JP2008133296A - Chemokines as adjuvant of immune response - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide materials and methods for treating disease states (including cancer and autoimmune disease) by facilitating or inhibiting the migration or activation of antigen-presenting dendritic cells, since dendritic cells play a critical role in antigen-specific immune responses. <P>SOLUTION: Materials and methods for treating disease states (including cancer and autoimmune disease) are provided by facilitating or inhibiting the migration or activation of antigen-presenting dendritic cells, since dendritic cells play a critical role in antigen-specific immune responses. In particular, chemokines are used to initiate, amplify or modulate an immune response. In one embodiment, chemokines are used to attract dendritic cells to the site of antigen delivery. An increased number of dendritic cells at the site of antigen delivery means more antigen uptake and a modified immune response. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  This application claims the benefit of European Patent Application No. 0 974 357 A1 (filed July 16, 1998 and published January 26, 2000).

(Field of Invention)
The present invention relates to the use of human chemokines in the treatment of disease states including cancer. Administered chemokines direct the migration of all antigen presenting dendritic cells or a specific subset of dendritic cells. In one embodiment, disease specific antigens and / or moieties designed to activate dendritic cells are administered with a chemokine.

(Background of the Invention)
Dendritic cells (DCs) are engaged in antigen uptake and their presentation to T cells. Thus, DC plays an important role in antigen-specific immune responses.

  DCs are represented by a diverse population of morphologically similar cell types that are widely distributed throughout the body in various lymphoid and non-lymphoid tissues (Caux et al., 1995, Immunology Today 16: 2; Steinman, 1991, Ann. Rev. Immunol. 9: 271-296). These cells include splenic lymphoid DCs and lymph nodes, epidermal Langerhans cells, and unclear cells in the blood circulation. DCs have some morphology molecules (B7-1 [CD80] and B7-2 [CD86]) that mediate their morphology, high levels of MHC class II expression on the surface, and T cell binding and costimulation (Inaba 1990, Inter. Rev. Immunol. 6: 197-206; Frententhal et al., 1990, Proc. Natl. Acad. Sci. USA 87: 7698), and on T cells, B cells, monocytes, and natural killer cells. Are grouped together as a group based on the absence of certain other surface markers expressed in

  DCs are derived from the bone marrow and migrate as precursors from the bloodstream to tissues where they become resident cells such as Langerhans cells in the epidermis.

  In the periphery, after pathogen invasion, immature DCs, such as fresh Langerhans cells, capture inflammatory sites that capture and process antigens (Kaplan et al., 1992, J. Exp. Med. 175: 1717-1728; McWilliam et al., 1994, J. Exp. Med. 179: 1331-1336) (Inaba et al., 1986. J. Exp. Med. 164: 605-613; Strilein et al., 1989, J. Immunol. 143: 3925-3933; Romani 1989, J. Exp. Med. 169: 1169-1178; Pure et al., 1990. J. Exp. Med. 172: 1459-1469; Schuler et al., 1985, J. Exp. Med. 161: 526-546) .

  The antigen-loaded DC then migrates from the peripheral tissue via lymphatic vessels to a region rich in lymph node T cells, where mature DCs are referred to as interconnected cells (IDCs) (Austyn et al. 1988, J. Exp. Med. 167: 646-651; Kupiec-Weglinski et al., 1988, J. Exp. Med.167: 632-645; Larsen et al., 1990, J. Exp.Med.172: 1483-1494. Fossum, S. 1988, Scand. J. Immunol.27: 97-105; Macatoria et al., 1987, J. Exp.Med.166: 1654-1667; Kripke et al., 1990., J. Immunol.145: 2833- 2838). At this site, they present the processed antigen to naive T cells and generate an antigen-specific initial T cell response (Liu et al., 1993, J. Exp. Med. 177: 1299-1307; Sornasse et al., 1992, J. Exp. Med. 175: 15-21; Heufler et al., 1988, J. Exp. Med. 167: 700-705).

  During these migrations from peripheral tissues to lymphoid organs, DC undergoes a maturation process that includes dramatic changes in phenotype and function (Larsen et al., 1990, J. Exp. Med. 172: 1483-1494; Strilein). 1990, Immunol. Rev. 117: 159-184; De Smedt et al., 1996, J. Exp. Med. 184: 1413-1424). In particular, the IDC of lymphoid organs, in contrast to immature DCs such as fresh Langerhans cells, are effective in efficiently capturing and processing soluble proteins and activating specific memory and effector T cells. Such mature DCs are remarkably effective in priming naïve T cells but poor in antigen capture and processing (Inaba et al., 1986. J. Exp. Med. 164: 605-613; Strilein et al. 1989, J. Immunol. 143: 3925-3933; Romani et al., 1989, J. Exp. Med. 169: 1169-1178; Pure et al., 1990, J. Exp. Med. 172: 1459-1469; 1995, J. Exp. Med. 182: 389- 00; Cella et al., 1997, Current Opin.Immunol.9: 10-16).

  The signals that control DC traffic patterns are complex and not well understood.

  It is known that signals provided by TNFα and LPS induce the migration of resident DCs from tissues to draining lymphoid organs in vivo (De Smedt et al., 1996, J. Exp. Med. 184: 1413-1424). MacPherson et al., 1995, J. Immunol.154: 1317-1322; Roake et al., 1995, J. Exp. Med.181: 2237-2247; Cumbatch et al., 1992, Immunology.75: 257-263; , Immunology. 84: 31-35).

  Chemokines are low molecular weight proteins that regulate leukocyte migration and activation (Openheim, 1993, Adv. Exp. Med. Biol. 351: 183-186; Schall et al., 1994, Curr. Opin. Immunol. 6: 865). Rollins, 1997, Blood 90: 909-928; Baggiolini et al., 1994, Adv. Immunol. 55: 97-179). These are secreted by activated leukocytes themselves, and by stromal cells including endothelial and epithelial cells upon inflammatory stimulation (Openheim, 1993, Adv. Exp. Med. Biol. 351: 183-186; Schall et al. 1994, Curr. Opin. Immunol. 6: 865-873; Rollins, 1997, Blood 90: 909-928; Baggiolini et al., 1994, Adv. Immunol. 55: 97-179). Response to chemokines is mediated by seven transmembrane G protein coupled receptors (Rollins, 1997, Blood 90: 909-928; Premac et al., 1996, Nat. Med. 2: 1174-1178; Murphy, PM et al. 1994, Ann. Rev. Immunol. 12: 593-633). Some chemokines such as monocyte chemotactic protein (MCP) -3, MCP-4, macrophage inflammatory protein (MIP) -1α, MIP-1β, RANTES (activation of expressed and secreted normal T cells SDF-1, Teck (thymus-expressing chemokine) and MDC (macrophage-derived chemokine)) have been reported to attract DCs in vitro (Sozani et al., 1995, J. Immunol. 155: 3292-3295; Sozzani et al., 1997, J. Immunol. 159: 1993-2000; Xu et al., 1996, J. Leukoc. Biol. 60: 365-371; MacPherson et al., 1995, J. Immunol. 154: 1317-1322. Roake et al. 1995, J. Exp. Med. 181: 2237-2247).

Recently, researchers have attempted to take advantage of DC activation in the treatment of cancer. In animal models, as few as 2 × 10 ⁵ antigen-pulsed DCs induce immunity when injected into naïve mice (Inaba et al., 1990, Inter. Rev. Immunol. 6: 197-206). Flamand et al. (Eur. J. Immunol, 1994, 24: 605-610) pulsed mouse DCs with idiotype antigens from B cell lymphomas and injected them into naïve mice. This treatment effectively protected recipient mice from subsequent tumor challenge and established a state of sustained immunity. Injection of antigen alone, or B cells pulsed with antigen, is ineffective, suggesting that it is a unique feature of DCs responsible for the anti-tumor response. Not only is DC capable of inducing anti-tumor immunity, it is postulated that DC is absolutely essential for the development of this process (Ostrand-Rosenberg, 1994, Current Opinion in Immunol. 6: 722). Grabbe et al., 1995, Immunol. Today 16: 117-120; Huang et al., 1994, Science 264: 961-965). Huang and co-workers (Huang et al., 1994, Science 264: 961-965) inoculated mice with B7-1 transfected tumors known to produce anti-tumor immunity. They demonstrated that only mice with MHC compatible APC can reject tumor challenge. Studies in humans have demonstrated a similar role for DC. Peptide-specific CTL has been reported to be easily derived from purified CD8 ⁺ T cells using peptide-pulsed DC, but not when using peptide-pulsed monocytes (Mehta-Damani et al. 1994, J. Immunology 153: 996-1003).

  Histological infiltration of dendritic cells into primary tumor lesions is associated with a very prolonged patient survival and reduced incidence of metastatic disease in patients with bladder, lung, esophageal, gastric and nasopharyngeal cancer It is very interesting clinically. In contrast, a relatively poor clinical prognosis is observed in patients with lesions that show diffuse infiltration with DC, with metastatic lesions frequently lacking DC infiltration (Becker, 1993, In Vivo 7: 187; Zeid et al., 1993, Pathology 25: 338; Furihaton et al., 1992, 61: 409; Tsujitani et al., 1990, Cancer 66: 2012; Gianni et al., 1991, Pathol.Res. Pract. 1993, J. Inv. Dermatol. 100: 3358). Recently, patients with advanced B-cell lymphoma were treated with DC pulsed with their own tumor idiotype (Hsu et al., 1996, Nature Medicine 2 (1): 52). This resulted in a measurable reduction in the patient's B cell lymphoma. Treatment of prostate cancer with DC pulsed with PSM antigen has been reported by Murphy et al. (The Prostate 1996 29: 371).

In response to granulocyte-macrophage colony stimulating factor (GM-CSF) in combination with either interleukin 4 (IL-4) or tumor necrosis factor alpha (TNFα), a number of circulating monocytes or CD34 hematopoietic progenitors Techniques for in vitro growth of DCs have recently emerged (Sallasto et al., 1994, J. Exp. Med. 179: 1109-1118; Romani et al., 1994, J. Exp. Med. 180: 83-93: Caux.
Et al., 1992, Nature 360: 258). The combination of GM-CSF and IL-4 induces peripheral blood monocytes to differentiate into potent DCs (Kiertscher and Roth, 1996, J. Leukocyte Biol. 59: 208-281). With the combination of these two cytokines, a 100-fold increase in DC yield from peripheral blood is achieved in vitro.

In mice, various groups have been shown that DCs generated in vitro loaded with tumor antigens prevent tumor development and, more importantly, induce regression of established tumors. A clinical trial was conducted in which melanoma patients were treated with GM-CSF activated APC pulsed with peptides derived from MAGE-1 tumor antigen (Mehta-Damani et al., 1994, J. Immunology 153: 996-1003). . Prior to immunization, tumor infiltrating lymphocytes from two patients were predominantly CD4 ⁺ and each lacked specific tumor reactivity. In contrast, after immunizing tumor infiltrating lymphocytes from the same patient, CD8 ⁺ was dominant, indicating MAGE-1 specific anti-tumor cytotoxicity. Thus, it is clear from these studies that DC has a unique and strong ability to stimulate an immune response.

