TW202104269A - Anti-cd38 antibodies and formulations - Google Patents
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Abstract
Description
本文提供了具有經改良之細胞毒活性的抗CD38抗體及其穩定調配物。Provided herein are anti-CD38 antibodies with improved cytotoxic activity and stable formulations thereof.
CD38是II型糖基化的45k道耳吞(kDa)膜蛋白,其被鑒別為淋巴細胞標記。CD38通過調節造血細胞存活和分化在白血球恆穩狀態中發揮作用(Richards JO,等人, Mol Cancer Ther. 2008; 7(8):2517-27)。CD38充當與CD31結合的受體,並且參與細胞黏附和信號轉導。CD38在信號轉導中的功能顯得多種多樣,這取決於細胞譜系、分化階段以及可能與不同共受體的締合(Richards JO,等人, 2008)。CD38也是催化將環腺苷二磷酸核糖(cADPR)從菸鹼醯胺腺嘌呤二核苷酸(NAD+)合成和水解為ADP-核糖的胞外酶(DiLillo DJ, Ravetch JV., Cell. 2015; 161(5):1035-45)。這些反應產物涉及鈣動員和細胞內信號傳導(Derer S,等人, MAbs. 2014; 6(2):409-21)。CD38 is a type II glycosylated 45k canal ear swallow (kDa) membrane protein, which is identified as a lymphocyte marker. CD38 plays a role in white blood cell homeostasis by regulating the survival and differentiation of hematopoietic cells (Richards JO, et al., Mol Cancer Ther. 2008; 7(8): 2517-27). CD38 acts as a receptor that binds to CD31, and is involved in cell adhesion and signal transduction. The functions of CD38 in signal transduction appear to be diverse, depending on the cell lineage, differentiation stage, and possible association with different co-receptors (Richards JO, et al., 2008). CD38 is also an extracellular enzyme that catalyzes the synthesis and hydrolysis of cyclic adenosine diphosphate ribose (cADPR) from nicotine amide adenine dinucleotide (NAD+) to ADP-ribose (DiLillo DJ, Ravetch JV., Cell. 2015; 161(5):1035-45). These reaction products are involved in calcium mobilization and intracellular signal transduction (Derer S, et al., MAbs. 2014; 6(2):409-21).
CD38在健康人類中的表現可以在NK細胞、單核細胞、樹突細胞、巨噬細胞、粒細胞、活化的T和B細胞、和漿細胞上檢測到。相比之下,在造血幹細胞、靜息T和B細胞或組織巨噬細胞中尚未檢測到表現。此外,若干種血液惡性腫瘤表現CD38,如惡性漿細胞病(例如,多發性骨髓瘤(MM)、澱粉樣變性)和其他造血源性癌症(包括、包括例如瓦爾登斯特倫氏病(Waldenstrom’s disease)、非霍奇金淋巴瘤(NHL)、急性淋巴細胞白血病(ALL)和急性骨髓性白血病(AML))。The expression of CD38 in healthy humans can be detected on NK cells, monocytes, dendritic cells, macrophages, granulocytes, activated T and B cells, and plasma cells. In contrast, no manifestations have been detected in hematopoietic stem cells, resting T and B cells, or tissue macrophages. In addition, several hematological malignancies express CD38, such as malignant plasma cell disease (for example, multiple myeloma (MM), amyloidosis) and other hematopoietic cancers (including, including, for example, Waldenstrom's disease). disease), non-Hodgkin’s lymphoma (NHL), acute lymphoblastic leukemia (ALL) and acute myelogenous leukemia (AML)).
CD38的表現在MM中尤其明顯,因為> 98%的患者對這種蛋白質呈陽性(Reinherz EL,等人, Proc Natl Acad Sci USA. 1980; 77(3):1588-92;Lin P,等人, Am J Clin Pathol. 2004; 121(4):482-8)。CD38在惡性選殖MM細胞上的強烈且一致的表現,與在正常細胞上有限的表現模式形成對比,這表明此抗原可用於特異性靶向腫瘤細胞。The performance of CD38 is particularly evident in MM, because> 98% of patients are positive for this protein (Reinherz EL, et al., Proc Natl Acad Sci USA. 1980; 77(3): 1588-92; Lin P, et al. , Am J Clin Pathol. 2004; 121(4):482-8). The strong and consistent expression of CD38 on malignant colonizing MM cells contrasts with the limited expression pattern on normal cells, which indicates that this antigen can be used to specifically target tumor cells.
MM是惡性漿細胞疾病,其特徵在於細胞表面上的CD38表現、骨髓(BM)中漿細胞的選殖增生和過量單株免疫球蛋白(通常為IgG或IgA型或遊離尿輕鏈(也稱為副蛋白、M蛋白或M組分)的產生。這是一種主要與年齡的增長有關的疾病,超過80%的患者年齡在60歲以上。MM is a malignant plasma cell disease characterized by CD38 expression on the cell surface, proliferation of plasma cells in the bone marrow (BM), and excess monoclonal immunoglobulin (usually IgG or IgA type or free urine light chain (also called It is the production of paraprotein, M protein or M component). This is a disease mainly related to aging, and more than 80% of patients are over 60 years old.
MM的病程隨疾病的侵襲性和相關預後因素而變化。多發性骨髓瘤的某些染色體異常已被證明與不良的臨床結果有關。高風險細胞遺傳學改變包括del(17p)、t(4;14)和t(14;16)、和1q增加等。在過去的二十年中,中位生存期從3年提高到6年;然而,一些患者可以活超過10年(Ocio EM,等人, Expert Rev Hematol. 2014; 7(1):127-41)。治療選擇和生存期取決於患者的年齡、健康和疾病狀況。年齡低於約65歲、身體健康良好、表現為症狀性活動性疾病的患者將通常首先接受自體幹細胞移植(ASCT)療法。為了在收集幹細胞之前實現疾病的細胞減少,投予誘導化療。誘導治療方案包括烷基化劑、單獨的地塞米松、沙利度胺加地塞米松,以及長春新鹼、Adriamycin®(阿黴素)和地塞米松(VAD;或對此方案的修改);然而,後兩種方案伴隨較高的毒性(Richardson PG,等人, Blood. 2010; 116(5):679-86;Arnulf B,等人, Haematologica. 2012; 97:1925-8)。使用單獨的Velcade®(硼替佐米)、硼替佐米組合以及Revlimid®(來那度胺)加地塞米松的治療作為誘導療法已顯示出改善的結果,並且這些藥劑顯示出更高的反應率和更低的毒性(Richardson PG 2010, Kumar S,等人, Blood. 2012; 119(19):4375-82;Roussel M,等人, J Clin Oncol. 2014; 32:2712-7;Durie BGM,等人, Lancet. 2017; 389:519-27)。另外核准的藥物包括泊馬度胺(與來那度胺屬於同一類IMiD® )以及卡非佐米和伊沙佐米(與硼替佐米屬於同一類蛋白酶體抑制劑)。除了這些新治療,單株抗體(特別是抗CD38抗體)也已經開始在骨髓瘤患者的治療中發揮重要作用。達雷木單抗是抗CD38抗體,其已被批准作為單一藥劑或與其他MM治療組合用於治療MM(Touzeau C, Moreau P., Expert Opin Biol Ther. 2017; 17(7):887-93;Tzogani K.等人, Oncologist. 2018; 23:1-11)。據報導,抗CD38抗體伊沙妥昔單抗在復發性或難治性多發性骨髓瘤中作為單一藥劑誘發出25%至29%反應率,且以組合療法的形式誘發出60%反應率(Martin T,等人, Blood. 2017; 129(25):3294-303);最近的3期研究結果指出,在復發性和難治性多發性骨髓瘤中,添加伊沙妥昔單抗能夠提高使用泊馬度胺-地塞米松治療的無進展生存期(J Clin Oncol 37, 2019 (增刊;摘要8004))。另外,抗Slam-F7抗體艾洛珠單抗已被批准與來那度胺和地塞米松組合用於治療接受過一至三種先前療法線(line)的成人MM患者,並且與泊馬度胺和地塞米松組合用於治療接受過至少兩種包括來那度胺和蛋白酶體抑制劑的先前療法的成人MM患者(Bristol-Myers Squibb Company. EMPLICITI®(艾洛珠單抗)[包裝說明書]。美國食品和藥物管理局網站: www-dot-accessdata-dot-fda.gov/drugsatfda_docs/label/2018/761035s008lbl.pdf,修訂於2018年11月。The course of MM varies with the aggressiveness of the disease and related prognostic factors. Certain chromosomal abnormalities in multiple myeloma have been shown to be related to poor clinical outcomes. High-risk cytogenetic changes include del(17p), t(4;14), t(14;16), and 1q increase. In the past two decades, the median survival has increased from 3 years to 6 years; however, some patients can live more than 10 years (Ocio EM, et al., Expert Rev Hematol. 2014; 7(1):127-41 ). Treatment options and survival depend on the patient’s age, health, and disease status. Patients younger than about 65 years old, in good health, and presenting with symptomatic active disease will usually receive autologous stem cell transplantation (ASCT) therapy first. In order to achieve cell reduction of the disease before collecting the stem cells, induction chemotherapy is administered. Induction treatment regimens include alkylating agents, dexamethasone alone, thalidomide plus dexamethasone, as well as vincristine, Adriamycin® (adriamycin) and dexamethasone (VAD; or a modification of this regimen); However, the latter two regimens are accompanied by higher toxicity (Richardson PG, et al., Blood. 2010; 116(5):679-86; Arnulf B, et al., Haematologica. 2012; 97:1925-8). The use of Velcade® (bortezomib), the combination of bortezomib and Revlimid® (lenalidomide) plus dexamethasone as induction therapy has shown improved results, and these agents have shown higher response rates and Lower toxicity (Richardson PG 2010, Kumar S, et al., Blood. 2012; 119(19):4375-82; Roussel M, et al., J Clin Oncol. 2014; 32:2712-7; Durie BGM, et al. People, Lancet. 2017; 389:519-27). Other approved drugs include pomalidomide (in the same class of IMiD ® as lenalidomide) and carfilzomib and ixazomib (in the same class of proteasome inhibitors as bortezomib). In addition to these new treatments, monoclonal antibodies (especially anti-CD38 antibodies) have also begun to play an important role in the treatment of myeloma patients. Darimumab is an anti-CD38 antibody, which has been approved as a single agent or in combination with other MM treatments for the treatment of MM (Touzeau C, Moreau P., Expert Opin Biol Ther. 2017; 17(7):887-93 ; Tzogani K. et al., Oncologist. 2018; 23:1-11). According to reports, the anti-CD38 antibody ixatuximab induces a response rate of 25% to 29% as a single agent in relapsed or refractory multiple myeloma, and a 60% response rate in the form of combination therapy (Martin T, et al., Blood. 2017; 129(25):3294-303); the results of the recent phase 3 study pointed out that in relapsed and refractory multiple myeloma, the addition of ixatuximab can increase the use of po Progression-free survival of maturamide-dexamethasone treatment (J Clin Oncol 37, 2019 (Supplement; Abstract 8004)). In addition, the anti-Slam-F7 antibody ilolizumab has been approved in combination with lenalidomide and dexamethasone for the treatment of adult MM patients who have received one to three previous treatment lines (line), and is combined with pomalidomide and pomalidomide and dexamethasone. The dexamethasone combination is used to treat adult MM patients who have received at least two previous therapies including lenalidomide and proteasome inhibitors (Bristol-Myers Squibb Company. EMPLICITI® (Ilocizumab) [package insert]. The US Food and Drug Administration website: www-dot-accessdata-dot-fda.gov/drugsatfda_docs/label/2018/761035s008lbl.pdf, revised in November 2018.
用於這些表現CD38的疾病的療法的當前目標,是盡可能有效地控制疾病、最大限度地提高生活品質和延長生存期。例如,MM患者在其一生中平均會接受4到8種不同的治療方案。因此,儘管更新療法改善了患者的預後,但MM仍然是致命的疾病。因此,對已經接受了包括抗體療法的當前療法並且進展的患者的治療仍然是未滿足醫療需求的重大挑戰。The current goals of therapies for these CD38-expressing diseases are to control the disease as effectively as possible, maximize the quality of life, and prolong survival. For example, MM patients will receive an average of 4 to 8 different treatment options during their lifetime. Therefore, although refresher therapy improves the patient's prognosis, MM is still a fatal disease. Therefore, the treatment of patients who have received current therapies including antibody therapy and progressed remains a major challenge for unmet medical needs.
與目前可用的表現CD38的疾病的治療(包括抗體治療)相比,設想到本文所提供的抗體、用途和治療方法可以提供所述疾病的優越的臨床反應或治療。本文提供了特異性結合人類CD38並介導對表現CD38的細胞的優越殺傷的抗體、包含所述抗體的調配物和單位劑型、製備所述抗體的方法以及使用所述抗體的方法。Compared with currently available treatments (including antibody treatments) for diseases expressing CD38, it is envisaged that the antibodies, uses and treatment methods provided herein can provide superior clinical responses or treatments for the diseases. Provided herein are antibodies that specifically bind to human CD38 and mediate superior killing of CD38-expressing cells, formulations and unit dosage forms comprising the antibodies, methods of preparing the antibodies, and methods of using the antibodies.
在一個態樣中,提供了特異性結合人類CD38的抗體。在一些實施例中,本文所提供的抗體包含具有選自SEQ ID NO: 7、8和9的胺基酸序列的輕鏈(LC)和具有選自SEQ ID NO: 2、3、4、5和6的胺基酸序列的重鏈(HC)。In one aspect, an antibody that specifically binds to human CD38 is provided. In some embodiments, the antibody provided herein comprises a light chain (LC) having an amino acid sequence selected from SEQ ID NO: 7, 8 and 9 and a light chain (LC) having an amino acid sequence selected from SEQ ID NO: 2, 3, 4, 5. And the heavy chain (HC) of the amino acid sequence of 6.
