TWI623324B - Antibody binding to fcrn for treating autoimmune diseases - Google Patents

Antibody binding to fcrn for treating autoimmune diseases Download PDF

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TWI623324B
TWI623324B TW104135358A TW104135358A TWI623324B TW I623324 B TWI623324 B TW I623324B TW 104135358 A TW104135358 A TW 104135358A TW 104135358 A TW104135358 A TW 104135358A TW I623324 B TWI623324 B TW I623324B
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antibody
fcrn
amino acid
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TW201714621A (en
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金聖旭
朴勝國
丁在甲
安惠敬
金旼善
金銀善
龍海英
申東玉
宋連情
柳泰亨
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韓諾生物製藥股份有限公司
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Abstract

本發明係關於一種分離的抗-FcRn抗體，是與FcRn（表示新生兒Fc受體，也稱為FcRP、FCRB或Brambell受體）結合的抗體，FcRn是對於IgG或其片段具有高親和性的受體，本發明還關於此抗體的製造方法、用於治療自體免疫病包含此抗體的組合物、以及使用此抗體治療和診斷自體免疫病的方法。根據本發明的FcRn特異性抗體與對於IgG具有非競爭性的FcRn結合，以降低血清致病的自體抗體的水平，並因此可用於自體免疫病的治療。The present invention relates to an isolated anti-FcRn antibody which is an antibody which binds to FcRn (representing a neonatal Fc receptor, also called FcRP, FCRB or Brambell receptor), and FcRn has high affinity for IgG or a fragment thereof. Receptor, the present invention also relates to a method for producing the antibody, a composition for treating an autoimmune disease comprising the antibody, and a method for treating and diagnosing an autoimmune disease using the antibody. The FcRn-specific antibody according to the present invention binds to FcRn which is non-competitive for IgG to lower the level of serum-pathogenic autoantibodies, and thus can be used for the treatment of autoimmune diseases.

Description

用於治療自體免疫病的與FCRN結合的抗體Antibody to FCRN for the treatment of autoimmune diseases

本發明係關於一種分離的抗-FcRn抗體，是與FcRn（表示新生兒Fc受體，也稱為FcRP、FCRB或Brambell受體）結合的抗體，FcRn是對於IgG或其片段具有高親和性的受體，本發明還關於此抗體的製造方法，用於治療自體免疫病包含此抗體的組合物，以及使用此抗體治療和診斷自體免疫病的方法。根據本發明的FcRn特異性抗體與對於IgG具有非競爭性的FcRn結合，以降低血清致病的自體抗體的水平，並因此可用於自體免疫病的治療。The present invention relates to an isolated anti-FcRn antibody which is an antibody which binds to FcRn (representing a neonatal Fc receptor, also called FcRP, FCRB or Brambell receptor), and FcRn has high affinity for IgG or a fragment thereof. Receptor, the present invention also relates to a method for producing the antibody, a composition for treating an autoimmune disease comprising the antibody, and a method for treating and diagnosing an autoimmune disease using the antibody. The FcRn-specific antibody according to the present invention binds to FcRn which is non-competitive for IgG to lower the level of serum-pathogenic autoantibodies, and thus can be used for the treatment of autoimmune diseases.

抗體為結合至特異性抗原的免疫蛋白。在大多數動物，包含人類和小鼠中，抗體由配對的重和輕多肽鏈構造且每條鏈係由稱為可變區和恆定區的兩個不同的區域組成。輕鏈和重鏈可變區顯示出抗體之間的顯著序列多樣性，並且負責結合目標抗原。恆定區顯示出較少的序列多樣性，並且負責結合一些天然蛋白質以引發重要的生物化學事件。An antibody is an immune protein that binds to a specific antigen. In most animals, including humans and mice, antibodies are constructed from paired heavy and light polypeptide chains and each chain consists of two distinct regions called variable and constant regions. The light and heavy chain variable regions display significant sequence diversity between antibodies and are responsible for binding to the antigen of interest. The constant regions show less sequence diversity and are responsible for binding some natural proteins to trigger important biochemical events.

在正常條件下，血清中不含IgG3的大多數IgG同種型的半衰期為人類中為大約22-23，這是相對於其他血漿蛋白的血清半衰期更長的一時間。對於IgG的這種延長的血清半衰期，通過胞吞作用進入的細胞的IgG在6.0的pH值可強烈結合至胞內體中的新生兒Fc受體（FcRn，一種Fc伽馬受體），以避免降解胞溶體途徑。當IgG-FcRn的複合循環到質膜時，在微鹼性pH值（〜7.4）的血液中IgG從FcRn快速解離。透過這種受體介導的循環機製，FcRn有效解救IgG免遭在胞溶體中降解，從而延長了IgG的半衰期（Roopenian等，免疫學170：3528，2003年）。Under normal conditions, the half-life of most IgG isoforms that do not contain IgG3 in serum is about 22-23 in humans, which is a longer period of serum half-life relative to other plasma proteins. For this extended serum half-life of IgG, the IgG of cells entering by endocytosis can strongly bind to the neonatal Fc receptor (FcRn, an Fc gamma receptor) in the endosome at a pH of 6.0, Avoid degradation of the cytosolic pathway. When the complex of IgG-FcRn is recycled to the plasma membrane, IgG is rapidly dissociated from FcRn in blood at a slightly alkaline pH (~7.4). Through this receptor-mediated circulatory mechanism, FcRn effectively rescues IgG from degradation in cytosols, thereby prolonging the half-life of IgG (Roopenian et al, Immunol. 170: 3528, 2003).

FcRn在新生鼠腸道中可識別出，在那裡它的功能是作為來自母乳IgG抗體的吸收體，並促進其運輸到循環系統。FcRn也從人類胎盤中分離出，在那裡它作為介導吸收母體的IgG且轉運到胎兒循環。在成年人中，FcRn出現於許多組織，包含肺、腸、腎的上皮組織、以及鼻、***、以及膽道樹表面。FcRn is recognized in the gut of neonatal mice, where it functions as an absorber from breast milk IgG antibodies and facilitates its transport to the circulatory system. FcRn is also isolated from the human placenta where it acts as an IgG that mediates the parent and transports it to the fetal circulation. In adults, FcRn occurs in many tissues, including the epithelial tissues of the lungs, intestines, and kidneys, as well as the nasal, vaginal, and biliary tree surfaces.

FcRn是通常駐留在內皮細胞和上皮細胞內體中的非共價異源二聚體。FcRn是具有三個重鏈α-域（α1、α2以及α3）和一單個可溶輕鏈β2微球蛋白（β2m）域的一膜結合受體。在結構上，它屬於具有β2m作為共同輕鏈的主要組織相容性複合物1類分子的一個家族。這種FcRn鏈具有大約46 kD的一分子量，並且由包含α1、α2、以及α3的重鏈結構域和β2m輕鏈結構域的胞外域組成且具有一單一糖鏈，一單次跨膜、以及一相對短細胞質尾。FcRn is a non-covalent heterodimer that typically resides in endothelial cells and epithelial endosomes. FcRn is a membrane-bound receptor with three heavy chain alpha-domains (α1, α2, and α3) and a single soluble light chain β2 microglobulin (β2m) domain. Structurally, it belongs to a family of major histocompatibility complex class 1 molecules with β2m as a common light chain. This FcRn chain has a molecular weight of approximately 46 kD and consists of an extracellular domain comprising a heavy chain domain of alpha 1, alpha 2, and alpha 3 and a beta 2 m light chain domain and has a single sugar chain, a single transmembrane, and A relatively short cytoplasmic tail.

為了研究FcRn對IgG動態平衡的貢獻，對小鼠進行改造，以使得至少一部分編碼為β2m和FcRn重鏈的基因已被「敲除」，使這些蛋白質不表現。在這些小鼠中，血清半衰期和IgG的濃度顯著地減少，這意味著對IgG動態平衡的一FcRn依賴機理。還意味著，抗人FcRn抗體可以在這些FcRn敲除小鼠中產生，並且這些抗體可以防止IgG與FcRn的結合。IgG抗體與FcRn之結合的抑制透過防止IgG的循環消極改變了IgG的血清半衰期，以使得可以治療自體抗體引起的自體免疫病。這種可能性表示於自體免疫性大疱性皮膚病的小鼠模型中（Li等人，臨床研究雜誌115：3440，2005年）。因此，阻止或抵抗IgG與FcRn之結合的藥劑可用於治療或預防由IgG介導的自體免疫和炎性疾病的方法中。To investigate the contribution of FcRn to IgG homeostasis, mice were engineered such that at least a portion of the genes encoding the β2m and FcRn heavy chains have been "knocked out" such that these proteins do not. In these mice, serum half-life and IgG concentration were significantly reduced, which means an FcRn-dependent mechanism for IgG homeostasis. It is also meant that anti-human FcRn antibodies can be produced in these FcRn knockout mice, and these antibodies can prevent the binding of IgG to FcRn. Inhibition of binding of IgG antibodies to FcRn negatively alters the serum half-life of IgG by preventing IgG cycling, such that autoimmune diseases caused by autoantibodies can be treated. This possibility is expressed in a mouse model of autoimmune bullous skin disease (Li et al., J. Clinical Research 115: 3440, 2005). Thus, agents that prevent or resist binding of IgG to FcRn can be used in methods of treating or preventing autoimmune and inflammatory diseases mediated by IgG.

「自體免疫病」包含當由於原因無法找到的免疫系統異常使得人體的免疫系統攻擊自身的正常組織、器官或其他的體內成分時發生的疾病。這些自體免疫病為可能出現在身體的幾乎所有部分，包含神經系統、胃腸系統、內分泌系統、皮膚、骨骼系統、以及血管組織的全身性疾病。已知的是，自體免疫病影響大約5-8％的世界人口，但是由於對自體免疫病的認識和用於診斷這些疾病的方法的限制，所報導的自體免疫病的發病率低於實際水平。"Autoimmune disease" includes diseases that occur when the immune system of the human body attacks its normal tissues, organs, or other internal components when an abnormality of the immune system cannot be found due to cause. These autoimmune diseases are systemic diseases that may occur in almost all parts of the body, including the nervous system, gastrointestinal system, endocrine system, skin, skeletal system, and vascular tissue. It is known that autoimmune diseases affect approximately 5-8% of the world's population, but the reported incidence of autoimmune diseases is low due to awareness of autoimmune diseases and limitations in the methods used to diagnose these diseases. At the actual level.

自體免疫病的原因在遺傳、環境以及免疫因子方面進行了長期的研究，但尚未明確確定。許多最近的研究表明，許多自體免疫病是由IgG型自體抗體導致。事實上，存在或不存在疾病特異性的自體抗體和自體免疫病治療之間的關係已廣泛地從對此病和自體免疫病的治療的研究中確定出。由此，已經確定疾病特異性的自體抗體和大量自體免疫病中其病理作用的存在，並且當所關心的自體抗體從血液中去除時，可以得到快速治療疾病的效果。The causes of autoimmune diseases have long been studied in terms of genetics, environment, and immune factors, but they have not been clearly established. Many recent studies have shown that many autoimmune diseases are caused by IgG-type autoantibodies. In fact, the relationship between the presence or absence of disease-specific autoantibodies and autoimmune disease treatment has been extensively determined from studies of the treatment of this disease and autoimmune diseases. Thus, the presence of disease-specific autoantibodies and their pathological effects in a large number of autoimmune diseases has been determined, and when the autologous antibodies of interest are removed from the blood, the effect of rapidly treating diseases can be obtained.

自體免疫病和免疫性疾病是由病原抗體介導的，並且其典型的實例包含免疫中性白細胞減少症、格林 - 巴利綜合症、癲癇、自體免疫性腦炎、Isaac綜合症、痣綜合徵、尋常天疱瘡、天疱瘡、大疱性類天疱瘡、後天性水疱性表皮鬆解症、妊娠期類天疱瘡、黏膜的天疱瘡樣、抗磷脂綜合徵、自體免疫性貧血、自體免疫性嚴重的疾病、古德帕斯徹氏綜合徵、重症肌無力、多發性硬化症、類風濕性關節炎、狼瘡、特發性血小板減少性紫癜（ITP）、狼瘡性腎炎或膜性腎病，或類似病。Autoimmune diseases and immune diseases are mediated by pathogenic antibodies, and typical examples thereof include immunological neutropenia, Guillain-Barré syndrome, epilepsy, autoimmune encephalitis, Isaac syndrome, sputum Syndrome, pemphigus vulgaris, pemphigus, bullous pemphigoid, acquired vesicular epidermolysis, pemphigoid in pregnancy, pemphigus-like mucosa, antiphospholipid syndrome, autoimmune anemia, self Severely immunized diseases, Goodpasch's syndrome, myasthenia gravis, multiple sclerosis, rheumatoid arthritis, lupus, idiopathic thrombocytopenic purpura (ITP), lupus nephritis or membranous Kidney disease, or similar disease.

舉例而言，已知的是，在重症肌無力（MG）的情況下，位於隨意肌的神經肌肉接合點的乙酰膽鹼受體（AChR）受到抵抗此受體的自體抗體的破壞或阻止，以削弱隨意肌的功能。此外，已知的是，當這樣的自體抗體減少時，肌肉的功能恢復。For example, it is known that in the case of myasthenia gravis (MG), the acetylcholine receptor (AChR) at the neuromuscular junction of the voluntary muscle is destroyed or prevented by the autoantibody against this receptor, Reduce the function of the free muscles. Furthermore, it is known that when such autoantibodies are reduced, the function of the muscles is restored.

至於ITP的情況，ITP為由於產生了結合到特定的血小板膜糖蛋白的自體抗體，由外週血小板破壞引起的疾病。抗血小板抗體對血小板發揮調理作用並透過網狀細胞（例如，巨噬細胞）導致快速的血小板破壞。As for the case of ITP, ITP is a disease caused by destruction of peripheral platelets due to the production of autoantibodies that bind to specific platelet membrane glycoproteins. Anti-platelet antibodies exert opsonization on platelets and cause rapid platelet destruction through reticulocytes (eg, macrophages).

通常，試圖治療ITP包含抑制免疫系統，並且因此引起血小板水平的增加。ITP對女性的影響多於男性，並且在兒童中相比較於成人更常見。發病率是10000人中有1人。慢性ITP是成人和兒童中一種主要的血液疾病。它是美國和世界各地的專門血液科中住院和治療成本的重要來源。每年在美國大約具有20000個新病例，並且ITP護理和特殊治療的成本是非常高的。大多數ITP患兒具有導致突發性出血的一非常低的血小板計數，典型症狀包含瘀傷、皮膚上的紅色小圓點、流鼻血以及牙齦出血。雖然孩子們有時可以不進行治療而恢復，但是許多醫生推薦仔細觀察和緩解出血，並且使用丙種球蛋白的靜脈注射來治療。In general, attempts to treat ITP involve suppression of the immune system and, therefore, an increase in platelet levels. ITP affects women more than men and is more common in children than adults. The incidence rate is 1 in 10,000 people. Chronic ITP is a major blood disorder in adults and children. It is an important source of hospitalization and treatment costs in specialized hematology in the United States and around the world. There are approximately 20,000 new cases in the United States each year, and the cost of ITP care and special treatment is very high. Most children with ITP have a very low platelet count that causes sudden bleeding. Typical symptoms include bruises, red dots on the skin, nosebleeds, and bleeding gums. Although children can sometimes recover without treatment, many doctors recommend careful observation and relief of bleeding and treatment with intravenous gamma globulin.

人們已知，一種自體免疫病，狼瘡性腎炎的重要的發病機理是增加的免疫複合物在全身臟器中累積以引起炎症反應，這種增加的免疫複合物可由於自體抗體，例如抗核抗體的不適當生產過剩而產生。大約40-70％的狼瘡患者涉及腎臟，並且大約30％的患者發展成為狼瘡性腎炎，這在狼瘡患者中已知為不良的預兆因素。雖然已經嘗試使用免疫抑製劑治療狼瘡性腎炎的方法，但是根據報導，即使使用了免疫抑製劑仍然有大約22％的狼瘡性腎炎患者未有緩解。另外，根據報導，即使症狀緩解，當使用免疫抑製劑減少時，10-65％的患者復發成狼瘡性腎炎。最終，5-15％的患有嚴重狼瘡性腎炎（世界衛生組織分類的III和IV級）的患者在10年後死亡，並且5-15％的患者導致末期腎病階段。因此，至今尚未報導狼瘡性腎炎適當的治療手段。It is known that an autoimmune disease, an important pathogenesis of lupus nephritis, is that an increased immune complex accumulates in the whole body to cause an inflammatory response. This increased immune complex can be due to autoantibodies, such as anti- An excessive production of nuclear antibodies is produced. Approximately 40-70% of lupus patients are involved in the kidney, and approximately 30% of patients develop lupus nephritis, which is known to be a poor predictor in lupus patients. Although attempts have been made to use immunosuppressive agents for the treatment of lupus nephritis, it has been reported that approximately 22% of patients with lupus nephritis have not been relieved even with the use of immunosuppressive agents. In addition, according to reports, even if the symptoms are alleviated, 10-65% of patients relapse into lupus nephritis when the immunosuppressive agent is used. Finally, 5-15% of patients with severe lupus nephritis (class III and IV of the World Health Organization classification) die 10 years later, and 5-15% of patients lead to the stage of end stage renal disease. Therefore, appropriate treatments for lupus nephritis have not been reported so far.

因此，使用透過清除致病的自體抗體具有治療自體免疫病的新機理的抗體期望對病原性IgG介導的自體免疫病具有治療作用，這些，自體免疫病例如為尋常性天疱瘡、視神經脊髓及重症肌無力，以及免疫複合物介導的腎小球疾病，例如狼瘡性腎炎或膜狀腎病。Therefore, an antibody having a novel mechanism for treating an autoimmune disease by removing a pathogenic autoantibody is expected to have a therapeutic effect on a pathogenic IgG-mediated autoimmune disease, such as an ordinary day. Pemphigus, optic nerve spinal cord and myasthenia gravis, and immune complex-mediated glomerular diseases such as lupus nephritis or membranous nephropathy.

透過IgG（IVIG）的大量靜脈給藥治療自體免疫病的方法已廣泛使用（Arnson自體免疫42：553，2009年）。IVIG作用透過各種機理加以解釋，但也透過FcRn的內源抗體IgG的競爭可提高致病抗體的清除率的機理來解釋。人免疫球蛋白（IVIG）的大量靜脈給藥已證明可增加患有免疫ITP的兒童的血小板數量，並且IVIG已證明因為對一些其他自體免疫病的治療是有益的。已經展開IVIG在自體免疫病的治療效果之機理的許多研究。關於ITP，早期研究得出的結論是，IVIG的效果主要是由於負責抗體調理的血小板吞噬作用的Fc受體的阻斷。隨後的研究表明，提供Fc貧化的IVIG製劑在一些ITP患者中增加了血小板數量，並且根據最近的報導，IVIG作用是由於對巨噬細胞表達的FcγRIIb的刺激，導致血小板吞噬作用的抑制。Methods for the treatment of autoimmune diseases by massive intravenous administration of IgG (IVIG) have been widely used (Arnson Autoimmune 42: 553, 2009). The role of IVIG is explained by various mechanisms, but it is also explained by the mechanism by which the competition of endogenous antibody IgG of FcRn can increase the clearance rate of pathogenic antibodies. A large number of intravenous administrations of human immunoglobulin (IVIG) have been shown to increase the number of platelets in children with immune ITP, and IVIG has proven to be beneficial for the treatment of some other autoimmune diseases. Many studies have been conducted on the mechanism of the therapeutic effects of IVIG in autoimmune diseases. Regarding ITP, early studies have concluded that the effect of IVIG is primarily due to the blockade of Fc receptors responsible for platelet phagocytosis by antibody conditioning. Subsequent studies have shown that the provision of Fc-depleted IVIG formulations increases the number of platelets in some ITP patients, and according to recent reports, IVIG effects are due to stimulation of macrophage-expressed FcyRIIb, leading to inhibition of platelet phagocytosis.

然而，這樣的IVIG治療具有相當大的副作用，並且用藥非常昂貴。此外，除了IVIG之外，用於治療自體免疫/同種免疫疾病的其他療法包含多克隆抗D免疫球蛋白、皮質類固醇、免疫抑製劑（包含化學治療劑）、細胞因子、血漿置換、體外抗體吸附（例如，使用Prosorba柱）、外科手術例如脾切除、以及其他療法。然而，類似於IVIG，這些療法也是複雜的，並且具有不完全的功效和高成本。此外，由於IVIG抑制FcRn與致病抗體結合（即競爭性抑制）的推定機理，並且由於這樣的事實︰在生理pH值（即，pH值7.2-7.4）下，IgG顯示出對FcRn非常低的親和性，並且IVIG的通常臨床劑量為大約2克/千克，因此需要非常高劑量的IVIG以產生致病抗體的清除率大幅度增加。However, such IVIG treatment has considerable side effects and is very expensive to administer. In addition, in addition to IVIG, other therapies for the treatment of autoimmune/incompatible immune diseases include polyclonal anti-D immunoglobulins, corticosteroids, immunosuppressants (including chemotherapeutic agents), cytokines, plasma exchange, in vitro antibodies Adsorption (eg, using a Prosorba column), surgery such as splenectomy, and other therapies. However, similar to IVIG, these therapies are also complex and have incomplete efficacy and high cost. Furthermore, due to the putative mechanism by which IVIG inhibits the binding (ie, competitive inhibition) of FcRn to pathogenic antibodies, and due to the fact that at physiological pH (ie, pH 7.2-7.4), IgG shows very low FcRn. Affinity, and the usual clinical dose of IVIG is about 2 grams per kilogram, so a very high dose of IVIG is required to produce a substantial increase in the clearance of pathogenic antibodies.

使用抑製劑以競爭性抑制IgG與FcRn的結合來治療自體免疫病的是一種有希望的治療方法。然而，由於內源性的IgG對FcRn的高親和性和血液中內源性IgG的高濃度，很可能FcRn的競爭性抑制需要非常高的劑量，並且因此具有類似於當前的IVIG治療的相同限制。The use of inhibitors to competitively inhibit the binding of IgG to FcRn for the treatment of autoimmune diseases is a promising therapeutic approach. However, due to the high affinity of endogenous IgG for FcRn and the high concentration of endogenous IgG in the blood, it is likely that competitive inhibition of FcRn requires very high doses and therefore has the same limitations similar to current IVIG treatments. .