  Thus, dendritic cell therapy represents a very promising approach to the treatment of diseases (particularly cancer). There continues to be improved materials and methods that not only proliferate and activate antigen-presenting dendritic cells, but can also be used to promote DC migration to be therapeutically useful and prophylactically useful. Is necessary.

(Summary of the Invention)
The present invention fulfills the aforementioned need by providing materials and methods for treating disease states by promoting or inhibiting the migration or activation of antigen presenting dendritic cells. At present, chemokines have been found to be useful therapeutic agents. Disease states that can be treated according to the present invention include parasitic infections, bacterial infections, viral infections, fungal infections, cancer, autoimmune diseases, graft rejection, and allergies.

  The present invention provides a method of treating a disease state, wherein the method provides an individual in need of treatment of the disease state with an amount of chemokine sufficient to increase migration of immature dendritic cells to the antigen delivery site. Administering. In one aspect of the invention, chemokines (eg, MCP-1, MCP-2, MCP-3, MCP-4, MIP-1α, MIP-3α, RANTES, SDF-1, Teck, DC tactin-β, 6Ckine , MDC, MIP-5, or combinations thereof). In preferred methods of the invention, a disease associated antigen (eg, a tumor associated antigen) is administered with a chemokine.

  Another aspect of the invention provides a method of treating a disease state that is sufficient for an individual in need of treating the disease state to reduce migration of immature dendritic cells to an antigen delivery site. Administering an amount of chemokine.

  In yet another aspect of the invention, cytokines (especially GM-CSF and IL-4) are administered in combination with chemokines, either before chemokines or simultaneously with chemokines. Administration of GM-CSF and IL-4 stimulates the production of DC from the precursor, thereby increasing the number of DC available to capture and process the antigen.

  Yet another aspect of the invention is that the activating agent (eg, an agonist of TNF-α, IFN-α, RANK-L or RANK, and an antagonist of a molecule of the toll-like receptor family) passes from the tissue through the draining lymph. Administered to provide a maturation signal that drives the movement of DC towards the lymphatic organs.

  The invention also provides a method of enhancing an immune response in a mammal, comprising administering to the mammal a chemokine MCP-4 or a biologically active fragment of MCP-4. Human MCP-4 (hMCP-4) is active against human blood dendritic cells and recruits dendritic cells and dendritic cell precursors from the blood. In a preferred aspect, the chemokine is a recombinant chemokine. Most preferably, the chemokine is administered with the antigen, for example, in the form of a recombinant chemokine-antigen fusion protein. Such an antigen can be a tumor associated antigen, a bacterial antigen, a viral antigen or a fungal antigen.

  Furthermore, the present invention provides a method of enhancing an immune response in a mammal, the method comprising administering to the mammal a chemokine 6Ckine or a biologically active fragment of 6Ckine. Human 6Ckine is active against human blood dendritic cells and recruits dendritic cells and dendritic cell precursors from the blood. Replenishment of dendritic cells indicates that chemokine 6Ckine acts as an antitumor agent and specifically exhibits an angiogenic effect on tumor blood vessels. In a preferred aspect, the chemokine is a recombinant chemokine. Most preferably, the chemokine is administered with the antigen, for example, in the form of a recombinant chemokine-antigen fusion protein. Such an antigen can be a tumor associated antigen, a bacterial antigen, a viral antigen or a fungal antigen.

  In yet another aspect of the invention, cytokines (especially GM-CSF and IL-4) are administered in combination with chemokines (either before chemokines or simultaneously with chemokines).

In a final aspect, the present invention provides a fusion protein comprising MCP-4 or a biologically active portion of MCP-4 and an antigen, and 6Ckine or a biologically active portion of 6Ckine and an antigen. These fusion proteins can be administered to the mammal in the form of a plasmid, viral vector, or in the form of a recombinant vector.
The present invention provides the following.
(1) Use of a chemokine that can direct the migration of dendritic cells in the manufacture of a medicament for the treatment of a disease state.
(2) The use according to item 1, wherein the chemokine is MCP-1, MCP-2, MCP-3, MCP-4, MIP-1α, MIP-1β, MIP-3α, RANTES, SDF-1 , Teck, DCactin-β, 6Ckine / SLC, LEC, MDC, and MIP-5.
(3) The use according to item 1, wherein the chemokine can direct the movement of dendritic cells to an antigen delivery site.
(4) The use according to item 1, wherein the chemokine can direct the migration of dendritic cells to lymphoid organs.
(5) Use according to item 1, wherein the disease state is bacterial infection, viral infection, fungal infection, parasitic infection or cancer.
(6) Use according to item 1, wherein the disease state is autoimmune disease, tissue rejection, or allergy.
(7) The use according to item 5, wherein the disease state is melanoma, breast cancer, pancreatic cancer, colon cancer, lung cancer, glioma, hepatocellular carcinoma, endometrial cancer, gastric cancer, intestinal cancer, kidney Cancer, prostate cancer, thyroid cancer, ovarian cancer, testicular cancer, liver cancer, head and neck cancer, colorectal cancer, esophageal cancer, stomach cancer, eye cancer, bladder cancer, glioblastoma type, and metastatic carcinoma Use wherein the cancer is selected from the group consisting of:
(8) The use according to item 3, wherein the dendritic cell is an immature dendritic cell.
(9) The use according to item 8, wherein the chemokine is MCP-1, MCP-2, MCP-3, MCP-4, MIP-1B, MDC, MIP-3α, MIP-1α, RANTES and MIP Use, selected from the group consisting of -5.
(10) Use according to item 4, wherein the chemokine is MIP-3β.
(11) The use according to item 3, further comprising the use of at least one disease-related antigen.
(12) The use according to item 11, wherein the antigen is a tumor-associated antigen.
(13) The use according to item 11, wherein the antigen is a bacterial antigen, a viral antigen, or a fungal antigen.
(14) The use according to item 12, wherein the tumor-associated antigen is Melan-A, tyrosinase, p97, β-HCG, GalNAc, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE -12, MART-1, MUC1, MUC2, MUC3, MUC4, MUC18, CEA, DDC, melanoma antigen gp75, Hker8, high molecular weight melanoma antigen, K19, Tyr1 and Tyr2, members of the pMel17 gene family, c-Met , PSA, PSM, α-fetoprotein, thyroid peroxidase, gp100, NY-ESO-1, telomerase and p53.
(15) The use according to item 14, wherein the cancer is prostate cancer and the tumor-associated antigen is PSA and / or PSM.
(16) The use according to item 14, wherein the disease state is melanoma and the tumor-associated antigen is Melan-A, gp100 or tyrosinase.
(17) The use according to item 1, further including the use of an activating drug.
(18) The use according to item 15, wherein the activating agent is selected from TNFα, RP-105, an anti-CD40 antibody, a nucleic acid containing an unmethylated CpG motif, or a toll-like receptor ligand. .
(19) The use according to item 1, further comprising the use of a combination of GM-CSF and IL-4 together with the chemokine.
(20) The use according to item 1, wherein the chemokine is administered intradermally, intramuscularly, subcutaneously, topically, or in the form of a vector.
(21) A method of enhancing an immune response in a mammal comprising the step of administering to the mammal a chemokine MCP-4 or a biologically active fraction of chemokine MCP-4. Method.
(22) The method according to item 21, wherein the chemokine is a recombinant chemokine.
(23) The method according to item 21, wherein the chemokine is a human chemokine.
(24) The method according to item 21, wherein the method further comprises the step of administering a substance that enables sustained release of the chemokine to a delivery site.
(25) The method according to item 21, wherein the method further comprises a step of administering an antigen together with the chemokine.
(26) The method according to item 25, wherein a fusion protein comprising MCP-4 and an antigen is administered to the mammal.
(27) The method according to item 25, wherein the antigen is a tumor-associated antigen.
(28) The method according to item 26, wherein the antigen is a tumor-associated antigen.
(29) The method according to item 25, wherein the antigen is a bacterial antigen, a viral antigen, or a fungal antigen.
(30) The method according to item 26, wherein the antigen is a bacterial antigen, a viral antigen or a fungal antigen.
(31) The method according to item 25, wherein the tumor-associated antigen is Melan-A, tyrosinase, p97, β-HCG, GalNAc, MAGE-1, MAGE-2, MAGE-3, MAGE-4, or MAGE. -12, MART-1, MUC1, MUC2, MUC3, MUC4, MUC18, CEA, DDC, melanoma antigen gp75, Hker8, high molecular weight melanoma antigen
, K19, Tyr1 and Tyr2, a member of the pMel 17 gene family, c-Met, PSA, PSM, α-fetoprotein, thyroid peroxidase, gp100, p53 and telomerase.
(32) The method according to item 26, wherein the tumor-associated antigen is Melan-A, tyrosinase, p97, β-HCG, GalNAc, MAGE-1, MAGE-2, MAGE-3, MAGE-4, or MAGE. -12, MART-1, MUC1, MUC2, MUC3, MUC4, MUC18, CEA, DDC, melanoma antigen gp75, Hker8, high molecular weight melanoma antigen, K19, Tyr1 and Tyr2, members of the pMel17 gene family, c-Met , PSA, PSM, α-fetoprotein, thyroid peroxidase, gp100, p53 and telomerase.
(33) The method according to item 25, further comprising administering a combination of GM-CSF and IL-4.
(34) The method according to item 26, further comprising administering a combination of GM-CSF and IL-4.
(35) The method according to item 21, further comprising the step of administering an activating drug together with the chemokine.
(36) The method according to item 21, wherein the chemokine is administered intradermally, intramuscularly, subcutaneously, topically, or in the form of a vector.
(37) A fusion protein comprising MCP-4 and an antigen.
(38) The fusion protein according to item 37, wherein the antigen is a tumor-associated antigen.
(39) The fusion protein according to item 38, wherein the tumor-associated antigen is Melan-A, tyrosinase, p97, β-HCG, GalNAc, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-12, MART-1, MUC1, MUC2, MUC3, MUC4, MUC18, CEA, DDC, melanoma antigen gp75, Hker8, high molecular weight melanoma antigen, K19, Tyr1 and Tyr2, members of the pMel17 gene family, c- A fusion protein selected from the group consisting of Met, PSA, PSM, α-fetoprotein, thyroid peroxidase, gp100, p53 and telomerase.
(40) The fusion protein according to item 38, wherein the antigen is a bacterial antigen, a viral antigen or a fungal antigen.
(41) A plasmid comprising the fusion protein according to item 37.
(42) The plasmid according to item 39, further comprising a promoter sequence particularly suitable for dendritic cells.
(43) A viral vector comprising the fusion protein according to item 37.
(44) A method of enhancing an immune response in a mammal, comprising the step of administering to the mammal a chemokine 6Ckine or a biologically active fraction of the chemokine 6Ckine.
(45) The method according to item 44, wherein the chemokine is a recombinant chemokine.
(46) A method according to item 44, wherein the chemokine is a human chemokine.
(47) A method according to item 44, wherein the method further comprises the step of administering a substance that enables sustained release of the chemokine to a delivery site.
(48) The method according to item 44, further comprising the step of administering an antigen together with the chemokine.
(49) The method according to item 48, wherein a fusion protein comprising 6Ckine and an antigen is administered to the mammal.
(50) The method according to item 48, wherein the antigen is a tumor-associated antigen.
(51) The method according to item 49, wherein the antigen is a tumor-associated antigen.
(52) A method according to item 48, wherein the antigen is a bacterial antigen, a viral antigen, or a fungal antigen.
(53) A method according to item 49, wherein the antigen is a bacterial antigen, a viral antigen, or a fungal antigen.
(54) The method according to item 48, wherein the tumor-associated antigen is Melan-A, tyrosinase, p97, β-HCG, GalNAc, MAGE-1, MAGE-2, MAGE-3, MAGE-4, or MAGE. -12, MART-1, MUC1, MUC2, MUC3, MUC4, MUC18, CEA, DDC, melanoma antigen gp75, Hker8, high molecular weight melanoma antigen, K19, Tyr1 and Tyr2, members of the pMel17 gene family, c-Met , PSA, PSM, α-fetoprotein, thyroid peroxidase, gp100, p53 and telomerase and C26 colon cancer.
(55) The method according to item 49, wherein the tumor-associated antigen is Melan-A, tyrosinase, p97, β-HCG, GalNAc, MAGE-1, MAGE-2, MAGE-3, MAGE-4, or MAGE. -12, MART-1, MUC1, MUC2, MUC3, MUC4, MUC18, CEA, DDC, melanoma antigen gp75, Hker8, high molecular weight melanoma antigen, K19, Tyr1 and Tyr2, members of the pMel17 gene family, c-Met , PSA, PSM, α-fetoprotein, thyroid peroxidase, gp100, p53 and telomerase and C26 colon cancer.
(56) The method according to item 48, further comprising administering a combination of GM-CSF and IL-4.
(57) The method according to item 49, further comprising administering a combination of GM-CSF and IL-4.
(58) The method according to item 44, further comprising administering an activating drug together with the chemokine.
(59) The method according to item 44, wherein the chemokine is administered intradermally, intramuscularly, subcutaneously.
, Administered topically, or in the form of a vector.
(60) A fusion protein comprising 6Ckine and an antigen.
(61) The fusion protein according to item 60, wherein the antigen is a tumor-associated antigen.
(62) The fusion protein according to item 61, wherein the tumor-associated antigen is Melan-A, tyrosinase, p97, β-HCG, GalNAc, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-12, MART-1, MUC1, MUC2, MUC3, MUC4, MUC18, CEA, DDC, melanoma antigen gp75, Hker8, high molecular weight melanoma antigen, K19, Tyr1 and Tyr2, members of the pMel17 gene family, c- A fusion protein selected from the group consisting of Met, PSA, PSM, α-fetoprotein, thyroid peroxidase, gp100, p53 and telomerase.
(63) The fusion protein according to item 61, wherein the antigen is a bacterial antigen, a viral antigen or a fungal antigen.
(64) A plasmid comprising the fusion protein according to item 60.
(65) The plasmid according to item 64, further comprising a promoter sequence particularly suitable for dendritic cells.
(66) A viral vector comprising the fusion protein according to item 60.