在一些實施例中,本文所提供的抗體包含具有SEQ ID NO: 7的胺基酸序列的LC和具有選自SEQ ID NO: 2、SEQ ID NO: 3和SEQ ID NO: 4的胺基酸序列的HC。在一些實施例中,本文所提供的抗體包含具有SEQ ID NO: 7的胺基酸序列的LC和具有SEQ ID NO: 2的胺基酸序列的HC。在一些實施例中,本文所提供的抗體包含具有SEQ ID NO: 7的胺基酸序列的LC和具有SEQ ID NO: 3的胺基酸序列的HC。在一些實施例中,本文所提供的抗體包含具有SEQ ID NO: 7的胺基酸序列的LC和具有SEQ ID NO: 4的胺基酸序列的HC。In some embodiments, the antibody provided herein comprises an LC having the amino acid sequence of SEQ ID NO: 7 and an amino acid selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. Sequence of HC. In some embodiments, the antibody provided herein comprises an LC having the amino acid sequence of SEQ ID NO: 7 and an HC having the amino acid sequence of SEQ ID NO: 2. In some embodiments, the antibody provided herein comprises an LC having the amino acid sequence of SEQ ID NO: 7 and an HC having the amino acid sequence of SEQ ID NO: 3. In some embodiments, the antibody provided herein comprises an LC having the amino acid sequence of SEQ ID NO: 7 and an HC having the amino acid sequence of SEQ ID NO: 4.
在一些實施例中,本文所提供的抗體包含具有SEQ ID NO: 8的胺基酸序列的LC和具有選自SEQ ID NO: 5和SEQ ID NO: 6的胺基酸序列的HC。在一些實施例中,本文所提供的抗體包含具有SEQ ID NO: 8的胺基酸序列的LC和具有SEQ ID NO: 5的胺基酸序列的HC。在一些實施例中,本文所提供的抗體包含具有SEQ ID NO: 8的胺基酸序列的LC和具有SEQ ID NO: 6的胺基酸序列的HC。In some embodiments, the antibody provided herein comprises an LC having an amino acid sequence of SEQ ID NO: 8 and an HC having an amino acid sequence selected from SEQ ID NO: 5 and SEQ ID NO: 6. In some embodiments, the antibody provided herein comprises an LC having the amino acid sequence of SEQ ID NO: 8 and an HC having the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody provided herein comprises an LC having the amino acid sequence of SEQ ID NO: 8 and an HC having the amino acid sequence of SEQ ID NO: 6.
在一些實施例中,本文所提供的抗體包含具有SEQ ID NO: 9的胺基酸序列的LC和具有選自SEQ ID NO: 6和SEQ ID NO: 7的胺基酸序列的HC。在一些實施例中,本文所提供的抗體包含具有SEQ ID NO: 9的胺基酸序列的LC和具有SEQ ID NO: 6的胺基酸序列的HC。在一些實施例中,本文所提供的抗體包含具有SEQ ID NO: 9的胺基酸序列的LC和具有SEQ ID NO: 7的胺基酸序列的HC。In some embodiments, the antibody provided herein comprises an LC having an amino acid sequence of SEQ ID NO: 9 and an HC having an amino acid sequence selected from SEQ ID NO: 6 and SEQ ID NO: 7. In some embodiments, the antibody provided herein comprises an LC having the amino acid sequence of SEQ ID NO: 9 and an HC having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody provided herein comprises an LC having the amino acid sequence of SEQ ID NO: 9 and an HC having the amino acid sequence of SEQ ID NO: 7.
在另一個態樣中,本文提供了包含經調配抗體的醫藥組合物。在所述醫藥組合物的一些實施例中,所述抗體包含具有如上所述的胺基酸序列的HC和LC,與蔗糖、L-組胺酸和聚山梨酯80組合。在一些實施例中,所述抗體以50 mg/mL的濃度存在,所述蔗糖以8%(w/v)的濃度存在,所述L-組胺酸以10 mM的濃度存在,所述聚山梨酯80(PS80)以0.05%(v/v)的濃度存在,並且所述調配物具有6.2的pH。在一些實施例中,所述醫藥組合物是凍乾的。In another aspect, provided herein is a pharmaceutical composition comprising a formulated antibody. In some embodiments of the pharmaceutical composition, the antibody comprises HC and LC having the amino acid sequence as described above, in combination with sucrose, L-histidine and polysorbate 80. In some embodiments, the antibody is present at a concentration of 50 mg/mL, the sucrose is present at a concentration of 8% (w/v), the L-histidine is present at a concentration of 10 mM, and the poly Sorbate 80 (PS80) is present at a concentration of 0.05% (v/v), and the formulation has a pH of 6.2. In some embodiments, the pharmaceutical composition is lyophilized.
在另一個態樣中,本文提供了包含經調配抗體的單位劑型。在一些實施例中,所述抗體包含具有如上所述的胺基酸序列的HC和LC,並且所述單位劑型包含215 mg的所述抗體、6.21 mg的L-組胺酸、344 mg的蔗糖和2.15 mg的聚山梨酯80。在一些實施例中,所述抗體的單位劑型是凍乾的。In another aspect, provided herein is a unit dosage form comprising the formulated antibody. In some embodiments, the antibody comprises HC and LC having the amino acid sequence as described above, and the unit dosage form comprises 215 mg of the antibody, 6.21 mg of L-histidine, and 344 mg of sucrose. And 2.15 mg of polysorbate 80. In some embodiments, the unit dosage form of the antibody is lyophilized.
在另一個態樣中,本文提供了用於製備醫藥組合物的方法,所述醫藥組合物包含特異性結合人類CD38的抗體。在一些實施例中,所述抗體包含具有如上所述的胺基酸序列的HC和LC,其中所述方法包括在細胞培養物中表現所述抗體,使所述抗體經受層析純化步驟和超過濾步驟中的至少一個以產生純化的抗體溶液;以及調節所述純化的抗體溶液以產生抗體調配物。在一些實施例中,所述抗體調配物包含所述純化的抗體、蔗糖、L-組胺酸和聚山梨酯80。在一些實施例中,抗體調配物包含濃度為50 mg/mL的抗體、濃度為8% w/v的蔗糖、濃度為10 mM的L-組胺酸和濃度為0.05% v/v的聚山梨酯80(PS80)。在一些實施例中,所述抗體調配物經製備以使得其具有6.2的pH。在用於製備所述抗體調配物的方法的一些實施例中,所述抗體調配物是凍乾的。In another aspect, provided herein is a method for preparing a pharmaceutical composition comprising an antibody that specifically binds to human CD38. In some embodiments, the antibody comprises HC and LC having amino acid sequences as described above, wherein the method includes expressing the antibody in cell culture, subjecting the antibody to a chromatographic purification step and ultra Filtering at least one of the steps to produce a purified antibody solution; and adjusting the purified antibody solution to produce an antibody formulation. In some embodiments, the antibody formulation comprises the purified antibody, sucrose, L-histidine, and polysorbate 80. In some embodiments, the antibody formulation comprises antibody at a concentration of 50 mg/mL, sucrose at a concentration of 8% w/v, L-histidine at a concentration of 10 mM, and polysorbate at a concentration of 0.05% v/v. Ester 80 (PS80). In some embodiments, the antibody formulation is prepared such that it has a pH of 6.2. In some embodiments of the method for preparing the antibody formulation, the antibody formulation is lyophilized.
在另一個態樣中,提供了用於製備重構的抗體調配物的方法。在一些實施例中,所述凍乾抗體調配物包含蔗糖、L-組胺酸、PS80和特異性結合人類CD38的抗體。在一些實施例中,所述抗體包含具有如上所述的胺基酸序列的HC和LC。在一些實施例中,所述凍乾的抗體調配物係在稀釋劑中重構,藉此製備所述重構的抗體調配物。在一些實施例中,所述重構的抗體調配物包含濃度為50 mg/mL的抗體、濃度為8% w/v的蔗糖、濃度為10 mM的L-組胺酸和濃度為0.05% v/v的PS80。在一些實施例中,所述重構的抗體調配物具有6.2的pH。In another aspect, a method for preparing a reconstituted antibody formulation is provided. In some embodiments, the lyophilized antibody formulation comprises sucrose, L-histidine, PS80, and an antibody that specifically binds to human CD38. In some embodiments, the antibody comprises HC and LC having amino acid sequences as described above. In some embodiments, the lyophilized antibody formulation is reconstituted in a diluent, thereby preparing the reconstituted antibody formulation. In some embodiments, the reconstituted antibody formulation comprises an antibody at a concentration of 50 mg/mL, sucrose at a concentration of 8% w/v, L-histidine at a concentration of 10 mM, and a concentration of 0.05% v. /v PS80. In some embodiments, the reconstituted antibody formulation has a pH of 6.2.
在另一個態樣中,提供了治療患有多發性骨髓瘤患者的方法。在一些實施例中,所述方法包括向所述患者投予一或多個劑量的特異性結合人類CD38的抗體,其中所述抗體包含具有如上所述的胺基酸序列的HC和LC。In another aspect, a method of treating a patient suffering from multiple myeloma is provided. In some embodiments, the method includes administering to the patient one or more doses of an antibody that specifically binds to human CD38, wherein the antibody comprises HC and LC having amino acid sequences as described above.
在另一個態樣中,提供了在治療多發性骨髓瘤的方法中使用的抗體。在一些實施例中,所述抗體特異性地結合人類CD38,其中所述抗體包含具有如上所述的胺基酸序列的HC和LC。In another aspect, an antibody for use in a method of treating multiple myeloma is provided. In some embodiments, the antibody specifically binds to human CD38, wherein the antibody comprises HC and LC having amino acid sequences as described above.
本文提供了抗CD38抗體,其對在細胞表面上表現高、中等和低量的CD38的細胞,具有優越的抗體依賴性細胞毒性(ADCC)。在一些實施例中,本文所提供的抗CD38抗體還對在細胞表面上表現CD38的細胞,具有優越的抗體依賴性細胞吞噬作用(ADCP)活性。在一些實施例中,本文所提供的抗體具有低於預期的等電點(pI)。儘管pI低於預期並且蛋白質在較低溫度下發生去折疊,這可能對抗體的穩定性(特別是在商業生產過程中)產生負面影響,但本文所提供的抗體係以適於向人類患者投予的穩定形式來提供。 抗體 Provided herein are anti-CD38 antibodies that have superior antibody-dependent cellular cytotoxicity (ADCC) to cells that exhibit high, medium, and low amounts of CD38 on the cell surface. In some embodiments, the anti-CD38 antibodies provided herein also have superior antibody-dependent cellular phagocytosis (ADCP) activity against cells expressing CD38 on the cell surface. In some embodiments, the antibodies provided herein have a lower than expected isoelectric point (pi). Although the pI is lower than expected and the protein unfolds at lower temperatures, which may have a negative impact on the stability of the antibody (especially in the commercial production process), the antibody system provided herein is suitable for administration to human patients. To provide it in a stable form. antibody
本文提供了特異性結合人類CD38的抗CD38抗體。可使用重組方法產生本文所提供的抗CD38抗體。為了重組產生抗抗原抗體,編碼抗體的核酸係經分離,並插入可複製載體中,以用於進一步選殖(DNA擴增)或表現。可以使用一般程序(例如,通過使用能夠與編碼抗體重鏈和輕鏈的基因特異性結合的寡核苷酸探針),輕易地分離編碼抗體的DNA並加以定序。許多載體是可用的。載體組分通常包括但不限於以下各項中的一或多個:信號序列、複製起點、一或多個標記基因、增強子元件、啟動子和轉錄終止序列。載體典型地被轉形(transform)到適用於核酸表現的宿主細胞中。在一些實施例中,宿主細胞是真核細胞或原核細胞。在一些實施例中,真核宿主細胞是哺乳動物細胞。有用的哺乳動物宿主細胞株的例子是由SV40轉形的猴腎CV1細胞株(COS-7,ATCC CRL 1651);人類胚腎細胞株(293或亞選殖用於在懸浮培養物中生長的293細胞, Graham等人, J. Gen Virol. 36:59 (1977));幼倉鼠腎細胞(BHK,ATCC CCL 10);小鼠睪丸支援細胞(TM4,Mather, Biol. Reprod. 23:243-251(1980));猴腎細胞(CV1,ATCC CCL 70);非洲綠猴腎細胞(VERO-76,ATCC CRL-1587);人類宮頸癌細胞(HELA,ATCC CCL 2);犬腎細胞(MDCK,ATCC CCL 34);buffalo大鼠肝細胞(BRL 3A,ATCC CRL 1442);人類肺細胞(W138,ATCC CCL 75);人類肝細胞(Hep G2,HB 8065);小鼠乳腺腫瘤(MMT 060562,ATCC CCL51);TRI細胞(Mather等人, Annals N.Y. Acad. Sci. 383:44-68 (1982));MRC 5細胞;FS4細胞;以及人類肝癌細胞株(Hep G2)。其他有用的哺乳動物宿主細胞株包括中國倉鼠卵巢(CHO)細胞,包括DHFR-CHO細胞(Urlaub等人, Proc. Natl. Acad. Sci. USA 77:4216 (1980));以及骨髓瘤細胞株,如NS0和Sp2/0。關於適用於抗體產生的某些哺乳動物宿主細胞株的綜述,參見例如Yazaki和Wu, Methods in Molecular Biology, 第248卷(B. K. C. Lo,編輯, Humana Press, Totowa, N.J., 2003), 第255-268頁。可使用例如羥基磷灰石層析、疏水相互作用層析、凝膠電泳、透析和親和層析(其中親和層析是一種通常較佳的純化步驟)來純化由細胞所製備的抗CD38抗體。通常,用於製備供研究、測試和臨床應用使用的抗體的各種方法,是業內已完善建立的、與上文所述方法是一致的,和/或被熟習此項技術者認為是適當的。Provided herein are anti-CD38 antibodies that specifically bind to human CD38. Recombinant methods can be used to produce the anti-CD38 antibodies provided herein. In order to recombinantly produce anti-antigen antibodies, the nucleic acid encoding the antibody is isolated and inserted into a replicable vector for further selection (DNA amplification) or performance. General procedures (for example, by using oligonucleotide probes that specifically bind to genes encoding antibody heavy and light chains) can be used to easily isolate and sequence antibody-encoding DNA. Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences. The vector is typically transformed into a host cell suitable for nucleic acid expression. In some embodiments, the host cell is a eukaryotic cell or a prokaryotic cell. In some embodiments, the eukaryotic host cell is a mammalian cell. Examples of useful mammalian host cell lines are monkey kidney CV1 cell lines (COS-7, ATCC CRL 1651) transformed by SV40; human embryonic kidney cell lines (293 or sub-selected for growth in suspension culture) 293 cells, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse testicular support cells (TM4, Mather, Biol. Reprod. 23:243- 251 (1980)); monkey kidney cells (CV1, ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK , ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse breast tumors (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982));
在一些實施例中,使用以饋料批次模式操作的500 L一次性生物反應器,由CHO 8D6宿主細胞株(DXB11衍生物)表現抗體。細胞培養過程是從解凍主細胞庫小瓶開始,接著是一系列種子培養(seed train)細胞擴增步驟。然後將細胞轉移到生物反應器中,並且使用無血清的化學成分確定的細胞培養基進行培養。10至14天後終止培養,以收取抗體。可以通過深層過濾從收穫的細胞培養物去除細胞和細胞培養物碎屑。In some embodiments, a 500 L disposable bioreactor operating in a feed batch mode was used to express antibodies from the CHO 8D6 host cell line (DXB11 derivative). The cell culture process starts with thawing the vial of the main cell bank, followed by a series of seed train cell expansion steps. The cells are then transferred to a bioreactor and cultured using a serum-free chemically defined cell culture medium. The culture was terminated after 10 to 14 days to collect the antibody. The cells and cell culture debris can be removed from the harvested cell culture by depth filtration.
