including receptor binding ligands, as for example interleukin2 (IL-2), GM- CSF or fragments thereof, and single-chain antibodies directed against dendritic cell surface receptors as for example DEC205 or DC-SIGN, preferably the cellular binding domain is IL-2 or a fragment thereof; and/or.
(iii) the toxin domain is directly or through a linker domain fused to the cellular binding domain, such linker including single amino acid residues or an oligopeptide; and/or
(iv) the toxin domain is fused at its C- or N-terminus, preferably at its N- terminus to the cellular binding domain.
3. The fusion protein for use as a medicament to tolerize DCs in a patient of claim 1 or 2, wherein the bacterial toxin domain comprises fragments A and B of diphtheria toxin, and the cellular binding domain comprises human interleukin 2 (IL-2) or a fragment thereof.
4. The fusion protein for use as a medicament to tolerize DCs in a patient according to any one of claims 1 to 3, wherein
(i) the bacterial toxin domain consists of fragments A and B of diphtheria toxin, preferably has the sequence of amino acid residues 1-387 of SEQ ID NO : l; and/or (ii) the cellular binding domain consists of the sequence of human IL-2, preferably has the sequence of amino acid residues 389 to 521 of SEQ ID NO : l; and/or
(iii) the toxin domain is directly fused at its N-terminus to the cellular binding domain.
5. The fusion protein for use as a medicament to tolerize DCs in a patient according to any one of claims 1 to 4, wherein the fusion protein has the sequence of SEQ ID NO : l .
6. The fusion protein for use as a medicament to tolerize DCs in a patient according to any one of claims 1 to 5, wherein the medicament is suitable
(i) to tolerize all or most dendritic cells in a patient; and/or
(ii) to be applied to the patient within a defined timeframe, such as a few days, which is determined by time of treatment (days), concentration of fusion protein (per kg bodyweight) and thorough numeric assessment of peripheral DC subpopulations; and/or
(iii) for tolerization to nominal antigen, including defined peptide epitopes, as well as alloantigens, including transplanted organ and graft-versus-host-disease (GVHD); and/or
(iv) for tolerization in vivo in the setting of organ transplantation requires treatment of donor, preferably also the recipient, with the fusion protein prior to transplantation, and optionally, after transplantation the recipient can (again) be treated with the fusion protein; and/or
(v) for tolerization for hematopoietic transplants/ hematopoietic stem cell transplantation; and/or
(vi) for antigen-specific tolerization in vivo, which is achievable by combining all possible vaccination procedures, including subcutaneous peptide injection or dendritic cell (DC) vaccination, with the fusion protein treatment, and is guided and controlled by antigen-specific peptide profiling (immunomonitoring) before and after fusion protein treatment and vaccination.
7. A method for tolerizing DCs in a patient, which comprises administering the patient a suitable amount of the fusion protein as defined in any one of claims 1 to 5.
8. A fusion protein as defined in any one of claims 1 to 5 for use in the treatment for immunological diseases, including autoimmune diseases that are induced and/or maintained by a DC-T cell interaction that causes detrimental T cell proliferation/activation and immunopathology.
9. A method for treating immunological diseases, including autoimmune diseases that are induced and/or maintained by a DC-T cell interaction that causes detrimental T cell proliferation/activation and immunopathology in a patient, which comprises administering the patient a suitable amount of the fusion protein as defined in any one of claims 1 to 5.
10. A method for generating tolerogenic antigen-loaded DCs ex vivo, which comprised loading DC with the fusion protein as defined in any one of claims 1 to 5.
11. A tolerogenic antigen-loaded DCs obtainable by the method of claim 10.
12. A tolerogenic antigen-loaded DCs of claim 11 for use in inducing antigen- specific T cell tolerance in a patient in vivo.
13. A method for inducing antigen-specific T cell tolerance in a patient in vivo, which comprises administering the patient a suitable amount of the antigen- loaded DC of claim 11.
14. Use of the tolerogenic antigen-loaded DCs of claim 11 for identifying molecules and mechanisms mediating the tolerogenic effect.
15. Use of the tolerogenic antigen-loaded DCs of claim 11 for co-culturing with T cells ex vivo to generate regulatory/tolerogenic T cells for adoptive transfer of tolerogenic T cell populations to induce antigen-specific tolerance in the patient.
16. The use of claim 15, wherein said regulatory T cells can be further expanded with or without cloning before adoptive transfer, and/or said regulatory T cells are suitable to clone TCR and indentify molecules mediating the tolerogenic effect(s) .
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015171965A2 (en) 2014-05-07 2015-11-12 Applied Molecular Transport Llc Cholix toxin-derived fusion molecules for oral delivery of biologically active cargo
EP3426785A4 (en) * 2016-03-10 2019-12-25 The Johns Hopkins University METHODS FOR PRODUCING DIPHTERIC TOXIN FUSION PROTEINS WITHOUT AGGREGATE AND THERAPEUTIC USES
US10617741B2 (en) 2010-09-15 2020-04-14 Applied Molecular Transport Inc. Compositions and methods for oral delivery of therapeutic cargo
US11160869B2 (en) 2019-08-16 2021-11-02 Applied Molecular Transport Inc. Compositions, formulations and interleukin production and purification
US11203626B2 (en) 2016-03-10 2021-12-21 The Johns Hopkins University Methods of producing aggregate-free monomeric diphtheria toxin fusion proteins and therapeutic uses
US11246915B2 (en) 2010-09-15 2022-02-15 Applied Molecular Transport Inc. Cholix toxin-derived fusion molecules for oral delivery of biologically active cargo
US11324833B2 (en) 2018-11-07 2022-05-10 Applied Molecular Transport Inc. Cholix-derived carriers for oral delivery of heterologous payload
US11426466B2 (en) 2018-03-08 2022-08-30 Applied Molecular Transport Inc. Toxin-derived delivery constructs for pulmonary delivery
US11965009B2 (en) 2016-03-10 2024-04-23 The Johns Hopkins University Methods of producing aggregate-free monomeric diphtheria toxin fusion proteins and therapeutic uses