因此，雖然抗-FcRn抗體在WO2006/118772、WO2007/087289、WO2009/131702、WO2012/167039中發明，但目前迫切需要開發具有對FcRn的高親和性的改進人類抗體，並且因而甚至可以在低劑量下清除致病抗體和降低免疫原性。Thus, although anti-FcRn antibodies were invented in WO2006/118772, WO2007/087289, WO2009/131702, WO2012/167039, there is an urgent need to develop improved human antibodies with high affinity for FcRn, and thus even at low doses Remove pathogenic antibodies and reduce immunogenicity.

技術問題technical problem

本發明人做出了大量的工作以解決上述問題，並且提供一種用於有效且從根本上治療自體免疫性病，包含特發性血小板減少性紫癜（ITP）的藥物，並且最後提供一種對FcRn具有高親和性的抗體或其片段及其製備方法。與FcRn結合的抗體或其片段以與pH無關的方式特異性地結合FcRn鏈，並且非競爭性干擾抗體的Fc與FcRn的結合，以透過減少體內的自體抗體來治療自體免疫病，而此自體抗體可能是自體免疫病的原因。The present inventors have made a great deal of work to solve the above problems, and provide a medicament for effectively and fundamentally treating autoimmune diseases, including idiopathic thrombocytopenic purpura (ITP), and finally providing a pair of FcRn. An antibody or fragment thereof having high affinity and a process for the preparation thereof. An antibody or fragment thereof that binds to FcRn specifically binds to an FcRn chain in a pH-independent manner, and non-competitively interferes with binding of an Fc of an antibody to FcRn to treat an autoimmune disease by reducing autoantibodies in vivo, and This autoantibody may be the cause of an autoimmune disease.

本發明的一個目的在於提供治療自體免疫病的藥物組合物，這種藥物組合物中抗體與FcRn相結合，其中自體免疫病免疫中性白細胞減少症、格林-巴利綜合症、癲癇、自體免疫性腦炎、Isaac綜合症、痣綜合徵、尋常天疱瘡、天疱瘡、大疱性類天疱瘡、後天性水疱性表皮鬆解症、妊娠期類天疱瘡、黏膜的天疱瘡樣、抗磷脂綜合徵、自體免疫性貧血、自體免疫性嚴重的疾病、古德帕斯徹氏綜合徵、重症肌無力、多發性硬化症、類風濕性關節炎、狼瘡、特發性血小板減少性紫癜、狼瘡性腎炎或膜性腎病或類似病。技術解決方案 It is an object of the present invention to provide a pharmaceutical composition for treating an autoimmune disease in which an antibody binds to FcRn, wherein autoimmune disease immunoneutral neutropenia, Guillain-Barré syndrome, epilepsy, Autoimmune encephalitis, Isaac syndrome, delirium syndrome, pemphigus vulgaris, pemphigus, bullous pemphigoid, acquired vesicular epidermolysis, pemphigoid in pregnancy, pemphigus-like mucosa, Antiphospholipid syndrome, autoimmune anemia, autoimmune disease, Goodpasch's syndrome, myasthenia gravis, multiple sclerosis, rheumatoid arthritis, lupus, idiopathic thrombocytopenia Sexual purpura, lupus nephritis or membranous nephropathy or the like. Technical solution

為了獲得上述目的，本發明提供一種分離的抗-FcRn抗體或其片段，包含： CDR1，具有選自以下序列號的組中的一種或多種氨基酸序列：21、24、27、30、33、36、39以及42； CDR2，具有選自以下序列號的組中的一種或多種氨基酸序列：22、25、28、31、34、37、40以及43；以及 CDR3，具有選自以下序列號的組中的一種或多種氨基酸序列：23、26、29、32、35、38、41以及44。To achieve the above object, the present invention provides an isolated anti-FcRn antibody or fragment thereof, comprising: CDR1, having one or more amino acid sequences selected from the group consisting of: 21, 24, 27, 30, 33, 36 , 39 and 42; CDR2, having one or more amino acid sequences selected from the group consisting of: 22, 25, 28, 31, 34, 37, 40 and 43; and CDR3 having a group selected from the following sequence numbers One or more amino acid sequences: 23, 26, 29, 32, 35, 38, 41, and 44.

此外，一種分離的抗-FcRn抗體或其片段，包含： CDR1，包含氨基酸序列，與選自以下序列號的組中的一種或多種氨基酸序列具有至少90％的同源性：21、24、27、30、33、36、39以及42； CDR2，包含氨基酸序列，與選自以下序列號的組中的一種或多種氨基酸序列具有至少90％的同源性：22、25、28、31、34、37、40以及43；以及 CDR3，包含氨基酸序列，與選自以下序列號的組中的一種或多種氨基酸序列具有至少90％的同源性：23、26、29、32、35、38、41以及44。Furthermore, an isolated anti-FcRn antibody or fragment thereof, comprising: CDR1 comprising an amino acid sequence having at least 90% homology to one or more amino acid sequences selected from the group consisting of: 21, 24, 27 , 30, 33, 36, 39 and 42; CDR2, comprising an amino acid sequence having at least 90% homology to one or more amino acid sequences selected from the group consisting of: 22, 25, 28, 31, 34 , 37, 40 and 43; and CDR3, comprising an amino acid sequence having at least 90% homology to one or more amino acid sequences selected from the group consisting of: 23, 26, 29, 32, 35, 38, 41 and 44.

另外，本發明提供了一種分離的抗-FcRn抗體或其片段，包含一種或多種重鏈可變區和輕鏈可變區，重鏈可變區和輕鏈可變區具有選自以下序列號的氨基酸序列所構成的組中的一種或多種氨基酸序列：2、4、6、8、10、12、14、16、18以及20。In addition, the invention provides an isolated anti-FcRn antibody or fragment thereof, comprising one or more heavy chain variable regions and a light chain variable region, the heavy chain variable region and the light chain variable region having a sequence number selected from the group consisting of One or more amino acid sequences in the group consisting of amino acid sequences: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20.

另外，本發明提供了一種分離的抗-FcRn抗體，包含具有氨基酸序列的一種或多種重鏈可變區和輕鏈可變區，氨基酸序列與選自以下序列號的氨基酸序列的組中一種或多種氨基酸序列具有至少90％的同源性：2、4、6、8、10、12、14、16、18以及20。Further, the present invention provides an isolated anti-FcRn antibody comprising one or more heavy chain variable regions and light chain variable regions having an amino acid sequence, one of a group of amino acid sequences and an amino acid sequence selected from the following sequence numbers or A variety of amino acid sequences have at least 90% homology: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20.

另外，一種編碼此抗-FcRn抗體或其片段的多核苷酸。In addition, a polynucleotide encoding the anti-FcRn antibody or a fragment thereof.

此外，本發明提供了一種編碼抗-FcRn抗體的多核苷酸，其中抗-FcRn抗體包含選自以下序列號的組中的一種或多種序列：1、3、5、7、9、11、13、15、17以及19。Furthermore, the present invention provides a polynucleotide encoding an anti-FcRn antibody, wherein the anti-FcRn antibody comprises one or more sequences selected from the group consisting of: 1, 3, 5, 7, 9, 11, 13 15, 17, and 19.

此外，本發明提供了一種編碼抗-FcRn抗體的多核苷酸，其中抗-FcRn抗體包含與選自以下序列號的組中的一種或多種序列具有至少90％的同源性的序列：1、3、5、7、9、11、13、15、17以及19。Furthermore, the invention provides a polynucleotide encoding an anti-FcRn antibody, wherein the anti-FcRn antibody comprises a sequence having at least 90% homology to one or more sequences selected from the group consisting of: 3, 5, 7, 9, 11, 13, 15, 17 and 19.

另外，本發明提供了一種包含多核苷酸的重組表達載體，使用重組表達載體橫切宿主細胞。本發明還提供了一種特異性結合至FcRn的抗體或其片段的製備方法，包括：培養此宿主細胞且由宿主細胞產生抗體；以及分離和純化產生的抗體，以恢復特異性結合至的此抗-FcRn的抗體。In addition, the invention provides a recombinant expression vector comprising a polynucleotide, the host cell being transected using a recombinant expression vector. The present invention also provides a method for producing an antibody or a fragment thereof which specifically binds to FcRn, comprising: culturing the host cell and producing an antibody from the host cell; and isolating and purifying the produced antibody to restore the specific binding to the antibody - an antibody to FcRn.

另外，本發明提供了一種藥物組合物以及一種或多種藥學上可接受的載體，其中藥物組合物包含此抗-FcRn的抗體或其片段。Additionally, the invention provides a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers, wherein the pharmaceutical composition comprises the anti-FcRn antibody or fragment thereof.

另外，本發明提供了一種自體免疫病患者的治療方法，包含施用此藥物組合物給患者。Additionally, the present invention provides a method of treating a patient with an autoimmune disease comprising administering the pharmaceutical composition to a patient.

另外，本發明提供了一種組合物，包含標記有一檢測標記的抗體。Additionally, the invention provides a composition comprising an antibody labeled with a detection label.

另外，本發明提供了一種在體內或體外使用抗體或其片段檢測FcRn的方法。有益效果 Further, the present invention provides a method for detecting FcRn using an antibody or a fragment thereof in vivo or in vitro. Beneficial effect

本發明特定於FcRn的抗體或其片段具有高親合性和特異性，其中FcRn係為對IgG具有高親和性的一受體，本發明的抗體或其片段導致很少或沒有免疫原性相關的問題，並且與IgG或類似物非競爭性地和FcRn結合，以降低血清自體抗體的水平。利用這種性質，抗體或其片段用於自體免疫病的治療和診斷。The FcRn-specific antibody or fragment thereof of the invention has high affinity and specificity, wherein the FcRn is a receptor having high affinity for IgG, and the antibody or fragment thereof of the invention causes little or no immunogenicity The problem, and non-competitive binding to FcRn with IgG or analogs to reduce the level of serum autoantibodies. Utilizing this property, antibodies or fragments thereof are used in the treatment and diagnosis of autoimmune diseases.

本發明的模式Mode of the invention

為了達到上述目的，本發明提供一種抗體，其可按照與pH值無關的方式以高親和性特異性地結合於FcRn且由來自人的序列組成，並且因此當體內給藥時引起很小或沒有免疫反應。In order to achieve the above object, the present invention provides an antibody which can specifically bind to FcRn with high affinity in a pH-independent manner and consists of a sequence derived from human, and thus causes little or no when administered in vivo. immune response.

根據本發明的抗體係為對FcRn分子具有特異性的結合分子。這些抗體可包含單克隆抗體（例如，具有免疫球蛋白Fc結構域的全長抗體）、具有多表位特異性的抗體組合、雙特異性抗體、雙抗體以及單鏈分子，以及抗體片段（例如，Fab、F(ab')2以及Fv），但不限於此。根據本發明的抗體可以是，例如，針對人FcRn的單克隆抗體。The anti-system according to the invention is a binding molecule specific for the FcRn molecule. These antibodies may comprise monoclonal antibodies (eg, full length antibodies with immunoglobulin Fc domains), antibody combinations with multi-epitope specificity, bispecific antibodies, diabody and single chain molecules, as well as antibody fragments (eg, Fab, F(ab') 2, and Fv), but are not limited thereto. The antibody according to the present invention may be, for example, a monoclonal antibody against human FcRn.

單克隆抗體包含鼠抗體。進一步而言，單克隆抗體包含「嵌合」抗體，「嵌合」抗體中重鏈與/或輕鏈的一部分等同或相同於從特定物種例如鼠或屬於特定抗體類別或亞類的抗體中得到的序列，而鏈的剩餘部分等同或相同於從另一物種或屬於另一抗體類別或亞類，例如人得到的抗體，以及此類抗體的片段的相應序列，只要它們展現出期望的生物活性。「人源化抗體」用作「嵌合抗體」的下游集。Monoclonal antibodies contain murine antibodies. Further, the monoclonal antibody comprises a "chimeric" antibody, and a portion of the heavy chain and/or light chain of the "chimeric" antibody is identical or identical to that obtained from a particular species, such as a mouse or an antibody belonging to a particular antibody class or subclass. Sequence, and the remainder of the strand is identical or identical to the antibody obtained from another species or belonging to another antibody class or subclass, such as a human, and the corresponding sequences of fragments of such antibodies, so long as they exhibit the desired biological activity . "Humanized antibodies" are used as a downstream set of "chimeric antibodies".

作為人源化的替代物，可以產生人抗體。「人抗體」係指由人類產生或具有與使用任何人抗體生產技術所產生的抗體相對應的氨基酸序列的抗體。人抗體可使用本領域已知的各種技術，包含噬菌體展示庫來製備。人抗體可以透過將抗原給予轉基因動物來製備，其中轉基因動物已被修改以響應於抗原攻擊以產生此類抗體，但其內源基因座已禁用，例如，免疫的轉基因鼠。根據本發明的抗體可以是例如人抗體的形式。As an alternative to humanization, human antibodies can be produced. "Human antibody" refers to an antibody produced by a human or having an amino acid sequence corresponding to an antibody produced using any human antibody production technique. Human antibodies can be prepared using a variety of techniques known in the art, including phage display libraries. Human antibodies can be prepared by administering an antigen to a transgenic animal, wherein the transgenic animal has been modified to produce such antibodies in response to antigenic challenge, but its endogenous locus has been disabled, for example, an immunized transgenic mouse. The antibody according to the invention may be in the form of, for example, a human antibody.

天然四鏈抗體為由兩條相同的輕（L）鏈和兩條相同的重（H）鏈組成的異四聚體糖蛋白。每條輕鏈在一端（VL）具有一可變結構域且在其另一端具有一恆定域。每條重鏈在N-末端具有一可變結構域（VH ），並且對於α和γ鏈具有三個恆定結構域（CH）且對於μ和ε同種型具有四個CH域。A native four-chain antibody is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain has a variable domain at one end (VL) and a constant domain at the other end. Each heavy chain has a variable domain ( VH ) at the N-terminus and three constant domains (CH) for the alpha and gamma chains and four CH domains for the μ and epsilon isoforms.

用語「可變」係指這樣的事實，可變結構域的某些部分的序列在抗體間之間廣泛不同。V結構域介導抗原結合並限定與其特定抗原的特定抗體的特異性。然而，可變性集中在稱為高度變異區（HVR）的三個區段中，即輕鏈和重鏈兩者的可變結構域中的CDR。可變結構域中更加高度保留的部分稱作框架區（FR）。輕鏈和重鏈可變結構域包含從N-至C-末端的結構域FR1、CDR1、FR2、CDR2、FR3、CDR3以及FR4。The term "variable" refers to the fact that the sequences of certain portions of the variable domains vary widely between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody to its particular antigen. However, variability is concentrated in three segments called hypervariable regions (HVR), the CDRs in the variable domains of both the light and heavy chains. The more highly reserved portion of the variable domain is called the frame region (FR). The light and heavy chain variable domains comprise the N- to C-terminal domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

在本發明中，對人FcRn具有親和性和特異性的抗體係使用人免疫球蛋白的轉基因動物獲得。轉基因動物可以透過使得動物Ig種系基因失活且移植人Ig種系基因座來產生。利用轉基因動物具有的一優點在於，抗體由動物的免疫系統自然優化而不需要親和性熟化，以可以在短時間內開發具有低免疫原性和高親和性的抗體藥物（US20090098134、US20100212035、Menoret等，歐洲免疫學雜誌，40：2932，2010年）。In the present invention, an anti-system having affinity and specificity for human FcRn is obtained using a transgenic animal of human immunoglobulin. Transgenic animals can be produced by inactivating the animal Ig germline gene and transplanting the human Ig germline locus. One advantage of using transgenic animals is that the antibodies are naturally optimized by the animal's immune system without the need for affinity maturation, so that antibody drugs with low immunogenicity and high affinity can be developed in a short time (US20090098134, US20100212035, Menoret, etc.) , European Journal of Immunology, 40: 2932, 2010).

在本發明中，使用具有人類免疫球蛋白轉基因鼠專利技術的OmniRatTM （OMT，美國）。OmniRatTM 可以有效地選擇對人FcRn具有高親和性的抗體，因為它包含由來自大鼠基因的CH2及CH3結構域，V、D以及J區，以及來自人基因的CH1結構域組成的重鏈，以及來自人的κ輕鏈和λ輕鏈，用以有效地選擇對人FcRn具有高親和性的抗體（Menoret等，歐洲免疫學雜誌，40：2932，2010年）。In the present invention, OmniRat (TM) (OMT, USA) having the patented technology of human immunoglobulin transgenic mice is used. OmniRat TM can effectively select human FcRn high affinity antibodies, because it contains the heavy chain CH2 and CH3 domains, V, D and J region genes from rat and human CH1 domain from gene consisting of And human kappa light chain and lambda light chain for efficient selection of antibodies with high affinity for human FcRn (Menoret et al., J. Immunol. 40: 2932, 2010).

為了得到對FcRn具有高親和性的單克隆抗體，透過在其中注入人FcRn使得轉基因鼠（OmniRatTM ）免疫，並且然後從細胞中提取B細胞並與骨髓瘤細胞融合以產生一融合瘤，然後將所產生的抗體從所生成的融合瘤進行純化。In order to obtain monoclonal antibodies with high affinity to FcRn, which is injected through the human FcRn transgenic mice that (OmniRat TM) immunized, and then extracted from the cells and B cells are fused with myeloma cells to produce a hybridoma, and then The produced antibody is purified from the resulting fusion tumor.

根據本發明的抗體可作為與FcRn結合的IgG的非競爭性抑製劑。本發明的抗體與FcRn的結合產生FcRn的致病抗體的抑制，這樣促進致病抗體從被檢體的清除率（即，去除），以減少致病抗體的半衰期。An antibody according to the invention acts as a non-competitive inhibitor of IgG that binds to FcRn. Binding of an antibody of the invention to FcRn produces inhibition of a FcRn-causing pathogenic antibody, which promotes clearance (i.e., removal) of the pathogenic antibody from the subject to reduce the half-life of the pathogenic antibody.

如在此所用，用語「致病抗體」係指引起病理狀況或疾病的抗體。此類抗體的實例包含但不限於，抗血小板抗體、抗乙酰膽鹼抗體、抗核酸抗體、抗磷脂抗體、抗膠原抗體、抗神經節苷脂抗體、抗橋粒芯蛋白抗體等。As used herein, the term "pathogenic antibody" refers to an antibody that causes a pathological condition or disease. Examples of such antibodies include, but are not limited to, anti-platelet antibodies, anti-acetylcholine antibodies, anti-nucleic acid antibodies, anti-phospholipid antibodies, anti-collagen antibodies, anti-ganglioside antibodies, anti-bridged core protein antibodies, and the like.

根據本發明的抗體或其片段具有的優點在於，使得可能非競爭性地抑制致病抗體與FcRn在生理pH下（即，pH值7.0-7.4）的結合。FcRn與其配體（即，IgG）相結合且在生理pH而不是酸性pH下不實質表示出與IgG的親和性。因此，在生理pH下特定結合至FcRn的抗-FcRn抗體充當IgG與FcRn結合的非競爭性抑製劑，並且在這種情況下，抗-FcRn抗體與FcRn的結合不因為IgG的存在而受到影響。因此，本發明的抗體以和pH無關的方式與IgG非競爭性地和FcRn結合，相比較於傳統的競爭性抑製劑（即，與IgG競爭性地和FcRn結合的抗體）具有的一優點在於︰透過FcRn介導的IgG的訊號，即使在顯著低濃度時也可治療疾病。此外，在結合於FcRn的狀態下，在細胞內遷移的過程中，根據本發明的抗-FcRn抗體在血液中與FcRn的結合保持相比較於IgG更高的親和性，因此即使在胞內體為酸性pH環境中，也可抑制IgG抗體與FcRn的結合，從而促進IgG的清除率。The antibody or fragment thereof according to the invention has the advantage of making it possible to non-competitively inhibit the binding of the pathogenic antibody to FcRn at physiological pH (ie pH pH 7.0-7.4). FcRn binds to its ligand (ie, IgG) and does not substantially express affinity for IgG at physiological pH rather than acidic pH. Thus, an anti-FcRn antibody that specifically binds to FcRn at physiological pH acts as a non-competitive inhibitor of IgG binding to FcRn, and in this case, the binding of the anti-FcRn antibody to FcRn is not affected by the presence of IgG. . Thus, the antibodies of the present invention bind non-competitively to FcRn in a pH-independent manner, with an advantage over conventional competitive inhibitors (i.e., antibodies that compete with IgG for binding to FcRn). .. through the FcRn-mediated IgG signal, the disease can be treated even at significantly low concentrations. Furthermore, in the state of binding to FcRn, the anti-FcRn antibody according to the present invention maintains a higher affinity for FcRn binding in the blood than IgG during intracellular migration, and thus even in the endosome In an acidic pH environment, binding of IgG antibodies to FcRn can also be inhibited, thereby promoting IgG clearance.

根據本發明的抗體即使在其中IgG不結合至FcRn的生理pH環境中（即，pH值7.0-7.4）也具有對FcRn的親和性。在pH6.0下，本發明的抗體相比較於血清IgG具有對FcRn更高的親和性，這表明它充當一非競爭性抑製劑。The antibody according to the present invention has an affinity for FcRn even in a physiological pH environment in which IgG does not bind to FcRn (i.e., pH 7.0-7.4). At pH 6.0, the antibody of the invention has a higher affinity for FcRn than serum IgG, indicating that it acts as a non-competitive inhibitor.

在本發明的一實施例中，本發明關於與FcRn特異性結合的抗體或其片段，此抗體或其片段包含： CDR1，包含氨基酸序列，與選自以下序列號的組中的一種或多種氨基酸序列具有至少90％的同源性：21、24、27、30、33、36、39以及42； CDR2，包含氨基酸序列，與選自以下序列號的組中的一種或多種氨基酸序列具有至少90％的同源性：22、25、28、31、34、37、40以及43；以及 CDR3，包含氨基酸序列，與選自以下序列號的組中的一種或多種氨基酸序列具有至少90％的同源性：23、26、29、32、35、38、41以及44。In one embodiment of the present invention, the present invention relates to an antibody or a fragment thereof which specifically binds to FcRn, the antibody or fragment thereof comprising: CDR1 comprising an amino acid sequence, and one or more amino acids selected from the group consisting of the following SEQ ID NO: The sequence has at least 90% homology: 21, 24, 27, 30, 33, 36, 39, and 42; CDR2, comprising an amino acid sequence having at least 90 of one or more amino acid sequences selected from the group consisting of the following SEQ ID NO: % homology: 22, 25, 28, 31, 34, 37, 40, and 43; and CDR3, comprising an amino acid sequence having at least 90% identical to one or more amino acid sequences selected from the group consisting of the following SEQ ID NO: Source: 23, 26, 29, 32, 35, 38, 41 and 44.