接著通過層析和過濾步驟進一步加工收穫的材料以產生純化的抗體。在液體溶液中配製純化的抗體並將其在 ≤ -30ºC下儲存。The harvested material is then further processed through chromatography and filtration steps to produce purified antibodies. Prepare purified antibodies in liquid solutions and store them at ≤ -30ºC.
在將經調配抗體分配到合適的小瓶中之前,將液體溶液解凍,並使用0.2 μm過濾裝置進行無菌過濾。將經調配抗體分配到合適的容器(如USP 1型玻璃小瓶,4.3 mL/小瓶)中。在一些實施例中,填充的小瓶包含0.3 mL過量的經調配抗體。在一些實施例中,經調配抗體在小瓶中凍乾。Before distributing the formulated antibody into suitable vials, the liquid solution is thawed and sterile filtered using a 0.2 μm filter device. Dispense the formulated antibody into a suitable container (
術語「抗體」通常是指包含兩條重鏈(HC)和兩條輕鏈(LC)的四聚體蛋白。每個這種四聚體通常由兩對相同的多肽鏈構成,每對多肽鏈具有一條LC(通常具有約25 kDa的分子量)和一條HC(通常具有約50-70 kDa的分子量)。如本文所用,術語「HC」和「LC」是指具有足以賦予對靶抗原的特異性的可變結構域序列的任何免疫球蛋白多肽。每條輕鏈和重鏈的胺基末端部分包含約100至110個或更多個胺基酸的可變結構域,所述可變結構域通常負責抗原識別。每條鏈的羧基末端部分通常定義負責效應子功能的恒定結構域。因此,在典型抗體中,全長HC免疫球蛋白多肽包含一個可變結構域(VH)和三個恒定結構域(CH1、CH2和CH3),其中所述VH結構域位於多肽的胺基末端並且所述CH3結構域位於羧基末端,並且全長LC免疫球蛋白多肽包含一個可變結構域(VL)和一個恒定結構域(CL),其中所述VL結構域位於多肽的胺基末端並且所述CL結構域位於羧基末端。The term "antibody" generally refers to a tetrameric protein containing two heavy chains (HC) and two light chains (LC). Each such tetramer is usually composed of two pairs of identical polypeptide chains, each pair of polypeptide chains having an LC (usually with a molecular weight of about 25 kDa) and an HC (usually with a molecular weight of about 50-70 kDa). As used herein, the terms "HC" and "LC" refer to any immunoglobulin polypeptide having a variable domain sequence sufficient to confer specificity for the target antigen. The amino terminal portion of each light chain and heavy chain contains a variable domain of about 100 to 110 or more amino acids, which is generally responsible for antigen recognition. The carboxy terminal part of each chain usually defines the constant domain responsible for effector functions. Therefore, in a typical antibody, a full-length HC immunoglobulin polypeptide contains one variable domain (VH) and three constant domains (CH1, CH2, and CH3), where the VH domain is located at the amino terminal end of the polypeptide and is The CH3 domain is located at the carboxy terminus, and the full-length LC immunoglobulin polypeptide comprises a variable domain (VL) and a constant domain (CL), wherein the VL domain is located at the amino terminus of the polypeptide and the CL structure The domain is located at the carboxy terminus.
術語「抗體」在本文中以最廣泛的意義使用並且具體地包括如上所述的典型抗體,其包括單株抗體和多特異性抗體(例如,二特異性和三特異性抗體,只要它們展現所需的生物活性(包括與CD38靶標的特異性結合以及觸發ADCC和ADCP的能力)即可)。The term "antibody" is used in the broadest sense herein and specifically includes typical antibodies as described above, which include monoclonal antibodies and multispecific antibodies (eg, bispecific and trispecific antibodies, as long as they exhibit all The required biological activity (including the specific binding to the CD38 target and the ability to trigger ADCC and ADCP).
如本文所用術語「Fc」是指包含由抗體消化得到或通過其他手段產生的非抗原結合片段的序列的分子,所述分子呈單體或多聚體形式,並且所述「Fc」可以含有鉸鏈區。Fc分子由可通過共價(即,雙硫鍵)和非共價結合連接成二聚體或多聚體形式的單體多肽組成。取決於類別(例如,IgG、IgA和IgE)或亞類(例如,IgG1、IgG2、IgG3、IgA1、IgGA2和IgG4),典型Fc分子的單體亞基之間分子間雙硫鍵的數量在1至4範圍內。Fc的一個例子是由IgG的木瓜蛋白酶消化產生的雙硫鍵鍵結的二聚體。As used herein, the term "Fc" refers to a molecule containing a sequence of a non-antigen-binding fragment obtained by digestion of an antibody or produced by other means. The molecule is in the form of a monomer or a multimer, and the "Fc" may contain a hinge Area. Fc molecules are composed of monomeric polypeptides that can be linked into dimers or multimers by covalent (ie, disulfide bonds) and non-covalent bonds. Depending on the class (eg, IgG, IgA, and IgE) or subclass (eg, IgG1, IgG2, IgG3, IgA1, IgGA2, and IgG4), the number of intermolecular disulfide bonds between monomer subunits of a typical Fc molecule is 1 To the range of 4. An example of Fc is a disulfide bond dimer produced by papain digestion of IgG.
本文所述的抗體可以是分離的。術語「分離的蛋白質」、「分離的多肽」或「分離的抗體」是指如下蛋白質、多肽或抗體,所述蛋白質、多肽或抗體由於其起源或衍生來源而:(1) 與在其天然狀態下伴隨其的天然締合組分非締合,(2) 基本上不含來自相同物種的其他蛋白質,(3) 由來自不同物種的細胞表現,和/或 (4) 在自然界中不存在。因此,化學合成或在與其天然起源的細胞不同的細胞系統中合成的多肽將與其天然締合組分「分離」。也可以使用業內熟知的蛋白質純化技術,通過分離使蛋白質基本上不含天然締合組分。The antibodies described herein can be isolated. The term "isolated protein", "isolated polypeptide" or "isolated antibody" refers to the following protein, polypeptide or antibody, said protein, polypeptide or antibody due to its origin or derived source: (1) and in its natural state Naturally associated components that accompany it are non-associative, (2) substantially free of other proteins from the same species, (3) expressed by cells from different species, and/or (4) not present in nature. Therefore, a polypeptide synthesized chemically or in a cell system different from the cell of its natural origin will be "separated" from its naturally associated components. It is also possible to use protein purification techniques well-known in the industry to make the protein substantially free of naturally associated components through separation.
在一些實施例中,本文所提供的抗體包含如表1中提供的HC和LC胺基酸序列。表 1
術語「親和力」是指兩種多肽(例如像受體/配體或抗原/抗體)之間吸引的量度。兩種多肽之間的內在吸引力可以表示為特定相互作用的結合親和力平衡常數(KD)。當KD ≤ 1 μM、較佳 ≤ 100 nM時,抗體被稱為與抗原特異性地結合。KD結合親和力常數可以例如通過表面電漿共振(SPR)(BIAcore®)或生物膜層干涉技術(Bio-Layer Interferometry)來量測。The term "affinity" refers to the measure of attraction between two polypeptides (such as receptors/ligands or antigens/antibodies). The intrinsic attractive force between two polypeptides can be expressed as the binding affinity equilibrium constant (KD) for a specific interaction. When KD ≤ 1 μM, preferably ≤ 100 nM, the antibody is said to specifically bind to the antigen. The KD binding affinity constant can be measured, for example, by Surface Plasma Resonance (SPR) (BIAcore®) or Bio-Layer Interferometry.
術語「koff 」是指特定的抗體-抗原相互作用的解離速率常數。koff 解離速率常數可以例如通過生物膜層干涉技術來量測。與 CD38 的結合 The term "k off "refers to the dissociation rate constant of a specific antibody-antigen interaction. The k off dissociation rate constant can be measured, for example, by the biofilm layer interference technique. Combination with CD38
在一些實施例中,本文所提供的抗體包含含有兩條HC和兩條LC的四聚體蛋白質,並且當通過如下所述的表面電漿共振(SPR)量測時,係以約1.91 x 10-10 M至約6.7 x 10-10 M的親和力或KD與人類CD38結合。在一些實施例中,HC和LC分別具有以下胺基酸序列:SEQ ID NO: 2和7(mAb 2)、3和7(mAb 3)、4和7(mAb 4)、5和8(mAb 5)、5和9(mAb 6)、6和8(mAb 7)、或6和9(mAb 8)。In some embodiments, the antibody provided herein comprises a tetrameric protein containing two HCs and two LCs, and when measured by surface plasmon resonance (SPR) as described below, it is approximately 1.91 x 10 -10 M to about 6.7 x 10 -10 M affinity or KD binds to human CD38. In some embodiments, HC and LC have the following amino acid sequences, respectively: SEQ ID NO: 2 and 7 (mAb 2), 3 and 7 (mAb 3), 4 and 7 (mAb 4), 5 and 8 (mAb 5), 5 and 9 (mAb 6), 6 and 8 (mAb 7), or 6 and 9 (mAb 8).
在一些實施例中,抗體包含分別具有SEQ ID NO: 3和7的胺基酸序列的HC和LC,並且抗體可以以約2 x 10-10 M的親和力或KD與人類CD38結合。在一些實施例中,抗體包含分別具有SEQ ID NO: 5和8的胺基酸序列的HC和LC,並且抗體可以以約6.7 x 10-10 M的親和力或KD與人類CD38結合。在一些實施例中,抗體包含分別具有SEQ ID NO: 5和9的胺基酸序列的HC和LC,並且抗體可以以約4.15 x 10-10 M的親和力或KD與人類CD38結合。在一些實施例中,抗體包含分別具有SEQ ID NO: 6和8的胺基酸序列的HC和LC,並且抗體可以以約3.85 x 10-10 M的親和力或KD與人類CD38結合。在一些實施例中,抗體包含分別具有SEQ ID NO: 6和9的胺基酸序列的HC和LC,並且抗體可以以約1.91 x 10-10 M的親和力或KD與人類CD38結合。與 FcγRIIIa ( CD16a )和 FcγRIIa ( CD32a )的結合 In some embodiments, the antibody comprises HC and LC having the amino acid sequences of SEQ ID NOs: 3 and 7, respectively, and the antibody can bind to human CD38 with an affinity or KD of about 2×10 -10 M. In some embodiments, the antibody comprises HC and LC having the amino acid sequences of SEQ ID NO: 5 and 8, respectively, and the antibody can bind to human CD38 with an affinity or KD of about 6.7 x 10 -10 M. In some embodiments, the antibody comprises HC and LC having the amino acid sequences of SEQ ID NOs: 5 and 9, respectively, and the antibody can bind to human CD38 with an affinity or KD of about 4.15 x 10 -10 M. In some embodiments, the antibody comprises HC and LC having the amino acid sequences of SEQ ID NO: 6 and 8, respectively, and the antibody can bind to human CD38 with an affinity or KD of about 3.85 x 10 -10 M. In some embodiments, the antibody comprises HC and LC having the amino acid sequences of SEQ ID NOs: 6 and 9, respectively, and the antibody can bind to human CD38 with an affinity or KD of about 1.91 x 10 -10 M. Combination with FcγRIIIa ( CD16a ) and FcγRIIa ( CD32a)
本文所提供的抗體還能夠以分別少於一個數量級內的KD和以如通過SPR所量測小於100 nM的KD,與在胺基酸位置158處具有苯丙胺酸(F)的較低親和力FcγRIIIa(CD16a)變異體(158F)結合,並且能夠與在胺基酸位置158處具有擷胺酸(V)的較高親和力FcγRIIIa(CD16a)變異體(158V)結合。在一些實施例中,本文所提供的抗體以如通過SPR所量測的小於100 nM的KD與FcγRIIIa(CD16a)的158F變異體和158V變異體結合,並且其中與158F和158V變異體的結合相差小於2倍。在一些實施例中,當通過例如SPR量測時,本文所提供的抗體以約59 nM或更小的KD與FcγRIIIa(CD16a)變異體(158F)結合。The antibodies provided herein can also have a KD of less than one order of magnitude and a KD of less than 100 nM as measured by SPR, and a lower affinity FcγRIIIa with phenylalanine (F) at the amino acid position 158 ( CD16a) variant (158F) binds and can bind to the higher affinity FcγRIIIa (CD16a) variant (158V) with amino acid (V) at position 158 of the amino acid. In some embodiments, the antibodies provided herein bind to the 158F and 158V variants of FcγRIIIa (CD16a) with a KD of less than 100 nM as measured by SPR, and the binding to the 158F and 158V variants is different Less than 2 times. In some embodiments, the antibodies provided herein bind to the FcγRIIIa (CD16a) variant (158F) with a KD of about 59 nM or less when measured by, for example, SPR.