(Detailed description of the invention)
All references cited herein are incorporated by reference in their entirety.

  The relationship between signals inducing DC migration in vivo and their response to chemokines has never been known. We have changed the pattern of chemokine receptors expressed by DCs according to their maturation stage, and chemokines can be used to drive migration of DC subsets, thereby controlling the initiation of immune responses. I found it to get. Chemokines can be used according to the present invention as adjuvants to selectively attract immature DC subsets to the antigen delivery site. In the context of autoimmune diseases, tissue rejection or allergies, the present invention provides for DC function by interfering with DC migration, for example through the generation of CCR6 agonists and antagonists, CCR7 agonists and antagonists, and CCR2 agonists and antagonists. Provide a way to block.

  Depending on the subset of DCs presenting the antigen to the immune system, the response varies dramatically. DC can induce tolerance. DCs found in the thymic medulla may play a role in negative selection to generate self-selective thymocytes (Bricker et al., 1997, J. Exp. Med. 185 (3): 541-550). DCs are also tolerated by autoreactive peripheral T cells (Kurts et al., 1997, J. Exp. Med. 186 (2): 239-245; Adler et al., 1998, J. Exp. Med. 187 (10): 1555-1564). A specific subset of mouse DCs (probably of lymphatic origin) has been proposed to induce immune tolerance (Ardavin, 1993, Nature 362 (6422): 761-763). Furthermore, the recent explanation that candidate human counterpart (DC-2) for lymphoid DC (Grouard et al., 1997, J. Exp. Med. 185 (6): 1101-1111) is unable to secrete IL-12 is This suggests that after presentation by the population, the immune response may be biased towards the TH-2 type.

  If the goal is to reduce the immune response, tolerance to DC (autoimmunity, allergy) is increased or the quality of the response is altered by specifically increasing DC-2 (TH2 Greater TH1, ie in allergies).

  Chemokines for use in the present invention are the body's natural proteins that are active against a limited subset of DCs, particularly immature DCs. Some of these chemokines have been identified by the present inventors, including but not limited to MIP-3α, Teck, MDC and MCP-4, and 6Ckine.

  The chemokine used in practicing the present invention can be derived from a recombinant protein having the same amino acid sequence as a natural product or a natural product, but the drug while retaining its original chemotaxis characteristics It can be a recombinant protein containing modifications that alter kinetic properties. The mode of delivery of chemokines can be by injection (including intradermal, intramuscular and subcutaneous) or topically (eg, ointment or patch).

  Chemokines can also be delivered as nucleic acid sequences by vectors (eg, viral vectors) (eg, adenoviruses, poxviruses, retroviruses, lentiviruses), or engineered plasmid DNA.

  As used herein, the term “chemokine” can include chemotactic factors. The chemotactic factor can be a small molecule compound, which is a selective agonist of the chemokine receptor expressed by immature DCs. CCR6 (the natural receptor for the chemokine MIP-3α) is an example of such a receptor.

  In a particularly preferred embodiment of the invention, the chemokine is administered with a disease associated antigen. The antigen can be any molecular moiety, against which an increase or decrease in immune response is examined. This includes antigens derived from organisms known to cause disease in humans or animals (eg, bacteria, viruses, parasites (eg, Leishmania), and fungi). This also includes antigens expressed by tumors (tumor-associated antigens) and plant antigens (allergens).

  Tumor associated antigens for use in the present invention include but are not limited to: Melan-A, tyrosinase, p97, β-HCG, GalNAc, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-12, MART-1, MUC1, MUC2, MUC3, MUC4, MUC18, CEA, DDC, melanoma antigen gp75, HKer 8, high molecular weight melanoma antigen, K19, Tyr1 and Tyr2, pMel 17 gene family C-Met, PSA, PSM, α-fetoprotein, thyroid peroxidase, gp100, NY-ESO-1, telomerase, and p53. This list is not intended to be exhaustive of the types of antigens that may be used in the practice of the present invention, but is merely exemplary.

  Different combinations of antigens can be used, which show optimal function according to different racial groups, gender, geographical distribution, and disease stage. In one embodiment of the invention, at least two or more different antigens are administered with the administration of the chemokine.

  The antigen can be delivered or administered at the same site and time as the chemokine or after a delay not exceeding 48 hours. Co-administration or combined administration, as used herein, causes the chemokine and antigen to differ (a) within time or (b) during the course of a general treatment schedule to the subject. Be administered at any time. In the latter case, the two compounds are administered within a time sufficiently close to achieve the intended effect. The antigen can be in the form of a protein, or one or several peptides, or in the form of a nucleic acid sequence contained in a delivery vector.

  Both primary and metastatic cancers can be treated according to the present invention. Types of cancer that can be treated include, but are not limited to: melanoma, breast cancer, pancreatic cancer, colon cancer, lung cancer, glioma, hepatocellular carcinoma, endometrial cancer, gastric cancer, intestinal cancer. , Kidney cancer, prostate cancer, thyroid cancer, ovarian cancer, testicular cancer, liver cancer, head and neck cancer, colorectal cancer, esophageal cancer, stomach cancer, eye cancer, bladder cancer, glioblastoma type, and metastasis Sexual cancer. The term “cancer” refers to a malignant tumor of epithelial or endocrine tissue, including respiratory, gastrointestinal, urogenital, prostate, endocrine, and melanoma. Metastatic is, as the term is used herein, defined as the spread of the tumor to a site away from the primary tumor (including local lymph nodes).

  Portions designed to achieve, induce or stimulate DC maturation can be advantageously administered. Such agents provide a maturation signal that promotes migration from tissue to lymph nodes. This moiety can be, for example, a bodily natural product, such as TNF-α or RP-105, or an agonist antibody (eg, an anti-CD-40 antibody) that recognizes a specific structure on DC, or another substance. . The activator can be an unmethylated CpG motif or a sequence of nucleic acids containing agonists of toll-like receptors known to stimulate DCs. In embodiments of the invention in which chemokines and / or antigens are delivered by plasmid vectors, these nucleic acid sequences can be part of the vector.

  GM-CSF and IL-4 can advantageously be administered in combination with chemokines and / or antigens. The combination of GM-CSF and IL-4 stimulates the production of DC from the precursor. GM-CSF and IL-4 can be administered to increase the number of circulating immature DCs. This can be replenished locally by subsequent injections of chemokines. This protocol includes systemic pretreatment for at least 5-7 days using GM-CSF and IL-4. Another method is advantageous for local differentiation of DC precursors (monocytes) into immature DCs, where antigens delivered to the same site can be picked up by local administration of GM-CSF and IL-4.

  Generally, the chemokine and / or antigen and / or activating agent and / or cytokine are administered as a pharmaceutical composition comprising an effective amount of the chemokine and / or antigen and / or activating agent and / or cytokine in a pharmaceutical carrier. Is done. These reagents can be combined with physiologically non-stimulating stabilizers and excipients, eg, in a conventional pharmaceutically acceptable carrier or excipient (eg, an immunogenic adjuvant), for additional active ingredients or Can be combined for therapeutic use with active ingredients. The pharmaceutical carrier can be any compatible, non-toxic substance suitable for delivering a composition of the invention to a patient.

The amount of reagent required for effective treatment depends on many different factors, including the means of administration, the target site, the physical condition of the patient, and other medications being administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Animals that test effective doses to treat certain cancers provide further predictive indicators of human dosage. Various considerations are described, for example, by Gilman et al. (Eds.) (1990) Goodman and.
Gilman's: The Pharmacological Bases of Therapeutics, 8th edition, Pergamon Press; and Remington's Pharmaceutical Sciences, 17th edition (1990), Mack Publishing Co., Ltd. , Easton, PA. Methods for administration are discussed therein and below, for example, for intravenous, intraperitoneal, or intramuscular administration, transdermal diffusion, and the like. Pharmaceutically acceptable carriers include water, saline, buffered solutions and, for example, the Merck Index, Merck & Co. , Rahway, New Jersey. Sustained release formulations or sustained release devices can be used for continuous administration.