本領域的技術人員可以理解，在所列出的上述序列號的氨基酸序列中一些氨基酸的缺失、添加或替換也落入本發明的範圍之內。It will be understood by those skilled in the art that deletions, additions or substitutions of some amino acids in the amino acid sequences of the above listed sequence numbers are also within the scope of the invention.

此外，對於在一定範圍內具有與本發明描述的核苷酸序列和氨基酸序列集同源性的序列也落入本發明的範圍之內。「同源性」係指與從序列號1至44組成的組中選擇的至少一個核苷酸序列或氨基酸序列的相似性，並且包含至少90％的同源性。較佳地，同源性可以是至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％。同源性的比較可視覺上或利用習知的比較程式，例如標準設定的BLAST算法來進行。一市售的程式可按照百分比來表示兩個或多個序列之間的同源性。可以對相鄰的序列來計算同源性（％）。Furthermore, sequences having homology to the nucleotide sequences and amino acid sequence sets described in the present invention within a certain range are also within the scope of the present invention. "Homology" refers to the similarity of at least one nucleotide sequence or amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 44, and comprises at least 90% homology. Preferably, the homology can be at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. Comparison of homology can be performed visually or using conventional comparison programs, such as the standard set BLAST algorithm. A commercially available program can represent the homology between two or more sequences in percentage. Homology (%) can be calculated for adjacent sequences.

此外，在pH 6.0和pH 7.4下具有0.01-2nM之KD（解離常數）的特異性結合FcRn的抗體也屬於本發明的範圍之內。在此所使用的「KD」係指抗體-抗原結合的平衡解離常數，並且可以使用下面的公式計算：KD = kd/ka，其中ka表示結合率常數，並且kd表示解離率常數。kd或ka的測量可以在25℃或37℃下進行。Furthermore, antibodies which specifically bind to FcRn having a KD (dissociation constant) of 0.01 to 2 nM at pH 6.0 and pH 7.4 are also within the scope of the present invention. As used herein, "KD" refers to the equilibrium dissociation constant of antibody-antigen binding and can be calculated using the formula: KD = kd/ka, where ka represents the binding rate constant and kd represents the dissociation rate constant. The measurement of kd or ka can be carried out at 25 ° C or 37 ° C.

在一個實例中，本發明的抗體包含：CDR1，包含序列號為22的氨基酸序列，CDR2，包含序列號為22的氨基酸序列，CDR3，包含序列號為23的氨基酸序列。 CDR1，包含序列號為27的氨基酸序列，CDR2，包含序列號為28的氨基酸序列，以及CDR3，包含序列號為29的氨基酸序列， CDR1，包含序列號為33的氨基酸序列，CDR2，包含序列號為34的氨基酸序列，CDR3，包含序列號為35的氨基酸序列，或 CDR1，包含序列號為39的氨基酸序列，CDR2，包含序列號為40的氨基酸序列，CDR3，包含序列號為41的氨基酸序列。In one example, an antibody of the invention comprises: CDR1 comprising the amino acid sequence of SEQ ID NO: 22, CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23. CDR1, comprising the amino acid sequence of SEQ ID NO: 27, CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and CDR3 comprising the amino acid sequence of SEQ ID NO: 29, CDR1 comprising the amino acid sequence of SEQ ID NO: 33, CDR2 comprising the sequence number Is the amino acid sequence of 34, CDR3, comprising the amino acid sequence of SEQ ID NO: 35, or CDR1, comprising the amino acid sequence of SEQ ID NO: 39, CDR2, comprising the amino acid sequence of SEQ ID NO: 40, CDR3, comprising the amino acid sequence of SEQ ID NO: 41 .

上述序列號中所列的氨基酸序列可以是對應於重鏈可變區的CDR1至CDR3的氨基酸序列。The amino acid sequence listed in the above sequence number may be the amino acid sequence corresponding to CDR1 to CDR3 of the heavy chain variable region.

在另一實例中，本發明的抗體或抗原結合片段包含： CDR1，包含序列號為24的氨基酸序列，CDR2，包含序列號為25的氨基酸序列，以及CDR3，包含序列號為26的氨基酸序列， CDR1，包含序列號為30的氨基酸序列，CDR2，包含序列號為31的氨基酸序列，以及CDR3，包含序列號為32的氨基酸序列， CDR1，包含序列號為36的氨基酸序列，CDR2，包含序列號為37的氨基酸序列，以及CDR3，包含序列號為38的氨基酸序列，或 CDR1，包含序列號為42的氨基酸序列，CDR2，包含序列號為43的氨基酸序列，以及CDR3，包含序列號為44的氨基酸序列。In another embodiment, an antibody or antigen-binding fragment of the invention comprises: CDR1 comprising the amino acid sequence of SEQ ID NO: 24, CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and CDR3 comprising the amino acid sequence of SEQ ID NO:26, CDR1, comprising the amino acid sequence of SEQ ID NO: 30, CDR2, comprising the amino acid sequence of SEQ ID NO: 31, and CDR3 comprising the amino acid sequence of SEQ ID NO: 32, CDR1 comprising the amino acid sequence of SEQ ID NO: 36, CDR2, comprising the SEQ ID NO: An amino acid sequence of 37, and CDR3, comprising the amino acid sequence of SEQ ID NO: 38, or CDR1, comprising the amino acid sequence of SEQ ID NO: 42, CDR2 comprising the amino acid sequence of SEQ ID NO: 43, and CDR3 comprising SEQ ID NO: 44 Amino acid sequence.

上述序列號中所列的氨基酸序列可以是對應於輕鏈可變區的CDR1至CDR3的氨基酸序列。The amino acid sequence listed in the above sequence number may be the amino acid sequence corresponding to CDR1 to CDR3 of the light chain variable region.

特別地，本發明的抗體或抗原結合片段包含：從以下組成的組中選擇的一種或多種重鏈可變區和輕鏈可變區： 重鏈可變區，包含序列號為21的氨基酸序列的CDR1，包含序列號為22的氨基酸序列的CDR2，以及包含序列號為23的氨基酸序列的CDR3，以及輕鏈可變區，包含序列號為24的氨基酸序列的CDR1，包含序列號為25的氨基酸序列的CDR2，以及包含序列號為26的氨基酸序列的CDR3； 重鏈可變區，包含序列號為27的氨基酸序列的CDR1，包含序列號為28的氨基酸序列的CDR2，以及包含序列號為29的氨基酸序列的CDR3，以及輕鏈可變區，包含序列號為30的氨基酸序列的CDR1，包含序列號為31的氨基酸序列的CDR2，以及包含序列號為32的氨基酸序列的CDR3； 重鏈可變區，包含序列號為33的氨基酸序列的CDR1，包含序列號為34的氨基酸序列的CDR2，以及包含序列號為35的氨基酸序列的CDR3，以及輕鏈可變區，包含序列號為36的氨基酸序列的CDR1，包含序列號為37的氨基酸序列的CDR2，以及包含序列號為38的氨基酸序列的CDR3；以及 重鏈可變區，包含序列號為39的氨基酸序列的CDR1，包含序列號為40的氨基酸序列的CDR2，以及包含序列號為41的氨基酸序列的CDR3，以及輕鏈可變區，包含序列號為42的氨基酸序列的CDR1，包含序列號為43的氨基酸序列的CDR2，以及包含序列號為44的氨基酸序列的CDR3。Particularly, the antibody or antigen-binding fragment of the present invention comprises: one or more heavy chain variable regions and light chain variable regions selected from the group consisting of: a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 21. CDR1, CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23, and a light chain variable region comprising CDR1 of amino acid sequence of SEQ ID NO: 24, comprising SEQ ID NO: 25. CDR2 of the amino acid sequence, and CDR3 comprising the amino acid sequence of SEQ ID NO: 26; a heavy chain variable region comprising CDR1 of amino acid sequence of SEQ ID NO: 27, CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and comprising the SEQ ID NO: CDR3 of amino acid sequence of 29, and light chain variable region, CDR1 comprising amino acid sequence of SEQ ID NO: 30, CDR2 comprising amino acid sequence of SEQ ID NO: 31, and CDR3 comprising amino acid sequence of SEQ ID NO: 32; heavy chain The variable region, CDR1 comprising the amino acid sequence of SEQ ID NO: 33, CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and CDR3 comprising the amino acid sequence of SEQ ID NO: 35, And a light chain variable region comprising CDR1 of amino acid sequence of SEQ ID NO: 36, CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and CDR3 comprising the amino acid sequence of SEQ ID NO: 38; and a heavy chain variable region comprising the sequence CDR1 of the amino acid sequence of 39, CDR2 comprising the amino acid sequence of SEQ ID NO: 40, and CDR3 comprising the amino acid sequence of SEQ ID NO: 41, and a light chain variable region comprising CDR1 of amino acid sequence of SEQ ID NO: 42, CDR2 comprising the amino acid sequence of SEQ ID NO: 43, and CDR3 comprising the amino acid sequence of SEQ ID NO: 44.

在一個實例中，本發明的抗體或抗原結合片段包含一種或多種重鏈可變區和輕鏈可變區，此重鏈可變區和此輕鏈可變區具有選自以下序列號的氨基酸序列所構成的組中的一種或多種氨基酸序列：2、4、6、8、10、12、14、16、18以及20。In one embodiment, an antibody or antigen-binding fragment of the invention comprises one or more heavy chain variable regions and a light chain variable region, the heavy chain variable region and the light chain variable region having an amino acid selected from the group consisting of One or more amino acid sequences in the set consisting of: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20.

特別地，本發明的抗體或抗原結合片段包含具有序列號為2、4、6、8、或10的氨基酸序列的重鏈可變區，與/或具有序列號為12、14、16、18或20的氨基酸序列的輕鏈可變區。In particular, the antibody or antigen-binding fragment of the invention comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10, and/or having a SEQ ID NO: 12, 14, 16, 18 Or a light chain variable region of the amino acid sequence of 20.

詳細而言，本發明的抗體或抗原結合片段包含選自以下所構成的組中的一種或多種重鏈可變區和輕鏈可變區： 包含序列號為2的氨基酸序列的重鏈可變區和包含序列號為12的氨基酸序列的輕鏈可變區； 包含序列號為4的氨基酸序列的重鏈可變區和包含序列號為14的氨基酸序列的輕鏈可變區； 包含序列號為6的氨基酸序列的重鏈可變區和包含序列號為16的氨基酸序列的輕鏈可變區； 包含序列號為8的氨基酸序列的重鏈可變區和包含序列號為18的氨基酸序列的輕鏈可變區；以及 包含序列號為10的氨基酸序列的重鏈可變區和包含序列號為20的氨基酸序列的輕鏈可變區。In particular, the antibody or antigen-binding fragment of the present invention comprises one or more heavy chain variable regions and light chain variable regions selected from the group consisting of: a heavy chain variable comprising an amino acid sequence of SEQ ID NO: a region and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14; a heavy chain variable region of amino acid sequence of 6 and a light chain variable region comprising amino acid sequence of SEQ ID NO: 16; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8 and an amino acid sequence comprising SEQ ID NO: 18. a light chain variable region; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20.

這裡所使用的關於一抗體的用語「片段」或「抗體片段」係指來自抗體多肽分子的多肽（例如，一抗體重鏈或輕鏈多肽），其不包含一全長抗體多肽，但仍包含全長抗體多肽的至少一部分。抗體片段通常包含具有全長抗體多肽的一裂解部分的多肽，儘管此用語並不限於這種裂解片段。由於在此使用的用語片段係關於抗體，因此包含來自抗體多肽（例如，重鏈或輕鏈抗體多肽）的單多肽鏈的片段，可以理解的是，抗體片段本身可不與抗原結合。The term "fragment" or "antibody fragment" as used herein with respect to an antibody refers to a polypeptide derived from an antibody polypeptide molecule (eg, an antibody heavy or light chain polypeptide) that does not comprise a full length antibody polypeptide but still comprises a full length At least a portion of an antibody polypeptide. Antibody fragments typically comprise a polypeptide having a cleavage portion of a full length antibody polypeptide, although the term is not limited to such cleavage fragments. Since the phrase used herein is directed to an antibody, thus comprising a fragment of a single polypeptide chain from an antibody polypeptide (eg, a heavy or light chain antibody polypeptide), it is understood that the antibody fragment itself may not bind to the antigen.

根據本發明的抗體片段包含但不限於具有與FcRn的結合能力的，單鏈抗體，雙特異性、三特異性、以及多特異性抗體，例如雙抗體、三抗體以及四抗體，Fab片段，F（ab'）2 片段，Fd，scFv，結構域抗體，雙特異性抗體，微型抗體，scap（固醇調節結合蛋白裂解激活蛋白），螯合劑重組抗體，三體（tribodies）或雙體（bibodies），胞內抗體，奈米抗體，小模塊immunopharmaceuticals（SMIP），結合結構域免疫球蛋白融合蛋白，駱駝源抗體，VHH含有抗體，IgD抗體，IgE抗體，IgM抗體，IgG1抗體，IgG2抗體，IgG3抗體，IgG4抗體，抗體恆定區中的衍生物，以及基於蛋白質支架的合成抗體。本領域的技術人員明顯可知，根據本發明的抗體的任何片段會表現出與根據本發明的抗體相同的性能。Antibody fragments according to the invention include, but are not limited to, single chain antibodies, bispecific, trispecific, and multispecific antibodies having binding ability to FcRn, such as diabodies, triabodies, and tetrabodies, Fab fragments, F (ab') 2 fragment, Fd, scFv, domain antibody, bispecific antibody, minibody, scap (sterol regulatory binding protein cleavage activator), chelating agent recombinant antibody, tribodies or bibodies ), intrabody antibody, nano antibody, small module immunopharmaceuticals (SMIP), binding domain immunoglobulin fusion protein, camel antibody, VHH antibody, IgD antibody, IgE antibody, IgM antibody, IgG1 antibody, IgG2 antibody, IgG3 Antibodies, IgG4 antibodies, derivatives in the constant region of antibodies, and synthetic antibodies based on protein scaffolds. It will be apparent to those skilled in the art that any fragment of an antibody according to the invention will exhibit the same properties as the antibody according to the invention.

另外，在可變區中具有突變的抗體也包含在本發明的範圍之內。這些抗體的實例包含在可變區中具有氨基酸殘基的保守性取代的抗體。如這裡所用，用語「保守性取代」係指使用具有相似於原始氨基酸殘基性質的另一氨基酸殘基的取代。舉例而言，賴氨酸、精氨酸以及組氨酸具有的相似性質在於它們具有一鹼性側鏈，並且天冬氨酸和谷氨酸具有的相似性質在於它們具有一酸性側鏈。此外，甘氨酸、aspargin、谷氨酰胺、絲氨酸、蘇氨酸、酪氨酸、半胱氨酸以及色氨酸具有的相似性質在於它們具有一不帶電的極性側鏈，並且丙氨酸、纈氨酸、亮氨酸、蘇氨酸、異亮氨酸、脯氨酸、苯丙氨酸以及甲硫氨酸具有的相似性質在於它們具有一非極性側鏈。此外，酪氨酸、苯丙氨酸、色氨酸以及組氨酸具有的相似性質在於它們具有一芳香族側鏈。因此，對於本領域的技術人員，顯而易見的是，即使發生了上述組中具有相似性質的氨基酸殘基的替代；性質上也會顯示沒有特別的變化。因此，具有可變區中保守性取代產生突變的抗體包含在本發明的範圍之內。In addition, antibodies having mutations in the variable region are also included in the scope of the present invention. Examples of such antibodies comprise conservatively substituted antibodies having amino acid residues in the variable region. As used herein, the term "conservative substitution" refers to the use of a substitution having another amino acid residue that is similar in nature to the original amino acid residue. For example, lysine, arginine, and histidine have similar properties in that they have a basic side chain, and aspartic acid and glutamic acid have similar properties in that they have an acidic side chain. In addition, glycine, aspargin, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have an uncharged polar side chain, and alanine, guanamine Acid, leucine, threonine, isoleucine, valine, phenylalanine, and methionine have similar properties in that they have a non-polar side chain. Furthermore, tyrosine, phenylalanine, tryptophan and histidine have similar properties in that they have an aromatic side chain. Thus, it will be apparent to those skilled in the art that even if substitutions of amino acid residues having similar properties in the above groups occur, there will be no particular change in properties. Thus, antibodies having a conservative substitution in the variable region to produce a mutation are encompassed within the scope of the invention.

此外，根據本發明的抗體或它的片段可用作與其他物質的結合物。可用作根據本發明的抗體或其片段的結合物的物質包含︰通常用於自體免疫病治療的治療劑，能夠抑制FcRn之活性的物質，以及物理上與抗體關聯的基團，以提高抗體在例如，血液、血清、淋巴，或其他組織中循環的穩定性與/或滯留。舉例而言，FcRn結合抗體可與一聚合物，例如一非抗原性聚合物，比如聚環氧烷或聚環氧乙烷相關。合適的聚合物重量上有很大的差別。可以使用具有分子數平均重量為從大約200至大約35000（或大約1000至大約15000，以及2000至大約12500）的聚合物。舉例而言，FcRn結合抗體可與水溶性聚合物，例如，親水的聚乙烯基聚合物，比如聚乙烯醇和聚乙烯吡咯烷酮共軛。這類聚合物的非限制性列表包含但不限於，例如聚乙二醇（PEG）或聚丙二醇的聚環氧烷均聚物，聚氧乙烯化多元醇，它們的共聚物和嵌段共聚物，前提是維持嵌段共聚物在水中的溶解度。Furthermore, the antibody or fragment thereof according to the invention can be used as a combination with other substances. Substances which can be used as a combination of an antibody or a fragment thereof according to the present invention include: a therapeutic agent generally used for the treatment of autoimmune diseases, a substance capable of inhibiting the activity of FcRn, and a group physically associated with the antibody to enhance The stability and/or retention of antibodies in, for example, blood, serum, lymph, or other tissues. For example, an FcRn binding antibody can be associated with a polymer, such as a non-antigenic polymer, such as a polyalkylene oxide or polyethylene oxide. There are large differences in the weight of suitable polymers. Polymers having an average molecular weight of from about 200 to about 35,000 (or from about 1000 to about 15,000, and from 2000 to about 12,500) can be used. For example, an FcRn-binding antibody can be conjugated to a water-soluble polymer, such as a hydrophilic polyvinyl polymer, such as polyvinyl alcohol and polyvinylpyrrolidone. A non-limiting list of such polymers includes, but is not limited to, polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycol, polyoxyethylated polyols, copolymers and block copolymers thereof. The premise is to maintain the solubility of the block copolymer in water.

在本發明的另一實施例中，本發明涉及一種用於治療自體免疫病的藥物組合物，這種藥物組合物包含抗FcRn的抗體，以及一種或多種可藥用載體。另外，本發明涉及一種自體免疫病的治療方法，這種方法包含將與FcRn特異性結合的有效量的抗體施加到患者。In another embodiment of the invention, the invention relates to a pharmaceutical composition for the treatment of an autoimmune disease, the pharmaceutical composition comprising an anti-FcRn antibody, and one or more pharmaceutically acceptable carriers. Additionally, the invention relates to a method of treating an autoimmune disease comprising administering to a patient an effective amount of an antibody that specifically binds to FcRn.

藥物組合物可包含藥學上可接受的載體、賦形劑等，這是本領域所習知的。藥學上可接受的載體應與根據本發明的活性成分例如抗體或其片段相容，並且可以是生理鹽水、無菌水、林格氏溶液、緩衝鹽水、右旋糖溶液、麥芽糊精溶液、甘油、乙醇、或其中兩種或更多種的混合物。此外，如果需要的話，本發明的藥物組合物可包含其他常規的添加劑，包含抗氧化劑、緩沖劑、以及抑菌劑。此外，本發明的藥物組合物在稀釋劑、分散劑、表面活性劑、黏合劑以及潤滑劑的輔助下，可以配製成可注射的形式例如水溶液、懸浮液或乳劑。此外，本發明的藥物組合物可配製成各種形式，例如粉末、片劑、膠囊、液體、注射劑、軟膏、糖漿等，以及單劑量或多劑量容器例如密封的充足劑量或提供小瓶。Pharmaceutical compositions may comprise pharmaceutically acceptable carriers, excipients, and the like, as is known in the art. The pharmaceutically acceptable carrier should be compatible with the active ingredient according to the invention, such as an antibody or fragment thereof, and can be physiological saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, Glycerin, ethanol, or a mixture of two or more thereof. Further, if desired, the pharmaceutical composition of the present invention may contain other conventional additives including antioxidants, buffers, and bacteriostatic agents. Further, the pharmaceutical composition of the present invention may be formulated into an injectable form such as an aqueous solution, suspension or emulsion with the aid of a diluent, a dispersing agent, a surfactant, a binder, and a lubricant. Further, the pharmaceutical composition of the present invention can be formulated into various forms such as powders, tablets, capsules, liquids, injections, ointments, syrups and the like, as well as single-dose or multi-dose containers such as a sealed sufficient dose or a vial.

本發明的藥物組合物可應用到由IgG和FcRn介導的所有自體免疫病，並且這些自體免疫病的典型實例包含但不限於，免疫中性白細胞減少症、格林-巴利綜合症、癲癇、自體免疫性腦炎、Isaac綜合症、痣綜合徵、尋常天疱瘡、天疱瘡、大疱性類天疱瘡、後天性水疱性表皮鬆解症、妊娠期類天疱瘡、黏膜的天疱瘡樣、抗磷脂綜合徵、自體免疫性貧血、自體免疫性嚴重的疾病、古德帕斯徹氏綜合徵、重症肌無力、多發性硬化症、類風濕性關節炎、狼瘡、特發性血小板減少性紫癜、狼瘡性腎炎以及膜性腎病。The pharmaceutical composition of the present invention can be applied to all autoimmune diseases mediated by IgG and FcRn, and typical examples of such autoimmune diseases include, but are not limited to, immunological neutropenia, Guillain-Barré syndrome, Epilepsy, autoimmune encephalitis, Isaac syndrome, sputum syndrome, pemphigus vulgaris, pemphigus, bullous pemphigoid, acquired vesicular epidermolysis, pemphigoid pregnancy, mucosal pemphigus , antiphospholipid syndrome, autoimmune anemia, autoimmune disease, Goodpasch's syndrome, myasthenia gravis, multiple sclerosis, rheumatoid arthritis, lupus, idiopathic Thrombocytopenic purpura, lupus nephritis, and membranous nephropathy.