在一些實施例中,抗體包含具有SEQ ID NO: 2的胺基酸序列的HC和具有SEQ ID NO: 7的胺基酸序列的LC,並且能夠以約53 nM的KD與FcγRIIIa(158F)結合並且以約47 nM的KD與FcγRIIIa(158V)結合,如通過例如SPR所量測。在一些實施例中,抗體包含具有SEQ ID NO: 3的胺基酸序列的HC和具有SEQ ID NO: 7的胺基酸序列的LC,並且能夠以約59 nM的KD與FcγRIIIa(158F)結合並且以約75 nM的KD與FcγRIIIa(158V)結合,如通過例如SPR所量測。在一些實施例中,抗體包含具有SEQ ID NO: 4的胺基酸序列的HC和具有SEQ ID NO: 7的胺基酸序列的LC,並且能夠以約51 nM的KD與FcγRIIIa(158F)結合並且以約47 nM的KD與FcγRIIIa(158V)結合,如通過例如SPR所量測。In some embodiments, the antibody comprises HC with the amino acid sequence of SEQ ID NO: 2 and LC with the amino acid sequence of SEQ ID NO: 7, and is capable of binding to FcγRIIIa (158F) with a KD of about 53 nM And it binds to FcγRIIIa (158V) with a KD of about 47 nM, as measured by, for example, SPR. In some embodiments, the antibody comprises HC with the amino acid sequence of SEQ ID NO: 3 and LC with the amino acid sequence of SEQ ID NO: 7, and is capable of binding to FcγRIIIa (158F) with a KD of about 59 nM And it binds to FcγRIIIa (158V) with a KD of about 75 nM, as measured by, for example, SPR. In some embodiments, the antibody comprises HC with the amino acid sequence of SEQ ID NO: 4 and LC with the amino acid sequence of SEQ ID NO: 7, and is capable of binding to FcγRIIIa (158F) with a KD of about 51 nM And it binds to FcγRIIIa (158V) with a KD of about 47 nM, as measured by, for example, SPR.
本文所提供的抗體還能夠以 ≤ 690 nM的KD與在胺基酸位置131處具有精胺酸的較低親和力FcγRIIa(CD32a)變異體(131R)結合,並且以小於約270 nM的KD與在位置131處具有組胺酸的較高親和力FcγRIIa(CD32a)變異體(131H)結合,如通過例如SPR所量測。The antibodies provided herein can also bind with a lower affinity FcγRIIa (CD32a) variant (131R) with arginine at position 131 of the amino acid with a KD of ≤ 690 nM, and with a KD of less than about 270 nM with The higher affinity FcγRIIa (CD32a) variant (131H) binding with histidine at position 131, as measured by, for example, SPR.
在一些實施例中,抗體包含具有SEQ ID NO: 2的胺基酸序列的HC和具有SEQ ID NO: 7的胺基酸序列的LC,並且能夠以約690 nM的KD與FcγRIIa(CD32a)(131R)結合,並且以約120 nM的KD與FcγRIIa(CD32a)(131H)結合,如通過例如SPR所量測。在一些實施例中,抗體包含具有SEQ ID NO: 3的胺基酸序列的HC和具有SEQ ID NO: 7的胺基酸序列的LC,並且能夠以 ≤ 約125 nM或 ≤ 100 nM的KD與FcγRIIa(CD32a)(131R)結合,並且以約220 nm的KD與FcγRIIa(CD32a)(131H)結合,如通過例如SPR所量測。在一些實施例中,抗體包含具有SEQ ID NO: 4的胺基酸序列的HC和具有SEQ ID NO: 7的胺基酸序列的LC,並且能夠以約510 nM的KD與FcγRIIa(CD32a)(131R)結合,並且以約60 nM的KD與FcγRIIa(CD32a)(131H)結合,如通過例如SPR所量測。In some embodiments, the antibody comprises HC with the amino acid sequence of SEQ ID NO: 2 and LC with the amino acid sequence of SEQ ID NO: 7, and is capable of interacting with FcγRIIa (CD32a) with a KD of about 690 nM ( 131R) binds and binds to FcγRIIa (CD32a) (131H) with a KD of about 120 nM, as measured by, for example, SPR. In some embodiments, the antibody comprises an HC having the amino acid sequence of SEQ ID NO: 3 and an LC having the amino acid sequence of SEQ ID NO: 7, and can be combined with a KD of ≤ about 125 nM or ≤ 100 nM. FcγRIIa (CD32a) (131R) binds and binds to FcγRIIa (CD32a) (131H) with a KD of about 220 nm, as measured by, for example, SPR. In some embodiments, the antibody comprises an HC having the amino acid sequence of SEQ ID NO: 4 and an LC having the amino acid sequence of SEQ ID NO: 7, and is capable of interacting with FcγRIIa (CD32a) with a KD of about 510 nM ( 131R) binds and binds to FcγRIIa (CD32a) (131H) with a KD of about 60 nM, as measured by, for example, SPR.
在一些實施例中,抗體還能夠以約70 nM或更小的表觀KD與FcγRIIIa(CD16a)158F結合,並且以約32 nM或更小的表觀KD與FcγRIIIa(CD16a)158V結合,如分別通過與表現FcγRIIIa(CD16a)158F或158V的HEK細胞的結合所量測。在一些實施例中,包含具有SEQ ID NO: 2的胺基酸序列的HC和具有SEQ ID NO: 7的胺基酸序列的LC的抗體還能夠以約70 nM的表觀KD與FcγRIIIa(CD16a)158F結合,並且以約18 nM的表觀KD與FcγRIIIa(CD16a)158V結合,如分別通過與表現FcγRIIIa(CD16a)158F或158V的HEK細胞的結合所量測。在一些實施例中,包含具有SEQ ID NO: 3的胺基酸序列的HC和具有SEQ ID NO: 7的胺基酸序列的LC的抗體還能夠以約60 nM的表觀KD與FcγRIIIa(CD16a)158F結合,並且以約32 nM的表觀KD與FcγRIIIa(CD16a)158V結合,如分別通過與表現FcγRIIIa(CD16a)158F或158V的HEK細胞的結合所量測。在一些實施例中,包含具有SEQ ID NO: 4的胺基酸序列的HC和具有SEQ ID NO: 7的胺基酸序列的LC的抗體還能夠以約44 nM的表觀KD與FcγRIIIa(CD16a)158F結合,並且以約28 nM的表觀KD與FcγRIIIa(CD16a)158V結合,如分別通過與表現FcγRIIIa(CD16a)158F或158V的HEK細胞的結合所量測。In some embodiments, the antibody can also bind to FcγRIIIa (CD16a) 158F with an apparent KD of about 70 nM or less, and bind to FcγRIIIa (CD16a) 158V with an apparent KD of about 32 nM or less, such as respectively Measured by binding to HEK cells expressing FcγRIIIa (CD16a) 158F or 158V. In some embodiments, the antibody comprising the HC having the amino acid sequence of SEQ ID NO: 2 and the LC having the amino acid sequence of SEQ ID NO: 7 is also capable of interacting with FcγRIIIa (CD16a) with an apparent KD of about 70 nM. ) 158F binds and binds to FcγRIIIa (CD16a) 158V with an apparent KD of about 18 nM, as measured by binding to HEK cells expressing FcγRIIIa (CD16a) 158F or 158V, respectively. In some embodiments, the antibody comprising the HC having the amino acid sequence of SEQ ID NO: 3 and the LC having the amino acid sequence of SEQ ID NO: 7 is also capable of interacting with FcγRIIIa (CD16a) with an apparent KD of about 60 nM. ) 158F binds and binds to FcγRIIIa (CD16a) 158V with an apparent KD of approximately 32 nM, as measured by binding to HEK cells expressing FcγRIIIa (CD16a) 158F or 158V, respectively. In some embodiments, the antibody comprising the HC having the amino acid sequence of SEQ ID NO: 4 and the LC having the amino acid sequence of SEQ ID NO: 7 is also capable of interacting with FcγRIIIa (CD16a) with an apparent KD of about 44 nM. ) 158F binds and binds to FcγRIIIa (CD16a) 158V with an apparent KD of about 28 nM, as measured by binding to HEK cells expressing FcγRIIIa (CD16a) 158F or 158V, respectively.
在一些實施例中,抗體還能夠以約890 nM或更小的表觀KD與在胺基酸位置131處具有精胺酸的FcγRIIa(CD32a)變異體(131R)結合,並且以約840 nM或更小的表觀KD與FcγRIIa(CD32a)變異體131H結合,如分別通過與表現FcγRIIa(CD32a)131R或131H的HEK細胞的結合所量測。在一些實施例中,包含具有SEQ ID NO: 2的胺基酸序列的HC和具有SEQ ID NO: 7的胺基酸序列的LC的抗體還能夠以約890 nM的表觀KD與FcγRIIa(CD32a)131R結合,並且以約840 nM的表觀KD與FcγRIIa(CD32a)131H結合,如分別通過與表現FcγRIIa(CD32a)131R或131H的HEK細胞的結合所量測。在一些實施例中,包含具有SEQ ID NO: 3的胺基酸序列的HC和具有SEQ ID NO: 7的胺基酸序列的LC的抗體還能夠以約87 nM的表觀KD與FcγRIIa(CD32a)131R結合,並且以約222 nM的表觀KD與FcγRIIa(CD32a)131H結合,如分別通過與表現FcγRIIa(CD32a)131R或131H的HEK細胞的結合所量測。在一些實施例中,包含具有SEQ ID NO: 4的胺基酸序列的HC和具有SEQ ID NO: 7的胺基酸序列的LC的抗體還能夠以約467 nM的表觀KD與FcγRIIa(CD32a)131R結合,並且以約544 nM的表觀KD與FcγRIIa(CD32a)131H結合,如分別通過與表現FcγRIIa(CD32a)131R或131H的HEK細胞的結合所量測。作用機制 In some embodiments, the antibody can also bind to the FcγRIIa (CD32a) variant (131R) with arginine at position 131 of the amino acid with an apparent KD of about 890 nM or less, and with an apparent KD of about 840 nM or The smaller apparent KD binds to the FcγRIIa (CD32a) variant 131H, as measured by binding to HEK cells expressing FcγRIIa (CD32a) 131R or 131H, respectively. In some embodiments, the antibody comprising the HC with the amino acid sequence of SEQ ID NO: 2 and the LC with the amino acid sequence of SEQ ID NO: 7 is also capable of interacting with FcγRIIa (CD32a) with an apparent KD of about 890 nM. ) 131R binds and binds to FcγRIIa (CD32a) 131H with an apparent KD of approximately 840 nM, as measured by binding to HEK cells expressing FcγRIIa (CD32a) 131R or 131H, respectively. In some embodiments, the antibody comprising the HC having the amino acid sequence of SEQ ID NO: 3 and the LC having the amino acid sequence of SEQ ID NO: 7 is also capable of interacting with FcγRIIa (CD32a) with an apparent KD of about 87 nM. ) 131R binds and binds to FcγRIIa (CD32a) 131H with an apparent KD of about 222 nM, as measured by binding to HEK cells expressing FcγRIIa (CD32a) 131R or 131H, respectively. In some embodiments, the antibody comprising the HC having the amino acid sequence of SEQ ID NO: 4 and the LC having the amino acid sequence of SEQ ID NO: 7 can also interact with FcγRIIa (CD32a) with an apparent KD of about 467 nM. ) 131R binds and binds to FcγRIIa (CD32a) 131H with an apparent KD of approximately 544 nM, as measured by binding to HEK cells expressing FcγRIIa (CD32a) 131R or 131H, respectively. Mechanism
本文所提供的抗體能夠通過在細胞表面上表現高量(約400,000個/細胞)、中等(約100,000個/細胞)和低量(約13,000個/細胞)的CD38分子的細胞的抗體依賴性細胞毒性,來殺滅表現CD38的細胞。本文所提供的抗體在存在自然殺手(NK)細胞的情況下觸發這種ADCC,所述NK細胞表現較低親和力的FcγIIIa(CD16a)受體變異體(158F)和/或表現較高親和力的FcγIIIa(CD16a)受體變異體(158V)。ADCC可以使用業內已知的方法量測,例如通過在基於細胞的效能分析中在存在抗體和效應細胞的情況下量測目標細胞溶解來量測。目標細胞可以是例如表現CD38的細胞,如KMS12-BM、RPMI-8226或MOLP-8。另外,效應細胞可以是例如可被誘導以通過ADCC殺傷目標細胞的任何細胞。在一些實施例中,效應細胞是從人類血液分離的原代細胞,如周邊血液單核球細胞(PBMC)或從PBMC純化的人類NK細胞。在另一個實施例中,細胞是自然殺手細胞株(如NK-92細胞),其在細胞表面上表現FcγRIIIa(158V)或FcγRIIIa(158F)(參見WO 06/023148)。在仍其他實施例中,NK細胞株是諸如Jurkat細胞株的細胞株,其已被工程化以表現FcγIIIa(CD16a)158F或158V(參見例如Promega, G701A)。The antibodies provided herein are capable of expressing high amounts (about 400,000/cell), medium (about 100,000/cell) and low amounts (about 13,000/cell) of CD38 molecule antibody-dependent cells on the cell surface. Toxic to kill cells that express CD38. The antibodies provided herein trigger this ADCC in the presence of natural killer (NK) cells that exhibit a lower affinity FcγIIIa (CD16a) receptor variant (158F) and/or a higher affinity FcγIIIa (CD16a) receptor variant (158V). ADCC can be measured using methods known in the industry, for example, by measuring target cell lysis in the presence of antibodies and effector cells in cell-based potency analysis. The target cell may be, for example, a cell expressing CD38, such as KMS12-BM, RPMI-8226, or MOLP-8. In addition, the effector cell may be any cell that can be induced to kill the target cell by ADCC, for example. In some embodiments, the effector cells are primary cells isolated from human blood, such as peripheral blood mononuclear cells (PBMC) or human NK cells purified from PBMC. In another embodiment, the cell is a natural killer cell line (eg, NK-92 cell), which expresses FcyRIIIa (158V) or FcyRIIIa (158F) on the cell surface (see WO 06/023148). In still other embodiments, the NK cell line is a cell line such as the Jurkat cell line, which has been engineered to express FcyIIIa (CD16a) 158F or 158V (see, for example, Promega, G701A).