  The dosage range for chemokines and / or antigens and / or activating agents is usually less than 1 mM, typically less than about 10 μM, usually less than about 100 nM, preferably with a suitable carrier, It is expected to be in an amount less than about 10 pM (picomolar), and most preferably less than about 1 fM (femtomolar). Generally, treatment is initiated with smaller dosages that are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. Determination of the appropriate dose and dosing regimen for a particular situation is within the skill of the art.

The preferred biologically active doses of GM-CSF and IL-4 in the practice of the present invention are the number of circulating CD14 ⁺ / CD13 ⁺ precursor cells; the antigen-presenting molecules on the surface of DC precursors and mature DCs Dosing combination that induces the greatest increase in stimulation of antigen-dependent T cell responses consistent with expression; antigen presentation activity to T cells; and / or mature DC function. In the practice of the present invention, the amount of IL-4 used for subcutaneous administration is typically about 0.05 to about 8.0 μg / kg / day, preferably 0.25 to 6.0 μg. / Kg / day, most preferably in the range of 0.50-4.0 μg / kg / day. The amount of GM-CSF used for subcutaneous administration is typically about 0.25 μg / kg / day to about 10.0 μg / kg / day, preferably about 1.0 to 8.0 μg / kg / day. Day, most preferably in the range of 2.5 to 5.0 μg / kg / day. An effective amount for a particular patient is established by measuring significant changes in one or more of the above parameters.

  Administration of a chemokine MCP-4 or biologically active fragment of MCP-4 promotes the recruitment of mouse dendritic cells in vivo in a dose-dependent manner and against human dendritic cells isolated from blood Even found to be active. Biologically active fragment means a portion of the MCP-4 molecule sufficient to stimulate a measurable immune response. This response can be measured as an enhancement of antigen-specific stimulation of immunoglobulin levels in the serum (typically known as the B cell response). Furthermore, biologically active fragments of MCP-4 stimulate the production of specific classes of immunoglobulins (eg, IgG2a that requires expansion in T cells). In addition, biologically active fragments of MCP-4 enhance antigen-specific anti-tumor responses. The enhanced response can be measured by delayed tumor growth or delayed tumor index after challenge with antigen expressing tumors. An enhanced immune response can also be measured by analysis of antigen-specific cytotoxic responses of a defined population of lymphocytes (blood, spleen, lymph node, tumor). Of course, it is recognized that small molecules that are CCR2 agonists (eg, found by drug discovery screening) also enhance antigen-specific anti-tumor responses. The underlying reason is that all MCPs (1-4) are natural CCR2 agonists, and subsequently artificial small molecule agonists can have the same effect. Many current therapeutic agents are small molecules obtained by organic chemical synthesis.

  A preferred embodiment is a recombinant hMCP-4 protein alone or a substance that allows sustained release at the delivery site (depot); a fusion protein consisting of hMCP-4 or a fraction of hMCP-4 and an antigen (a peptide larger than 9 amino acids or A protein); a DNA or viral vector (peptide or protein larger than 9 amino acids) encoding hMCP-4 or a fraction of hMCP-4 (with or without antigen), or combined with a nucleic acid sequence contained in a delivery vector The recombinant hMCP-4 protein is not limited thereto.

  Human MCP-4 (hMCP-4) belongs to the CC family of chemokines. The sequence was first published in 1996 (Uguccioni et al., 1996, Monocyte Chemical Protein 4 (MCP-4), A Novel Structural and Functional Analog of MCP-3 and Eotax. -2394). Human MCP-4 is an 8.6 kDa peptide consisting of 75 amino acid residues (FIG. 3). This is also known as CK-β-10, SCY-A13 and NCC-1 (Swiss-Prot accession number Q99616) and was renamed CCL13 in the new chemokine nomenclature (Zlotnik et al., 2000, Chemokines: A New Classification System and Their Role In Immunity, Immunity, 12: 121-127).

6Ckine belongs to the CC family of chemokines (Hedrick et al., 1997, J. Immunol. 159: 1589-1593). CK-β-9, exodus-2 and SLC (Swiss-Prot accession number 000585 for human protein
) And was renamed CCL21. Human 6Ckine (h6Ckine) binds to the chemokine CCR7, while mouse 6Ckine (m6Ckine) binds to CCR7 and CXCR3 receptors, but with low affinity (Jenh et al., 1999, J. Immunol. 162: 3765-3769). . Mouse 6Ckine has been shown to have an anti-tumor effect when injected into a tumor in a mouse (Sharma et al., 2000, J. Immunol. 164: 4558-4563).

6Ckine-like MIP-3β and MCP-4 induce the migration of mature DCs. Interestingly, 6Ckine and MIP-3β are found in all human DC populations (CD1a ⁺ Langerhans cells, CD14 ⁺ stromal DC, monocyte-derived DC, circulating blood CD11c ⁺ DC, monocytes, and circulating blood CD11c ⁻ after maturation. Migration of plasma cell-like DCs). Responses to 6Ckine are observed after maturation induced by several DC activators (including CD40-L, TNF-α, and LPS). As seen in the case of MIP-3β, CCR7 is upregulated during DC activation via 6Ckine, possibly explaining the response to 6Ckine.

Thus, it has been proposed that the chemokine h6Ckine can be used in cancer treatment. Preferred embodiments consist of, but are not limited to: recombinant h6Ckine protein alone or in combination with a substance that allows its sustained release at the delivery site (depot at the tumor site); fusion protein or h6Ckine Or a construct made by chemical ligation, consisting of a fraction of h6Ckine and a targeting moiety (eg, antibody or antibody fragment, protein ligand, peptide larger than 10 amino acids) that allows delivery of the construct to the tumor; h6Ckine Or a DNA or viral vector (eg, adenovirus) encoding a fraction of h6Ckine (with or without the targeting moiety described above).

  The invention may be illustrated by the following non-limiting examples. The following examples can be readily understood by reference to the following materials and methods.

(Hematopoietic factors, reagents and cell lines) Recombinant GM-CSF (specific activity: 2.10 ⁶ U / mg
, Schering-Plough Research Institute, Kenilworth, NJ) at a saturating concentration of 100 ng / ml. Recombinant human TNFα (specific activity: 2 × 10 ⁷ U / mg, Genzyme, Boston, Mass.) Was used at an optimal concentration of 2.5 ng / m. Recombinant human SCF (specific activity: 4 × 10 ⁵ U / mg, R & D Abington, UK) was used at an optimal concentration of 25 ng / ml. Recombinant human IL-4 (specific activity: 2.10 ⁷ U / mg, Schering-Plough Research Institute, Kenilworth, NJ) was used at a saturating concentration of 50 U / ml. Recombinant human chemokine MIP-1α (specific activity: 2 × 10 ⁵ U / mg, 9 × 10 ¹² U / M), RANTES (specific activity: 1 × 10 ⁴ U / mg, 8 × 10 ¹⁰ U / M), MIP-3α (specific activity: 4 × 10 ⁵ U / mg, 3 × 10 ¹² U / M) and MIP-3β (specific activity: 1 × 10 ⁴ U / mg, 9 × 10 ¹⁰ U / M), R & D (Abington, UK). LPS was used at 10 ng / ml (Sigma).
A mouse CD40 ligand transfected cell line (CD40-L L cells) was used as a stimulator of DC maturation.

Generation of DC from cord blood CD34 ⁺ HPC Umbilical cord blood samples were obtained after full period delivery. Cells with CD34 ⁺ antigen, as described (Caux et al., 1996, J.Exp.Med.184: 695-706; Caux et al, 1990, Blood.75: 2292-2298), anti-CD34 ⁺ monoclonal antibodies ( Immu-133.3, Immunotech Marseille, France), goat anti-mouse IgG coated microbeads (Miltenyi Biotec GmBH, Bergish Gladbach, Germany) and Minimacs separation column (Miltenyi Biotec) via positive selection using a microsphere Isolated from minutes. In all experiments, isolated cells were 80% -99% CD34 ⁺ . After purification, CD34 ⁺ cells were cryopreserved in 10% DMSO.

Endotoxin-free medium (10% (v / v) inactivated as described (Caux et al., 1996, J. Exp. Med. 184: 695-706) in the presence of SCF, GM-CSF and TNFα. Fetal bovine serum (FBS) (Life Technologies, France, Irvine, UK), 10 mM Hepes, 2 mM L-glutamine, 5 × 10 ⁻⁵ M D-mercaptoethanol, 100 μg / ml gentamicin (Schering-Plough, Levallois, France) Replenished RPMI
1640 (consisting of Gibco, Grand Island, NY) (also called complete medium). After thawing, CD34 ⁺ cells were placed in a 25-75 cm ² culture vessel (
(Linbro, ICN Biomedicals, Acron, OH) for seeding to grow to 2 × 10 ⁴ cells / ml. Optimal conditions were maintained by dividing these cultures on days 5 and 10 using media containing fresh GM-CSF and TNFα (cell concentration: 1-3 × 10 ⁵ cells / cell). ml). CD1a ⁺ DC is between 70-90% of cells on day 12.

(Isolation of immature and mature DCs following CD86 expression by FACS sorting)
After 12 days of culture in the presence of GM-CSF and TNFα, cells were harvested and FITC-conjugated OKT6 (CD1a) (Ortho Diagnostics System, Raritan, NJ) and PE-conjugated IT2.2 (CD86) ( Pharmingen, San Diego, CA). Cells were transferred to immature CD1a ⁺ CD86 ⁻ DC population, and mature CD1a ⁺ CD86 ⁺ DC population according to CD1a and CD86 expression, FACStarplus® (laser setting: output 250 mW, excitation wavelength 488 nm, Becton-Dickinson, Sunnyvale, CA). All staining and sorting procedures were performed with 0.5 mM to avoid cell aggregation.
Performed in the presence of EDTA. Re-analysis of the sorted population showed a purity> 98%.
Generation of DC from peripheral blood monocytes. Monocytes were purified by PBMC preparation followed by 52% Percoll gradient by immunomagnetic removal (Dynabeads, Dynal Oslo, Norway). Removal of anti-CD3 monoclonal antibody (OKT3), anti-CD19 monoclonal antibody (4G2), anti-CD8 monoclonal antibody (OKT8), anti-CD56 monoclonal antibody (NKH1, Coulter Corporation, Hialeah, FL) and anti-CD16 monoclonal antibody (ION16, Immunotech) ). Monocyte-derived dendritic cells were generated by culturing purified monocytes for 6-7 days in the presence of GM-CSF and IL-4 (Sallusto et al., 1994, J. Exp. Med. 179: 1109-1118. ).

Induction of maturation of DC generated in vitro CD34 ⁺ HPCs were cultured in the presence of GM-CSF + TNFα up to day 6 and their un-exposed from day 6 to day 12 in the presence of GM-CSF alone. Cultured to maintain maturity. CD34 ⁺ HPC-derived immature DCs or monocyte-derived DCs in the presence of TNFα (2.5 ng / ml) or LPS (10 ng / ml) or CD40L transfected L cells (1 L cell for 5DCs) 3 Activated as described (Caux et al., 1994, J. Exp. Med. 180: 1263-1272) between hours and 72 hours.