在根據本發明的治療方法中，抗體的劑量可透過考慮患者的嚴重程度、病情、年齡、病史等適當地確定。舉例而言，抗體可確定為1毫克/千克至2克/千克的劑量。此抗體可以一次或數次給藥。In the treatment method according to the present invention, the dose of the antibody can be appropriately determined by considering the severity, condition, age, medical history, and the like of the patient. For example, the antibody can be determined to be a dose from 1 mg/kg to 2 g/kg. This antibody can be administered once or several times.

本發明還提供了用於改善自體免疫性或同種免疫狀況的方法，此種方法包含將本發明的抗體或抗體片段提供給需要治療的患者。本發明還提供了一種特定的抗-FcRn療法。The invention also provides methods for improving autoimmune or alloimmunicy conditions, the methods comprising providing an antibody or antibody fragment of the invention to a patient in need of treatment. The invention also provides a specific anti-FcRn therapy.

本發明用於改善自體免疫性或同種免疫病的方法或本發明的抗-FcRn療法可以透過將本發明的藥物組合物作用於患者來實現。本發明的藥物組合物可以口服或腸胃外給藥。根據本發明的藥物組合物可透過各種途徑給藥，包含但不限於，口服、靜脈內、肌內、動脈內、髓內、硬膜內、心臟內、經皮、皮下、腹膜內、胃腸、舌下、以及局部給藥。本發明的組合物的劑量可根據各種因素，例如病人的體重、年齡、性別、健康狀況以及飲食、給藥時間和方式、***率、以及疾病的嚴重性而變化，並且可容易地由本領域的普通技術人員來確定。通常，1-200毫克/千克，較佳1-40毫克/千克的組合物可施藥給患有自體免疫病或同種免疫病的患者，並且這些方案優選地設計為以使得血清內源性IgG的濃度減少到小於75 ％的預處理值。可根據患者的狀況，應用間歇與/或慢性（連續）的給藥策略。The method of the present invention for improving autoimmune or alloimmunopathy or the anti-FcRn therapy of the present invention can be achieved by administering the pharmaceutical composition of the present invention to a patient. The pharmaceutical composition of the present invention can be administered orally or parenterally. The pharmaceutical composition according to the present invention can be administered by various routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, gastrointestinal, Sublingual, as well as topical administration. The dosage of the composition of the present invention may vary depending on various factors such as the patient's weight, age, sex, health status and diet, time and manner of administration, excretion rate, and severity of the disease, and may be readily made by the art. Ordinary technicians to determine. Typically, a composition of 1-200 mg/kg, preferably 1-40 mg/kg, can be administered to a patient having an autoimmune disease or an allogeneic immune disease, and these protocols are preferably designed to render the serum endogenous The concentration of IgG was reduced to a pretreatment value of less than 75%. Intermittent and/or chronic (continuous) dosing strategies can be applied depending on the condition of the patient.

在本發明的另一實施例中，本發明還提供了一種診斷組合物以及使用此診斷組合物的診斷方法，其中診斷組合物包含本發明的抗體或其片段。換句話而言，本發明與FcRn結合的抗體或其片段具有體外和體內的診斷效用。In another embodiment of the invention, the invention also provides a diagnostic composition and diagnostic method using the diagnostic composition, wherein the diagnostic composition comprises an antibody of the invention or a fragment thereof. In other words, the antibody or fragment thereof of the present invention that binds to FcRn has diagnostic utility in vitro and in vivo.

在本發明的另一實施例中，本發明涉及一種組合物，用於檢測包含抗-FcRn抗體或其片段的FcRn。本發明還提供了一種方法、系統或裝置，用於在體內或體外檢測包含處理抗-FcRn抗體的FcRn。In another embodiment of the present invention, the present invention relates to a composition for detecting FcRn comprising an anti-FcRn antibody or a fragment thereof. The invention also provides a method, system or device for detecting FcRn comprising an anti-FcRn antibody for treatment in vivo or in vitro.

這種體外檢測方法、系統或裝置例如可包含：（1）使一樣本與FcRn結合抗體相接觸；（2）檢測FcRn-結合抗體和樣本之間複合物的形成；與/或（3）使得一參考樣本（例如，對照樣本）與抗體相接觸；以及（4）透過與參考樣本中的比較，確定抗體和樣本之間複合物的形成程度。樣本或患者中複合物的形成與對照樣本或患者中相比的變化（例如，統計上的顯著變化）表明FcRn在樣本中的存在。Such an in vitro detection method, system or device may, for example, comprise: (1) contacting the same with an FcRn-binding antibody; (2) detecting the formation of a complex between the FcRn-binding antibody and the sample; and/or (3) A reference sample (eg, a control sample) is contacted with the antibody; and (4) a degree of formation of the complex between the antibody and the sample is determined by comparison with the reference sample. A change (eg, a statistically significant change) in the formation of a complex in a sample or patient compared to a control sample or patient indicates the presence of FcRn in the sample.

體內檢測方法、系統或裝置可包含：（1）將FcRn-結合抗體應用於一對象；以及（2）檢測FcRn-結合抗體和對象之間複合物的形成。這個檢測可包含確定複合物形成的位置或時間。FcRn-結合抗體可直接或間接標記有一可檢測物質以便於檢測抗體的結合或未結合。合適的可檢測物質包含各種酶、輔基、熒光材料、發光材料、以及放射性材料。FcRn-結合抗體和FcRn之間複合物的形成可透過測量或可視化抗體結合或沒有結合至FcRn來檢測。可使用一常規的檢測分析，例如，酵素免疫法（ELISA）、放射免疫測定（RIA）或組織免疫組織化學。除了FcRn-結合抗體的標記之外，可透過使用標記有可檢測物質的一標準和未標記的FcRn-結合抗體之競爭免疫來測定樣本中FcRn的存在。在這種測定的一個實例中，生物樣本、標記的標準以及FcRn-結合抗體相結合，並且確定未結合至FcRn的標記的標準之量。生物樣本中的FcRn的量與未結合至FcRn的標記的標準之量成反比。An in vivo detection method, system or device can comprise: (1) applying an FcRn-binding antibody to a subject; and (2) detecting the formation of a complex between the FcRn-binding antibody and the subject. This detection can include determining the location or time at which the complex is formed. The FcRn-binding antibody can be directly or indirectly labeled with a detectable substance to facilitate detection of binding or unbinding of the antibody. Suitable detectable materials include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials. The formation of a complex between the FcRn-binding antibody and FcRn can be detected by measuring or visualizing antibody binding or not binding to FcRn. A conventional assay can be used, for example, enzyme immunoassay (ELISA), radioimmunoassay (RIA), or tissue immunohistochemistry. In addition to the labeling of the FcRn-binding antibody, the presence of FcRn in the sample can be determined by competitive immunization using a standard and unlabeled FcRn-binding antibody labeled with a detectable substance. In one example of such an assay, the biological sample, the labeled standard, and the FcRn-binding antibody are combined and the standard amount of the label that is not bound to the FcRn is determined. The amount of FcRn in the biological sample is inversely proportional to the amount of standard that does not bind to the label of FcRn.

為了檢測之目的，本發明的抗體或其片段可標記有熒光團和發色團。因為抗體和其他蛋白質吸收具有波長高達大約310奈米的光線，因此熒光部分應當選擇為在波長高於310奈米，較佳高於400奈米時真正吸收光線。本發明的抗體或其片段可標記有多種合適的熒光劑和發色團。一組熒光劑是二苯并（口比）喃染料，包含熒光素和玫瑰紅。另一組熒光化合物為萘胺。一旦標記了熒光團或發色團，抗體可例如使用熒光顯微鏡術（例如共焦或解卷積顯微術）用於檢測樣本中FcRn的存在或定位。For the purpose of detection, an antibody or fragment thereof of the invention may be labeled with a fluorophore and a chromophore. Since antibodies and other proteins absorb light having a wavelength of up to about 310 nm, the fluorescent portion should be selected to actually absorb light at wavelengths above 310 nm, preferably above 400 nm. The antibodies or fragments thereof of the invention may be labeled with a variety of suitable fluorescers and chromophores. One group of fluorescers is a dibenzo(n)pyran dye containing fluorescein and rose bengal. Another group of fluorescent compounds is naphthylamine. Once the fluorophore or chromophore is labeled, the antibody can be used, for example, to detect the presence or localization of FcRn in the sample using fluorescence microscopy (eg, confocal or deconvolution microscopy).

使用本發明的抗體或其片段檢測FcRn的存在或位置可透過各種方法，例如組織學分析、蛋白質陣列以及螢光流式細胞分選（FACS）進行。Detection of the presence or location of FcRn using the antibodies or fragments thereof of the invention can be performed by a variety of methods, such as histological analysis, protein arrays, and fluorescent flow cytometric sorting (FACS).

在本發明中，體內FcRn或FcRn-表達組織的存在可透過一體內成像法執行。此種方法包含（i）對一對象（例如，具有自體免疫病的患者）施用一抗-FcRn抗體，其中抗-FcRn抗體與一檢測標記共軛；以及（ii）使得對象暴露於一裝置，此裝置用於檢測FcRn-表達組織或細胞的可檢測標記。舉例而言，此對象例如透過NMR或其他斷層裝置進行成像。用於診斷成像的標記之實例包含放射性標記、熒光標記、正電子發射同位素、化學發光劑以及酶標記。一放射標記的抗體也可用於體外診斷測試。一同位素標記的抗體的活性比度取決於半衰期、放射性標記的同位素純度、以及標記如何摻入抗體。In the present invention, the presence of FcRn or FcRn-expressing tissues in vivo can be performed by an integrated internal imaging method. Such methods comprise (i) administering to a subject (eg, a patient having an autoimmune disease) an anti-FcRn antibody, wherein the anti-FcRn antibody is conjugated to a detection marker; and (ii) exposing the subject to a device This device is used to detect detectable markers of FcRn-expressing tissues or cells. For example, the object is imaged, for example, by NMR or other tomographic device. Examples of labels for diagnostic imaging include radioactive labels, fluorescent labels, positron emitting isotope, chemiluminescent agents, and enzyme labels. A radiolabeled antibody can also be used in in vitro diagnostic tests. The activity ratio of an isotopically labeled antibody depends on the half-life, the radiolabeled isotope purity, and how the label is incorporated into the antibody.

本發明還提供一種試劑盒，此試劑盒包含其片段與FcRn結合的抗體以及用於診斷用途的指令，例如，使用FcRn-結合抗體或其片段以在體外，例如在一樣本，比如具有自體免疫病的患者的活組織或細胞中，或在體內，例如透過對一對象成像來檢測FcRn。試劑盒可進一步包含至少一種額外試劑，例如標記或附加診斷劑。對於體內使用，可以將抗體配製為藥物組合物。The invention also provides a kit comprising an antibody whose fragment binds to FcRn and instructions for diagnostic use, for example, using an FcRn-binding antibody or fragment thereof, in vitro, for example in a sample, such as having an autologous FcRn is detected in living tissue or cells of a patient with an immunological disease, or in vivo, for example by imaging an object. The kit may further comprise at least one additional agent, such as a label or additional diagnostic agent. For in vivo use, the antibody can be formulated into a pharmaceutical composition.

在本發明的另一實施例中，本發明涉及編碼本發明的抗體或其片段的多核苷酸序列。In another embodiment of the invention, the invention relates to a polynucleotide sequence encoding an antibody of the invention or a fragment thereof.

在一實例中，編碼本發明的抗體或其片段的一多核苷酸序列係為這樣的序列，其與選自序列號1、3、5、7、9、11、13、15、17以及19的組中的一種或多種序列具有至少90％的同源性，或者相比較於上述序列具有大於90％同源性的序列。In one embodiment, a polynucleotide sequence encoding an antibody of the invention or a fragment thereof is a sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 and One or more sequences in the set of 19 have at least 90% homology, or a sequence having greater than 90% homology compared to the above sequences.

特別地，本發明的抗體或其片段的一多核苷酸序列係為這樣的序列，其編碼本發明之抗體的重鏈的序列號是1、3、5、7或9，與/或其編碼本發明的抗體之輕鏈的序列號是11、13、15、17或19。In particular, a polynucleotide sequence of an antibody or fragment thereof of the invention is a sequence encoding a heavy chain of an antibody of the invention having a sequence number of 1, 3, 5, 7 or 9, and/or The light chain encoding the light chain of the antibody of the present invention is 11, 13, 15, 17 or 19.

在本發明的另一實施例中，本發明涉及一種重組表達載體，其包含多核苷酸、橫切此重組表達載體的宿主細胞，並且本發明還涉及一種透過使用重組表達載體和宿主細胞，特異性結合至FcRn的抗體或其片段的製備方法。In another embodiment of the present invention, the present invention relates to a recombinant expression vector comprising a polynucleotide, a host cell transecting the recombinant expression vector, and the present invention also relates to the use of a recombinant expression vector and a host cell, specifically A method of producing an antibody or fragment thereof that binds sexually to FcRn.

在本發明的一實施例中，根據本發明的抗體或其片段較佳透過使用一基因重組方法表達和純化來製備。特別地，編碼本發明的特異性結合至FcRn之抗體的可變區透在單獨宿主細胞或同時在一單個宿主細胞中表達來產生。In an embodiment of the invention, the antibody or fragment thereof according to the invention is preferably produced by expression and purification using a genetic recombination method. In particular, a variable region encoding an antibody of the invention that specifically binds to FcRn is produced by expression in a single host cell or simultaneously in a single host cell.

如在此所用，用語「重組載體」係指能夠表達一合適宿主細胞中感興趣的蛋白質的表達載體，並且表示DNA構建體，此DNA構建體具有可操作地鏈接的必需調節要素以表達核酸***。如在此所用，「可操作地鏈接」表示一核酸表達調控序列功能性地鏈接至編碼感興趣之蛋白質的一核酸序列，以便執行一般功能。可以使用本領域中習知的基因重組技術來進行重組載體可操作的鏈接，而位點特異性DNA切割和連接可使用本領域習知的酶來容易進行。As used herein, the term "recombinant vector" refers to an expression vector capable of expressing a protein of interest in a suitable host cell, and represents a DNA construct having the necessary regulatory elements operably linked to express nucleic acid insertions. . As used herein, "operably linked" means that a nucleic acid expression control sequence is functionally linked to a nucleic acid sequence encoding a protein of interest to perform a general function. Recombinant vector-operable linkages can be performed using genetic recombination techniques well known in the art, while site-specific DNA cleavage and ligation can be readily performed using enzymes well known in the art.

可使用於本發明中的合適的表達載體可包含表達調節要素，例如一啟動子、一操縱子、一起始密碼子、一終止密碼子、一多腺苷酸化訊號、以及一增強子，以及用於膜靶向或分泌的一訊號序列。起始和終止密碼子通常認為是編碼免疫原靶蛋白的核苷酸序列的一部分，並且對於已進行遺傳構造的個體發揮功能是必要的，並且必須與編碼序列在一個框架中。啟動子通常可以是組成型或誘導型。原核啟動子包含但不限於，lac、tac、T3以及T7啟動子。真核啟動子包含但不限於，猿猴病毒40（SV40）啟動子、小鼠乳腺腫瘤病毒（MMTV）啟動子、人免疫缺陷病毒（HIV）啟動子例如HIV長末端重複序列（LTR）啟動子、莫洛尼病毒啟動子、巨細胞病毒（CMV）啟動子、愛潑斯坦巴爾病毒（EBV）啟動子、勞氏肉瘤病毒（RSV）啟動子，以及來自人基因的啟動子，例如人β肌動蛋白、人血紅蛋白、人肌肉肌酸以及人金屬硫蛋白。表達載體可包含一選擇標記，選擇標記允許選擇具有此載體的宿主細胞。對賦予可選擇的表型，例如抗藥性、營養要求、耐細胞毒性劑或表面蛋白質表達的產品編號的基因用作通常的選擇標記。由於只有表達一可選擇標記的細胞在選擇性藥劑處理的環境中存活，因此能夠選擇轉化的細胞。此外，可複製的表達載體可以包含一複製起點，啟動複製的一特定的核酸序列。在本發明中可使用的重組表達載體包含各種載體，例如質粒、病毒以及黏粒。重組載體的類型沒有特別的限制，並且重組載體功能上可以表達期望的基因且產生在各種宿主細胞例如原核和真核細胞中所希望的蛋白質。然而，優選使用可產生大量類似於天然蛋白質的異性蛋白同時具有表現出強烈活性的啟動子的強表達能力的載體。Suitable expression vectors for use in the present invention may comprise expression regulating elements such as a promoter, an operon, a start codon, a stop codon, a polyadenylation signal, and an enhancer, and A signal sequence that is targeted or secreted by the membrane. The start and stop codons are generally considered to be part of the nucleotide sequence encoding the immunogenic target protein and are essential for individuals who have undergone genetic construction and must be in a framework with the coding sequence. Promoters can usually be constitutive or inducible. Prokaryotic promoters include, but are not limited to, the lac, tac, T3, and T7 promoters. Eukaryotic promoters include, but are not limited to, simian virus 40 (SV40) promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) promoter such as the HIV long terminal repeat (LTR) promoter, Moloney virus promoter, cytomegalovirus (CMV) promoter, Epstein Barr virus (EBV) promoter, Rous sarcoma virus (RSV) promoter, and promoter from human genes, such as human β-acting Protein, human hemoglobin, human muscle creatine, and human metallothionein. The expression vector can comprise a selectable marker which allows selection of a host cell having such a vector. Genes that confer a selectable phenotype, such as drug resistance, nutritional requirements, cytotoxic agent or surface protein expression, are used as the usual selection marker. Since only cells expressing a selectable marker survive in a selective agent treated environment, the transformed cells can be selected. In addition, the replicable expression vector can comprise an origin of replication that initiates replication of a particular nucleic acid sequence. The recombinant expression vector which can be used in the present invention contains various vectors such as a plasmid, a virus, and a cosmid. The type of recombinant vector is not particularly limited, and the recombinant vector functionally expresses a desired gene and produces a desired protein in various host cells such as prokaryotic and eukaryotic cells. However, it is preferred to use a carrier which can produce a large amount of a heterologous protein similar to a natural protein while having a strong expression ability of a promoter exhibiting a strong activity.

在本發明，各種表達宿主/載體組合可用以表達根據本發明的抗體或其片段。舉例而言，適合於真核宿主的表達載體包含但不限於SV40、牛乳頭瘤病毒、腺病毒、腺相關病毒、巨細胞病毒、以及逆轉錄病毒。可用於細菌宿主的表達載體包含細菌質粒，例如pET、pRSET、pBluescript、pGEX2T、pUC、colE1、pCR1、pBR322、pMB9及其衍生物，具有較寬的宿主範圍的質粒，例如RP4，描述為不同的噬菌體lambda衍生物例如gt10，gt11以及NM989的噬菌體DNA，以及其他的DNA噬菌體，例如M13和絲狀單鏈DNA噬菌體。在酵母細胞中有用的表達載體包含2μm的質粒及其衍生物。在昆蟲細胞中有用的一載體為pVL941。In the present invention, various expression host/vector combinations can be used to express an antibody or fragment thereof according to the invention. For example, expression vectors suitable for eukaryotic hosts include, but are not limited to, SV40, bovine papilloma virus, adenovirus, adeno-associated virus, cytomegalovirus, and retrovirus. Expression vectors useful in bacterial hosts include bacterial plasmids such as pET, pRSET, pBluescript, pGEX2T, pUC, colE1, pCR1, pBR322, pMB9 and derivatives thereof, and plasmids having a broad host range, such as RP4, are described as being different. Phage lambda derivatives such as ph10, gt11 and phage DNA of NM989, as well as other DNA phages, such as M13 and filamentous single-stranded DNA phage. An expression vector useful in yeast cells comprises a 2 μm plasmid and its derivatives. One vector useful in insect cells is pVL941.

重組載體引入至宿主細胞中以形成一轉化體。適合使用於本發明用的宿主細胞包含原核細胞，例如大腸桿菌、枯草芽孢桿菌、鏈黴菌屬、假單胞菌屬、變形桿菌以及金黃色葡萄球菌屬、真菌，例如曲霉，酵母，例如畢赤酵母、釀酒酵母、裂殖屬、以及粗糙脈孢菌，以及真核細胞，例如低級真核細胞，以及更高等的真核細胞例如昆蟲細胞。The recombinant vector is introduced into a host cell to form a transformant. Host cells suitable for use in the present invention comprise prokaryotic cells, such as Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus, and Staphylococcus aureus, fungi, such as Aspergillus, yeast, such as Pichia Yeast, Saccharomyces cerevisiae, Schizophyllum, and Neurospora crassa, as well as eukaryotic cells, such as lower eukaryotic cells, and higher eukaryotic cells such as insect cells.

可以在本發明中使用的宿主細胞較佳選自植物和哺乳動物，其實例包含但不限於猴腎細胞（COS7）、NSO細胞、SP2/0、中國倉鼠卵巢（CHO）細胞、W138、幼倉鼠腎（BHK）細胞、MDCK、骨髓瘤細胞、HuT78細胞以及HEK293細胞。優選地，使用CHO細胞。The host cell which can be used in the present invention is preferably selected from plants and mammals, and examples thereof include, but are not limited to, monkey kidney cells (COS7), NSO cells, SP2/0, Chinese hamster ovary (CHO) cells, W138, baby hamsters. Kidney (BHK) cells, MDCK, myeloma cells, HuT78 cells, and HEK293 cells. Preferably, CHO cells are used.

