English:
[0001] The present invention relates to methods and compositions useful to diagnose and treat conditions correlated with an abnormal level of interferon-α (IFNα) expression in a subject.
[0002] Human interferons (IFNs) are functionally-related cytokines that modulate both innate and adaptive immune responses. They are categorized in two groups, largely on the basis of sequence homology, as Type I and II. Type I IFNs include six types (IFN-α, IFN-β, IFN-ω, IFN- K, IFN-ε, and IFN-λ). IFN-α, β, ω, and K act through an identical IFN receptor, IFNAR. IFN-λ associates with a distinct receptor, IFNLR. The receptor for IFN-ε is currently unknown. Type II IFN consists of a single type (IFN-γ) and associates with the receptor IFNGR. While Type I IFNs are strongly induced during viral infections, Type II IFN is induced primarily in response to immune and inflammatory stimuli, and thus IFN-γ is frequently referred to as "immune IFN". The most studied of the numerous Type I IFNs include IFN-α, IFN-β, and IFN-ω. Of these, IFN-α is the most complex, includes at least fifteen distinct protein subtypes exhibiting upwards of 75% sequence homology. Diaz (1995) Semin. Virol. 6:143-149; Weissmann et al. (1986) Prog Nucl Acid Res MoI Biol 33:251; J Interferon Res (1993) 13:443-444; Roberts et al. (1998) J Interferon Cytokine Res 18:805-816. In addition to having structural similarity, the IFN-α genes and their products show functional similarities. For example, they are induced by dsRNA or virus, and can interact with the same receptor, the IFNα/β receptor IFNAR. Mogensen, et al. (1999) J. Interferons and other Regulatory Cytokines, John Wiley & Sons. IFNα also inhibits apoptosis, promotes the survival and differentiation of antigen-activated T helper cells and promotes the maturation of functionally efficient monocyte-derived dendritic cells.
[0003] Many types of cells produce IFNα when exposed to viruses and dsRNA. Specialized leukocytes (called interferon-α producing cells ("IPCs") produce IFNα in response to a wider variety of stimuli, e.g., viruses, bacteria and protozoa. Several in vitro studies indicate that the various IFN-α subtypes are produced to different extents by distinct IFN-α-secreting cell lines or in a virus type-specific manner following infection of human peripheral blood mononuclear cells (PBMCs), and that these patterns are often associated with subtype-dependent differences in antiproliferative, anti-viral, and anti-tumor activities. However, the physiological significance of the individual subtypes and their synergistic or antagonist activities with one another in vivo remains undefined.
[0004] IFN-α has been implicated as a mediator of the pathology seen in several autoimmune diseases. Moreover, it can cause autoimmune disease development in patients treated with IFN-α for cancer and viral infections. Increased expression of IFN-α has been observed in the disease- localized tissues of patients with insulin-dependent diabetes mellitus (DDDM or type I diabetes), psoriasis, Crohn's disease, and celiac disease. Over expression of IFN-α has been observed in patients with systemic lupus erythematosus (SLE), IDDM and AIDS. Ih the case of SLE, which is characterized by an abundance of both autoreactive B and T cells, IFN-α expression is observed in not only tissue lesions but circulating within the blood of afflicted individuals. Furthermore, the IFN-α serum levels tend to correlate with the clinical disease activity index. This is believed to stem from cyclical induction of normally quiescent monocytes into potent antigen-presenting dendritic cells (DCs) as triggered by upregulation of IFN-α production by plasmacytoid DCs (pDCs). Indeed, the present inventors have previously demonstrated via oligonucleotide microarray analysis that SLE can be distinguished by "signatures" of unregulated genes involved in granulopoiesis and IFN induction; these signatures revert to normal upon high-dose infusion of glucocorticoids (U.S. patent application 11/228,586, the content of which is incorporated by reference hereto).
[0005] Systemic lupus erythematosus (SLE) is a systemic autoimmune rheumatic disease that is particularly aggressive in children and characterized by flares of high morbidity. Autoimmune diseases such as SLE often act in self-perpetuating cycles of relapse and remission. These cycles are often defined by phases of treatment with generally therapeutic regimens administered to quench the SLE disease cycle. FDA-approved treatment options for SLE include corticosteroids, nonsteroidal immune suppressants, antimalarials, and nonsteroidal anti-inflammatory drugs. These drugs abrogate the integrity of all immune effector responses rather than acting upon those specific to the pathogenesis of SLE. SLE represents an unmet medical need since these treatments are only partially effective with moderate to severe side-effects including bone thinning, weight gain, acne, anemia, sterility, rashes diarrhea, hair loss, and nausea. Furthermore, no new therapeutics for SLE have been approved in 40 years.