The CD11c ⁺ DC (CD11c ⁺ Purification of DC from peripheral blood or tonsil) were prepared as previously described from peripheral blood or tonsils (Grouard, et al., 1996, Nature
384: 364-367). Briefly, tonsils obtained from children who had undergone tonsils removal were minced and digested using collagenase IV and DNase I (Sigma). Harvested cells were centrifuged through Ficoll-Hypaque containing SRBC (BioMerieux, Lyon, France) at 500 rpm for 15 minutes and then at 2000 rpm for 30 minutes. Peripheral blood monocytes (PBMC) were isolated by Ficoll-Hypaque. CD3 ⁺ T cells (OKT3), CD19 ⁺ B cells (4G7), and CD14 ⁺ monocytes (MOP9) were removed from the resulting low density cells by magnetic beads (anti-mouse Ig coated Dynabeads, Dynal). A second removal was performed using anti-NKH1, anti-glycophorin A (Immunotech) and anti-CD20 (1F54). The remaining cells were stained with the following mAbs: anti-CD1a FITC (OKT6); anti-CD14 FITC, anti-CD57 FITC, anti-CD16 FITC, anti-CD7 FITC, anti-CD20 FITC, anti-CD3 FITC (Becton Dickinson, Mountain Wine , CA); anti-CD4 PE-Cy5 (Immunotech) as well as anti-CD11c PE (Becton Dickinson). CD4 ⁺ CD11c ⁺ series ⁻ DCs were isolated by cell sorting using a FACStarPlus® (laser setting: output 250 mW, excitation wavelength 488 nm). All removal, staining and sorting procedures were performed in the presence of 0.5 mM EDTA. A re-analysis of the sorted population showed a purity> 97%.

Chemotaxis assay Cell migration was performed using chemotaxis microchamber technology (48-well Boyden microchamber, Neuroprobe, Pleasanton, CA) (Bacon et al., 1988, Br. J. Pharmacol. 95: 966-974). evaluated. Briefly, human recombinant MIP-3α and MIP-3β, MIP-1α and RANTES are diluted in RPMI 1640 medium to concentrations ranging from 1 ng / ml to 1000 ng / ml, and the lower well of the chemotaxis chamber Added to. 10 ⁵ cells / well (or 5 × 10 ⁴ cells / well for CD11c ⁺ DC) in 50 μl of RPMI 1640 medium, and a standard 5 μm pore polyvinylpyrrolidone free polycarbonate filter (Neuroprobe separating the lower wells). ) Was applied to the upper well of the chamber. The chamber was incubated for 1 hour at 37 ° C. in humidified air with 5% CO ₂ . Cells that migrated to the lower surface of the filter were then stained using Field's A and Field's B (BDH, Dorset, England) in two randomly selected low power fields (magnification x20). Counting was done using an image analyzer (software: Vision Explorer and ETC 3000, Graphtek, Mirmande, France). Each assay was performed in duplicate and results were expressed as the mean ± SD of migrating cells per 2 fields.

Total RNA Extraction and cDNA Synthesis Cells were prepared as described above and total RNA was extracted by the guanidinium thiocyanate method (RNAgents total RNA isolation system, Promega) as noted by the manufacturer. Following DNAse I (RQ1 RNAse-free DNAse, Promega) treatment, RNA was quantified with a spectrophotometer and quality was assessed by electrophoresis under formaldehyde denaturing conditions. First strand cDNA was synthesized from total RNA extracted under RNAse-free conditions. This reaction was performed as described by the manufacturer, with 5 μg total RNA, 25 ng / μl oligo dT _12-18 primer (Pharmacia, Orsay, France) and Superscript kit (SuperScript II RNase H ^- Reverse TransRB, G). Made using. For all samples, cDNA synthesis was controlled and calibrated by 21 cycles of RT-PCR using β-actin primers.

  (RT-PCR analysis) Semi-quantitative PCR was performed with a final volume of 100 μl of reaction mixture (2.5 U AmpliTaq enzyme (5 U / μl, Perkin Elmer, Paris, France) with its 1 × buffer, 0.2 mM of each dNTP ( Perkin Elmer, Paris, France), 5% DMSO, and 1 μM each forward and reverse primer) in a Perkin Elmer 9600 thermal cycler. CCR6 (registration number Z79784) and CCR7 (registration number L08176) primers were designated within the region of lowest homology between chemokine receptors. Less than:

Was used for RT-PCR and sequencing. For both chemokine receptors, the reaction mixture was subjected to 30 and 35 cycles of PCR using the following conditions: 94 ° C for 1 minute, 61.5 ° C for 2 minutes, and 72 ° C for 3 minutes. PCR products were visualized on a 1.2% agarose gel containing 0.5 μg / ml ethidium bromide. Estimated size (CCR
The reaction product migrated to 1,021 bp for 6 and 1,067 bp for CCR7) was gel purified and sequenced on a ABI 373A sequencer (Applied Biosystems, Foster City, CA.) using dye terminator technology. For confirmation, it was subcloned into the pCRII TA cloning vector (Invitrogen, Leek, The Netherlands). Two other oligonucleotides:

Was used as a probe for hybridization with PCR products separated on a 1.2% agarose gel and blotted to Hybond N ⁺ membrane (Amersham, Les Ulis, France).

(Calcium Fluorescence Measurement) Intracellular Ca ²⁺ concentration was measured using the fluorescent probe Indo-1 according to the technique reported by Grinkiewicz et al. (J. Biol. Chem., 1985, 260: 3440-3450). Briefly, cells were washed in PBS and resuspended at 10 ⁷ cells / ml in complete RPMI 1640 medium (see above). Cells were then incubated for 45 minutes at room temperature with 3 μg / ml Indo-1 AM (Molecular Probes) protected from light. Following incubation, cells were washed and resuspended at 10 ⁷ cells / ml in HBSS / 1% FCS. Prior to measurement of intracellular Ca ²⁺ concentration, cells were diluted 10-fold in HBSS / 10 mM Hepes / 1.6 mM CaCl ₂ pre-warmed to 39 ° C. Samples were excited at 330 nm with continuous agitation, and Indo-1 fluorescence was complexed with 405 nm (Ca ²⁺ ) in an 810 photomultiplier tube detection system (Software: Felix, Photon Technology International, Monjunto Junction, NJ). Dye) and 485 nm (Ca ²⁺ -free medium) as a function of time. Results are expressed as the ratio of the values obtained at the two emission wavelengths.

  In Situ Hybridization In situ hybridization was performed as described (Pechmaur et al., 1990, Am. J. Pathol. 136: 383-390). Two pairs of primers were used to amplify most of the open reading frame of the MIP-3α gene (registration number D86955) and MIP-3β gene (registration number U77180) by RT-PCR.

Was used as described above at an annealing temperature of 62 ° C. The PCR product was then cloned into the pCRII TA cloning vector (Invitrogen, Leek, The Netherlands) for generation of sense and antisense probes with a suitable promoter. MIP-3α and MIP-3β ³⁵ S-labeled sense and antisense probes were obtained by replicating the transcripts of the 367 bp and 435 bp fragments, respectively. Sections of 6 μm human tonsil were fixed in acetone and 4% paraformaldehyde, followed by 0.1 M triethanolamine / 0.25% acetic anhydride. The sections were hybridized overnight, treated with RNAse A and exposed for 24 days. After development, the sections were stained with hematoxylin.

Example 1: Differential responsiveness to MIP-3α and MIP-3β during development of CD34 ⁺ derived DC
To understand the regulation of DC trafficking, the response of DC to various chemokines at different stages of maturation was studied. DCs were generated from CD34 ⁺ HPC cultured in the presence of GM-CSF and TNFα and tested for their ability to migrate in response to chemokines in Boyden microchambers at different culture days. MIP-3α and MIP-3β increased CD34 + -derived DCs 2-3 fold over MIP-1α or RANTES. However, DCs attracted by MIP-3α and MIP-3β were collected at different time points in culture. Responses to MIP-3α were already detected on day 4 and were maximal on days 5-6 and lasted until day 10. On days 13-14, the response to MIP-3α was usually lost. In contrast, a response corresponding to MIP-3β could not be detected before day 10, peaked at day 13, and could persist beyond day 15. At earlier time points, if the majority of cells in culture were still DC precursors (CD1a ^- CD86 ⁻ ), the response to MIP-3α was a concentration of 1-10 ng / ml (depending on the experiment) Could be detected. In contrast, after 4 days, if almost all cells were immature DC (CD1a ⁺ CD86 ⁻ ), over 300 ng / ml was required to attract the cells, which means that during maturation Suggests continuous desensitization of cells. A relatively high concentration of MIP-3β (300 ng / ml) was also required to increase mature DC (CD1a ⁺ CD86 ⁺ ). Checkerboard analysis established that MIP-3α and MIP-3β induce chemotaxis but not DC chemokinesis.

To confirm the relationship between the stage of maturation and the response to MIP-3α and MIP-3β, CD34 ⁺ derived DCs were subjected to immature DC (CD1a ⁺ CD86 ⁻ ) and mature DC (CD1a ⁺ according to CD86 expression). CD86 ⁺ ) was sorted by FACS on day 10 of culture. CD1a ⁺ CD86 ⁻ responded exclusively to MIP-3α, whereas CD1a ⁺ CD86 ⁺ responded mainly to MIP-3β. These observations also confirmed that the cells increased by MIP-3α and MIP-3β were actually DC (CD1a ⁺ ). The correlation between DC maturation and chemokine responsiveness is maintained by removal of TNFα from day 6 to day 12, and their maturity is determined by the addition of TNFα, LPS or CD40L. It is further illustrated when synchronized by The response to MIP-3α was strongly diminished during 48 hours maturation using TNFα, LPS and CD40L. At the same time, the response to MIP-3β was induced by all three signals, CD40L and LPS were more potent than TNFα. In kinetic experiments, the response to MIP-3α was reduced by 50-70% only 24 hours after CD40 activation and was completely lost at 72 hours. The response to MIP-3β was already maximal 24 hours after CD40 activation and required relatively high concentrations (100-300 ng / ml at 48 hours) of chemokines.

  Taken together, these results establish that immature CD34 + -derived DCs respond to MIP-3α while mature DCs respond to MIP-3β.

(Example 2: Responses to MIP-3α and MIP-3β parallel the expression of the respective receptors CCR6 and CCR7 on CD34 ⁺ derived DC)
To elucidate the mechanism of regulation of MIP-3α and MIP-3β responsiveness, their respective receptor CCR6 mRNA (Power et al., 1997, J. Exp. Med. 186: 825-835; Greaves et al., 1997, J. Exp. Med. 186: 837-844; Baba et al., 1997, J. Biol.Chem.272: 14893-14898; Liao et al., 1997, Biochem.Biophys.Res.Commun. 236: 212-217) and CCR7 The expression of mRNA (Yoshida et al., 199, J. Biol. Chem. 272: 13803-13809) was studied by semi-quantitative RT-PCR. During DC development from CD34 ⁺ HPC, CCR6 mRNA was first detected on day 6 and increased by day 10 and then decreased but slightly detectable on day 14. . In contrast, CCR7 mRNA appeared on day 10 and increased reliably until day 14. Furthermore, CD40L-dependent maturation induced continuous downregulation of CCR6 mRNA (which was almost detectable after 72 hours) and induced upregulation of CCR7 mRNA as early as 24 hours. Similar results were obtained after inducible DC maturation with either LPS or TNFα. Upregulation of CCR7 mRNA after activation was confirmed by Southern blot analysis of the cDNA library.