在本發明中，轉染或轉化到宿主細胞包含核酸可引入至生物體、細胞、組織或器官中的任何方法，而且，正如本領域所習知，可以使用根據宿主細胞的類型選擇的合適的標準技術。這些方法包含但不限於，電穿孔、原生質體融合、磷酸鈣（CaPO4 ）沉澱、氯化鈣（CaCl2 ）沉澱、攪拌用碳化矽纖維、以及農桿菌-、PEG-、葡聚醣硫酸鹽-、lipofectamine-以及乾燥/抑制-介導的轉化。In the present invention, transfection or transformation into a host cell comprises any method in which the nucleic acid can be introduced into an organism, cell, tissue or organ, and, as is known in the art, a suitable one selected according to the type of host cell can be used. Standard technology. These methods include, but are not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, barium carbide fiber for agitation, and Agrobacterium-, PEG-, dextran sulfate -, lipofectamine- and dry/inhibitory-mediated transformation.

根據本發明的FcRn特異性抗體或其片段可以透過在營養培養基中培養包含重組載體的轉化體大量的製造，並且本發明中使用的培養基和培養條件可以根據宿主細胞的類型進行適當的選擇。在培養期間，培養條件，包含溫度、培養基的pH、以及培養時間可以控制以便適合於細胞生長和蛋白質的大量製造。透過上述重組方法產生的抗體或抗體片段能夠從培養基或細胞溶解物收集並可以透過常規生化分離技術（Sambrook等，分子克隆：實驗指南，第二版，冷泉港實驗室出版社（1989）；Deuscher，M，蛋白質酶學純化方法，第182卷，學術出版社公司，聖地亞哥，加利福尼亞州（1990））分離和純化。這些技術包含但不限於電泳、離心、凝膠過濾、沉澱、滲析、層析法（離子交換層析、親和層析、免疫層析、大小排阻層析等）、等電點聚焦、以及其各種變型和組合。優選地，使用蛋白A分離和純化抗體或抗體片段。The FcRn-specific antibody or fragment thereof according to the present invention can be produced by culturing a transformant comprising a recombinant vector in a nutrient medium in a large amount, and the medium and culture conditions used in the present invention can be appropriately selected depending on the type of the host cell. During the culture, the culture conditions, including the temperature, the pH of the medium, and the culture time can be controlled so as to be suitable for cell growth and mass production of proteins. Antibodies or antibody fragments produced by the above recombinant methods can be collected from culture media or cell lysates and can be passed through conventional biochemical separation techniques (Sambrook et al., Molecular Cloning: A Laboratory Guide, Second Edition, Cold Spring Harbor Laboratory Press (1989); Deuscher , M, Protein Enzymatic Purification Methods, Vol. 182, Academic Press, San Diego, Calif. (1990)) Isolation and purification. These techniques include, but are not limited to, electrophoresis, centrifugation, gel filtration, precipitation, dialysis, chromatography (ion exchange chromatography, affinity chromatography, immunochromatography, size exclusion chromatography, etc.), isoelectric focusing, and Various variants and combinations. Preferably, protein A is used to isolate and purify antibodies or antibody fragments.

本發明的抗體表現出在pH 7.4時抗原結合能力（KD值）為從大約300pM或更少至大約2nM或更小，並且也表現出在pH 6.0時KD值為從2nM或更少至900pM更小。本發明的抗體具有0.01-2nM的強hFcRn結合親和性，並且因此可以相信甚至它們結合胞內體時，結合於細胞外的抗體會維持，這表明這些抗體具有阻斷自體抗體與hFcRn結合的優異效果。此外，自體抗體與hFcRn結合的阻斷效果也在使用人FcRn表達細胞和螢光流式細胞分選儀（FACS）執行的一阻斷分析中得到證實。 實例 The antibody of the present invention exhibits an antigen binding ability (KD value) of from about 300 pM or less to about 2 nM or less at pH 7.4, and also exhibits a KD value of from 2 nM or less to 900 pM at pH 6.0. small. The antibodies of the present invention have strong hFcRn binding affinity of 0.01-2 nM, and thus it is believed that even when they bind to endosomes, antibodies that bind to extracellular cells are maintained, indicating that these antibodies have the ability to block autoantibody binding to hFcRn. Excellent results. Furthermore, the blocking effect of autoantibody binding to hFcRn was also confirmed in a blocking assay performed using human FcRn expressing cells and a fluorescent flow cytometry (FACS). Instance

以下，將參照實例對本發明進行更詳細的說明。本領域的普通技術人員明顯可知，這些實例僅用於說明之目的，並且不應解釋為限制本發明的範圍。實例 1 ：使用轉基因大鼠構建抗 -FcRn- 表達庫 Hereinafter, the present invention will be described in more detail with reference to examples. It is apparent to those skilled in the art that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention. Example 1 : Construction of an anti- FcRn- expression library using transgenic rats

採用共6個轉基因大鼠（OmniRat® ，OMT）進行免疫接種。使用人FcRn作為一免疫原。使用0.0075毫克的人FcRn（每次）連同一佐劑以3天的時間間隔進行24天，對大鼠的兩個腳墊免疫八次。在第28天，使用在PBS緩衝液稀釋的5-10微克免疫原將大鼠免疫。在第28天，收集大鼠血清並用於測量抗體滴定度。在第31天，將大鼠安樂死，並且回收膕淋巴結和腹股溝淋巴結用於和P3X63 / AG8.653骨髓瘤細胞融合。A total of 6 transgenic rats (OmniRat ® , OMT) were used for immunization. Human FcRn was used as an immunogen. The two foot pads of the rats were immunized eight times using 0.0075 mg of human FcRn (each time) with the same adjuvant for 24 days at 3 day intervals. On day 28, rats were immunized with 5-10 micrograms of immunogen diluted in PBS buffer. On day 28, rat serum was collected and used to measure antibody titers. On day 31, rats were euthanized and axillary and inguinal lymph nodes were recovered for fusion with P3X63 / AG8.653 myeloma cells.

進行ELISA分析以測量大鼠血清中的抗體滴定度。特別地，人FcRn在PBS（pH為6.0或pH 7.4）緩衝液中稀釋，以製成2微克/毫升的溶液，並且100微升（μl）的此溶液塗佈在96-孔平板的每個孔中，並且然後在4℃下孵育至少18小時。使用300微升的洗滌緩衝液（PBS中0.05％的Tween-20）將各孔洗滌三次，以去除未結合的人FcRn，然後200微升的封閉緩衝液加入至每一孔中並在室溫下孵育2小時。一測試血清樣本稀釋至1/100，並且然後將溶液連續2倍稀釋，以生成具有1/100至1/256000的稀釋因子的10個試驗樣本。在封閉之後，每一孔使用300微升的洗滌緩衝液洗滌，並且然後將每一測試樣本加入到每一孔中並在室溫下孵育2小時。在洗滌三次之後，將PBS緩衝液中100微升的1︰50000稀釋的二次檢測抗體加入至每一孔中並在室溫下孵育2小時。再次洗滌三次之後，100微升的TMB溶液添加至每一孔中並使其在室溫下反應10分鐘，並且然後50微升的1M含硫酸終止溶液加入至每一孔中以終止反應，之後使用酶標儀測定在450奈米處的OD值。關於免疫所護得之抗-hFcRn的IgG的滴定度相比較於未免疫的大鼠免疫前血清中的更高，這表明大鼠獲得了良好的免疫，其中未免疫的大鼠在1/100稀釋條件下在450奈米處的OD值為1.0或更高。An ELISA assay was performed to measure antibody titers in rat serum. Specifically, human FcRn was diluted in PBS (pH 6.0 or pH 7.4) buffer to prepare a solution of 2 μg/ml, and 100 μl (μl) of this solution was coated on each of 96-well plates. In the wells, and then incubate at 4 ° C for at least 18 hours. Each well was washed three times with 300 microliters of wash buffer (0.05% Tween-20 in PBS) to remove unbound human FcRn, then 200 microliters of blocking buffer was added to each well and at room temperature Incubate for 2 hours. A test serum sample was diluted to 1/100, and then the solution was serially diluted 2-fold to generate 10 test samples having a dilution factor of 1/100 to 1/256,000. After blocking, each well was washed with 300 microliters of wash buffer, and then each test sample was added to each well and incubated for 2 hours at room temperature. After washing three times, 100 μl of a 1.50000 diluted secondary detection antibody in PBS buffer was added to each well and incubated at room temperature for 2 hours. After washing three more times, 100 μl of TMB solution was added to each well and allowed to react at room temperature for 10 minutes, and then 50 μl of a 1 M sulfuric acid-containing stop solution was added to each well to terminate the reaction, after which the reaction was terminated. The OD value at 450 nm was measured using a microplate reader. The titer of the IgG against the anti-hFcRn protected by immunization was higher than that in the unimmunized rat pre-immune serum, indicating that the rat obtained good immunity, wherein the unimmunized rat was at 1/100. The OD value at 450 nm under dilution conditions was 1.0 or higher.

產生使用聚乙二醇融合的總共三個融合瘤庫A、B以及C。特別地，轉基因大鼠1和5用來產生融合瘤庫A，大鼠2和6用來產生融合瘤庫B，以及大鼠3和4用來產生融合瘤庫C。用於構建每一融合瘤庫的一融合瘤庫融合混合物在含HAT的培養基中培養7天，以使得僅選擇融合至HAT的細胞。收集HAT培養基中的存活的融合瘤細胞並且在HT培養基中培養大約6天，並且然後收集上清液，並且使用大鼠IgG ELISA試劑盒（RD-生物技術）測量在上清液中大鼠IgG的量。特別地，將每一樣本稀釋為1：100，並且100微升的稀釋物添加到一ELISA板的每一孔中並與過氧化物酶結合的抗-大鼠IgG混合，接著在室溫下反應15分鐘。100微升的TMB溶液添加至每一孔中並且使其在室溫下反應10分鐘，並且然後50微升的1M含硫酸終止溶液加入到每一孔中以終止反應。接著，用酶標儀測定在450奈米處的OD值。實例 2 ：抗原結合親和性與融合瘤庫的抗 -hFcRn 抗體的 IgG 結合阻擋能力的評價 A total of three fusion tumor pools A, B, and C were generated using polyethylene glycol fusion. In particular, transgenic rats 1 and 5 were used to generate fusion tumor pool A, rats 2 and 6 were used to generate fusion tumor pool B, and rats 3 and 4 were used to generate fusion tumor pool C. A fusion tumor pool fusion mixture used to construct each fusion tumor pool was cultured for 7 days in HAT-containing medium so that only cells fused to HAT were selected. Surviving fusion tumor cells in HAT medium were collected and cultured in HT medium for about 6 days, and then the supernatant was collected, and rat IgG in the supernatant was measured using a rat IgG ELISA kit (RD-biotechnology). The amount. Specifically, each sample was diluted to 1:100 and 100 microliters of the dilution was added to each well of an ELISA plate and mixed with peroxidase-conjugated anti-rat IgG, then at room temperature Reaction for 15 minutes. One hundred microliters of the TMB solution was added to each well and allowed to react at room temperature for 10 minutes, and then 50 microliters of a 1 M sulfuric acid-containing stop solution was added to each well to terminate the reaction. Next, the OD value at 450 nm was measured with a microplate reader. Example 2 : Evaluation of antigen binding affinity and IgG binding blocking ability of anti- hFcRn antibody of fusion tumor bank

為了分析抗體與人FcRn的結合，執行與上述相同的ELISA分析（在pH6.0和pH7.4）。三個融合瘤庫A、B以及C的hFcRn結合的評估結果表明，hFcRn的結合親和性在pH值6.0和pH值7.4兩種情況下均是A＞C＞B的順序。To analyze the binding of the antibody to human FcRn, the same ELISA assay as described above (at pH 6.0 and pH 7.4) was performed. The evaluation of hFcRn binding of the three fusion tumor pools A, B and C showed that the binding affinity of hFcRn was in the order of A>C>B in both pH 6.0 and pH 7.4.

使用三種融合瘤庫的培養上清液，在pH 6.0和pH 7.4條件下透過螢光流式細胞分選儀（FACS）在5奈克/毫升和25奈克/毫升時執行hFcRn的結合親和性的評估。人FcRn穩定表達的HEK293細胞從燒瓶中分離，然後在反應緩衝液（PBS中0.05％的BSA，pH6.0或pH7.4）中懸浮。將懸浮液稀釋至2×106 個細胞/毫升的細胞密度，並且50微升的稀釋液加入到96-孔平板的每一孔中。然後，50微升的稀釋到分別為10奈克/毫升和50奈克/毫升的融合瘤庫培養上清液加入到每一孔中且懸浮，以允許抗體結合。A488兔抗-IgG山羊抗體在1：200的反應緩衝液中稀釋，並且100微升的稀釋液添加至每一孔中並混合細胞團塊以進行結合反應，然後150微升的反應緩衝液加入每一孔中。在螢光流式細胞分選儀（FACS）進行測定（BD）。類似於ELISA結果，可以看出融合瘤庫A表現出最高的結合親和性。The binding affinity of hFcRn was performed at 5 ng/ml and 25 ng/ml by fluorescence flow cytometry (FACS) at pH 6.0 and pH 7.4 using culture supernatants of three fusion rum banks. evaluation of. Human FcRn stably expressed HEK293 cells were isolated from the flask and then suspended in reaction buffer (0.05% BSA in PBS, pH 6.0 or pH 7.4). The suspension was diluted to a cell density of 2 x 10 6 cells/ml, and 50 microliters of the dilution was added to each well of a 96-well plate. Then, 50 microliters of the fusion tumor pool culture supernatant diluted to 10 ng/ml and 50 ng/ml, respectively, was added to each well and suspended to allow antibody binding. A488 rabbit anti-IgG goat antibody was diluted in 1:200 reaction buffer, and 100 μl of the dilution was added to each well and the cell pellet was mixed for binding reaction, then 150 μl of reaction buffer was added. In each hole. The measurement (BD) was carried out by a fluorescent flow cytometer (FACS). Similar to the ELISA results, it can be seen that the fusion tumor library A exhibits the highest binding affinity.

在pH6.0下，透過螢光流式細胞分選儀（FACS）執行融合瘤細胞庫的人FcRn的阻斷能力的評估。特別地，naïve HEK293細胞和人FcRn-過表達HEK293細胞在反應緩衝液（0.05% BSA in PBS，pH 6.0）中懸浮。1×105 個細胞添加至96-孔平板中，並且使用每個4nM的每個融合瘤庫培養上清液和0.4nM的10倍稀釋的上清液處理。為了確認hIgG的阻擋能力，100nM的A488-hIgG1加入到每一孔中，然後在冰上孵育90分鐘。反應完成之後，將細胞團塊使用100微升的反應緩衝液洗滌，並且轉移到一U-形圓底管中，隨後透過螢光流式細胞分選儀（FACS）測量。測定保留在人FcRn-過表達穩定細胞中的100nM的A488-hIgG1的量，並且然後計算阻塞（％）。作為一同種型對照，使用hIgG1，並且作為一陽性對照，以前開發的HL161-1Ag抗體用以比較評估抗體阻斷作用。每一對照在1μM和2μM的濃度下進行分析，並且融合瘤庫樣本在0.4nM和4nM兩種濃度下測量。結果，發現融合瘤庫A顯示出最高的阻斷作用。實例 3 ：融合瘤克隆透過 螢光流式細胞分選儀 （ FACS ）的分離和人抗體的選擇 Evaluation of the blocking ability of human FcRn of the fusion tumor cell bank was performed by a fluorescence flow cytometer (FACS) at pH 6.0. Specifically, naïve HEK293 cells and human FcRn-overexpressing HEK293 cells were suspended in reaction buffer (0.05% BSA in PBS, pH 6.0). 1 x 10 5 cells were added to 96-well plates and treated with each 4 nM of each of the fusion tumor library culture supernatants and 0.4 nM of 10-fold diluted supernatant. To confirm the blocking ability of hIgG, 100 nM of A488-hIgG1 was added to each well and then incubated on ice for 90 minutes. After the reaction was completed, the cell pellet was washed with 100 μl of reaction buffer and transferred to a U-shaped round bottom tube, followed by measurement by a fluorescence flow cytometry (FACS). The amount of 100 nM of A488-hIgG1 remaining in human FcRn-overexpressing stable cells was determined, and then blocking (%) was calculated. As an isotype control, hIgG1 was used, and as a positive control, the previously developed HL161-1Ag antibody was used to compare antibody blocking effects. Each control was analyzed at concentrations of 1 μM and 2 μM, and the fusion tumor pool samples were measured at both 0.4 nM and 4 nM concentrations. As a result, it was found that the fusion tumor library A showed the highest blocking effect. Example 3 : Isolation of fusion tumor clones by fluorescence flow cytometry ( FACS ) and selection of human antibodies

使用顯示出最高的人FcRn結合親和性和阻斷作用融合瘤庫A，透過螢光流式細胞分選儀（FACS）（流式細胞術）分離出克隆，從而得到總共442個的單個克隆。分離的單克隆分別在HT培養基中培養，並且收集上清液。透過螢光流式細胞分選儀（FACS）選擇上清液中結合hFcRn的抗體-表達融合瘤克隆。結果，可以看出100個克隆（M1-M100）強烈結合至hFcRn-表達的HEK293細胞。Clones were isolated by fluorescence flow cytometry (FACS) (flow cytometry) using the highest shown human FcRn binding affinity and blocking effect, resulting in a total of 442 individual clones. The isolated monoclonals were separately cultured in HT medium, and the supernatant was collected. The antibody-expressing fusion tumor clone that binds hFcRn in the supernatant was selected by a fluorescent flow cytometry (FACS). As a result, it can be seen that 100 clones (M1-M100) strongly bind to hFcRn-expressing HEK293 cells.

RNA從透過螢光流式細胞分選儀（FACS）分析所選擇出的這100個克隆中分離且分離的RNA進行測序。在第一步驟測序中，100個克隆的88個進行測序，並根據氨基酸序列劃分成總共35組（G1至G38）。排除其介質不可用的兩個克隆（G33及G35）之外的33組代表性克隆培養的上清液在100奈克/毫升的濃度下稀釋，並且對於hFcRn的結合親和性透過ELISA來評估。RNA was isolated and isolated from the 100 clones selected by fluorescence flow cytometry (FACS) analysis. In the first step of sequencing, 88 of 100 clones were sequenced and divided into a total of 35 groups (G1 to G38) according to the amino acid sequence. The supernatants of 33 representative clones cultured except for two clones (G33 and G35) whose media were not available were diluted at a concentration of 100 Ng/ml, and the binding affinity for hFcRn was evaluated by ELISA.

按照與上述相同的方式，在pH 6.0和7.4條件下，執行透過螢光流式細胞分選儀（FACS）的hFcRn結合親和性的評估。克隆的結合親和性的次序在pH值之間是相似的，並且結合強度以不同的水平出現。Evaluation of hFcRn binding affinity by fluorescence flow cytometry (FACS) was performed in the same manner as above under conditions of pH 6.0 and 7.4. The order of binding affinity of the clones is similar between pH values, and the binding strength occurs at different levels.

另外，在pH 6.0條件下，透過螢光流式細胞分選儀（FACS）執行33個克隆的hFcRn的阻斷作用的評估。阻塞（％）基於測到的MFI值計算出。基於在1667 pM濃度下的阻斷％分析的結果，克隆一共劃分為以下四組：A組：70％-100％；B組：30-70％；C組：10-30％；以及D組：10％或以下。In addition, the evaluation of the blocking effect of hFcRn of 33 clones was performed by a fluorescence flow cytometry (FACS) under pH 6.0. Blocking (%) is calculated based on the measured MFI value. Based on the results of % block analysis at 1667 pM, clones were divided into the following four groups: group A: 70%-100%; group B: 30-70%; group C: 10-30%; and group D : 10% or less.

對於透過SPR的融合瘤克隆的動力學分析，固定人FcRn，然後使用融合瘤培養作為一分析物進行分析。除了幾個克隆之外，大部分的克隆顯示出106 M或更高的一kon 以及10-3 M或更低的一koff 值。總之，結果表明，所有的克隆具有10-9 至10-11 M的一KD值。For kinetic analysis of fusion tumor clones transfected with SPR, human FcRn was immobilized and then analyzed using fusion tumor culture as an analyte. Several clones addition, most of the clones showed 10 6 M or higher k on a 10 -3 M or less and a k off value. In summary, the results indicate that all clones have a KD value of 10 -9 to 10 -11 M.

在這五個融合瘤克隆中，根據hFcRn阻斷作用分析結果劃分的A及B組的CDR序列中，不具有N-糖基化位點或游離半胱氨酸的18個克隆的基因轉化為整個人IgG序列。Among the five fusion tumor clones, the genes of the 18 clones without the N-glycosylation site or the free cysteine in the CDR sequences of the A and B groups classified according to the hFcRn blocking analysis result were converted into Whole human IgG sequence.

特別地，18個選定抗體的VH及VL和人種系抗體組之間的氨基酸序列的相似性使用NCBI網頁的Ig BLAST程式進行檢查。In particular, the amino acid sequence similarity between the VH and VL of the 18 selected antibodies and the human germline antibody set was examined using the Ig BLAST program of the NCBI webpage.

為了克隆這18個人抗體基因，限制性內切酶識別位點按照以下的方式***至基因的兩端。EcoRI/ApaI***至重鏈可變結構域（VH）中；EcoRI/XhoI***至輕鏈λ可變結構域（VL（λ））中；EcoRI/NheI限制性酶認可位點***至輕鏈κ可變結構域（VL（κ））中。在輕鏈可變結構域的情況下，在基因克隆期間，輕鏈λ可變結構域（VL（λ））的基因序列鏈接至人輕鏈恆定（LC（λ））域基因，以及輕鏈κ可變結構域（VL（κ））基因序列鏈接至人輕鏈恆定（LC（κ））域基因。To clone these 18 human antibody genes, restriction endonuclease recognition sites were inserted into both ends of the gene in the following manner. EcoRI/ApaI is inserted into the heavy chain variable domain (VH); EcoRI/XhoI is inserted into the light chain lambda variable domain (VL(λ)); EcoRI/NheI restriction enzyme recognition site is inserted into the light chain κ Variable domain (VL(κ)). In the case of a light chain variable domain, the gene sequence of the light chain lambda variable domain (VL(λ)) is linked to the human light chain constant (LC(λ)) domain gene, as well as the light chain, during gene cloning. The kappa variable domain (VL(k)) gene sequence is linked to the human light chain constant (LC (kappa)) domain gene.