本文所提供的抗體能夠通過抗體依賴性細胞吞噬作用(ADCP)殺滅在細胞表面上表現高量的CD38的細胞。本文所提供的抗體在存在人類PBMC的情況下觸發這種ADCP。在一個實施例中,本文所提供的抗體以212.6 pM的相對EC50通過表現FcγRIIIa(158V)的人類PMBC觸發對表現CD38的細胞的約41%吞噬作用。醫藥組合物和經調配抗體 The antibodies provided herein can kill cells that exhibit high amounts of CD38 on the cell surface through antibody-dependent cellular phagocytosis (ADCP). The antibodies provided herein trigger this ADCP in the presence of human PBMC. In one example, the antibody provided herein triggers about 41% phagocytosis of CD38-expressing cells by human PMBC expressing FcγRIIIa (158V) with a relative EC50 of 212.6 pM. Pharmaceutical composition and formulated antibody
術語「醫藥調配物」是指以下的製劑:其處於使得活性成分的生物活性有效的形式,並且不含對接受製劑的受試者具有不可接受的毒性的額外組分。此類調配物通常是無菌的。「醫藥上可接受的」賦形劑(媒劑、添加劑)是那些可以合理地投予受試哺乳動物以提供有效劑量之所用活性成分。The term "pharmaceutical formulation" refers to a preparation that is in a form that makes the biological activity of the active ingredient effective and does not contain additional components that have unacceptable toxicity to the subject receiving the preparation. Such formulations are usually sterile. "Pharmaceutically acceptable" excipients (vehicles, additives) are those active ingredients that can be reasonably administered to the tested mammal to provide an effective dose.
本文所提供的抗體的醫藥組合物和調配物可以通過將具有所需純度的抗體與一種或多種任選的醫藥上可接受的載劑(Remington’s Pharmaceutical Sciences 第16版, Osol, A.編輯(1980))混合來製備,並且可以以凍乾調配物或水溶液的形式提供。The pharmaceutical compositions and formulations of the antibodies provided herein can be prepared by combining an antibody of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th Edition, Osol, A. Edited (1980). )) It is prepared by mixing, and can be provided in the form of a lyophilized formulation or an aqueous solution.
在一些實施例中,本文所提供的醫藥組合物包含經調配抗體,所述經調配抗體包含抗體和一種或多種下列賦形劑:蔗糖、L-組胺酸和聚山梨酯80(PS80)。在一些實施例中,抗體包含表1中提供的任一種單株抗體的HC和LC胺基酸序列。在一些實施例中,抗體以5 mg/mL至50 mg/mL的濃度存在於醫藥組合物中。在一些實施例中,抗體以5 mg/mL的濃度存在於醫藥組合物中。在一些實施例中,抗體以10 mg/mL的濃度存在於醫藥組合物中。在一些實施例中,抗體以20 mg/mL的濃度存在於醫藥組合物中。在一些實施例中,抗體以50 mg/mL的濃度存在於醫藥組合物中。選擇每種賦形劑的濃度使得醫藥組合物可被稀釋用於通過輸注投予。例如,在一些實施例中,蔗糖以8%(w/v)至10%(w/v)的濃度存在。在一些實施例中,蔗糖以8%(w/v)的濃度存在。在一些實施例中,蔗糖以10%(w/v)的濃度存在。在一些實施例中,L-組胺酸以10 mM至20 mM的濃度存在。在一些實施例中,L-組胺酸以10 mM的濃度存在。在一些實施例中,L-組胺酸以20 mM的濃度存在。在一些實施例中,PS80以0.005%(v/v)至0.05%(v/v)的濃度存在。在一些實施例中,PS80以0.005%(v/v)的濃度存在。在一些實施例中,PS80以0.02%(v/v)的濃度存在。在一些實施例中,PS80以0.05%(v/v)的濃度存在。在一些實施例中,將醫藥組合物的pH優化以維持例如在醫藥組合物呈液體形式時抗體的穩定性。在一些實施例中,經調配抗體的pH具有約6.0至約6.5的pH。在一些實施例中,經調配抗體具有約6.0的pH。在一些實施例中,經調配抗體具有約6.2的pH。在一些實施例中,經調配抗體具有約6.5的pH。在一些實施例中,經調配抗體具有6.0的pH。在一些實施例中,經調配抗體具有6.2的pH。在一些實施例中,經調配抗體具有6.5的pH。In some embodiments, the pharmaceutical compositions provided herein comprise a formulated antibody comprising the antibody and one or more of the following excipients: sucrose, L-histidine, and polysorbate 80 (PS80). In some embodiments, the antibody comprises the HC and LC amino acid sequences of any of the monoclonal antibodies provided in Table 1. In some embodiments, the antibody is present in the pharmaceutical composition at a concentration of 5 mg/mL to 50 mg/mL. In some embodiments, the antibody is present in the pharmaceutical composition at a concentration of 5 mg/mL. In some embodiments, the antibody is present in the pharmaceutical composition at a concentration of 10 mg/mL. In some embodiments, the antibody is present in the pharmaceutical composition at a concentration of 20 mg/mL. In some embodiments, the antibody is present in the pharmaceutical composition at a concentration of 50 mg/mL. The concentration of each excipient is selected so that the pharmaceutical composition can be diluted for administration by infusion. For example, in some embodiments, sucrose is present at a concentration of 8% (w/v) to 10% (w/v). In some embodiments, sucrose is present at a concentration of 8% (w/v). In some embodiments, sucrose is present at a concentration of 10% (w/v). In some embodiments, L-histidine is present at a concentration of 10 mM to 20 mM. In some embodiments, L-histidine is present at a concentration of 10 mM. In some embodiments, L-histidine is present at a concentration of 20 mM. In some embodiments, PS80 is present at a concentration of 0.005% (v/v) to 0.05% (v/v). In some embodiments, PS80 is present at a concentration of 0.005% (v/v). In some embodiments, PS80 is present at a concentration of 0.02% (v/v). In some embodiments, PS80 is present at a concentration of 0.05% (v/v). In some embodiments, the pH of the pharmaceutical composition is optimized to maintain the stability of the antibody when the pharmaceutical composition is in liquid form, for example. In some embodiments, the pH of the formulated antibody has a pH of about 6.0 to about 6.5. In some embodiments, the formulated antibody has a pH of about 6.0. In some embodiments, the formulated antibody has a pH of about 6.2. In some embodiments, the formulated antibody has a pH of about 6.5. In some embodiments, the formulated antibody has a pH of 6.0. In some embodiments, the formulated antibody has a pH of 6.2. In some embodiments, the formulated antibody has a pH of 6.5.
在一些實施例中,本文所提供的醫藥組合物的抗體和/或經調配抗體包含一種或多種帶電同種型。帶電同種型通常被描述為主要同種型、酸性同種型和鹼性同種型。主要同種型是指在給定批次的抗體中最普遍的同種型。一種或多種酸性同種型與主要同種型相比具有較低的等電點(pI),並且鹼性同種型與主要同種型相比具有較高的pH。帶電同種型可以通過對HC和/或LC的胺基酸序列的翻譯後修飾而產生。例如,去醯胺基、糖化和唾液酸化的增加可能導致抗體pI的降低。N末端麩胺酸向焦麩胺酸(或pyroQ)的轉化導致失去一個正電荷,這可能導致pI的降低。另外,C末端賴胺酸的存在可能導致抗體pI的增加。此外,C末端脯胺酸的醯胺化可能導致抗體pI的增加。In some embodiments, the antibodies and/or formulated antibodies of the pharmaceutical compositions provided herein comprise one or more charged isotypes. Charged isoforms are usually described as major isoforms, acidic isoforms, and basic isoforms. The major isotype refers to the most common isotype in a given batch of antibodies. One or more acidic isotypes have a lower isoelectric point (pI) compared to the main isotype, and the basic isotype has a higher pH than the main isotype. Charged isoforms can be generated by post-translational modification of the amino acid sequence of HC and/or LC. For example, an increase in deamidation, glycation, and sialylation may lead to a decrease in antibody pI. The conversion of N-terminal glutamic acid to pyroglutamic acid (or pyroQ) results in the loss of a positive charge, which may lead to a decrease in pI. In addition, the presence of C-terminal lysine may lead to an increase in antibody pI. In addition, the amidation of C-terminal proline may lead to an increase in antibody pI.
在本文所提供的抗體(包括醫藥組合物中的抗體和經調配抗體)的一些實施例中,在HC胺基酸序列的位置1處的胺基酸是焦麩醯胺酸。在本文所提供的抗體的一些實施例中,HC具有由甘胺酸組成的C端胺基酸。In some embodiments of the antibodies (including antibodies in pharmaceutical compositions and formulated antibodies) provided herein, the amino acid at
本文所提供的抗體的帶電同種型可以使用業內已知的用於基於電荷分離多肽的方法來分離和量化。例如,在一些實施例中,可以在具有磷酸鹽/氯化鈉梯度緩衝液的高效液相層析(HPLC)系統中使用弱陽離子交換管柱。在此系統中,將抗體裝載到管柱上之後,抗體的酸性同種型會先沖提出來,接著是抗體的主要同種型,然後是酸性同種型。在另一個實施例中,可以使用毛細管等電聚焦(cIEF)。毛細管等電聚焦是將蛋白質通過其等電點(pI)值來分離的方法。在cIEF中,抗體樣品在毛細管內的pH梯度上遷移到其等電點,從而沿毛細管長度解析不同的帶電同種型。為了鑒定和表徵cIEF變異體,可以通過強陽離子交換層析(SCX)分離帶電同種型,並通過cIEF對其進行分析。採用全管柱檢測和量測在280 nm處的吸光度,使用電荷耦合器件照相機來採集毛細管中解析出的帶電同種型的圖像。使用cIEF將測試樣品的帶電同種型分佈與參考標準相比較,以鑒定抗體的帶電同種型。在一些實施例中,cIEF被用作品質控制措施以便在商業生產期間確認抗體的身份,並通過與參考標準相比(例如,具有在預先定義的分佈型式內的帶電同種型分佈型式的測試抗體)或與參考標準的分佈型式相比確定抗體的帶電同種型分佈。The charged isotypes of the antibodies provided herein can be separated and quantified using methods known in the art for separating polypeptides based on charge. For example, in some embodiments, a weak cation exchange column may be used in a high performance liquid chromatography (HPLC) system with a phosphate/sodium chloride gradient buffer. In this system, after the antibody is loaded onto the column, the acidic isotype of the antibody will be extracted first, followed by the main isotype of the antibody, and then the acidic isotype. In another embodiment, capillary isoelectric focusing (cIEF) can be used. Capillary isoelectric focusing is a method of separating proteins by their isoelectric point (pI) value. In cIEF, the antibody sample migrates to its isoelectric point on the pH gradient in the capillary, thereby resolving different charged isotypes along the length of the capillary. In order to identify and characterize cIEF variants, the charged isoforms can be separated by strong cation exchange chromatography (SCX) and analyzed by cIEF. The entire column is used to detect and measure the absorbance at 280 nm, and a charge-coupled device camera is used to collect images of the charged isoforms resolved in the capillary. Use cIEF to compare the charged isotype distribution of the test sample with a reference standard to identify the charged isotype of the antibody. In some embodiments, cIEF is used as a quality control measure to confirm the identity of the antibody during commercial production and pass comparison with a reference standard (for example, a test antibody with a charged isotype distribution pattern within a predefined distribution pattern ) Or compared with the distribution pattern of the reference standard to determine the distribution of the charged isotype of the antibody.