Consistent with the migration assay and regulation of CCR6 and CCR7 expression, MIP-3α induced Ca ²⁺ flux exclusively in resting / immature DCs, and MIP-3β induced Ca ²⁺ flux only in mature DCs. Maximum Ca ²⁺ flux was observed for immature and mature DCs using 30 ng / ml MIP-3α and 30 ng / ml MIP-3β, respectively.

These results indicate that the change in responsiveness to MIP-3α and MIP-3β is CCR6
mRNA and CCR7 are shown to be involved in the regulation of mRNA expression, suggesting that CCR6 and CCR7 are the major functional receptors expressed on DCs for MIP-3α and MIP-3β, respectively.

(Example 3: Response to MIP-3β is also induced upon maturation of monocyte-derived DCs)
Monocyte-derived DCs generated by culturing monocytes in the presence of GM-CSF + IL-4 for 6 days are typically immature DCs (CD1a ⁺ , CD14 ⁻ , CD80 ^low , CD86 ^low , CD83). ^- ) (Cella et al., 1997, Current Opin. Immunol. 9: 10-16; Sallusto et al., 1994, J. Exp. Med. 179: 1109-1118). They migrated in response to MIP-1α and RANTES, but did not respond to MIP-3α or MIP-3β, and did not migrate. The lack of response of monocyte-derived DCs to MIP-3α follows the absence of CCR6 expression on those cells (Power et al., 1997, J. Exp. Med. 186: 825-835; Greaves et al., 1997, J Exp. Med. 186: 837-844). Upon maturation induced by TNFα, LPS, or CD40L, responses to MIP-1α and RANTES were lost, but responses to MIP-3β were induced. As with CD34 ⁺ derived DCs, responses to MIP-3β correlated with the upregulation of CCR7 mRNA expression observed upon maturation induced by TNFα, LPS or CD40L. To reiterate, CCR7 upregulation after TNFR or CD40 signaling occurred at an early time point (3h). Furthermore, migration and chemokine receptor expression data were consistent with Ca ²⁺ flux results.

  These results extend the notion that DCs lose their responsiveness to various chemokines upon maturation, but become sensitive to one chemokine MIP-3β to monocyte-derived DCs.

(Example 4: Peripheral blood CD11c ⁺ DC migrates in response to MIP-3β after maturation) MIP-3α and MIP-3β run on immature CD11c ⁺ DC isolated from peripheral blood (or tonsils) Chemoactive activity was also studied. Freshly isolated DCs did not migrate in response to MIP-3α but migrated in response to MIP-3β, and a correlation between the absence of CCR6 and CCR7 mRNA expression in these cells was observed. . However, maturation known to occur after overnight culture with GM-CSF was directed to the response of CD11c ⁺ DC to MIP-3β but not to the response to MIP-3α. Again, the response to MIP-3β correlated with the induction of CCR7 mRNA expression.

Thus, even though immature CD11c ⁺ DC freshly isolated from blood cannot respond to MIP-3α, these results indicate that maturation dependent on responsiveness to MIP-3β is also isolated ex vivo. It is applied to DC.

(Example 5: MIP-3α is expressed in inflamed epithelium in vivo, and MIP-3β is expressed in the T cell-enriched region of tonsils)
The physical relevance of the findings reported in Example 4 was addressed through analysis of MIP-3α and MIP-3β mRNA expression by in situ hybridization on inflamed tonsil sections. MIP-3α mRNA was detected at high levels in inflamed epithelial crypts, but not in the T cell enriched region or in the B cell follicles. Indeed, MIP-3α expression was restricted to cells covering epithelial crypts. In contrast, MIP-3β mRNA expression was restricted to the T cell enriched region. The strongest signal was present in scattered cells and the distribution overlapped with that of IDC. Outside the paracortical area, no signal was detected in either B cell follicles or epithelial crypts. Serial sections showed a clear absence of MIP-3β expression in epithelial crypts that are richly expressed in MIP-3α. MIP-3α and MIP-3β sense probes did not produce background hybridization.

  Thus, MIP-3α expression is restricted to the inflamed epithelium at the site of antigen entry to be supplemented with immature DCs. In contrast, MIP-3β is only detected in the subcortical region, where mature IDCs home and generate a first T cell response.

Example 6 In Vivo Chemokine MIP-3α Administration in a Mouse Model
MIP-3α has shown that we are a chemotactic factor for mouse immature dendritic cells in vitro, and the chemokine MIP-3α attracts immature DCs in vivo and antigen-specific for tumors in vivo The ability to modulate the immune response was studied. If the tumor associated antigen is delivered at the same time, more DC is available to capture the antigen. Thus, the antigen specific response to this antigen should be increased.

Chemokines are delivered in vivo via a plasmid vector (pcDNA3, InVitrogen), which contains a cDNA encoding mouse MIP-3α under the control of a CMV promoter (PMIP-3α). The antigen used was E. coli. It was β-galactosidase isolated from E. coli. This antigen was delivered in vivo via the same plasmid vector pcDNA3 (referred to as pLacz). The tumor was C26 colon cancer in syngeneic BALB / c mice stably transfected with a gene encoding β-galactosidase. Thus, in this system, β-galactosidase defines the tumor associated antigen.

  Groups of 6 6 week old female mice were injected with either an empty pcDNA3 plasmid (negative control), a plasmid pLacz encoding the antigen alone, or a mixture of pLacz and PMIP-3α. Injections (50 μg total plasmid) were made weekly for 4 weeks in the hind footpad. The mice were then injected subcutaneously with a C26 tumor cell line expressing β-galactosidase. Typically, all mice developed tumors subcutaneously after 10 days. The appearance of tumors in these groups of mice was monitored. Tumor appearance was delayed after injection of pLacz and pLacz + PMIP-α (FIG. 1). This indicates that immunization with a plasmid encoding a tumor associated antigen has a protective effect against tumor transplantation. This delay was greater with pLacz + PMIP-3α than with pLacz. This suggests that the chemokine MIP-3α increases the tumor-associated antigen-specific immune response when delivered with the antigen.

  An excellent anti-tumor response is thought to be associated with strong T cell-mediated antigen-specific cytotoxicity (CTL activity). Therefore, CTL activity in the same group of mice was analyzed 30 days after tumor inoculation. Splenocytes were removed and stimulated for 5 days with irradiated syngeneic DCs and immunodominant CTL peptides derived from β-galactosidase in the presence of interleukin-2. Their ability to lyse cell lines stably transfected with the gene encoding β-galactosidase (P13.1) is then paralleled with their ability to lyse the parent cell line P815 that does not express β-galactosidase. Was measured (FIG. 2). This was done using different ratios of effector (spleen cells) to target (P13.1 or P815). This result shows that mice injected with pLacz + P-MIP-3α prior to tumor challenge have greater CTL activity against tumor associated antigen β-galactosidase than mice injected only with pLacz or PCDNA3 alone.

(Example 7: Chemokine hMCP-4 administration in an in vivo mouse model)
We have shown that local injection of hMCP-4 can promote dendritic cell recruitment in vivo in mice in a dose-dependent manner (FIG. 4).

Female BALB / c mice, 6-10 weeks old, were purchased from Charles River (Iffa-Credo, L'Arbresle, France) and maintained at our facility under standard conditions. The procedures for animals and their care were in accordance with the instructions of the EEC (European Economic Community) meeting (86/609, OJL 358, 1, 12/1987). Recombinant human MCP-4 protein (> 97% purity (Figure 3)) was obtained from Peprotech and resuspended in PBS (Gibco-BRL). Groups of 3 mice were injected intradermally with PBS alone or various amounts of human MCP-4 in PBS at a volume of 50 μ in the right hind footpad. Mice were sacrificed 2-20 hours later and the skin at the injection site and popliteal lymph nodes draining from the injection site were removed. Local cell recruitment in the skin was examined by immunohistochemistry using specific monoclonal antibodies according to standard techniques. Cell suspensions were prepared from lymph nodes in RPMI 1640 and 10% fetal calf serum (FCS) (Gibco-BRL). Cells are counted and biotin-CD11c antibody and FITC-CD11b antibody (Becton Dickinson) followed by PBS containing PE-streptavidin (Dako) and 2% in PBS and 2% FCS according to standard techniques Stained in FCS. Expression of CD11b and CD11c (which defines a population of mouse dendritic cells) was analyzed on a Facscan flow cytometer (Becton Dickinson) using CellQuest software. From this analysis and count, the number of CD11b ⁺ CD11c ⁺ in each lymph node was determined. These experiments show that: (A) Local injection of hMCP-4 can induce recruitment of CD11b expressing cells at the injection site after a short period (2 hours). These cells can be mouse blood dendritic cells or dendritic cell precursors (eg, monocytes). Because both can express CD11b. In mice, circulating blood dendritic cells were not identified due to technical limitations. However, in humans, blood dendritic cells can be isolated and they respond to a chemotaxis assay for hMCP-4 (FIG. 3). (B) When an antigen-specific immune response is initiated in draining lymph nodes, hMCP-4 induces recruitment of dendritic cells identified by co-expression of CD11b and CD11c, but for long periods (20 hours) Only after. This delay is most likely to correspond to the maturation and migration time required for dendritic cells or their precursors that are initially recruited in the skin to migrate to draining lymph nodes.

(Example 8: Response of human blood-derived dendritic cells to hMCP-4)
hMCP-4 is also active against human dendritic cells, including dendritic cells isolated from blood (FIG. 5).

Panel A: Human circulating blood CD11c ⁺ DC was enriched by magnetic bead removal and studied in a transwell (5 μm pore size) migration assay in response to various chemokines. This migration was revealed 2 hours later by triple staining: lineage markers FITC, HLA-DR tricolor and CD11c PE and stained with Facs. Each chemokine is tested over a wide range of concentrations (1-1000 ng / ml) and shows only the optimal response. Results are expressed as migration index and show average values obtained from 3-10 independent experiments. Blood CD11c ⁺ responds primarily to MCP-4 and MCP1, MCP2 and MCP3 (not shown). SDF-1 lacks selectivity and is the only other chemokine that is strongly active against CD11c ⁺ DC.

Panel B: Different populations of human DCs and DC precursors (including blood CD11c ⁺ DC, monocytes, monocyte-derived DCs, CD1a ⁺ Langerhans cell precursors and CD14 ⁺ stromal DC precursors), MCP-1 and MCP -4 in a transwell (5 μm pore size) migration assay. All populations respond to MCP-4 with the exception of CD1a ⁺ Langerhans cell precursors. Furthermore, monocyte-derived DCs respond to MCP-4 through receptors different from CCR2, but do not respond to MCP-1.