在克隆至作為動物細胞中抗體表達的pCHO1.0表達載體期間，在使用EcoRV、PacI、AvrⅡ以及BstZ17I限制酶裂解之後，***輕鏈和重鏈基因。為了檢驗含有18個選定人抗體基因的pCHO1.0表達載體是否與合成基因序列一致，進行DNA測序。During cloning into the pCHO1.0 expression vector for antibody expression in animal cells, the light and heavy chain genes were inserted after cleavage with EcoRV, PacI, AvrII and BstZ17I restriction enzymes. To test whether the pCHO1.0 expression vector containing 18 selected human antibody genes was identical to the synthetic gene sequence, DNA sequencing was performed.

使用含有所有抗體輕鏈和重鏈基因的動物細胞表達系統的pCHO1.0表達載體，整個的人IgG得到表達。透過將每一抗體的質粒DNA瞬時轉染到CHO-S細胞和透過蛋白A柱純化分泌到培養基中的抗體，獲得人抗體。The entire human IgG was expressed using the pCHO1.0 expression vector containing the animal cell expression system of all antibody light and heavy chain genes. A human antibody is obtained by transiently transfecting plasmid DNA of each antibody into CHO-S cells and purifying the antibody secreted into the medium through a protein A column.

人IgG注入至hFcRn-表達Tg32（hFcRn+/+、hβ2m+/+、mFcRn-/-、mβ2m-/-）小鼠（Jackson實驗室）中，然後轉化為人IgG序列的18個人抗體作用至小鼠，以檢查抗體是否會影響人IgG的代謝。Human IgG was injected into hFcRn-expressing Tg32 (hFcRn+/+, hβ2m+/+, mFcRn-/-, mβ2m-/-) mice (Jackson Laboratories), and then 18 human antibodies transformed into human IgG sequences were applied to mice. To check if antibodies affect the metabolism of human IgG.

基於對抗原的結合親和性（KD）和人FcRn結合親和性的分析以及透過螢光流式細胞分選儀（FACS）的阻斷作用的體外分析結果，以及人IgG分解代謝的體內分析，選擇最有效作用的四種人抗-FcRn抗體蛋白（HL161A、HL161B、HL161C以及HL161D）（圖1）。另外，透過使用賴氨酸（K）取代HL161B抗體的重鏈可變框架的位置83的天冬醯胺酸（N），準備不具有N-糖基化位點的一HL161BK抗體。每一抗體的輕鏈和重鏈可變區的核苷酸序列、氨基酸序列以及CDR序列表示於表1、2以及3。表 1 ：選定的人 FcRn 抗體的重鏈和輕鏈可變結構域的多核苷酸序列 表 2 ：選定的人 FcRn 抗體的重鏈和輕鏈可變結構域的氨基酸序列 表 3 ：選定的人 FcRn 抗體的重鏈和輕鏈可變結構域的 CDR 序列 實例 4 ︰透過 SPR 對 HL161A/HL161B/HL161C/HL161D 抗體的抗原結合親和性的測定 Based on analysis of binding affinity (KD) of antigen and binding affinity of human FcRn, and in vitro analysis by blocking of fluorescent flow cytometry (FACS), and in vivo analysis of human IgG catabolism, selection The four most potent human anti-FcRn antibody proteins (HL161A, HL161B, HL161C, and HL161D) (Fig. 1). Further, an HL161BK antibody having no N-glycosylation site was prepared by substituting lysine (N) at position 83 of the heavy chain variable frame of HL161B antibody with lysine (K). The nucleotide sequence, amino acid sequence and CDR sequences of the light and heavy chain variable regions of each antibody are shown in Tables 1, 2 and 3. Table 1 : Polynucleotide sequences of the heavy and light chain variable domains of selected human FcRn antibodies Table 2 : Amino acid sequences of the heavy and light chain variable domains of selected human FcRn antibodies Table 3 : CDR sequences of the heavy and light chain variable domains of selected human FcRn antibodies Examples 4 through the SPR to ︰ HL161A / HL161B / HL161C / HL161D affinity of the antigen binding assay

透過SPR的HL161A、HL161B、HL161C以及HL161D抗體的結合親和性透過將水溶性的hFcRn在Proteon GLC晶片（Bio-Rad公司）上固定為一配體且測定親和性來測定。使用Proteon XPR36系統執行動力學分析。shFcRn固定於一GLC晶片上，並且一抗體樣本允許在5的濃度下進行反應，並且獲得sensogram結果。在動力學分析中，使用1：1的Langmuir結合模型，分別在pH 6.0和pH 7.4下，分析重複6次，並且計算平均KD值。後面是固定化步驟中，在EDAC/NHS 0.5X，30微升/分鐘和300秒的條件下激活此晶片。為了固定化，shFcRn在醋酸鹽緩衝液（pH5.5）中稀釋至2微克/毫升和250微升的濃度，並將稀釋液在30微升/分鐘的速率下在晶片上流過。當到達200-300 RU的固定化水平時停止反應。然後，在30微升/分鐘的速率下使用乙醇胺300秒執行失活。每一HL161抗體連續2倍稀釋，從10nM到5nM、2.5nM、1.25 nM、0.625 nM、0.312 nM等，從而製備樣本。在每一pH下使用1X PBST（pH7.4）或1X PBST（pH6.0）進行樣本稀釋。為了試樣分析，在50微升/分鐘的條件下進行200秒的聯合，並且在50微升/分鐘條件下執行600秒的解離步驟，此後，使用甘氨酸緩衝液（pH 2.5）在100微升/分鐘的條件下執行18秒的再生。每一樣本的動力學分析重複6次，然後測定平均抗原結合親和性（KD）。由SPR分析產生的抗體動力學參數表示於以下的表4中（圖2a至2h）。表 4 透過人 FcRn 固定化的 SPR 的抗體動力學分析的結果 實例 5 ：透過 螢光流式細胞分選儀 （ FACS ） HL161A/HL161B 抗體結合至人 FcRn 的分析 The binding affinity of the HL161A, HL161B, HL161C, and HL161D antibodies that passed through SPR was determined by fixing the water-soluble hFcRn to a ligand on a Proteon GLC wafer (Bio-Rad) and measuring the affinity. Kinetic analysis was performed using the Proteon XPR36 system. shFcRn was immobilized on a GLC wafer, and an antibody sample allowed the reaction to proceed at a concentration of 5, and a sensogram result was obtained. In the kinetic analysis, the analysis was repeated 6 times at pH 6.0 and pH 7.4 using a 1:1 Langmuir binding model, and the average KD values were calculated. Following the immobilization step, the wafer was activated under EDAC/NHS 0.5X, 30 microliters/minute and 300 seconds. For immobilization, shFcRn was diluted to a concentration of 2 μg/ml and 250 μl in acetate buffer (pH 5.5), and the dilution was passed over the wafer at a rate of 30 μl/min. The reaction was stopped when the immobilization level of 200-300 RU was reached. Then, deactivation was performed using ethanolamine at a rate of 30 μl/min for 300 seconds. Each HL161 antibody was serially diluted 2-fold, from 10 nM to 5 nM, 2.5 nM, 1.25 nM, 0.625 nM, 0.312 nM, etc., to prepare a sample. Sample dilution was performed using 1X PBST (pH 7.4) or 1X PBST (pH 6.0) at each pH. For sample analysis, a 200 second combination was performed at 50 μl/min, and a 600 second dissociation step was performed at 50 μl/min, after which a glycine buffer (pH 2.5) was used at 100 μl. Perform 18 seconds of regeneration under conditions of /min. The kinetic analysis of each sample was repeated 6 times and then the average antigen binding affinity (KD) was determined. The antibody kinetic parameters generated by SPR analysis are shown in Table 4 below (Figures 2a to 2h). Table 4 Results of antibody kinetic analysis of SPR immobilized by human FcRn Example 5 : Analysis of binding of HL161A/HL161B antibody to human FcRn by fluorescence flow cytometry ( FACS )

使用螢光流式細胞分選儀（FACS）系統，使用人FcRn-表達穩定的HEK293細胞，分析在每一pH下結合至FcRn。使用螢光流式細胞分選儀（FACS）的FcRn結合試驗在pH 6.0和pH 7.4下在反應緩衝液中執行。特別地，100000個人FcRn-表達穩定的HEK293細胞使用PBS緩衝液進行洗滌，並且在一桌面微型離心機中以4500 rpm的轉速執行5分鐘的離心以得到細胞團塊。將抗體加入到100微升的pH 6.0或pH7.4的PBS/10mM EDTA。剩餘的細胞團塊在反應緩衝液中懸浮，並且進行細胞計數。10微升（μL）的細胞懸浮液加載到一載玻片上，並且在一TC10系統中計數細胞懸浮液中細胞的數目，其後，細胞懸浮液使用反應緩衝液稀釋至2×106 個細胞/毫升的細胞濃度。每一抗體樣本稀釋到500nM。對於在pH 6.0的分析，稀釋液在一96-孔v底面板中稀釋至20 nM，並且50微升（μL）的稀釋液加入到每一孔中。對於在pH 7.4的分析，500nM的抗體樣本透過3倍連續的稀釋而稀釋，並且在從250nM至0.11nM的濃度範圍內進行分析。稀釋至2×106 個細胞/毫升的50微升（μL）的細胞添加至每一孔中且懸浮。將此面板安裝在旋轉器中，在4℃條件下以15°的一角度並以10rpm進行90分鐘的旋轉。反應完成之後，將此面板從旋轉器取出且以2000rpm離心10分鐘，並且除去上清液。A488抗-hIgG羊抗體在反應緩衝液中稀釋為1：200，並且100微升（μL）的抗體稀釋液添加至每一孔中並懸浮。接著，將此面板再次安裝於旋轉器中，在4℃條件下以15°的一角度並以10rpm進行90分鐘的旋轉。反應完成之後，將面板從旋轉器取出且以2000rpm的轉速離心10分鐘，並且除去上清液。再一次執行洗滌過程之後，100微升的反應緩衝液添加到每一孔中以溶解細胞團塊，並且面板轉移到一藍色試管中。接著，200微升的反應緩衝液加入到每一孔中，然後在螢光流式細胞分選儀（FACS）中執行測定。在下列條件下執行螢光流式細胞分選儀（FACS）測量：FS 108伏、SS 426伏、FL1 324伏、FL2 300伏。透過使用BD FACSDivaTM v6.1.3軟體（BD Bioscience公司）的螢光流式細胞分選儀（FACS）對這些細胞進行分析。結果表示為平均熒光強度（MFI）（圖3）。在10nM的濃度和pH6.0時，HL161A和HL161B抗體顯示的MFI值分別為10.59和8.34。在pH7.4和0.11-250nM的濃度下，透過使用MFI值的4參數邏輯回歸作為分析，抗體分別顯示出2.46nM和1.20nM的EC 50（有效濃度50％）值。實例 6 ：透過 螢光流式細胞分選儀 （ FACS ）對 HL161A/HL161B 抗體的阻斷作用的分析 Binding to FcRn at each pH was analyzed using a fluorescent flow cytometry (FACS) system using human FcRn-expressing HEK293 cells. The FcRn binding assay using a fluorescent flow cytometer (FACS) was performed in reaction buffer at pH 6.0 and pH 7.4. Specifically, 100,000 human FcRn-expressing HEK293 cells were washed with PBS buffer, and centrifugation was performed at 4500 rpm for 5 minutes in a tabletop microcentrifuge to obtain cell pellets. The antibody was added to 100 microliters of PBS/10 mM EDTA at pH 6.0 or pH 7.4. The remaining cell pellet was suspended in reaction buffer and cell counts were performed. Ten microliters (μL) of the cell suspension was loaded onto a glass slide, and the number of cells in the cell suspension was counted in a TC10 system, after which the cell suspension was diluted to 2 × 10 6 cells using a reaction buffer. /ml of cell concentration. Each antibody sample was diluted to 500 nM. For the analysis at pH 6.0, the dilution was diluted to 20 nM in a 96-well v bottom panel and 50 microliters (μL) of the dilution was added to each well. For the analysis at pH 7.4, 500 nM antibody samples were diluted by 3 fold continuous dilution and analyzed over a concentration range from 250 nM to 0.11 nM. 50 microliters (μL) of cells diluted to 2 × 10 6 cells/ml were added to each well and suspended. The panel was mounted in a rotator and rotated at an angle of 15° and at 10 rpm for 90 minutes at 4 °C. After the reaction was completed, the panel was taken out from the spinner and centrifuged at 2000 rpm for 10 minutes, and the supernatant was removed. The A488 anti-hIgG sheep antibody was diluted 1:200 in reaction buffer and 100 microliters (μL) of antibody dilution was added to each well and suspended. Next, the panel was again mounted in a rotator and rotated at an angle of 15° and at 10 rpm for 90 minutes at 4 °C. After the reaction was completed, the panel was taken out from the spinner and centrifuged at 2000 rpm for 10 minutes, and the supernatant was removed. After performing the washing process again, 100 microliters of reaction buffer was added to each well to dissolve the cell mass, and the panel was transferred to a blue test tube. Next, 200 μl of reaction buffer was added to each well, and then the assay was performed in a fluorescent flow cytometer (FACS). Fluorescence flow cytometry (FACS) measurements were performed under the following conditions: FS 108 volts, SS 426 volts, FL1 324 volts, FL2 300 volts. By using fluorescent BD FACSDiva TM v6.1.3 software (BD Bioscience Inc.) flow sorter (FACS) analysis on these cells. Results are expressed as mean fluorescence intensity (MFI) (Figure 3). At a concentration of 10 nM and a pH of 6.0, the HL161A and HL161B antibodies showed MFI values of 10.59 and 8.34, respectively. At a concentration of pH 7.4 and 0.11-250 nM, the antibody was subjected to a 4-parameter logistic regression using MFI values, and the antibodies showed EC50 (effective concentration 50%) values of 2.46 nM and 1.20 nM, respectively. Example 6 : Analysis of blocking effect of HL161A/HL161B antibody by fluorescence flow cytometry ( FACS )

在細胞表面上表達hFcRn的HEK293細胞使用兩種抗體進行處理，這兩種抗體對於細胞表面人FcRn的結合親和性進行了分析，並且抗體的阻斷作用基於Alexa-Fluo-488標記的hIgG1的結合降低來檢查。分析過程按照以下的方式進行。HEK293 cells expressing hFcRn on the cell surface were treated with two antibodies whose binding affinity for cell surface human FcRn was analyzed, and the blocking effect of the antibody was based on the binding of Alexa-Fluo-488-labeled hIgG1. Lower to check. The analysis process is carried out in the following manner.

2毫升的1×TE加入至naïve HEK293細胞和人FcRn-過表達穩定HEK293細胞的每一類型之中，其中這兩種細胞在5％的二氧化碳培養箱中在37℃條件下孵育了1分鐘。將細胞從燒瓶中回收，並且8毫升的反應緩衝液（pH6.0）加入其中，然後將細胞轉移到一50毫升的cornical管中。將細胞懸浮液以2000rpm離心5分鐘以除去上清液，並且1毫升的反應緩衝液（pH 6.0）加入到每一細胞團塊中。然後，將細胞懸浮液轉移到一新的1.5毫升的Eppendorf管中。接著，將細胞懸浮液以4000 rpm離心5分鐘，並且除去上清液。然後，反應緩衝液（pH 6.0）加入到剩餘的細胞團塊中，並且將細胞懸浮液的細胞數進行計數。最後，使用反應緩衝液將細胞懸浮液稀釋至2.5×106 細胞/毫升的細胞濃度。2 ml of 1 x TE was added to each of the naïve HEK293 cells and human FcRn-overexpressing stable HEK293 cells, which were incubated for 1 minute at 37 ° C in a 5% carbon dioxide incubator. The cells were recovered from the flask, and 8 ml of a reaction buffer (pH 6.0) was added thereto, and then the cells were transferred to a 50 ml cornical tube. The cell suspension was centrifuged at 2000 rpm for 5 minutes to remove the supernatant, and 1 ml of a reaction buffer (pH 6.0) was added to each cell pellet. The cell suspension was then transferred to a new 1.5 ml Eppendorf tube. Next, the cell suspension was centrifuged at 4000 rpm for 5 minutes, and the supernatant was removed. Then, a reaction buffer (pH 6.0) was added to the remaining cell pellet, and the number of cells of the cell suspension was counted. Finally, the cell suspension was diluted to a cell concentration of 2.5 x 10 6 cells/ml using reaction buffer.

每一抗體樣本稀釋至400nM，然後在一96-孔v-底的面板中進行4倍連續稀釋液的稀釋。稀釋到200nM至0.01nM的一最終濃度的50微升（μL）的樣本加入到每一孔中。然後，使用1μM的反應緩衝液（pH 6.0）稀釋的10微升（μL）的Alex488-hIgG1加入至每一孔中。最後，稀釋至2.5×106 細胞/毫升的濃度細胞的40微升（μL）的細胞加入到每一孔中並懸浮。將面板安裝在旋轉器中，在4℃條件下以15°的一角度並以10rpm進行90分鐘的旋轉。反應完成之後，將面板從旋轉器取出且以2000rpm離心10分鐘以除去上清液。100微升（μL）的反應緩衝液添加到每一孔以溶解細胞團塊，並且此面板轉移到一藍色試管中。然後，200微升（μL）的反應緩衝液加入到每一孔中，然後在螢光流式細胞分選儀（FACS）中執行測定。在下列條件下執行螢光流式細胞分選儀（FACS）測量：FS 108伏、SS 426伏、FL1 324伏、FL2 300伏。透過使用BD FACSDivaTM v6.1.3軟體（BD Bioscience公司）的螢光流式細胞分選儀（FACS）對這些細胞進行分析。結果表示為平均熒光強度（MFI）。在減去單獨的細胞所測得的MFI值（背景訊號）之後進行處理試驗組的MFI。相對於對照管（單獨為Alexa Fluor 488，並且無競爭者）的100％，計算含競爭者的管的MFI的百分比。 Each antibody sample was diluted to 400 nM and then diluted in 4-fold serial dilutions in a 96-well v-bottom panel. A 50 microliter (μL) sample diluted to a final concentration of 200 nM to 0.01 nM was added to each well. Then, 10 μl (μL) of Alex488-hIgG1 diluted with 1 μM of reaction buffer (pH 6.0) was added to each well. Finally, 40 μL (μL) of cells diluted to a concentration of 2.5 × 10 6 cells/ml were added to each well and suspended. The panel was mounted in a rotator and rotated at an angle of 15° and at 10 rpm for 90 minutes at 4 °C. After the reaction was completed, the panel was taken out from the spinner and centrifuged at 2000 rpm for 10 minutes to remove the supernatant. One hundred microliters (μL) of reaction buffer was added to each well to dissolve the cell mass, and the panel was transferred to a blue test tube. Then, 200 μl (μL) of the reaction buffer was added to each well, and then the assay was performed in a fluorescent flow cytometer (FACS). Fluorescence flow cytometry (FACS) measurements were performed under the following conditions: FS 108 volts, SS 426 volts, FL1 324 volts, FL2 300 volts. By using fluorescent BD FACSDiva TM v6.1.3 software (BD Bioscience Inc.) flow sorter (FACS) analysis on these cells. Results are expressed as mean fluorescence intensity (MFI). The MFI of the test group was treated after subtracting the MFI value (background signal) measured by the individual cells. The percentage of MFI of the competitor-containing tube was calculated relative to 100% of the control tube (Alexa Fluor 488 alone, and no competitor).

當此MFI相比較於人IgG1含競爭者的管的MFI更低時，競爭抗體測定為具有高競爭率。基於在pH6.0和0.01-200nM濃度的條件下測定的HL161A和HL161B抗體的阻斷作用（％），進行4參數邏輯回歸。結果，顯示出HL161A及HL161B抗體分別顯示出0.92nM及2.24nM的IC 50（抑制濃度50％）值（圖4）。實例 7 ：測試在 mFcRn-/- hFcRn 轉基因 32 （ Tg32 ）鼠中 HL161A/HL161B 的效果 When this MFI is lower than the MFI of the human IgG1 competitor-containing tube, the competing antibody is determined to have a high competition rate. A 4-parameter logistic regression was performed based on the blocking effect (%) of HL161A and HL161B antibodies measured at pH 6.0 and a concentration of 0.01-200 nM. As a result, it was revealed that the HL161A and HL161B antibodies showed IC50 (inhibitory concentration 50%) values of 0.92 nM and 2.24 nM, respectively (Fig. 4). Example 7 : Testing the effect of HL161A/HL161B in mFcRn-/- hFcRn transgenic 32 ( Tg32 ) mice

人IgG注入至人FcRn-表達Tg32（hFcRn+/+、hβ2m+/+、mFcRn-/-、mβ2m-/-）鼠（Jackson實驗室）中，然後HL161A及HL161B與人IgG一起施用給小鼠，以檢查是否抗體會影響人IgG的代謝。Human IgG was injected into human FcRn-expressing Tg32 (hFcRn+/+, hβ2m+/+, mFcRn-/-, mβ2m-/-) mice (Jackson Laboratories), and then HL161A and HL161B were administered to mice together with human IgG to Check if antibodies affect the metabolism of human IgG.

HL161A及HL161B抗體和人IgG（Greencross綠十字，IVglobulinS）在5、10以及20毫克/千克的劑量下分配為4天施用且存儲，並且PBS（磷酸鹽緩衝鹽水）緩衝液（pH7.4）用作載體以及一20毫克/千克IgG1對照。人FcRn Tg32鼠適應大約7天，並給予水和隨意的餵養。溫度（23±2℃）、濕度（55±5％）以及12小時光照/12小時黑暗循環進行自動控制。每一動物組由4只鼠組成。為了使用人IgG作為示蹤物，使用試劑盒（Pierce，目錄號︰21327）製備生物素-共軛的hIgG。在0小時，5毫克/千克的生物素-hIgG和495毫克/千克的人IgG腹膜內施用達到體內的飽和IgG。在生物素-IgG給藥之後的24、48、72以及96小時，每一藥物在腹腔內每日一次注入，劑量為5、10和以及毫克/千克。為了採血，將小鼠使用異氟烷（JW藥物）輕度麻醉，然後在給藥生物素-IgG之後的24、48、72、96、120以及168小時，使用肝素化的微血細胞比容毛細管（費舍爾）從眼眶叢取血。在24、48、72以及96小時，在取血之後施用藥物。在0.1 mL的全血接收於Eppendorf管中之後，血漿立即透過離心而分離且在-70℃存儲於一深冷凍機（熱）直至分析。HL161A and HL161B antibodies and human IgG (Greencross Green Cross, IVglobulinS) were dispensed at 4, 10 and 20 mg/kg for 4 days and stored, and used in PBS (phosphate buffered saline) buffer (pH 7.4). As a vector and a 20 mg/kg IgG1 control. Human FcRn Tg32 mice were acclimated for approximately 7 days and given water and ad libitum feeding. Temperature (23 ± 2 ° C), humidity (55 ± 5%) and 12 hours light / 12 hours dark cycle for automatic control. Each animal group consisted of 4 rats. To use human IgG as a tracer, biotin-conjugated hIgG was prepared using a kit (Pierce, catalog number: 21327). At 0 hours, 5 mg/kg of biotin-hIgG and 495 mg/kg of human IgG were administered intraperitoneally to achieve saturated IgG in vivo. At 24, 48, 72, and 96 hours after biotin-IgG administration, each drug was injected once daily in the peritoneal cavity at doses of 5, 10, and mg/kg. For blood collection, mice were lightly anesthetized with isoflurane (JW drug) and then heparinized microhematocrit capillaries were used at 24, 48, 72, 96, 120 and 168 hours after administration of biotin-IgG. (Fisher) Take blood from the eyelids. At 24, 48, 72 and 96 hours, the drug was administered after the blood was taken. After 0.1 mL of whole blood was received in the Eppendorf tube, the plasma was immediately separated by centrifugation and stored at -70 ° C in a deep freezer (heat) until analysis.