在一些實施例中,抗體(包括醫藥組合物中的抗體和經調配抗體)包含表1中提供的任一種單株抗體的HC和LC胺基酸序列,以及選自至少約70%主要同種型、不多於約30%酸性同種型和小於4%鹼性同種型的一種或多種帶電同種型參數。在一些實施例中,抗體包含71%主要同種型、28%酸性同種型和小於4%鹼性同種型。在一些實施例中,抗體含有具有在約5.8至約9.0範圍內的等電點的帶電同種型。在一些實施例中,抗體含有具有在約5.85至約8.97範圍內的等電點的帶電同種型。In some embodiments, the antibody (including the antibody in the pharmaceutical composition and the formulated antibody) comprises the HC and LC amino acid sequences of any of the monoclonal antibodies provided in Table 1, and is selected from at least about 70% of the major isotypes , No more than about 30% acid isotype and less than 4% basic isotype one or more charged isotype parameters. In some embodiments, the antibody comprises 71% major isotype, 28% acidic isotype, and less than 4% basic isotype. In some embodiments, the antibody contains a charged isotype with an isoelectric point in the range of about 5.8 to about 9.0. In some embodiments, the antibody contains a charged isotype having an isoelectric point in the range of about 5.85 to about 8.97.
在一些實施例中,抗體(包括醫藥組合物中的抗體和經調配抗體)是凍乾的。抗體(包括醫藥組合物中的抗體和經調配抗體)可以在容器(例如小瓶)中使得可從容器移取處方劑量的抗體以用於向患者投予。In some embodiments, antibodies (including antibodies and formulated antibodies in pharmaceutical compositions) are lyophilized. Antibodies (including antibodies and formulated antibodies in pharmaceutical compositions) can be in a container (eg, vial) so that a prescribed dose of antibody can be removed from the container for administration to a patient.
本文提供了特異性結合CD38的經調配抗體的單位劑型。在一些實施例中,抗體包含表1中提供的任一種單株抗體的HC和LC胺基酸序列,並且單位劑型包含抗體和一或多種賦形劑,該賦形劑係選自蔗糖、L-組胺酸和聚山梨酯80。在一些實施例中,單位劑型包含約215 mg抗體、約6.21 mg的L-組胺酸、約344 mg的蔗糖和約2.15 mg的聚山梨酯80。在一些實施例中,單位劑型包含215 mg抗體、6.21 mg的L-組胺酸、344 mg的蔗糖和2.15 mg的聚山梨酯80。在一些實施例中,單位劑型是凍乾的。單位劑型可以在容器(例如小瓶)中使得可從容器移取處方劑量的抗體以用於向患者投予。在一些實施例中,抗體、醫藥調配物和/或經調配抗體是在裝有彈性封閉件(closure)的玻璃小瓶中。在一些實施例中,小瓶含有約215 mg抗體。在一些實施例中,小瓶含有215 mg抗體。在一些實施例中,已確定小瓶的填充體積,以使得能夠移取約4 mL。在一些實施例中,已確定小瓶的填充體積,以使得能夠移取4 mL。製備方法 Provided herein are unit dosage forms of formulated antibodies that specifically bind CD38. In some embodiments, the antibody comprises the HC and LC amino acid sequences of any of the monoclonal antibodies provided in Table 1, and the unit dosage form comprises the antibody and one or more excipients, the excipient being selected from sucrose, L -Histidine and Polysorbate 80. In some embodiments, the unit dosage form contains about 215 mg of antibody, about 6.21 mg of L-histidine, about 344 mg of sucrose, and about 2.15 mg of polysorbate 80. In some embodiments, the unit dosage form contains 215 mg of antibody, 6.21 mg of L-histidine, 344 mg of sucrose, and 2.15 mg of polysorbate 80. In some embodiments, the unit dosage form is lyophilized. The unit dosage form can be in a container (eg, vial) such that a prescribed dose of antibody can be removed from the container for administration to a patient. In some embodiments, the antibodies, pharmaceutical formulations, and/or formulated antibodies are in glass vials equipped with elastic closures. In some embodiments, the vial contains about 215 mg of antibody. In some embodiments, the vial contains 215 mg of antibody. In some embodiments, the fill volume of the vial has been determined so that about 4 mL can be removed. In some embodiments, the fill volume of the vial has been determined so that 4 mL can be removed. Preparation
本文提供了用於製備抗體、醫藥組合物和單位劑型的方法。在一些實施例中,抗體包含表1中提供的任一種單株抗體的HC和LC胺基酸序列。在一個實施例中,方法包括在合適的細胞培養物中表現抗體。使抗體經受至少一個層析步驟和至少一個超過濾步驟,從而產生純化的抗體溶液。對純化的抗體溶液進行調節以濃縮抗體並添加賦形劑和調節pH。在一個實施例中,抗體的濃度係調節至約50 mg/ml;添加蔗糖、L-組胺酸和聚山梨酯80,且pH係調節至至少約6.0。在一個實施例中,抗體的濃度係調節至50 mg/ml;添加蔗糖、L-組胺酸和聚山梨酯80,且pH係調節至至少6.0。在一些實施例中,pH為約6.2。在一些實施例中,pH為6.2。在一些實施例中,蔗糖的濃度為約8% w/v,L-組胺酸的濃度為約10 mM;並且聚山梨酯80的濃度為約0.05% v/v。在一些實施例中,蔗糖的濃度為8% w/v,L-組胺酸的濃度為10 mM;並且聚山梨酯80的濃度為0.05% v/v。在一些實施例中,抗體、醫藥組合物和/或經調配抗體是凍乾的。Provided herein are methods for preparing antibodies, pharmaceutical compositions, and unit dosage forms. In some embodiments, the antibody comprises the HC and LC amino acid sequences of any of the monoclonal antibodies provided in Table 1. In one embodiment, the method includes expressing the antibody in a suitable cell culture. The antibody is subjected to at least one chromatography step and at least one ultrafiltration step, thereby producing a purified antibody solution. The purified antibody solution is adjusted to concentrate the antibody and add excipients and adjust the pH. In one embodiment, the concentration of the antibody is adjusted to about 50 mg/ml; sucrose, L-histidine and polysorbate 80 are added, and the pH is adjusted to at least about 6.0. In one embodiment, the concentration of the antibody is adjusted to 50 mg/ml; sucrose, L-histidine and polysorbate 80 are added, and the pH is adjusted to at least 6.0. In some embodiments, the pH is about 6.2. In some embodiments, the pH is 6.2. In some embodiments, the concentration of sucrose is about 8% w/v, the concentration of L-histidine is about 10 mM; and the concentration of polysorbate 80 is about 0.05% v/v. In some embodiments, the concentration of sucrose is 8% w/v, the concentration of L-histidine is 10 mM; and the concentration of polysorbate 80 is 0.05% v/v. In some embodiments, the antibody, pharmaceutical composition, and/or formulated antibody is lyophilized.
在一些實施例中,提供了製備重構的經調配抗體的方法,其中經調配抗體是以凍乾形式提供,並且包含表1中所提供之任一個單株抗體的HC和LC胺基酸序列、濃度為約8% w/v的蔗糖、濃度為約10 mM的L-組胺酸,和濃度為約0.05% v/v的聚山梨酯80。在一些實施例中,提供了製備重構的經調配抗體的方法,其中經調配抗體是以凍乾形式提供,並且包含表1中所提供之任一個單株抗體的HC和LC胺基酸序列、濃度為8% w/v的蔗糖、濃度為10 mM的L-組胺酸,和濃度為0.05% v/v的聚山梨酯80。在一些實施例中,在使用之前,在合適體積的注射用水中重構經調配抗體以產生溶液,所述溶液包含濃度為約50 mg/mL的抗體、濃度為約8% w/v的蔗糖、濃度為約10 mM的L-組胺酸和濃度為約0.05% v/v的聚山梨酯80,其中重構的抗體的pH為約6.2。在一些實施例中,在使用之前,在合適體積的注射用水中重構經調配抗體以產生包含溶液,所述溶液包含濃度為50 mg/mL的抗體、濃度為8% w/v的蔗糖、濃度為10 mM的L-組胺酸和濃度為0.05% v/v的聚山梨酯80,其中重構的抗體的pH為6.2。In some embodiments, a method for preparing a reconstituted formulated antibody is provided, wherein the formulated antibody is provided in a lyophilized form and contains the HC and LC amino acid sequences of any of the monoclonal antibodies provided in Table 1. , Sucrose at a concentration of about 8% w/v, L-histidine at a concentration of about 10 mM, and Polysorbate 80 at a concentration of about 0.05% v/v. In some embodiments, a method for preparing a reconstituted formulated antibody is provided, wherein the formulated antibody is provided in a lyophilized form and contains the HC and LC amino acid sequences of any of the monoclonal antibodies provided in Table 1. , Sucrose at a concentration of 8% w/v, L-histidine at a concentration of 10 mM, and Polysorbate 80 at a concentration of 0.05% v/v. In some embodiments, prior to use, the formulated antibody is reconstituted in a suitable volume of water for injection to produce a solution containing the antibody at a concentration of about 50 mg/mL and sucrose at a concentration of about 8% w/v. , L-histidine at a concentration of about 10 mM and polysorbate 80 at a concentration of about 0.05% v/v, wherein the pH of the reconstituted antibody is about 6.2. In some embodiments, prior to use, the formulated antibody is reconstituted in a suitable volume of water for injection to produce a containing solution containing the antibody at a concentration of 50 mg/mL, sucrose at a concentration of 8% w/v, L-histidine at a concentration of 10 mM and polysorbate 80 at a concentration of 0.05% v/v, wherein the pH of the reconstituted antibody is 6.2.
如本文所用,術語「治療(treatment)」或「治療(treating)」是指被設計為改變臨床病理學過程中被治療的個體或細胞的自然進程的臨床幹預。希望的治療效果包括降低疾病進展速率、改善或緩和疾病狀態、以及消退或者預後改善。例如,如果與癌症相關的一種或多種症狀減輕或消除,則成功「治療」個體,包括但不限於減少癌細胞的增生、破壞癌細胞、減少由所述疾病產生的症狀、提高患所述疾病的那些個體的生活品質、減少治療所述疾病所需要的其他藥物的劑量和/或延長個體的生存期。As used herein, the term "treatment" or "treating" refers to a clinical intervention designed to change the natural progression of an individual or cell being treated in the course of clinical pathology. The desired therapeutic effects include reducing the rate of disease progression, improving or alleviating the disease state, and remission or improvement in prognosis. For example, if one or more symptoms associated with cancer are alleviated or eliminated, the individual will be successfully "treated", including but not limited to reducing the proliferation of cancer cells, destroying cancer cells, reducing symptoms caused by the disease, and increasing the risk of the disease. The quality of life of those individuals, reducing the dosage of other drugs needed to treat the disease, and/or prolonging the individual’s survival.
本文提供了用於治療個體的多發性骨髓(如復發性多發性骨髓瘤或復發性和難治性多發性骨髓瘤)或延遲其進展的方法,所述方法包括向有需要的受試者投予有效量的本文所提供的抗CD38抗體。在一些實施例中,抗體包含表1中所提供之任一個單株抗體的HC和LC胺基酸序列。在一些實施例中,抗體能夠提高鼠類MM模型中小鼠的生存期,其中模型中的小鼠表現出低親和力之人類CD16a變異體(158F),並且其中已向小鼠注射500,000個表現人類CD38的EL4細胞。在一些實施例中,一或多個劑量的抗CD38抗體是以凍乾調配物的形式提供,其中在投予之前,凍乾調配物係經重構從而形成重構的抗體調配物,使得重構的抗體調配物具有6.2的pH,並且在液體中(該液體包含約10 mM L-組胺酸、約8% w/v蔗糖和約0.05% v/v聚山梨酯80)包含濃度為約50 mg/mL的抗體。在一些實施例中,一或多個劑量的抗CD38抗體是以凍乾調配物的形式提供,其中在投予之前,凍乾調配物係經重構從而形成重構的抗體調配物,使得重構的抗體調配物具有6.2的pH,並且在液體中(該液體包含10 mM L-組胺酸、8% w/v蔗糖和0.05% v/v聚山梨酯80)包含濃度為50 mg/mL的抗體。Provided herein is a method for treating or delaying the progression of multiple bone marrow (such as relapsed multiple myeloma or relapsed and refractory multiple myeloma) in an individual, the method comprising administering to a subject in need An effective amount of the anti-CD38 antibody provided herein. In some embodiments, the antibody comprises the HC and LC amino acid sequences of any of the monoclonal antibodies provided in Table 1. In some embodiments, the antibody can improve the survival time of the mouse in the murine MM model, where the mouse in the model exhibits a low-affinity human CD16a variant (158F), and where 500,000 expressing human CD38 have been injected into the mouse EL4 cells. In some embodiments, one or more doses of anti-CD38 antibody are provided in the form of a lyophilized formulation, wherein the lyophilized formulation is reconstituted to form a reconstituted antibody formulation before administration. The antibody formulation has a pH of 6.2 and contains a concentration of about 10 mM L-histidine, about 8% w/v sucrose, and about 0.05% v/v polysorbate 80 in a liquid 50 mg/mL antibody. In some embodiments, one or more doses of anti-CD38 antibody are provided in the form of a lyophilized formulation, wherein the lyophilized formulation is reconstituted to form a reconstituted antibody formulation before administration. The structured antibody formulation has a pH of 6.2 and contains a concentration of 50 mg/mL in a liquid (the liquid contains 10 mM L-histidine, 8% w/v sucrose and 0.05% v/v polysorbate 80) Of antibodies.