Importantly, MCP-4 is active against human DCs. In particular, compared to other chemokines, MCP-4 is the most potent chemokine that induces migration of circulating blood CD11c ⁺ DC. MCP-1, and MCP-2 and MCP-3 show similar activity against blood DC. MCP appears to recruit blood DC via CCR2 expressed on these cells. In addition, MCP-4, except for CD1a ⁺ Langerhans cell precursors that do not express CCR2, is a population of all DCs or DC precursors (blood CD11c ⁺ DC, monocytes, monocyte-derived DCs, CD14 ⁺ stromal DCs Active on the precursor). Finally, MCP-4 (not MCP-1) probably induces migration of monocyte-derived DCs via a different receptor than CCR2.

Example 9: Administration of hMCP-4 and β-galactosidase in an in vivo mouse model
In addition, hMCP-4 can be used as an adjuvant for antigen-specific immune responses induced by plasmid DNA vaccination. Furthermore, when hMCP-4 is used as an adjuvant for plasmid DNA vaccination, this may increase the protection of mice subsequently challenged with tumors that express the antigen encoded by the plasmid DNA.

  Groups of 7 female BALB / c mice (Iffa-Credo, L'Arbresle, France) 6-8 weeks old were given PBS alone or 100 ng human MCP-4 in PBS in a volume of 50 μl, right back The footpad was injected subcutaneously. Three hours later, 50 μg of control pcDNA3 plasmid (InVitrogen) or 50 μg of pcDNA3 plasmid encoding β-galactosidase under the CMV promoter (pLacz, InVitrogen) was injected into the same site of the mouse under a volume of PBS of 50 μl. This immunization protocol was repeated four times at weekly intervals.

  Serum was collected one day before the first immunization and one week after the last immunization. The level of β-galactosidase specific immunoglobulin in the serum was measured using a specific ELISA assay as previously described (Mendoza et al., 1997, J. Immunol. 159: 5777-5781).

As seen in FIG. 6, MCP-4 injection increased antigen-specific humoral response after β-galactosidase DNA immunization (100 ng hMCP-4 injection in the right hind footpad and 50 μg DNA injection 3 hours later) To do. FIG. 6 shows anti-β-galactosidase antibodies measured after 4 immunizations [significance of hMCP-4 + pLacz when compared to PBS + pLacz: Student test].

One week after the last immunization, groups of mice were treated with 5 × 10 ⁴ C26-BAG colon cancer cells expressing β-galactosidase under a 100 μl volume of RPMI-1640 (kindly, Mario Combo, Instituto). Nazionale Tumori, Milan, Italy) was challenged by subcutaneous injection in the right flank. Tumor development was assessed 3 times a week by palpation.

As can be seen in FIG. 7, MCP-4 injection resulted in β-galactosidase DNA immunization (100 ng hMCP-4 in the right hind footpad) when mice were challenged with a C26 colon cancer cell line expressing β-galactosidase. Increased the antitumor effect induced by injection of 50 μg of DNA 3 hours after injection, 4 immunizations before tumor challenge [PBS + pLac
Significance of hMCP-4 + pLacz when compared to z: p <0.05, logrank
MCP-4 challenge (op): hMCP-4 injected at different sites].

  Thus, Examples 7-9 show that chemokine hMCP-4 can be used as an adjuvant for immune responses, particularly anti-tumor responses. The enhanced immune response mediated by MCP-4 administration was measured as the enhancement of antigen-specific immunoglobulin levels in the serum. Thus, there is clearly an enhancement of the B cell response to MCP-4 administration. Furthermore, as the subclass of immunoglobulin (eg, IgG2a) that requires T cell mediated assistance for switching increases, the T cell mediated response also appears to increase.

(Example 10: Response of human dendritic cells to h6Ckine chemokine)
In this example, the inventors have shown that human 6Ckine (h6Ckine) is a chemotactic factor for all known subsets of human dendritic cells in vitro. In particular, h6Ckine is active against human blood dendritic cells after a short incubation period of 3 hours with GM-CSF, IL-3 and CD40L (FIG. 8).

Different human DC populations ^- a (CD1a ⁺ Langerhans cells, CD14 ⁺ stromal DC, monocyte-derived DC, circulating blood CD11c ⁺ DC, monocytes, and circulating blood CD11c including plasma cell-like DC), prior to maturation and later, Studyed in a migration assay that responds to human 6Ckine. CD34-derived DC precursors were isolated by Facs sorting according to CD1a and CD14 expression after culturing in the presence of GM-CSF + TNF and SCF for 6 days. Cells were cultured for up to day 12 in GM-CSF alone (immature) or in the last 2 days in GM-CSF + CD40-L (mature). Monocyte-derived DCs were generated by culturing monocytes in the presence of GM-CSF + IL-4 for 5 days and were activated (mature) or active for the last 2 days using CD40-L (Immature). Human circulating blood CD11c ⁺ DC and CD11c ^- plasma cell-like DC, enriched by removal of the magnetic beads, using a triple staining, lineage markers FITC-negative, HLA-DR tricolor positive, as well as CD11c PE positive and CD11c PE negative facs sorted. CD11c ⁺ DC and CD11c ^- plasma cell like DC, in the presence of GM-CSF + IL-3, together with CD40L (mature) or without CD40L (immature), and cultured for 3 hours.

  Migration assays were performed for 1 to 3 hours using Transwells (6.5 mm diameter, COSTAR, Cambridge, Mass.) With 5 μm pore size or 8 μm pore size and revealed by facs analysis. All populations responded to 6Ckine only after CD40-L activation.

(Example 11: Chemokine m6Ckine gene transfer in tumor model)
In this example, we showed the following:
C26 colon cancer tumor cells engineered to express m6Ckine are less tumorigenic, and this effect depends on CD8 ⁺ cells and natural killer cell activity in vivo (FIG. 9);
C26 tumors expressing m6Ckine are significantly infiltrated by dendritic cells and CD8 + T cells compared to the parent tumor (FIG. 10); and C26 colon cancer tumor cells engineered to express m6Ckine are parental Less antigenic than the C26 tumor (Figure 11).

Female BALB / c (H-2 ^d ) mice, 6-10 weeks old, were purchased from Charles River (Iffa-Credo, L'Arbresle, France) and maintained under standard conditions. The procedures for animals and their care were in accordance with the instructions of the EEC (European Economic Community) meeting (86/609, OJL 358, 1, 12/1987). All tumor cell cultures were treated with 10% FCS (Gibco-BRL), 1 mM hepes (Gibco-BRL), Gentallin (Schering-Plough, Union, NJ), 2 × 10 ⁻⁵ M β-2 mercaptoethanol (Sigma, St (Luis, MO) in DMEM (Gibco-BRL, Life Technologies, Paisley Park, Scotland) supplemented. All cell cultures were performed in a humidified incubator with 5% CO ₂ at 37 ° C. A cDNA encoding mouse 6Ckine / SLC (m6Ckine / SLC) was cloned into a pcDNA3 vector (InVitrogen, Carlsbad, CA) containing a CMV promoter. C26 colon cancer tumor cells (kindly received from Mario P. Colombo, Instituto Nazionale per lo Studio e la Cura dei Tumori, Milano, Italy) according to the manufacturer's instructions, Fugene reagent (RumolMilemolehleMolehleMolehleMolehleMolehleMoL Germany) and transfected with this construct. A single C26 clone expressing m6Ckine / SLC mRNA (C26-6CK) was obtained after neomycin (Sigma) selection at 800 μg / ml. C26 tumor cells or C26-6CK tumor cells were injected into the right flank in 100 μl DMEM. C. Injections and tumor growth was monitored by palpating 3 times per week. For antibody removal, one day prior to tumor inoculation, 0.5 mg anti-CD8 (clone 2.43) purified antibody, rat control (GL113) purified antibody or 200 μl rabbit anti-asialo GM1 serum (Wako) in 200 μl PBS. Pure Chemicals, Osaka, Japan) i. p. Then, 0.2 mg antibody or 100 μl of anti-asialo GM1 serum was injected 3 days later and once a week during the course of the experiment. FIG. 10 shows that subcutaneous C26-6CK cell injection results in a significantly delayed tumor uptake (p <0.01) by logrank analysis compared to parental tumor cells (A <A).
And B: C26 ⁺ control vs. C26 ^- 6CK ⁺ control). Removal of CD8 ⁺ cells (A) or natural killer cell activity (B) with certain antibodies in vivo partially returns the delayed tumorigenicity of C26-6CK tumor cells. This indicates that CD8 ⁺ cells and NK cells play a role in tumor growth delay.

Tumors were removed by surgery when they reached a size of about 1 cm. The tumor mass was minced into small fragments and incubated in collagenase A (Roche Molecular Biochemicals) solution at 37 ° C. for 30 minutes with agitation. The suspension was then washed several times in DMEM. Cell suspension staining was performed in PBS and 5% FCS. Prior to incubation with FITC-labeled specific antibody, biotin-labeled specific antibody or PE-labeled specific antibody, Fc receptors were blocked using Fc-block ^™ CD16 / CD32 antibody (PharMingen, San Diego, Calif.). The various antibodies used in this study (all from PharMingen) are CD8β (53-5.8), CD11c (HL3), anti-MHC class II IA ^d / IE ^d (269), CD3 (145 -2C11). Biotinylated antibodies were revealed using PE streptavidin (Becton Dickinson). Phenotypic parameters were acquired with FacScan (Becton Dickinson, Mountain View, CA) and analyzed using CellQuest software (Becton Dickinson). In FIG. 10, C26 wild type tumors or C26-6CK tumors expressing m6Ckine were analyzed for infiltration of CD8 T cells and CD11c ⁺ MHC class II ⁺ dendritic cells (DC) by flow cytometric analysis of the entire tumor suspension. (N = 7). Data show significant recruitment of leukocyte subsets in both C26-6CK tumors compared to C26 tumors (Student t test). These results show that m6Ckine gene transfer into tumors recruits dendritic cells, which are essential cells to initiate immune responses (including anti-tumor responses), and CD8 T cells, which are effector cells of adaptive immune responses. Suggest to promote both.

In some experiments, tumors were removed from animals, embedded in OCT compounds (Miles laboratory, Elkhart, Ind.), Snap frozen in liquid nitrogen, and stored at −80 ° C. prior to immunohistochemistry procedures. 5 μm cryostat sections applied to glass slides were fixed in acetone and incubated with 1% H ₂ O _{2 for} 10 minutes at room temperature. The slides were then incubated with biotin block ^TM reagent and avidin block ^TM reagent (both Vector, Burlingame, CA). All incubations were performed after three 2 minute washes in PBS (Gibco-BRL). The slides were then preincubated for 30 minutes with a 1/10 dilution of serum from the same species of secondary antibody (Dako, Glostrup, Denmark). Slides were then serialized with 5 μg / ml purified CD105 (clone MJ7 / 18, PharMingen, San Diego, Calif.), Biotinylated secondary antibody (Vector rabbit anti-rat), streptavidin-alkali (Vector ABC kit). And incubated. Enzyme reactions were developed using the corresponding Vector substrate. Angiogenesis assays were performed by determining the hemoglobin content of Matrigel (Becton Dickinson, Bedford, Mass.) Pellets containing tumor cells developing in vivo. BALB / c mice were mixed with 2 × 10 ⁵ C26 cells or C26-6CK cells with 0.5 ml Matrigel at the midline of the abdomen. c. Injected. After 9 days, the Matrigel pellet was removed, the surrounding connective tissue was dissected and removed, and the pellet was dissolved in MatriSperse solution v / v (Becton Dickinson) for 90 minutes at 4 ° C. The hemoglobin content was determined by the Drabkin method (Sigma reagent). FIG. 11A shows that C26-6CK tumors form less blood vessels than the parent C26 tumor. FIG. 11B shows that C26-6CK tumor cells form less vessels than C26 cells in the Matrigel assay. Overall, these results indicate that gene transfer of m6Ckine chemokine into tumors has an angiostatic effect on tumor blood vessels.