[0006] SLE has recently been closely linked to unabated IFNα production. Shi et al. (1987) Br. J. Dermatol. 117(2):155-159. IFNα is present at elevated levels in SLE serum (Crow et al. (2004) Curr. Opin. Rheumatol. 16(5):541-547) and plasmacytoid DCs (pDCs), the primary source of IFNα, accumulate in SLE skin. Farkas et al. (2001) Am. J. Pathol. 159(l):237-243. Moreover, it has been observed that some patients treated with IFNα have developed lupus (Okanoue et al. (1996) J. Hepatol. 25(3):283-291; Tothova et al. (2002) Neoplasma 49(2):91-94; and Raanani et al. (2002) Acta Haematol. 107(3):133-144) and that lupus patients that present with IFNα antibodies have been shown to display a milder form of the disease. Von Wussow et al. (1988) Rheumatol. Int. 8(5):225-230. IFNα may act via the differentiation of monocytes into functional dendritic cells (DCs) which in turn mediates the etiopathogenesis of SLE. Pascual V. et al. (2003) Curr. Opin. Rheumatol. 15(5):548-556. A proposed approach for the treatment of SLE is neutralization of IFNα (see Banchereau et al., PCT/US02/00343, the contents of which is incorporated by reference).
[0007] Although monoclonal antibodies (MAbs) that can block human IFN-α bioactivity have been produced, none have been reported to date that can neutralize all fifteen known subtypes, and few can neutralize naturally-derived, IFN-α-containing leukocyte IFN. PBL Biomedical Laboratories offers ten mouse monoclonal antibodies that bind to multiple human IFNα gene subtypes (see the world wide web at interferonsource.com/relativespecificity.html). However, each of the PBL antibodies bind the IFNα protein subtype encoded by human IFNα gene subtype 1 (IFNα protein subtype D) and from up to one to twelve other IFNα subtypes. U.S. Patent Publ. No. 2003/0166228 Al discloses a monoclonal antibody (designated 9F3) that was derived from immunization of mice with leukocyte IFNα (which includes all of the IFNα protein subtypes). The 9F3 MAb binds and neutralizes the anti-viral activity of the proteins encoded by seven human IFNα gene subtypes 1, 2, 4, 5, 8, 10 and 21 (which encode IFNα protein subtypes D, A, 4, G, B2, C and F, respectively), without neutralizing the antiviral activity of human IFNβ. This publication does not disclose whether the 9F3 antibody binds and inactivates the other eight IFNα gene subtypes, nor whether it binds to one or both of IFNα protein subtypes 4a and 4b. The PCT publication suggests using the monoclonal antibodies to treat disorders associated with increased expression of IFNα's, in particular, autoimmune disorders such as insulin-dependent diabetes mellitus and SLE. However, it is not known whether the 9F3 antibody is able to sufficiently neutralize the biological activity of the IFNα protein subtypes found in SLE serum.
[0008] Because IFNα is a multi-functional mediator of the immune response and has beneficial antiviral activity, complete inhibition or significant down-regulation of all IFNα subtypes is not an optimal therapeutic approach. Thus, a need exists for agents that will selectively neutralize the IFNα subtypes associated with pathological conditions. This invention satisfies this need and provides related advantages as well.
[0009] The invention provides monoclonal antibodies and derivatives thereof that neutralize specific IFNα subtypes. The antibodies of the invention are useful in the amelioration and treatment of conditions associated with increased expression of IFNα, such as SLE, psoriasis, type I diabetes, AIDS, Graft versus Host Disease and other autoimmune diseases. In one aspect, the invention includes an antibody that selectively neutralizes a bioactivity of at least two interferon alpha ("IFNα") protein subtypes such as subtypes A, 2, B2, C, F, G, H2, 1, Jl, K, 4a, 4b and WA, but does not significantly neutralize at least one bioactivity of IFNα protein subtype D; wherein the bioactivity is activation of the MxA promoter or antiviral activity. Preferably, the antibody is a monoclonal antibody. In one embodiment, the antibody does not significantly neutralize H7Na protein subtype D nor 1. In one embodiment, the monoclonal antibody is ACO-I or ACO-2. Preferably, the antibodies of the invention inactivate the ability of serum isolated from systemic lupus erythematosus (SLE) patients to stimulate the differentiation of monocytes into dendritic cells.