在一些實施例中,向患者投予一或多個劑量的抗體。在一些實施例中,劑量可以是約0.1、約0.2、約0.3、約0.5、約1.0、約2.5、約5、約10或約20 mg/kg的抗體。在一些實施例中,劑量可以是0.1、0.2、0.3、0.5、1.0、2.5、5、10和20 mg/kg的抗體。在一些實施例中,抗體是靜脈內投予的。在一些實施例中,抗體是皮下投予的。定義 In some embodiments, one or more doses of antibody are administered to the patient. In some embodiments, the dosage may be about 0.1, about 0.2, about 0.3, about 0.5, about 1.0, about 2.5, about 5, about 10, or about 20 mg/kg of antibody. In some embodiments, the dosage may be 0.1, 0.2, 0.3, 0.5, 1.0, 2.5, 5, 10, and 20 mg/kg of antibody. In some embodiments, the antibody is administered intravenously. In some embodiments, the antibody is administered subcutaneously. definition
除非上下文另有明確說明,否則如在本說明書和所附申請專利範圍中所使用的,單數形式「一種/一個(a)」、「一種/一個(an)」和「所述」包括複數指示物。因此,例如,提及「一種分子」任選地包括兩種或更多種這樣的分子的組合等。Unless the context clearly dictates otherwise, as used in this specification and the appended claims, the singular forms "one/one (a)", "one/one (an)" and "the" include plural indications Things. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules and the like.
如本文所用的術語「約」是指熟習此項技術者容易知道的相應值的通常誤差範圍。本文對「約」某一值或參數的提及包括(並描述)涉及所述值或參數本身的實施例。在一些實施例中,術語「約」表示規定值加上或減去所述值的10%;例如,「約10 mg/kg」可以涵蓋9至11 mg/kg。在一些實施例中,術語「約」表示規定值加上或減去所述值的5%;例如,「約20 mg/kg」可以涵蓋19至21 mg/kg。The term "about" as used herein refers to the usual error range of the corresponding value that is easily known by those familiar with the art. References herein to "about" a certain value or parameter include (and describe) embodiments related to the value or parameter itself. In some embodiments, the term "about" means the specified value plus or minus 10% of the stated value; for example, "about 10 mg/kg" can encompass 9 to 11 mg/kg. In some embodiments, the term "about" means the specified value plus or minus 5% of the stated value; for example, "about 20 mg/kg" can encompass 19 to 21 mg/kg.
「受試者」或「個體」出於治療目的是指分類為哺乳動物的任何動物,包括人類、家禽和農場動物,以及動物園動物、運動項目用動物或寵物諸如狗、馬、貓、牛等。較佳地,哺乳動物是人類。"Subject" or "individual" for therapeutic purposes refers to any animal classified as a mammal, including humans, poultry and farm animals, as well as zoo animals, sports animals or pets such as dogs, horses, cats, cows, etc. . Preferably, the mammal is a human.
為了說明和描述本發明的某些具體實施例,下面闡明瞭實例。然而,申請專利範圍的範疇不應以任何方式受到本文所闡明的實例的限制。 實例 1. 抗體設計: In order to illustrate and describe some specific embodiments of the present invention, examples are set forth below. However, the scope of the patent application should not be limited by the examples set forth herein in any way. Example 1. Antibody design:
生成了Fc工程化抗CD38抗體,其在抗體的Fc部分中具有突變,以提高對於效應細胞上的活化FcγRIIIa和FcγRIIa受體的親和力。An Fc engineered anti-CD38 antibody was generated with mutations in the Fc portion of the antibody to increase the affinity for activated FcγRIIIa and FcγRIIa receptors on effector cells.
兩條人類化重鏈(HC)(SEQ ID NO: 5和SEQ ID NO: 6)在框架區攜帶14和10個突變(與SEQ ID NO: 1相比),這導致智人生殖(germinality)得分從74.49%分別增至88.78%和84.69%。兩條人類化輕鏈(LC)(SEQ ID NO: 8和SEQ ID NO: 9)在框架區攜帶17和15個突變(與SEQ ID NO: 7相比),這導致智人生殖得分從64.36%分別增至81.19%和79.21%。另外,在LC 2和3中,用白胺酸取代位置11處的甲硫胺酸,以避免潛在的有問題的甲硫胺酸氧化。HC和LC序列提供於表2中,並且mAb HC與LC對係提供於表3中。表 2
在一個實驗中,使用Biacore®儀器通過SPR量測mAb 1-8對於人類CD38的結合親和力和動力學常數。簡言之,將抗CD38抗體捕獲至已由抗Fc抗體共價結合的CM5晶片。在被測試的捕獲抗體上注射多種濃度的huCD38。使用Biacore評價軟體生成結合曲線(感測圖)並進行動力學分析。In one experiment, the binding affinity and kinetic constant of mAb 1-8 for human CD38 were measured by SPR using a Biacore® instrument. In short, the anti-CD38 antibody is captured to the CM5 chip that has been covalently bound by the anti-Fc antibody. Various concentrations of huCD38 were injected on the capture antibody to be tested. Use Biacore evaluation software to generate binding curve (sensing map) and perform kinetic analysis.
如表4所示,mAb 3和8對於人類CD38具有最高親和力(針對每種變異體的T檢定:p值 > 2)。表 4 對於人類 CD38 的親和力( n = 3 )
在另一個實驗中,如上所述使用的Biacore®儀器通過SPR量測mAb 1和3對於人類CD38的結合親和力。mAb 1具有0.22 nM的Kd,並且mAb 3具有0.20 nM的Kd。In another experiment, the Biacore® instrument used as described above measured the binding affinity of
熱應力對人類CD38的結合親和力的影響:使用Biacore®儀器通過SPR量測在mAb經受熱應力(在40ºC下,在10 mM組胺酸pH 6、8%蔗糖、0.02% PS80中14天)之後mAb 1-8對於人類CD38的結合親和力和動力學常數。已使用以上段落中所述的相同方案。The effect of thermal stress on the binding affinity of human CD38: SPR measurement using Biacore® instrument after mAb subjected to thermal stress (at 40ºC, in 10
如表5所示,經受熱應力的樣品顯示與相應的非應力樣品CD38(表4)(針對每種變異體的T檢定:p值 > 2)非常相似的親和力和動力學常數。表 5 熱應力對針對人類 CD38 的親和力的影響( n = 3 )
在一個實驗中,使用表面電漿共振(SPR)量測mAb對於FcγRIIIa 158V受體或FcγRIIIa 158F受體的親和力。根據供應商(ThermoFisher Scientific)通過暫態轉染在HEK293細胞中產生mAb,並且根據供應商(GE Healthcare)通過蛋白A親和層析將其純化。根據供應商(Ni-Sepharose GE, Healthcare)通過暫態表現在HEK293細胞中產生帶有C端histag的具有V158或F158的FcγRIIIa(CD16a)胞外結構域,並通過固定金屬親和層析(IMAC)將其純化。使用在CM5晶片上固定的抗His IgG,通過捕獲分析在BIACore 2000(GE Healthcare)上經由SPR量測親和力。然後將FcγRIIIa-histag添加至運行緩衝液中,接著是濃度在8至256 nM範圍內的抗CD38 mAb。使用BIAevaluation Software 4_1(GE Healthcare),利用兩態反應進行分析,其中可接受的χ2 值小於5(意味最高測試濃度的2%)並且最大理論共振單位的百分比大於25(意味超過¼生產性相互作用)。In one experiment, surface plasmon resonance (SPR) was used to measure the affinity of mAb for FcγRIIIa 158V receptor or FcγRIIIa 158F receptor. MAb was produced in HEK293 cells by transient transfection according to the supplier (ThermoFisher Scientific), and purified by protein A affinity chromatography according to the supplier (GE Healthcare). According to the supplier (Ni-Sepharose GE, Healthcare), the extracellular domain of FcγRIIIa (CD16a) with V158 or F158 with C-terminal histag was produced in HEK293 cells through transient performance, and passed through immobilized metal affinity chromatography (IMAC) Purify it. Using anti-His IgG immobilized on a CM5 chip, the affinity was measured by SPR on BIACore 2000 (GE Healthcare) by capture analysis. FcyRIIIa-histag was then added to the running buffer, followed by anti-CD38 mAb in a concentration ranging from 8 to 256 nM. Using BIAevaluation Software 4_1 (GE Healthcare), the two-state reaction is used for analysis, where the acceptable χ 2 value is less than 5 (meaning 2% of the highest test concentration) and the percentage of the maximum theoretical resonance unit is greater than 25 (meaning more than ¼ productive interaction effect).
如表6所示,與mAb 1相比,mAb 2-4對於FcγRIIIa 158V具有高9倍的親和力,並且與mAb 1相比,mAb 2-4對於FcγRIIIa 158F受體具有高超過27倍的親和力。表 6
在另一個實驗中,使用如上所述的SPR量測mAb 1和3對於FcγRIIIa 158V受體或FcγRIIIa 158F受體的親和力。mAb 3以75 nM的KD結合FcγRIIIa 158V,並且mAb 3以59 nM的KD結合FcγRIIIa 158F。In another experiment, the affinity of
使用AlphaScreen親近分析來量測mAb對於在胺基酸位置131處具有精胺酸(FcγRIIa 131R)或在胺基酸位置131處具有組胺酸(FcγRIIa 131H)的FcγRIIa受體的親和力。The AlphaScreen affinity analysis was used to measure the affinity of the mAb for the FcγRIIa receptor with arginine at amino acid position 131 (FcγRIIa 131R) or histidine at amino acid position 131 (FcγRIIa 131H).
分別由R&D System(批次1330-CD)或Biorbyt(批次ORB138408)提供具有R131或H131的FcγRIIa胞外結構域。分析是由供應商(Perkin Elmer)所描述的基於珠的近接分析,其允許通過與具有天然IgG1的抗CD38競爭對抗CD38 Fc變異體進行排序。在供體珠(donor bead)上捕獲具有天然IgG1的生物素化抗CD38,並且在受體珠(acceptor bead)上捕獲FcγRIIa histag蛋白。當抗CD38與FcγRIIa相互作用時,珠會靠近。在680 nm處供體珠的激發引起單線態氧的釋放,當處於近接狀態時,所述單線態氧擴散並觸發來自受體珠的520至620 nm處的光放射。光的量與相互作用的程度成正比,並且用EnVision多模式讀板器(Perkin Elmer)來量測。在競爭分析期間,未標記的競爭性mAb抗CD38 Fc變異體DE(S239D/I332E,Eu編號)、ADE(G236A/S239D/I332E,Eu編號)或ADLE(G236A/S239D/F243L/I332E,Eu編號)用於競爭具有天然IgG1的生物素化抗CD38與FcγRIIa-histag蛋白之間的相互作用。生物素化抗CD38的濃度是由預先完成的校準曲線設定。未標記的競爭性mAb是在不同水準下使用,以使得遞增濃度的未標記的mAb可以取代生物素化抗CD38並且降低信號。關於最大信號將資料進行歸一化。運行了兩個實驗並且通過BIOST@T-SPEED-LTS 2.1.0評估IC50。The FcyRIIa extracellular domain with R131 or H131 is provided by R&D System (batch 1330-CD) or Biorbyt (batch ORB138408), respectively. The analysis is a bead-based proximity analysis described by the supplier (Perkin Elmer), which allows ranking by anti-CD38 Fc variants by competing with anti-CD38 with native IgG1. The biotinylated anti-CD38 with native IgG1 was captured on the donor bead, and the FcγRIIa histag protein was captured on the acceptor bead. When anti-CD38 interacts with FcγRIIa, the beads will approach. Excitation of the donor beads at 680 nm causes the release of singlet oxygen, which when in the close state diffuses and triggers light emission at 520 to 620 nm from the acceptor beads. The amount of light is proportional to the degree of interaction and is measured with EnVision multi-mode plate reader (Perkin Elmer). During the competition analysis, unlabeled competitive mAb anti-CD38 Fc variant DE (S239D/I332E, Eu numbering), ADE (G236A/S239D/I332E, Eu numbering) or ADLE (G236A/S239D/F243L/I332E, Eu numbering) ) Used to compete for the interaction between biotinylated anti-CD38 with natural IgG1 and FcγRIIa-histag protein. The concentration of biotinylated anti-CD38 is set by a pre-completed calibration curve. Unlabeled competitive mAb is used at different levels, so that increasing concentrations of unlabeled mAb can replace biotinylated anti-CD38 and reduce signal. Normalize the data with respect to the maximum signal. Two experiments were run and IC50 was evaluated through BIOST@T-SPEED-LTS 2.1.0.