所收集的血液中的生物素-hIgG1的水平透過ELISA按照以下的方式進行分析。100微升（μl）的中性親和素（Neutravidin）（Pierce，31000）加入到一96-孔面板（Costar公司，目錄號：2592）中，以形成1.0微克/毫升的濃度，然後在4℃條件下塗上16小時。使用緩衝液A（0.05％ Tween-20，10 mM的PBS，pH 7.4）將面板洗滌三次，然後在室溫下在1％含BSA的PBS（pH 7.4）緩衝液中孵育2小時。接著，使用緩衝液A將面板洗滌三次，然後一中性親和素（Neutravidin）面板使用0.5％含BSA的PBS（pH7.4）緩衝液製備，以便對應於1微克/毫升。血液樣本在緩衝液B（100mM的MES、150mM的氯化鈉、0.5％無IgG的BSA、0.05％ Tween-20，pH6.0）中連續稀釋500-1000倍，並且150微升（μl）的稀釋液加入到面板的每一孔中。所添加的樣本在室溫下反應1小時。接著，將面板使用緩衝液A洗滌三次，然後200微升（μl）的1nM HRP-結合的抗-入IgG羊抗體加到每一孔中，在37℃下孵育2小時。接著，將面板使用冰冷的緩衝液B洗滌三此，然後將100微升(μl)的底物溶液四甲基聯苯胺(RnD，目錄號：DY999)加入到每一孔中並允許在室溫下反應15分鐘。50微升的1.0M硫酸溶液(Samchun，目錄號：S2129)加入到每一孔中以終止反應，其後測定在450奈米處的吸光度。 The level of biotin-hIgG1 in the collected blood was analyzed by ELISA in the following manner. 100 microliters (μl) of Neutravidin (Pierce, 31000) was added to a 96-well panel (Costar, catalog number: 2592) to form a concentration of 1.0 μg/ml, then at 4 °C. Apply for 16 hours under conditions. The panels were washed three times with buffer A (0.05% Tween-20, 10 mM PBS, pH 7.4) and then incubated for 2 hours at room temperature in 1% BSA-containing PBS (pH 7.4) buffer. Next, the panel was washed three times with buffer A, and then a Neutravidin panel was prepared using 0.5% BSA-containing PBS (pH 7.4) buffer to correspond to 1 μg/ml. Blood samples were serially diluted 500-1000 fold and 150 microliters (μl) in buffer B (100 mM MES, 150 mM sodium chloride, 0.5% IgG-free BSA, 0.05% Tween-20, pH 6.0). The dilution is added to each well of the panel. The added sample was reacted at room temperature for 1 hour. Next, the panel was washed three times with buffer A, and then 200 μl (μl) of 1 nM HRP-conjugated anti-injection IgG goat antibody was added to each well and incubated at 37 ° C for 2 hours. Next, the panel was washed three times with ice-cold buffer B, and then 100 microliters (μl) of the substrate solution tetramethylbenzidine (RnD, catalog number: DY999) was added to each well and allowed at room temperature. The reaction was carried out for 15 minutes. 50 microliters of a 1.0 M sulfuric acid solution (Samchun, catalog number: S2129) was added to each well to terminate the reaction, after which the absorbance at 450 nm was measured.

24小時之後(小鼠中生物素-IgG的大約最高溫度；在生物素-IgG的分解代謝發生之前)生物素-IgG的濃度設定為100%，並且相對於24小時的濃度，其他時間點的濃度百分比表示於圖5a與5b中。分析的結果表明，載體和20毫克/千克的IgG1對照物的半衰期分別為103小時和118小時。然而，在體外分析中表現出優異人FcRn的結合親和性和阻斷作用的HL161A抗體之血液IgG的半衰期，以及在人FcRn轉基因Tg32小鼠中最快的IgG分解代謝在不同劑量下為30、23以及18小時。此外，HL161B抗體顯示出41、22以及21小時的IgG半衰期。這表明，對於hFcRn的pH非依賴性和Fc-非競爭性抗體具有增加內源性抗體的分解代謝的效果(圖5a及5b)。 After 24 hours (the approximate maximum temperature of biotin-IgG in mice; before the catabolism of biotin-IgG occurs) the concentration of biotin-IgG was set to 100%, and relative to the concentration of 24 hours, at other time points The percent concentration is shown in Figures 5a and 5b. The results of the analysis showed that the half lifes of the vector and the 20 mg/kg IgG1 control were 103 hours and 118 hours, respectively. However, the half-life of blood IgG showing excellent binding affinity and blocking effect of human FcRn in in vitro assays, and the fastest IgG catabolism in human FcRn transgenic Tg32 mice was 30 at different doses. 23 and 18 hours. In addition, the HL161B antibody showed an IgG half-life of 41, 22, and 21 hours. This indicates that pH-independent and Fc-non-competitive antibodies to hFcRn have an effect of increasing catabolism of endogenous antibodies (Figs. 5a and 5b).

實例8：測試猴子中HL161A/HL161B的效果Example 8: Testing the effect of HL161A/HL161B in monkeys

使用具有對人FcRn具有96%同源性的獼猴，透過施用HL161A及HL161B抗體對猴子的IgG、IgA、IgM以及白蛋白水平進行分析，並且分析抗體的藥物動力學(PK)特性。 Monkeys were analyzed for IgG, IgA, IgM, and albumin levels by administration of HL161A and HL161B antibodies using rhesus monkeys with 96% homology to human FcRn, and the pharmacokinetic (PK) properties of the antibodies were analyzed.

1)猴的血液中免疫球蛋白G的表達變化分析1) Analysis of the expression changes of immunoglobulin G in the blood of monkeys

首先，透過ELISA分析測定猴IgG中的變化。100微升(μL)的抗-人IgG Fc抗體(BethylLab，A80-104A)裝載入一96-孔面板(Costar公司，目錄號：2592)的每一孔中，以具有4.0微克/毫升的濃度，然後在4℃下進行16小時的塗佈。使用洗滌緩衝液(0.05% Tween-20，含10mM的PBS，pH 7.4)將面板洗滌三次，然後在室溫下使用1%的含BSA的PBS(pH7.4)緩衝液孵育2小時。標準的猴IgG在3.9-500奈克/毫升的濃度下使用，並且血液樣本在1%含BSA的PBS（pH7.4）緩衝液中稀釋80000倍，並且稀釋液裝載入面板並在室溫下孵育2小時。接著，使用洗滌緩衝液將面板洗滌三次，然後100微升（μl）的20000倍稀釋的抗-hIgG抗體（Biorad公司，201005）裝載入面板中，並使其在室溫下反應1小時。在每一面板洗滌之後，將100微升（μl）的底物溶液3,3',5,5'-四甲基聯苯胺（RnD，目錄號：DY999）裝載入此面板中，並使其在室溫下反應7分鐘，在此之後50微升（μL）的1.0M硫酸溶液（Samchun，目錄號：S2129）加入至每一孔中以終止反應。為了分析，使用450奈米和540奈米的吸光度讀數器（MD：模式︰VersaMax）測定吸光度（OD）。結果，表明當HL161A及HL161B抗體每週一次，劑量為5及20毫克/千克分別靜脈注射到獼猴中時，猴的IgG水平以劑量依賴的方式降低，並且HL161抗體有效地阻斷IgG-FcRn的相互作用。5毫克/千克的HL161A在第9天將猴IgG的水平降低到47.1％，並且20毫克/千克的HL161A在第10天將猴IgG的水平降低到29.6％。5毫克/千克的HL161B在第10天將猴IgG的水平降低到53.6 ％，並且20毫克/千克的HL161B在第9天將猴IgG的水平降低至31％，這表明兩種抗體顯示類似的結果（表5和圖6a及6c）。此外，透過HL161A和HL161B的靜脈內給藥猴IgG水平的變化在個體之間進行比較，並且結果表明，猴IgG水平在個體之間以非常相似的方式下降。表 5. 透過施用 HL161A 及 HL161B 猴 IgG 水平的變化（％） 2 ）猴的血液中 HL161A/HL161B 的藥物動力學特性分析 First, changes in monkey IgG were determined by ELISA analysis. 100 microliters (μL) of anti-human IgG Fc antibody (BethylLab, A80-104A) was loaded into each well of a 96-well panel (Costar, catalog number: 2592) to have a thickness of 4.0 μg/ml. The concentration was then applied at 4 ° C for 16 hours. The panels were washed three times with wash buffer (0.05% Tween-20, 10 mM PBS, pH 7.4) and then incubated with 1% BSA-containing PBS (pH 7.4) buffer for 2 hours at room temperature. Standard monkey IgG was used at a concentration of 3.9-500 Ng/ml, and blood samples were diluted 80,000 times in 1% BSA-containing PBS (pH 7.4) buffer, and the dilution was loaded into the panel at room temperature. Incubate for 2 hours. Next, the panel was washed three times with washing buffer, and then 100 μl (μl) of 20,000-fold diluted anti-hIgG antibody (Biorad, 201005) was loaded into the panel, and allowed to react at room temperature for 1 hour. After each panel wash, 100 microliters (μl) of the substrate solution 3,3',5,5'-tetramethylbenzidine (RnD, catalog number: DY999) was loaded into the panel and It was reacted at room temperature for 7 minutes, after which 50 μl of a 1.0 M sulfuric acid solution (Samchun, catalog number: S2129) was added to each well to terminate the reaction. For analysis, the absorbance (OD) was measured using an absorbance reader (MD: mode: VersaMax) of 450 nm and 540 nm. As a result, it was shown that when the HL161A and HL161B antibodies were administered once a week to a dose of 5 and 20 mg/kg, respectively, the IgG levels of the monkeys were decreased in a dose-dependent manner, and the HL161 antibody effectively blocked the IgG-FcRn. interaction. 5 mg/kg of HL161A reduced monkey IgG levels to 47.1% on day 9, and 20 mg/kg of HL161A reduced monkey IgG levels to 29.6% on day 10. 5 mg/kg of HL161B reduced monkey IgG levels to 53.6% on day 10, and 20 mg/kg of HL161B reduced monkey IgG levels to 31% on day 9, indicating that both antibodies showed similar results. (Table 5 and Figures 6a and 6c). Furthermore, changes in monkey IgG levels administered intravenously through HL161A and HL161B were compared between individuals and the results indicated that monkey IgG levels decreased in a very similar manner between individuals. Table 5. Changes in IgG levels by administration of HL161A and HL161B monkeys (%) 2 ) Analysis of pharmacokinetic properties of HL161A/HL161B in monkey blood

在靜脈內給藥之後，透過競爭性ELISA分析HL161A及HL161B的時間依賴性藥物動力學特性（PK）。特別地，準備2微克/毫升的中性親和素（Neutravidin）溶液，並且100微升（µL）的此溶液的塗佈於96-孔面板的每一孔中，然後在4℃條件下孵育18小時。使用300微升（µL）的洗滌緩衝液（0.05％ Tween 20， 含10mM的PBS，pH 7.4）將面板洗滌三次，然後使用1％的含BSA的PBS（pH7.4）緩衝液在25℃條件下將每一孔孵育2小時。生物素化的hFcRn使用PBS稀釋至1微克/毫升，然後100微升（µL）的稀釋液加入到96-孔面板的每一孔中在25℃條件下孵育1小時。接著，使用300微升（μl）的洗滌緩衝液將面板洗滌三次，以去除未結合的hFcRn，然後一標準樣本（0.156-20奈克/毫升）加入至每一孔中並在25℃條件下孵育2小時。接著，使用洗滌緩衝液將面板洗滌三次，並且100微升（μL）的PBS中1:10000稀釋的檢測抗體加入到每一孔中並在25℃條件下孵育1.5小時。將面板最後洗滌三次，並且100微升（μL）的TMB溶液添加到每一緩衝器並在室溫下孵育5分鐘，之後，50微升（μL）的1M硫酸作為反應終止溶液加入每一孔中以使反應停止。接著，使用酶標儀測量450奈米處的吸光度。HL161A及HL161B的分析結果表示於以下的表6中，並且在其中看到，抗體的藥物動力學特性以劑量依賴的方式增加。抗體的半衰期（T1/2）係為大約6-12天，這相比較於通常已知抗體的半衰期更短。此外，顯示出當總體來看時，HL161B相比較於HL161A半衰期、AUC以及Cmax更高（圖7a及7b）。 表6. 不同劑量下HL161A及HL161B的藥物動力學特性的分析結果。 3 ）猴的血液中 IgM 及 IgA 抗體水平的變化分析 Time-dependent pharmacokinetic properties (PK) of HL161A and HL161B were analyzed by competitive ELISA after intravenous administration. Specifically, prepare a 2 μg/ml Neutravidin solution, and apply 100 μl of this solution to each well of a 96-well panel, then incubate at 4 °C. hour. The panel was washed three times with 300 microliters (μL) of wash buffer (0.05% Tween 20, containing 10 mM PBS, pH 7.4) and then used in 1% BSA-containing PBS (pH 7.4) buffer at 25 °C. Each well was incubated for 2 hours. The biotinylated hFcRn was diluted to 1 μg/ml with PBS, and then 100 μl (μL) of the dilution was added to each well of a 96-well panel and incubated at 25 ° C for 1 hour. Next, the panel was washed three times with 300 microliters (μl) of wash buffer to remove unbound hFcRn, then a standard sample (0.156-20 ng/ml) was added to each well at 25 °C. Incubate for 2 hours. Next, the panel was washed three times with washing buffer, and a 1:10000 diluted detection antibody in 100 μl (μL) of PBS was added to each well and incubated at 25 ° C for 1.5 hours. The panel was washed three times at the end, and 100 microliters (μL) of TMB solution was added to each buffer and incubated for 5 minutes at room temperature, after which 50 μl (μL) of 1 M sulfuric acid was added as a reaction stop solution to each well. In order to stop the reaction. Next, the absorbance at 450 nm was measured using a microplate reader. The analysis results of HL161A and HL161B are shown in Table 6 below, and it is seen therein that the pharmacokinetic properties of the antibody are increased in a dose-dependent manner. The half-life (T1/2) of the antibody is about 6-12 days, which is shorter than the half-life of commonly known antibodies. Furthermore, it is shown that when viewed in general, HL161B is higher in half-life, AUC, and Cmax than HL161A (Figs. 7a and 7b). Table 6. Analytical results of pharmacokinetic properties of HL161A and HL161B at different doses. 3 ) Analysis of changes in IgM and IgA antibody levels in monkey blood

按照類似於測定IgG水平的ELISA方法的方式，執行用於測定猴血中IgM及IgA水平的ELISA分析。特別地，100微升（μL）的抗-猴IgM抗體（Alpha Diagnostic公司，70033）或IgA抗體（Alpha Diagnostic公司，70043）加入到96-孔面板的每一孔中，以獲得2.0微克/毫升的濃度，然後在4 ℃條件下塗佈16小時。使用洗滌緩衝液（0.05％ Tween-20 含10mM的PBS，pH 7.4）將面板洗滌三次，然後在室溫下使用1％的含BSA的PBS（pH7.4）緩衝液孵育2小時。標準猴IgM在7.8-1000奈克/毫升的濃度下進行分析，並且IgA在15.6-2000奈克/毫升的濃度下進行分析。血液樣本在1％的含BSA的PBS（pH7.4）緩衝液中稀釋10000-或20000倍，並將稀釋液加入到每一孔中且在室溫下孵育2小時。接著，使用洗滌緩衝液將面板洗滌三次，然後，抗-猴IgM第二抗體（Alpha Diagnostic公司，70031）和抗-猴IgA第二抗體（KPL，074-11-011）分別100微升（μL）的5000倍稀釋液加入至每一孔中並允許在室溫下反應1小時。面板最後洗滌三次，並且100微升（μL）的底物溶液3,3',5,5'-四甲基聯（RnD，目錄號：DY999）加入至每一孔中並允許在室溫下反應7分鐘。接著， 50微升（μL）的1.0M的硫溶液（Samchun，目錄號：S2129）加入至每一孔中以終止反應。使用450奈米和540奈米的吸光度讀數器（MD：模式︰VersaMax）測定吸光度每一孔的吸光度。4 ）猴的血液中白蛋白水平變化的分析 An ELISA assay for determining IgM and IgA levels in monkey blood was performed in a manner similar to an ELISA method for determining IgG levels. Specifically, 100 microliters (μL) of anti-monkey IgM antibody (Alpha Diagnostic, 70033) or IgA antibody (Alpha Diagnostic, 70043) was added to each well of a 96-well panel to obtain 2.0 μg/ml. The concentration was then coated at 4 ° C for 16 hours. The panels were washed three times with wash buffer (0.05% Tween-20 containing 10 mM PBS, pH 7.4) and then incubated with 1% BSA-containing PBS (pH 7.4) buffer for 2 hours at room temperature. Standard monkey IgM was analyzed at a concentration of 7.8-1000 Ng/ml, and IgA was analyzed at a concentration of 15.6-2000 Ng/ml. Blood samples were diluted 10,000- or 20,000-fold in 1% BSA-containing PBS (pH 7.4) buffer, and the dilutions were added to each well and incubated for 2 hours at room temperature. Next, the panel was washed three times with washing buffer, and then, anti-monkey IgM secondary antibody (Alpha Diagnostic, 70031) and anti-monkey IgA secondary antibody (KPL, 074-11-011) were respectively 100 μl (μL A 5000-fold dilution was added to each well and allowed to react at room temperature for 1 hour. The panel was washed three times at the end, and 100 microliters (μL) of substrate solution 3,3',5,5'-tetramethyl linkage (RnD, catalog number: DY999) was added to each well and allowed at room temperature. Reaction for 7 minutes. Next, 50 microliters (μL) of a 1.0 M sulfur solution (Samchun, catalog number: S2129) was added to each well to terminate the reaction. The absorbance of each well of the absorbance was measured using an absorbance reader of 450 nm and 540 nm (MD: mode: VersaMax). 4 ) Analysis of changes in albumin levels in the blood of monkeys

使用一商業ELISA試劑盒（Assaypro，目錄號：EKA2201-1）執行猴血中白蛋白水平中變化的分析。簡而言之，將猴血清作為一測試樣本進行4000倍稀釋，並且將25微升（μL）的稀釋液添加至96-孔面板的每一孔中，其中此面板塗覆有能夠結合猴白蛋白的抗體。25微升（μL）的生物素化猴白蛋白溶液加入至每一孔中並在25℃條件下孵育2小時。使用200微升（μL）的洗滌緩衝液將面板最後洗滌三次，然後50微升（μL）的1：100稀釋的鏈狀抗生物素-過氧化物酶結合的抗體加入到每一孔中並在25℃條件下孵育30分鐘。將面板最終洗滌三次，然後50微升（μL）的一底物加入至每一孔中，並且在室溫下孵育10分鐘。接著，50微升（μL）的反應終止液加入到每一孔中，並測定450奈米處的吸光度。結果，透過施用HL161A及HL161B抗體在猴IgM、IgA以及白蛋白水平中的清晰變化在整個試驗期間中未觀察到（圖8a至8c）。因此，可以得出結論，抗體HL161僅參與IgG水平，並且不影響IgM及IgA的水平，這表明透過免疫球蛋白水平的降低對於免疫的降低沒有顯著的影響。此外，觀察到在整個試驗期間猴白蛋白水平沒有顯著變化，這表明HL161A及HL161B抗體特異性僅阻止IgG-FcRn的相互作用。5 ）血生化水平和尿成分的分析 Analysis of changes in albumin levels in monkey blood was performed using a commercial ELISA kit (Assaypro, catalog number: EKA2201-1). Briefly, monkey serum was diluted 4000-fold as a test sample, and 25 microliters (μL) of the dilution was added to each well of a 96-well panel, which was coated with a monkey white Protein antibody. 25 microliters (μL) of biotinylated monkey albumin solution was added to each well and incubated for 2 hours at 25 °C. The panel was washed three times with 200 microliters (μL) of wash buffer, and then 50 microliters (μL) of a 1:100 dilution of streptavidin-peroxidase-conjugated antibody was added to each well. Incubate for 30 minutes at 25 °C. The panel was finally washed three times, then 50 microliters (μL) of a substrate was added to each well and incubated for 10 minutes at room temperature. Next, 50 microliters (μL) of the reaction stop solution was added to each well, and the absorbance at 450 nm was measured. As a result, clear changes in monkey IgM, IgA, and albumin levels by administration of HL161A and HL161B antibodies were not observed throughout the experiment period (Figs. 8a to 8c). Therefore, it can be concluded that antibody HL161 is only involved in IgG levels and does not affect the levels of IgM and IgA, indicating that a decrease in the level of immunoglobulin has no significant effect on the reduction of immunity. Furthermore, no significant changes in monkey albumin levels were observed throughout the trial, indicating that HL161A and HL161B antibody specificity only prevented IgG-FcRn interaction. 5 ) Analysis of blood biochemical level and urine composition

最後，使用樣本在測試的第14天進行透過施用抗體的血液生化分析和尿分析。血液生化標記物，包含天門冬氨酸轉氨酶（AST）、丙氨酸轉氨酶（ALT）、鹼性磷酸酶（ALP）、肌酸磷酸激酶（CPK）、總膽紅素（TBIL）、葡萄糖（GLU）、總膽固醇（TCHO）、甘油三酯（TG）、總蛋白（TP）、白蛋白（Alb）、白蛋白/球蛋白（A/G）、血液尿素氮（BUN）、肌酸酐（CRE）、無機磷（IP）、鈣（Ca）、鈉（Na）、鉀（K）以及氯（Cl），使用日立7180系統進行分析。另外，尿分析的標記物，包含白細胞（LEU）、硝酸（NIT）、尿膽素原（URO）、蛋白質（PRO）、pH、潛血（BLO）、比重（SG）、酮體（KET）、nilirubin（BIL）、葡萄糖（GLU）以及抗壞血酸（ASC），使用Mission U120系統進行分析。雖然水平上具有微小變化，但是測量的水平包含於獼猴的正常水平的範圍內。Finally, blood biochemical analysis and urinalysis by administration of antibodies were performed on the 14th day of the test using the samples. Blood biochemical markers containing aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatine phosphokinase (CPK), total bilirubin (TBIL), glucose (GLU) ), total cholesterol (TCHO), triglyceride (TG), total protein (TP), albumin (Alb), albumin/globulin (A/G), blood urea nitrogen (BUN), creatinine (CRE) Inorganic phosphorus (IP), calcium (Ca), sodium (Na), potassium (K), and chlorine (Cl) were analyzed using a Hitachi 7180 system. In addition, markers for urinalysis include white blood cells (LEU), nitric acid (NIT), urobilinogen (URO), protein (PRO), pH, occult blood (BLO), specific gravity (SG), ketone body (KET), Nilirubin (BIL), glucose (GLU), and ascorbic acid (ASC) were analyzed using the Mission U120 system. Although there are minor changes in the level, the measured levels are included in the range of normal levels of macaques.