中文:
[0001]  本发明涉及用于诊断和治疗患者的干扰素-α(IFNα)表达异常水平相关疾病的方法和组合物。
[0002]  人干扰素(IFN)是功能相关的细胞因子,可以调节先天性及适应性免疫应答。主要根据序列同源性,将它们分为两组:I型和II型。I型IFN包括6类(IFN-α、IFN-β、IFN-ω、IFN-κ、IFN-ε和IFN-λ)。IFN-α、β、ω和κ通过相同IFN受体IFNAR起作用。IFN-λ与不同受体IFNLR结合。IFN-ε的受体目前尚不知道。II型IFN由单独一类(IFN-γ)组成,与受体IFNGR结合。尽管在病毒感染期间I型IFN被强烈诱导,但II型IFN却主要是在响应免疫和炎性刺激时而被诱导,因此IFN-γ经常被称为“免疫IFN”。众多I型IFN中研究最多的包括IFN-α、IFN-β和IFN-ω。其中IFN-α最复杂,包括至少15种不同蛋白亚型,表现出超过75%的序列同源性。Diaz(1995)Semin.Virol.6:143-149;Weissmann等(1986)Prog Nucl Acid Res Mol Biol33:251;J Interferon Res(1993)13:443-444;Roberts等(1998)J InterferonCytokine Res 18:805-816。IFN-α基因及其产物除了具有结构相似性外,也表现出功能相似性。例如,它们由dsRNA或病毒诱导,可以与相同受体(IFNα/β受体IFNAR)相互作用。Mogensen等(1999)JInterferons and other Regulatory Cytokine,John Wiley&Sons。IFNα也抑制细胞凋亡,促进抗原活化T辅助细胞的存活和分化,并促进功能有效单核细胞衍生的树突细胞的成熟。
[0003]  当接触病毒和dsRNA时,许多细胞类型都可产生IFNα。特化的白细胞(称为干扰素-α产生细胞(“IPC”)在响应各种刺激(例如病毒、细菌和原生动物)时产生IFNα。一些体外研究表明,在人外周血单核细胞(PBMC)感染后,不同IFN-α亚型在不同程度上由各种IFN-α-分泌细胞系产生或者以病毒类型特异性方式产生,并且这些方式通常与抗增殖、抗病毒和抗肿瘤活性上的亚型依赖性差异相关。然而,不同亚型及其彼此在体内的协同或拮抗活性的生理意义仍未确定。
[0004]  在几种自身免疫性疾病中观察到IFN-α是病理介导物。此外,它在用IFN-α治疗癌症和病毒感染的患者中可引起自身免疫性疾病发展。在胰岛素依赖型糖尿病(IDDM或I型糖尿病)、银屑病、节段性回肠炎(Crohn’s disease)和乳糜泻患者的局部患病组织中都观察到IFN-α表达增加。在系统性红斑狼疮(systemic lupuserythematosus,SLE)、IDDM和AIDS患者中观察到IFN-α的过量表达。就以富含自身反应型B和T细胞为特征的SLE而言,不仅在组织病灶中而且在受累个体血液循环中都观察到IFN-的表达。此外,IFN-α血清水平倾向于与临床疾病活性指数相关。人们认为,这是因为当IFN-α的产生受到浆细胞样DC(plasmacytoid DC,pDC)上调时而触发正常静息单核细胞经循环诱导,生成强效抗原呈递树突细胞(DC)所致。的确,本发明人先前已经通过寡核苷酸微阵列分析证明,通过涉及粒细胞生成和IFN诱导的未调节基因的“标记(signature)”而区别SLE;当高剂量输注糖皮质激素时,这些标记恢复正常(美国专利申请11/228,586,所述文献通过引用结合到本文中)。
[0005]  系统性红斑狼疮(SLE)是全身性自身免疫性风湿性疾病,尤其会侵袭儿童,其特征是突发高致病率。自身免疫性疾病例如SLE通常表现出复发和缓解的自身延续性循环(self-perpetuatingcycle)。这些循环通常按照给予通用治疗方案以平息SLE疾病循环的治疗期来定义。FDA批准的SLE治疗方案包括皮质类固醇、非甾体免疫抑制药、抗疟药和非甾体抗炎药。这些药物破坏了所有免疫效应物反应的完整性,而不是专门针对SLE发病机制起作用。SLE成为无法满足的医学要求,因为这些治疗仅部分有效,且具有中度至严重副作用,包括骨质疏松(bone thinning)、体重增加、痤疮、贫血、不育、皮疹、腹泻、脱发和恶心。此外,在40年内没有新型SLE治疗药获得批准。