  These results indicate that the chemokine 6Ckine can be used for the treatment of cancer through gene transfer. Preferred embodiments consist of, but are not limited to: m6Ckine or h6Ckine or DNA encoding a fraction of m6Ckine or h6Ckine or a viral vector (eg, adenovirus) (with targeting moiety (peptide or antibody)) Or not).

Example 12: Local delivery of chemokine 6Ckine to tumors in vivo
In this example, we have shown that injection of recombinant human or recombinant mouse 6Ckine protein into an existing C26 tumor increases the survival of tumor-bearing mice. h6Ckine injection delays tumor growth (FIG. 12).

Female BALB / c (H-2 ^d ) mice, 6-10 weeks old, were purchased from Charles River (Iffa-Credo, L'Arbresle, France) and maintained under standard conditions. The procedures for animals and their care were in accordance with the instructions of the EEC (European Economic Community) meeting (86/609, OJL 358, 1, 12/1987). All tumor cell cultures were treated with 10% FCS (Gibco-BRL), 1 mM hepes (Gibco-BRL), Gentallin (Schering-Plough, Union, NJ), 2 × 10 ⁻⁵ M β-2 mercaptoethanol (Sigma, St (Luis, MO) in DMEM (Gibco-BRL, Life Technologies, Paisley Park, Scotland) supplemented. All cell cultures were performed in a humidified incubator with 5% CO ₂ at 37 ° C. C26 cells were obtained from Mario P. Acquired from Columbo (Milano, Italy).

C26 tumor cells were injected s. On the right flank with 100 μl DMEM. c. Injections and tumor growth was monitored by palpating 3 times per week. In some experiments, tumor volume was monitored using a caliper and predicted as follows: tumor volume = small diameter ² × large diameter × 0.4. For treatment with recombinant chemokines, mice were treated with 10 ng> 97% purity recombinant human or mouse 6Ckine / SLC (R & D) under 50 μl PBS.
(Systems, Minneapolis, MN) was injected intratumorally. FIG. 1 shows that mice injected using h6Ckine or m6Ckine show improved survival when compared to PBS vehicle alone (A). h6Ckine injection also reduced tumor growth (B). These data indicate that intratumoral delivery of recombinant 6Ckine chemokine has an antitumor effect.

Example 13: Mitigation of rAd / 6Ckine metastatic tumors
In female mice (BALB / c ByJ; Jackson Laboratories), 3 × 10 ¹⁵ 4T1-p53 mammary tumor cells (syngeneic) in a volume of 0.2 ml (medium) were used by subcutaneous route on the left flank of the animals. Injected. When tumors grew to a size of 50-100 mm ³ , animals were injected intratumorally with 100 μl CMCB (1e10 PN / injection) in VPBS. Mice were injected 3 times per week (Monday, Wednesday, Friday) for 2 weeks. Tumors were measured three times a week (length, width, depth) using calipers and tumor volume was calculated according to the following formula: V = 4 / 3r ³
Here, r = (W (mm) + L (mm) + D (mm)) / 6
Animals were sacrificed when the tumor exceeded 1000 mm ³ .

Three mice from each group were sacrificed, starting when tumors reached 50 mm ³ (typically day 10), and tumors and lungs were analyzed for biochemical analysis as described below. Excised for tissue processing and assessed for the presence of metastases by gross and histochemical means.

  As shown in FIG. 13, 6Ckine inhibits tumor growth and inhibits spontaneous metastasis in established tumors by increasing immunity and suppressing angiogenesis.

  Many modifications and variations of this invention can be made without departing from its spirit and scope, and will be apparent to those skilled in the art. The specific embodiments described herein are provided by way of illustration only and are limited only by the accompanying claims, along with the full scope of equivalents to which such claims are entitled. Should be.

FIG. 1 shows that immunization with a plasmid containing MIP-3α and tumor-associated antigen has a protective effect against tumor transplantation. FIG. 2 shows the greater CTL activity associated with administration of the chemokine MIP-3α. FIG. 3 shows the nucleotide and partial amino acid sequence of chemokine hMCP-4. FIG. 4 shows that hMCP-4 injection promotes dendritic cell recruitment in vivo in a dose-dependent manner in mice. FIG. 5 shows that hMCP-4 is active in recruiting dendritic cells in human blood. FIG. 6 shows that MCP-4 injection increases the antigen-specific humoral response after β-galactosidase DNA immunization. FIG. 7 shows that MCP-4 increases the anti-tumor effect induced by β-galactosidase DNA immunization when mice are challenged with a C26 colon cancer cell line expressing β-galactosidase. FIG. 8 shows that h6Ckine is active in recruiting dendritic cells in human blood. FIG. 9 shows that C26 colon cancer tumor cells engineered to express m6Ckine are less tumorigenic and this effect is dependent on CD8 ⁺ cells and natural killer cell activity in vivo. FIG. 10 shows that C26 tumors expressing m6Ckine are significantly infiltrated by dendritic cells and CD8 ⁺ T cells when compared to parental tumors. FIG. 11 shows that C26 colon cancer tumor cells engineered to express m6Ckine are less antigenic than the parent C26 tumor. FIG. 12 shows that h6Ckine injection delays tumor growth in mice in vivo. FIG. 13 shows that 6Ckine inhibits tumor growth and spontaneous metastasis of established tumors in vivo.

Claims (35)

Use of a chemokine capable of directing dendritic cell migration in the manufacture of a medicament for the treatment of a disease state, said chemokine comprising MCP-1, MCP-2, MCP-3, MCP-4, MIP- Use, selected from the group consisting of 1α, MIP-1β, MIP-3α, RANTES, SDF-1, Teck, DCtactin-β, 6Ckine / SLC, LEC, MDC, and MIP-5. The use according to claim 1, wherein the dendritic cells are immature dendritic cells. The use according to claim 2, wherein the chemokine is MCP-1, MCP-2, MCP-3, MCP-4, MIP-1B, MDC, MIP-3α, MIP-1α, RANTES and MIP-5. Use, selected from the group consisting of: The use according to claim 1, wherein the chemokine is MIP-3β. A method of enhancing an immune response in a mammal comprising administering to the mammal a chemokine MCP-4 or a biologically active fraction of chemokine MCP-4. 6. The method of claim 5, wherein the chemokine is a recombinant chemokine. 6. The method of claim 5, wherein the chemokine is a human chemokine. 6. The method of claim 5, further comprising the step of administering to the delivery site a substance that enables sustained release of the chemokine. 6. The method of claim 5, further comprising administering an antigen with the chemokine. 10. The method of claim 9, wherein a fusion protein comprising MCP-4 and an antigen is administered to the mammal. 10. The method of claim 9, wherein the antigen is a tumor associated antigen. 11. The method of claim 10, wherein the antigen is a tumor associated antigen. 10. The method according to claim 9, wherein the antigen is a bacterial, viral or fungal antigen. 11. A method according to claim 10, wherein the antigen is a bacterial, viral or fungal antigen. The method according to claim 9, wherein the tumor-associated antigen is Melan-A, tyrosinase, p97, β-HCG, GalNAc, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-12. MART-1, MUC1, MUC2, MUC3, MUC4, MUC18, CEA, DDC, melanoma antigen gp75, Hker8, high molecular weight melanoma antigen, K19, Tyr1 and Tyr2, members of the pMel 17 gene family, c-Met, PSA , PSM, α-fetoprotein, thyroid peroxidase, gp100, p53 and telomerase. The method according to claim 10, wherein the tumor-associated antigen is Melan-A, tyrosinase, p97, β-HCG, GalNAc, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-12. MART-1, MUC1, MUC2, MUC3, MUC4, MUC18, CEA, DDC, melanoma antigen gp75, Hker8, high molecular weight melanoma antigen, K19, Tyr1 and Tyr2, members of the pMel 17 gene family, c-Met, PSA , PSM, α-fetoprotein, thyroid peroxidase, gp100, p53 and telomerase. 10. The method of claim 9, further comprising administering a combination of GM-CSF and IL-4. 11. The method of claim 10, further comprising administering a combination of GM-CSF and IL-4. 6. The method of claim 5, further comprising administering an activating agent with the chemokine. 6. The method of claim 5, wherein the chemokine is administered intradermally, intramuscularly, subcutaneously, topically, or in the form of a vector. A fusion protein comprising MCP-4 and an antigen. The fusion protein according to claim 21, wherein the antigen is a tumor-associated antigen. 23. The fusion protein according to claim 22, wherein the tumor-associated antigen is Melan-A, tyrosinase, p97, β-HCG, GalNAc, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-. 12, MART-1, MUC1, MUC2, MUC3, MUC4, MUC18, CEA, DDC, melanoma antigen gp75, Hker8, high molecular weight melanoma antigen, K19, Tyr1 and Tyr2, members of the pMel 17 gene family, c-Met, A fusion protein selected from the group consisting of PSA, PSM, α-fetoprotein, thyroid peroxidase, gp100, p53 and telomerase. 23. A fusion protein according to claim 22, wherein the antigen is a bacterial, viral or fungal antigen. A plasmid comprising the fusion protein of claim 21. 24. A plasmid according to claim 23, further comprising a promoter sequence particularly suitable for dendritic cells. A viral vector comprising the fusion protein of claim 21. A fusion protein comprising 6Ckine and an antigen. 30. The fusion protein of claim 28, wherein the antigen is a tumor associated antigen. 30. The fusion protein according to claim 29, wherein the tumor-associated antigen is Melan-A, tyrosinase, p97, β-HCG, GalNAc, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-. 12, MART-1, MUC1, MUC2, MUC3, MUC4, MUC18, CEA, DDC, melanoma antigen gp75, Hker8, high molecular weight melanoma antigen, K19, Tyr1 and Tyr2, members of the pMel 17 gene family, c-Met, A fusion protein selected from the group consisting of PSA, PSM, α-fetoprotein, thyroid peroxidase, gp100, p53 and telomerase. 30. A fusion protein according to claim 29, wherein the antigen is a bacterial, viral or fungal antigen. A plasmid comprising the fusion protein of claim 28. 33. The plasmid of claim 32, further comprising a promoter sequence particularly suitable for dendritic cells. A viral vector comprising the fusion protein of claim 28. Chemokines and uses as described in the specification.