雖然本發明參照具體特徵揭露如上，然而本領域的技術人員顯然可知這些描述僅是為了說明之目的，並不限制本發明的範圍。因此，本發明的實質範圍將由所附的專利申請範圍及其等同範圍來限定。While the invention has been described above with reference to the specific features thereof, it is apparent that the description is intended to be illustrative only and not limiting the scope of the invention. Therefore, the scope of the invention is to be defined by the scope of the appended claims and their equivalents.

無no

圖1係為表示在還原或非還原條件下，在SDS-PAGE凝膠上透過蛋白A純化得到的CHO-S細胞中抗體的表達，以及HL161A、HL161B、HL161C以及HL161D抗體蛋白的分析結果。結果表明，非還原條件下，每一HL161抗體具有大約160 kDa的大小的整體人IgG1型結構，並且在還原條件下，重鏈具有大約55 kDa的大小，以及輕鏈具有大約25kDa的大小，這表明抗體係由典型的抗體亞單位組成。在圖1中，通道1表示分子量（M.W.）標記物，通道2表示2微克的非還原（*NEM處理的）抗體，通道3表示2微克的還原的抗體。 圖2a-2h表示使用SPR系統執行的以確定結合FcRn的四種抗-FcRn抗體（HL161A、HL161B、HL161C以及HL161D）的動力學解離（KD）的分析結果。圖2a-2h中的結果透過在pH 6.0和pH 7.4下使用Proteon氣液色譜晶片及Proteon XPR36（Bio-Rad公司）系統分析人FcRn和HL161A、HL161B、HL161C以及HL161D之間的相互作用獲得： 圖2a表示在pH 6.0時人FcRn與HL161A抗體之間的相互作用的分析結果。 圖2b表示在pH 7.4時人FcRn與HL161A抗體之間的相互作用的分析結果。 圖2c表示在pH 6.0時人FcRn與HL161B抗體之間的相互作用的分析結果。 圖2d表示在pH 7.4時人FcRn與HL161B抗體之間的相互作用的分析結果。 圖2e表示在pH 6.0時人FcRn與HL161C抗體之間的相互作用的分析結果。 圖2f表示在pH 7.4時人FcRn與HL161C抗體之間的相互作用的分析結果。 圖2g表示在pH 6.0時人FcRn與HL161D抗體之間的相互作用的分析結果。 圖2h表示在pH 7.4時人FcRn與HL161D抗體之間的相互作用的分析結果。 圖3表示兩個所選抗體結合到細胞表面的能力，並且表示在pH 6.0和pH 7.4條件下，透過使用所選的結合至細胞表面上人FcRn的HL161A及HL161B抗體處理人FcRn-過度表達HEK293細胞，並且分析結合於細胞表面的這些抗體所獲得的結果。HL161A及HL161B抗體分別與人FcRn的結合表示為在不同pH下處理細胞後，透過使用Alexa488-標記的抗-人山羊抗體執行螢光流式細胞分選儀（FACS）獲得的平均熒光強度（MFI）值。 圖4表示在pH 6.0條件下阻斷人IgG與人FcRn-表達細胞結合的能力的分析結果，並且表示是否結合到細胞表面的人FcRn的兩個所選抗體在細胞水平下可以阻斷人IgG與人FcRn結合的觀察結果。透過從200nM連續4倍分別稀釋證實結合人FcRn-過度表達HEK293細胞的HL161A和HL161B抗體，獲得阻斷Alexa488-標記的人IgG與人FcRn結合的能力的特性。 圖5a及圖5b表示HL161A及HL161B抗體對hIgG1的分解代謝效果的分析結果，其中HL161A及HL161B抗體從人FcRn-表達的轉基因小鼠Tg32（hFcRn+/+、hβ2m+/+、mFcRn-/-、mβ2m-/-）中選擇。在0小時，5毫克/千克的生物素-hIgG和495毫克/千克的人IgG腹膜內施用達到體內的飽和IgG。關於藥物施用，在生物素-IgG給藥之後的24、48、72以及96小時，IgG1、HL161A、HL161B或PBS在腹腔內每日一次注入，劑量為5、10以及20毫克/千克。在給藥生物素-IgG之後的24、48、72、96、120以及168小時收集樣品。在24、48、72以及96小時，在藥物施用之前收集血液，並且生物素-IgG的剩餘量透過ELISA法進行分析。結果表示為24小時的時候收集的血液樣本中剩餘量為100％，在每一時間點的剩餘量的比例。 圖6a至圖6c表示對人FcRn具有96％的序列同源性的獼猴施用兩種抗體（HL161A及HL161B）引起的猴IgG抗體之血液水平變化的分析結果。HL161A及HL161B抗體每日一次對獼猴靜脈內給藥，劑量為5及20毫克/千克，並且結果表明，猴IgG相比較於0小時降低高達70％，並且到29天降低大約30％。 圖6a表示HL161A及HL161B抗體在不同抗體濃度下的血清IgG還原效果。 圖6b表示在猴個體中HL161A及HL161B抗體（濃度︰（5毫克/千克））的血清IgG還原效果。 圖6c表示在猴個體中HL161A及HL161B抗體（濃度︰（20毫克/千克））的血清IgG還原效果。 圖7a及圖7b表示在使用獼猴執行的一實驗中HL161A及HL161B的藥物動力學曲線的分析結果。結果表明HL161B相比較於HL161A具有一更高的半衰期AUC以及Cmax。 圖8a至圖8c表示在使用獼猴執行的一實驗中，透過施用HL161A及HL161B抗體引起的猴IgM、IgA以及白蛋白的血液水平變化的分析結果。猴IgM、IgA以及白蛋白的血液水平具有微小的變化，這樣的變化是獼猴的正常範圍內，表明這樣的變化係由個體之間的差別造成而不是試驗物質的影響造成。 圖8a表示猴的血清IgM水平的變化。 圖8b表示猴的血清IgA水平的變化。以及 圖8c表示猴的血清白蛋白水平的變化。Figure 1 is a graph showing the expression of antibodies in CHO-S cells purified by protein A on SDS-PAGE gel under reduced or non-reducing conditions, and the analysis results of HL161A, HL161B, HL161C and HL161D antibody proteins. The results indicate that under non-reducing conditions, each HL161 antibody has an overall human IgGl type structure of a size of about 160 kDa, and under reducing conditions, the heavy chain has a size of about 55 kDa and the light chain has a size of about 25 kDa, which It is shown that the anti-system consists of typical antibody subunits. In Figure 1, channel 1 represents a molecular weight (M.W.) label, channel 2 represents 2 micrograms of non-reduced (*NEM treated) antibody, and channel 3 represents 2 micrograms of reduced antibody. Figures 2a-2h show the results of analysis of the kinetic dissociation (KD) performed using the SPR system to determine the four anti-FcRn antibodies (HL161A, HL161B, HL161C, and HL161D) that bind to FcRn. The results in Figures 2a-2h were obtained by analyzing the interaction between human FcRn and HL161A, HL161B, HL161C and HL161D using a Proteon gas chromatography chip and a Proteon XPR36 (Bio-Rad) system at pH 6.0 and pH 7.4: 2a represents the result of analysis of the interaction between human FcRn and HL161A antibody at pH 6.0. Figure 2b shows the results of analysis of the interaction between human FcRn and HL161A antibodies at pH 7.4. Figure 2c shows the results of an analysis of the interaction between human FcRn and HL161B antibodies at pH 6.0. Figure 2d shows the results of analysis of the interaction between human FcRn and HL161B antibodies at pH 7.4. Figure 2e shows the results of analysis of the interaction between human FcRn and HL161C antibodies at pH 6.0. Figure 2f shows the results of analysis of the interaction between human FcRn and HL161C antibody at pH 7.4. Figure 2g shows the results of analysis of the interaction between human FcRn and HL161D antibody at pH 6.0. Figure 2h shows the results of analysis of the interaction between human FcRn and HL161D antibodies at pH 7.4. Figure 3 shows the ability of two selected antibodies to bind to the cell surface and indicates that human FcRn-overexpressing HEK293 is treated by using selected HL161A and HL161B antibodies that bind to human FcRn on the cell surface at pH 6.0 and pH 7.4. Cells, and the results obtained by binding these antibodies to the cell surface were analyzed. Binding of the HL161A and HL161B antibodies to human FcRn, respectively, is expressed as the mean fluorescence intensity (MFI) obtained by performing a fluorescence flow cytometry (FACS) using Alexa488-labeled anti-human goat antibody after treatment of cells at different pHs. )value. Figure 4 shows the results of analysis of the ability to block the binding of human IgG to human FcRn-expressing cells under pH 6.0, and to indicate whether two selected antibodies of human FcRn binding to the cell surface can block human IgG at the cellular level. Observations of binding to human FcRn. The ability to block the binding of Alexa488-labeled human IgG to human FcRn was obtained by HL161A and HL161B antibodies that bind to human FcRn-overexpressing HEK293 cells, respectively, by serial dilutions from 200 nM. Fig. 5a and Fig. 5b show the results of analysis of the catabolism effect of HL161A and HL161B antibodies on hIgG1 from human FcRn-transgenic mouse Tg32 (hFcRn+/+, hβ2m+/+, mFcRn-/-, mβ2m). -/-) Select. At 0 hours, 5 mg/kg of biotin-hIgG and 495 mg/kg of human IgG were administered intraperitoneally to achieve saturated IgG in vivo. For drug administration, IgGl, HL161A, HL161B or PBS were injected once daily in the peritoneal cavity at doses of 5, 10 and 20 mg/kg at 24, 48, 72 and 96 hours after biotin-IgG administration. Samples were collected at 24, 48, 72, 96, 120, and 168 hours after the administration of biotin-IgG. At 24, 48, 72, and 96 hours, blood was collected prior to drug administration, and the remaining amount of biotin-IgG was analyzed by ELISA. The results are expressed as the ratio of the remaining amount in the blood sample collected at 24 hours, the remaining amount at each time point. Fig. 6a to Fig. 6c show the results of analysis of changes in blood levels of monkey IgG antibodies caused by administration of two antibodies (HL161A and HL161B) to rhesus monkeys having 96% sequence homology to human FcRn. HL161A and HL161B antibodies were administered to cynomolgus monkeys once daily at doses of 5 and 20 mg/kg, and the results showed that monkey IgG decreased by up to 70% compared to 0 hours and by about 30% by 29 days. Figure 6a shows the serum IgG reduction effect of HL161A and HL161B antibodies at different antibody concentrations. Figure 6b shows the serum IgG reducing effect of HL161A and HL161B antibodies (concentration: (5 mg/kg)) in monkey individuals. Figure 6c shows the serum IgG reducing effect of HL161A and HL161B antibodies (concentration: (20 mg/kg)) in monkey individuals. Figures 7a and 7b show the results of analysis of the pharmacokinetic profiles of HL161A and HL161B in an experiment performed using macaques. The results indicate that HL161B has a higher half-life AUC and Cmax compared to HL161A. Figures 8a to 8c show the results of analysis of changes in blood levels of monkey IgM, IgA, and albumin by administration of HL161A and HL161B antibodies in an experiment performed using macaques. The blood levels of monkey IgM, IgA, and albumin have minor changes, such changes are within the normal range of macaques, indicating that such changes are caused by differences between individuals rather than by test substances. Figure 8a shows the change in serum IgM levels in monkeys. Figure 8b shows the change in serum IgA levels in monkeys. And Figure 8c shows the change in serum albumin levels in monkeys.

Claims (19)

一種分離的抗-FcRn抗體或其片段，包含：重鏈可變區，包含具有序列號為21的氨基酸序列的CDR1，具有序列號為22的氨基酸序列的CDR2，具有序列號為23的氨基酸序列的CDR3；以及輕鏈可變區，包含具有序列號為24的氨基酸序列的CDR1，具有序列號為25的氨基酸序列的CDR2，具有序列號為26的氨基酸序列的CDR3。 An isolated anti-FcRn antibody or fragment thereof comprising: a heavy chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 21, CDR2 having the amino acid sequence of SEQ ID NO: 22, having the amino acid sequence of SEQ ID NO: 23. CDR3; and a light chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 24, CDR2 having the amino acid sequence of SEQ ID NO: 25, and CDR3 having the amino acid sequence of SEQ ID NO: 26. 一種分離的抗-FcRn抗體或其片段，包含：重鏈可變區，包含具有序列號為27的氨基酸序列的CDR1，具有序列號為28的氨基酸序列的CDR2，以及具有序列號為29的氨基酸序列的CDR3；以及輕鏈可變區，包含具有序列號為30的氨基酸序列的CDR1，具有序列號為31的氨基酸序列的CDR2，以及具有序列號為32的氨基酸序列的CDR3。 An isolated anti-FcRn antibody or fragment thereof comprising: a heavy chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 27, CDR2 having the amino acid sequence of SEQ ID NO: 28, and amino acid having SEQ ID NO: 29. The CDR3 of the sequence; and the light chain variable region, comprising CDR1 having the amino acid sequence of SEQ ID NO: 30, CDR2 having the amino acid sequence of SEQ ID NO: 31, and CDR3 having the amino acid sequence of SEQ ID NO: 32. 一種分離的抗-FcRn抗體或其片段，包含具有序列號為2的氨基酸序列的重鏈可變區和具有序列號為12的氨基酸序列的輕鏈可變區。 An isolated anti-FcRn antibody or fragment thereof comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2 and a light chain variable region having the amino acid sequence of SEQ ID NO: 12. 一種分離的抗-FcRn抗體或其片段，包含具有序列號為4的氨基酸序列的重鏈可變區和具有序列號為14的氨基酸序列的輕鏈可變區。 An isolated anti-FcRn antibody or fragment thereof, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 4 and a light chain variable region having the amino acid sequence of SEQ ID NO: 14. 一種分離的抗-FcRn抗體或其片段，包含具有序列號為6的氨基酸序列的重鏈可變區和具有序列號為16的氨基酸序列的輕鏈可變區。 An isolated anti-FcRn antibody or fragment thereof comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 6 and a light chain variable region having the amino acid sequence of SEQ ID NO: 16. 如請求項1至5中任意一項所述的抗-FcRn抗體或其片段，其中該抗體在pH 6.0和pH 7.4的條件下，具有0.01-2nM的KD(解離常數)值與FcRn相結合。 The anti-FcRn antibody or fragment thereof according to any one of claims 1 to 5, wherein the antibody has a KD (dissociation constant) value of 0.01 to 2 nM in combination with FcRn under conditions of pH 6.0 and pH 7.4. 如請求項1至5中任意一項所述的抗-FcRn抗體或其片段，其中該抗體係為單克隆抗體、鼠抗體、嵌合抗體、人源化抗體或人抗體。 The anti-FcRn antibody or fragment thereof according to any one of claims 1 to 5, wherein the anti-system is a monoclonal antibody, a murine antibody, a chimeric antibody, a humanized antibody or a human antibody. 如請求項1至5中任意一項所述的抗-FcRn抗體或其片段，其中該抗體或其片段包含全長抗體、Fab、F(ab')2、Fd、scFv、結構域抗體、雙特異性抗體、雙體(bibodies)、微型抗體、三體(tribodies)、scap(固醇調節結合蛋白裂解激活蛋白)、雙特異性抗體、三特異性抗體、多特異性抗體、雙抗體、三抗體、四抗體、胞內抗體、奈米抗體、小模塊immunopharmaceuticals(SMIP)、結合結構域免疫球蛋白融合蛋白、駱駝源抗體、VHH含有抗體。 The anti-FcRn antibody or fragment thereof according to any one of claims 1 to 5, wherein the antibody or fragment thereof comprises a full length antibody, Fab, F(ab') 2 , Fd, scFv, domain antibody, bispecific Antibodies, bibodies, minibodies, tribodies, scap (sterol-regulated binding protein cleavage activator), bispecific antibodies, trispecific antibodies, multispecific antibodies, diabodies, triabodies , four antibodies, intracellular antibodies, nano antibodies, small modules of immunopharmaceuticals (SMIP), binding domain immunoglobulin fusion proteins, camel-derived antibodies, VHH containing antibodies. 如請求項1至5中任意一項所述的抗-FcRn抗體或其片段，其中該抗體包含IgD抗體、IgE抗體、IgM抗體、IgG1抗體、IgG2抗體、IgG3抗體、IgG4抗體。 The anti-FcRn antibody or fragment thereof according to any one of claims 1 to 5, wherein the antibody comprises an IgD antibody, an IgE antibody, an IgM antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody. 一種編碼如請求項1至5中任意一項所述的抗-FcRn抗體或其片段的多核苷酸。 A polynucleotide encoding the anti-FcRn antibody or fragment thereof according to any one of claims 1 to 5. 一種編碼抗-FcRn抗體或其片段的多核苷酸，包含以下其中一組序列：編碼重鏈的序列號是1，編碼輕鏈的序列號是11；編碼重鏈的序列號是3，編碼輕鏈的序列號是13；或編碼重鏈的序列號是5，編碼輕鏈的序列號是15。 A polynucleotide encoding an anti-FcRn antibody or a fragment thereof, comprising one of the following sequences: the sequence number encoding the heavy chain is 1, the sequence number encoding the light chain is 11; the sequence number encoding the heavy chain is 3, the coding is light The sequence number of the strand is 13; or the sequence number encoding the heavy chain is 5, and the sequence number encoding the light chain is 15. 一種重組表達載體，包含如請求項10或11所述的多核甘酸。 A recombinant expression vector comprising the polynucleotide as claimed in claim 10 or 11. 一種宿主細胞，使用如請求項12所述的重組表達載體橫切。 A host cell transected using the recombinant expression vector of claim 12. 一種抗-FcRn抗體或其片段的製備方法，包括：培養如請求項13所述的宿主細胞且由該宿主細胞產生抗體；以及分離和純化產生的該抗體，以恢復特異性結合至FcRn的該抗體。 A method for producing an anti-FcRn antibody or a fragment thereof, comprising: cultivating a host cell according to claim 13 and producing an antibody from the host cell; and isolating and purifying the produced antibody to restore specific binding to FcRn antibody. 一種藥物組合物以及一種或多種藥學上可接受的載體，其中該藥物組合物包含如請求項1至5中任意一項所述之抗體或其片段。 A pharmaceutical composition and one or more pharmaceutically acceptable carriers, wherein the pharmaceutical composition comprises the antibody or fragment thereof according to any one of claims 1 to 5. 一種用於自體免疫病患者的治療方法之藥物組合物，包含如請求項1至5中任意一項所述之抗體或其片段以及一種或多種藥學上可接受的載體。 A pharmaceutical composition for use in a method of treating a patient with an autoimmune disease, comprising the antibody of any one of claims 1 to 5, or a fragment thereof, and one or more pharmaceutically acceptable carriers. 如請求項16所述的藥物組合物，其中該自體免疫病從包含以下的組中選擇：免疫中性白細胞減少症、格林-巴利綜合症、癲癇、自體免疫性腦炎、Isaac綜合症、痣綜合徵、尋常天疱瘡、天疱瘡、大疱性類天疱瘡、後天性水疱性表皮鬆解症、妊娠期類天疱瘡、黏膜的天疱瘡樣、抗磷脂綜合徵、自體免疫性貧血、自體免疫性嚴重的疾病、古德帕斯徹氏綜合徵、重症肌無力、多發性硬化症、類風濕性關節炎、狼瘡、特發性血小板減少性紫癜、狼瘡性腎炎以及膜性腎病。 The pharmaceutical composition according to claim 16, wherein the autoimmune disease is selected from the group consisting of: immunological neutropenia, Guillain-Barré syndrome, epilepsy, autoimmune encephalitis, Isaac synthesis Symptoms, hernia syndrome, pemphigus vulgaris, pemphigus, bullous pemphigoid, acquired vesicular epidermolysis, pemphigoids in pregnancy, pemphigus-like mucosa, antiphospholipid syndrome, autoimmune Anemia, autoimmune disease, Goodpasch's syndrome, myasthenia gravis, multiple sclerosis, rheumatoid arthritis, lupus, idiopathic thrombocytopenic purpura, lupus nephritis, and membranous Kidney disease. 一種組合物，包含標記有一檢測標記的如請求項1至5中任意一項所述的抗體或其片段。 A composition comprising an antibody or a fragment thereof according to any one of claims 1 to 5, which is labeled with a detection marker. 一種在體外使用如請求項1至5中任意一項所述的抗體或其片段檢測FcRn的方法。 A method of detecting FcRn using an antibody or a fragment thereof according to any one of claims 1 to 5 in vitro.
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