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02169521A (en) * 1988-12-22 1990-06-29 Ajinomoto Co Inc Remedy for autoimmune disease
WO2009014835A2 (en) * 2007-06-21 2009-01-29 Angelica Therapeutics, Inc. Modified toxins

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02169521A (en) * 1988-12-22 1990-06-29 Ajinomoto Co Inc Remedy for autoimmune disease
WO2009014835A2 (en) * 2007-06-21 2009-01-29 Angelica Therapeutics, Inc. Modified toxins

Non-Patent Citations (22)

* Cited by examiner, † Cited by third party
Title
BAECHER-ALLAN CLARE ET AL: "Regulatory cells and human cancer.", SEMINARS IN CANCER BIOLOGY APR 2006 LNKD- PUBMED:16378733, vol. 16, no. 2, April 2006 (2006-04-01), pages 98 - 105, XP005274570, ISSN: 1044-579X *
CHENG F. ET AL., IMMUNITY., vol. 19, no. 3, 2003, pages 425 - 36
CHENG, F. ET AL., IMMUNITY., vol. 19, 2003, pages 425 - 436
DATABASE WPI Week 199032, Derwent World Patents Index; AN 1990-242906, XP002644604 *
DRAGHICI, S. ET AL., GENOME RES., vol. 17, 2007, pages 1537 - 1545
FOLIGNE B. ET AL., PLOS ONE., vol. 2, no. 3, 21 December 2006 (2006-12-21), pages 313
HERRMANN, A. ET AL., CANCER RES., vol. 70, 2010, pages 7455 - 7464
KELLEY V E ET AL: "Interleukin 2-diphtheria toxin fusion protein can abolish cell-mediated immunity in vivo.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA JUN 1988 LNKD- PUBMED:3131768, vol. 85, no. 11, June 1988 (1988-06-01), pages 3980 - 3984, XP002644606, ISSN: 0027-8424 *
KORTYLEWSKI, M. ET AL., NAT. MED., vol. 11, 2005, pages 1314 - 1321
KORTYLEWSKI, M.; YU, H., CURR. OPIN. IMMUNOL., vol. 20, 2008, pages 228 - 233
LICHTNER M. ET AL., AIDS RES HUM RETROVIRUSES., vol. 20, no. 2, 2004, pages 175 - 82
MAGALHAES, J.G. ET AL., J. IMMUNOL., vol. 181, 2008, pages 7925 - 7935
MALDONADO, R.A.; VON ANDRIAN, U.H., ADV. IMMUNOL., vol. 108, 2010, pages 111 - 165
MANICASSAMY S. ET AL., SCIENCE, vol. 329, no. 5993, 2010, pages 849 - 53
MANICASSAMY, S. ET AL., SCIENCE, vol. 329, 2010, pages 849 - 853
MELILLO, J.A. ET AL., J. IMMUNOL., vol. 184, 2010, pages 2638 - 2645
PANKEWYCZ O ET AL: "Interleukin-2-diphtheria toxin fusion protein prolongs murine islet cell engraftment.", TRANSPLANTATION FEB 1989 LNKD- PUBMED:2493178, vol. 47, no. 2, February 1989 (1989-02-01), pages 318 - 322, XP002644607, ISSN: 0041-1337 *
PULENDRAN, B. ET AL., NAT. IMMUNOL., vol. 11, 2010, pages 647 - 655
SCHUBERT, W. ET AL., NAT. BIOTECHNOL., vol. 24, 2006, pages 1270 - 1278
STEINMAN, R.M ET AL., ANNU. REV. IMMUNOL., vol. 21, 2003, pages 685 - 711
VIDALAIN P. O. ET AL., J VIROL., vol. 74, no. 1, 2000, pages 556 - 9
WALSH SCOTT R A ET AL: "Psoriasis and the new biologic agents: interrupting a T-AP dance.", CMAJ : CANADIAN MEDICAL ASSOCIATION JOURNAL = JOURNAL DE L'ASSOCIATION MEDICALE CANADIENNE 22 JUN 2004 LNKD- PUBMED:15210644, vol. 170, no. 13, 22 June 2004 (2004-06-22), pages 1933 - 1941, XP002644605, ISSN: 0820-3946 *

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