[0006]  目前已将SLE与未减弱的IFNα的产生密切联系起来。Shi等(1987)Br.J.Dermatol.117(2):155-159。IFNα在SLE血清(Crow等(2004)Curr.Opin.Rheumatol.16(5):541-547)和作为IFNα的主要来源的浆细胞样DC(pDC)中以升高水平存在,在SLE皮肤中累积。Farkas等(2001)Am.J.Pathol.159(1):237-243。此外,已经观察到经IFNα治疗的某些患者狼疮进一步生长(Okanoue等(1996)J.Hepatol.25(3):283-291;Tothova等(2002)Neoplasma 49(2):91-94;和Raanani等(2002)Acta Haematol.107(3):133-144),并且具有IFNα抗体的狼疮患者表现出更温和的疾病形式。Von Wussow等(1988)Rheumatol.Int.8(5):225-230。IFNα可通过单核细胞向功能性树突细胞(DC)分化而起作用,这样的分化反过来又介导SLE发病机理。Pascual V.等(2003)Curr.Opin.Rheumatol.15(5):548-556。治疗SLE的一个建议性方法是中和IFNα(参见Banchereau等,PCT/US02/00343,所述文献通过引用结合到本文中)。
[0007]  尽管已制备出可阻断人IFN-α生物活性的单克隆抗体,但是迄今为止并无它可中和所有15种已知亚型的相关报道,而且也很少能中和天然来源的含IFN-α白细胞IFN。PBL BiomedicalLaboratories提供10种小鼠单克隆抗体,它们可结合多种人IFNα基因亚型(参见万维网Interferonsource.com/relativespecificity.html)。然而,每种PBL抗体都可结合由人IFNα基因亚型1编码的IFNα蛋白亚型(IFNα蛋白亚型D)并且可结合1-12种其它IFNα亚型。美国专利公布号2003/0166228A1公开了来自用白细胞IFNα(其包括所有IFNα蛋白亚型)免疫小鼠的单克隆抗体(称为9F3)。9F3 Mab可结合并中和由7种人IFNα基因亚型1、2、4、5、8、10和21(分别编码IFNα蛋白亚型D、A、4、G、B2、C和F)所编码的蛋白质的抗病毒活性,而不中和人IFNβ的抗病毒活性。该专利公布说明书没有公开9F3抗体是否能结合并钝化其它8种IFNα基因亚型,也没有公开它是否与IFNα蛋白亚型4a和4b中的一种或两种都结合。PCT公布说明书提出用单克隆抗体治疗IFNα表达增加的相关疾病,特别是自身免疫病例如胰岛素依赖型糖尿病和SLE。然而,尚不知道9F3抗体是否足以中和SLE血清中存在的IFNα蛋白亚型的生物活性。
[0008]  因为IFNα是免疫应答的多功能介导物,具有有益的抗病毒活性,所以对所有IFNα亚型的完全抑制或明显下调并非最佳治疗方法。因此,需要能够选择性中和病理状况相关的IFNα亚型的药物。本发明即可满足该要求并提供相关优势。
[0009]  本发明提供能中和特定IFNα亚型的单克隆抗体及其衍生物。本发明的抗体可用于改善和治疗IFNα表达增加的相关疾病,例如SLE、银屑病、I型糖尿病、AIDS、移植物抗宿主病和其它自身免疫性疾病。一方面,本发明包括这样的抗体:所述抗体能选择性中和至少两种干扰素α(“IFNα”)蛋白亚型(例如亚型A、2、B2、C、F、G、H2、I、J1、K、4a、4b和WA)的生物活性,但不明显地中和IFNα蛋白亚型D的至少一种生物活性;其中所述生物活性是MxA启动子的活化或抗病毒活性。优选的抗体是单克隆抗体。在一个实施方案中,抗体不明显地中和IFNα蛋白亚型D,也不明显地中和IFNα蛋白亚型1。在一个实施方案中,单克隆抗体是ACO-1或ACO-2。优选的本发明抗体钝化自系统性红斑狼疮(SLE)患者体内分离出的血清刺激单核细胞分化成树突细胞的能力。