如表7所示,與mAb 1相比,mAb 3顯示分別對於FcγRIIa 131R和FcγRIIa 131H的高超過14倍和18倍的親和力。表 7 對於 FcγRIIa R131 和 FcγRIIa H131 的親和力
將HEK293T-FcγRIIIa-158F、HEK293T-FcγRIIIa-158V、HEK293T-FcγRIIa-131H、HEK293T-FcγRIIa-131R、HEK293T-FcγRI或HEK293T-FcγRIIb以105
個細胞/孔接種在96孔微型盤中。將這些盤在800 g下離心1分鐘,並在4ºC下重新懸浮於含有不同濃度的抗體的50 μL PBS中,持續30分鐘。然後添加200 μL PBS 1% FBS以洗滌細胞,接著在800 g下離心1分鐘。總共重複洗滌步驟三次,之後在4ºC下將細胞用與FITC綴合的二抗(片段山羊抗人類IgG(H+L)FITC,編號109-546-088 Jackson ImmunoResearch,最終10 μg/mL)染色30分鐘。用200 μL PBS 1% FBS洗滌細胞三次,並將其重新懸浮於100 μL PBS中,之後在MACSVYB(B1通道)上獲取。如表8所示,與mAb 1相比,mAb 2、3和4對於在HEK細胞上表現的FcγRIIIa 158F分別具有高32倍、38倍和51倍的親和力,並且與mAb 1相比,mAb 2、3和4對於在HEK細胞上表現的FcγRIIIa 158V分別具有高9倍、5倍和6倍的親和力。表 8
如表9所示,與mAb 1相比,mAb 3對於在HEK細胞上表現的FcγRIIa 131H具有高約5倍的親和力,並且與mAb 1相比,mAb 3對於在HEK細胞上表現的FcγRIIa 131R具有高約16倍的親和力。表 9
如表10所示,與mAb 1相比,mAb 2、3和4對於FcγRI具有相似的結合親和力水準,並且與mAb 1相比,mAb 2、3和4對於在HEK細胞上表現的FcγRIIb具有低1.8至3.9倍的親和力。表 10
使用鈣黃綠素-乙醯氧基甲基(Calcein-AM;Invitrogen)釋放分析來檢查使用被工程化以過表現FcγRIIIa受體的NK92細胞株作為效應細胞的抗體依賴性細胞毒性(ADCC)分析。在活細胞中,將非螢光鈣黃綠素AM轉化成綠色螢光鈣黃綠素。將標記的目標細胞與SAR442085抗體和NK-92效應細胞一起孵育。通過ADCC對目標細胞的溶解導致螢光鈣黃綠素的釋放。螢光強度與所存在的ADCC活性程度成比例。Calcein-acetoxymethyl (Calcein-AM; Invitrogen) release analysis was used to examine the antibody-dependent cytotoxicity (ADCC) analysis using the NK92 cell line engineered to overexpress the FcγRIIIa receptor as effector cells. In living cells, non-fluorescent calcein AM is converted into green fluorescent calcein. The labeled target cells were incubated with SAR442085 antibody and NK-92 effector cells. The lysis of target cells by ADCC results in the release of fluorescent calcein. The fluorescence intensity is proportional to the degree of ADCC activity present.
將多發性骨髓瘤目標細胞(MOLP-8、RPMI 8226、MM1R或KMS12-BM)用鈣黃綠素-AM(50 µg稀釋於25 µL DMSO中,然後將10 µL鈣黃綠素稀釋於4 mL RPMI 1640 + 1% FBS + 1%丙磺舒中,用於4 × 106 個細胞的染色)標記30 min,然後洗滌並以2 × 104 個細胞/孔的密度鋪於96孔圓底盤上。添加從10 μg/mL至0.01 pg/mL的不同濃度的所示測試mAb或對照同型mAb持續30 min以允許調理作用,之後添加用所示FcγRIIIa變異體轉導的自然殺手(NK)細胞。以5 : 1的E : T比率(1 × 105 個NK細胞對於2 × 104 個目標細胞)添加表現158F或158V的NK細胞作為效應細胞。然後將這些盤在37ºC下在具有5% CO2 的濕潤培養箱中孵育1 h,並且收穫100 µL上清液,並將其轉移到不透明的96孔微型盤中,以供在Tecan Infinite M1000上使用螢光測定法進行分析以量測鈣黃綠素釋放(激勵濾光片:492 nm;放射:515 nm)。為了最大釋放,用2% Triton X-100溶解細胞。從實驗釋放(A)、目標細胞自發釋放(B)和目標細胞最大釋放(C)的螢光值中減去培養基背景的螢光值。使用下式計算每種盤(一式兩份)的細胞毒性和ADCC百分比: 細胞毒性(%) = (A - B)/(C - B)×100%Target multiple myeloma cells (MOLP-8, RPMI 8226, MM1R or KMS12-BM) with calcein-AM (50 µg diluted in 25 µL DMSO, and then 10 µL calcein diluted in 4 mL RPMI 1640 + 1 % FBS + 1% probenecid, used for staining 4 × 10 6 cells) label for 30 min, then wash and spread on a 96-well circular dish at a density of 2 × 10 4 cells/well. Different concentrations of the indicated test mAb or control isotype mAb from 10 μg/mL to 0.01 pg/mL were added for 30 min to allow opsonization, after which natural killer (NK) cells transduced with the indicated FcγRIIIa variant were added. With an E: T ratio of 5:1 (1 × 10 5 NK cells to 2 × 10 4 target cells), NK cells expressing 158F or 158V are added as effector cells. The plates were then incubated at 37ºC in a humidified incubator with 5% CO 2 for 1 h, and 100 µL of the supernatant was harvested and transferred to an opaque 96-well microplate for use on Tecan Infinite M1000 Analyze by fluorometry to measure calcein release (excitation filter: 492 nm; emission: 515 nm). For maximum release, use 2% Triton X-100 to lyse the cells. Subtract the fluorescence value of the medium background from the fluorescence values of the experimental release (A), spontaneous release of target cells (B), and maximum release of target cells (C). Use the following formula to calculate the cytotoxicity and ADCC percentage of each disc (in duplicate): Cytotoxicity (%) = (A-B)/(C-B)×100%
每個實驗至少重複3次。通過使用GraphPad Prism 5(GraphPad Software, Inc.,聖地牙哥,加利福尼亞州)將資料點擬合至4參數方程來計算半最大有效濃度(EC50)值。高 CD38 受體密度目標細胞 Each experiment is repeated at least 3 times. The half maximum effective concentration (EC50) value was calculated by fitting the data points to a 4-parameter equation using GraphPad Prism 5 (GraphPad Software, Inc., San Diego, California). High CD38 receptor density target cells
如上所述在活體外評估多發性骨髓瘤細胞株MOLP-8的ADCC。MOLP-8細胞展現出在細胞表面上的高CD38受體密度(約400,000個/細胞)。The ADCC of the multiple myeloma cell line MOLP-8 was evaluated in vitro as described above. MOLP-8 cells exhibit a high CD38 receptor density (approximately 400,000 per cell) on the cell surface.
表11示出了在存在表現158V的NK細胞的情況下每種測試抗體針對MOLP-8細胞的EC50。如表11所示,在存在表現高親和力FcγRIIIa變異體(158V)的NK細胞的情況下,mAb 2、3和4以比mAb 1低24至33倍的EC50觸發MOLP-8細胞的ADCC。表 11
表12示出了在存在表現158F的NK細胞的情況下每種測試抗體針對MOLP-8細胞的EC50。如表12所示,在存在表現低親和力FcγRIIIa變異體(158F)的NK細胞的情況下,mAb 4顯示出針對MOLP-8細胞的最強ADCC活性,其中EC50比mAb 1低約97倍。在存在表現低親和力FcγRIIIa變異體(158F)的NK細胞的情況下,mAb 2和3以比mAb 1低約59至69倍的EC50觸發MOLP-8細胞的ADCC。表 12
如上所述在活體外評估多發性骨髓瘤細胞株RPMI-8226的ADCC。RPMI-8226細胞展現出在細胞表面上的中等CD38受體密度(約70,000個/細胞)。The ADCC of the multiple myeloma cell line RPMI-8226 was evaluated in vitro as described above. RPMI-8226 cells exhibit a moderate CD38 receptor density (approximately 70,000 per cell) on the cell surface.
表13示出了在存在表現158V的NK細胞的情況下每種測試抗體針對RPMI-8226細胞的EC50。如表13所示,在存在表現較高親和力FcγRIIIa變異體(158V)的NK細胞的情況下,mAb 2至4顯示出相似提高的觸發針對表現中等CD38受體密度的MM細胞的ADCC的能力水準,其中與mAb 1相比,EC50低27倍。表 13
表14示出了在存在表現158F的NK細胞的情況下每種測試抗體針對RPMI-8226細胞的EC50。在存在表現低親和力FcγRIIIa變異體的NK細胞的情況下,與mAb 1相比,mAb 4顯示出約73倍提高的觸發針對表現中等CD38受體密度的MM細胞的ADCC的能力;而在存在表現低親和力FcγRIIIa變異體的NK細胞的情況下,與mAb 1相比,mAb 3顯示出約65倍提高的觸發針對表現中等CD38受體密度的MM細胞的ADCC的能力並且mAb 2顯示出約49倍提高的所述觸發的能力。表 14
如上所述在活體外評估多發性骨髓瘤細胞株KMS-12BM的ADCC。KMS-12BM細胞展現出在細胞表面上的低CD38受體密度(約13,000個/細胞)。The ADCC of the multiple myeloma cell line KMS-12BM was evaluated in vitro as described above. KMS-12BM cells exhibit a low CD38 receptor density (approximately 13,000/cell) on the cell surface.
表15示出了在存在表現較高親和力FcγRIIIa變異體(158V)的NK細胞的情況下每種測試抗體針對KMS-12BM細胞的EC50。有趣的是,如表15所示,在存在表現較高親和力FcγRIIIa變異體(158V)的NK細胞的情況下,與mAb 1相比,mAb 2、3和4顯示出提高的觸發針對表現低CD38受體密度的MM細胞的ADCC的能力。mAb 3具有比mAb 1低約69倍的EC50,並且mAb 4具有比mAb 1低約91倍的EC50。表 15
表16示出了在存在表現較低親和力FcγRIIIa變異體(158F)的NK細胞的情況下每種測試抗體針對KMS-12BM細胞的EC50。顯著地,如表16所示,在存在表現較低親和力FcγRIIIa變異體(158F)的NK細胞的情況下,與mAb 1相比,mAb 2、3和4顯示出提高的觸發針對表現低CD38受體密度的MM細胞的ADCC的能力。mAb 3具有比mAb 1低約145倍的EC50,並且mAb 4具有比mAb 1低約269倍的EC50。表 16
首先從來自法國血液研究機構(Etablissement Français du Sang)的七名健康供體的血沉棕黃層分離人類PBMC。所有這些供體都展現出相同的FCGR3A和FCGR2A基因型(FcγRIIIa- 158V/V和FcγRIIa- 131H/H)。首先將血液收集在50 mL Falcon試管中。將15 mL Ficoll-PlaqueTM
PLUS 96%輕輕地添加到Sepmate試管的底部,然後將血液緩慢地添加到試管中,之後在1,300 g下離心10分鐘。然後收集PBMC環,並將其用50 mL PBS洗滌,並且在300 g下進行另一輪離心持續5分鐘。重複洗滌過程兩次,之後根據製造商說明書(Miltenyi;編號130-050-201)用抗CD14磁珠對PBMC染色:在4ºC下孵育時間基本上為15分鐘,之後經由AutoMACS pro(Posseld選擇)進行陽性選擇。收集單核細胞,然後將其用50 mL PBS洗滌。將單核細胞在T75燒瓶中的RPMI1640、10% FBS、2%滅活的人類血清、50 ng/ml GM-CSF中在37ºC、5% CO2
下培養5天。5天培養之後,單核細胞分化為巨噬細胞。為了收集巨噬細胞,將細胞消化液(ThermoFisher,編號:A1110501)在37ºC下置於燒瓶中持續15分鐘,以分離細胞,然後添加20 mL RPMI 10% FBS 1%麩醯胺酸,以終止細胞消化液活性。將所收集的細胞在350 g下離心10分鐘,接著在20 mL PBS中洗滌,並在300 g下進行另一輪離心持續10分鐘。對於兩個實驗,所用的巨噬細胞是來自在液氮中冷凍的巨噬細胞批次。First, human PBMCs were isolated from the buffy coat of seven healthy donors from the French Institute of Blood Research (Etablissement Français du Sang). All these donors exhibited the same FCGR3A and FCGR2A genotypes (FcγRIIIa-158V/V and FcγRIIa-131H/H). First collect the blood in a 50 mL Falcon test tube. Gently add 15 mL of Ficoll-Plaque TM PLUS 96% to the bottom of the Sepmate test tube, then slowly add blood to the test tube, and then centrifuge at 1,300 g for 10 minutes. The PBMC loop was then collected, washed with 50 mL PBS, and subjected to another round of centrifugation at 300 g for 5 minutes. Repeat the washing process twice, then stain PBMC with anti-CD14 magnetic beads according to the manufacturer's instructions (Miltenyi; No. 130-050-201): incubate at 4ºC for basically 15 minutes, and then use AutoMACS pro (Posseld selection) Positive selection. Collect monocytes and wash them with 50 mL PBS. Monocytes were cultured in RPMI1640, 10% FBS, 2% inactivated human serum, 50 ng/ml GM-CSF in T75 flasks at 37ºC and 5% CO 2 for 5 days. After 5 days of culture, monocytes differentiate into macrophages. To collect macrophages, place the cell digest (ThermoFisher, number: A1110501) in a flask at 37ºC for 15 minutes to separate the cells, and then add 20 mL of RPMI 10
將巨噬細胞以20 x 106
個細胞/mL重新懸浮於稀釋劑C中(在來自Sigma Aldrich的製造商染色套組號PKH26GL-1KT中提供)。對於1體積的巨噬細胞,將1體積的PKH26(20 μL稀釋於1 mL稀釋劑C中)添加至細胞中,以在黑暗中孵育5分鐘。然後添加5 mL FBS以使染色過程失活,並持續1分鐘,之後用RPMI1640 10% FBS補充至50 mL。將表現CD38的MOLP-8細胞用PKH67螢光染料染色,並將由純化的單核細胞得到的巨噬細胞用PKH26染色,使得可通過流式細胞術對它們進行鑒別。然後將MOLP-8細胞和巨噬細胞在存在mAb 1或mAb 3的情況下置於培養物中過夜,之後通過流式細胞術進行雙陽性群體(吞噬MOLP-8細胞的巨噬細胞)分析。The macrophages were 20 x 10 6 cells / mL were resuspended in a diluent in C (provided in the kit manufacturer staining PKH26GL-1KT number of from Sigma Aldrich). For 1 volume of macrophages, add 1 volume of PKH26 (20 μL diluted in 1 mL of Diluent C) to the cells to incubate in the dark for 5 minutes. Then add 5 mL FBS to inactivate the staining process and continue for 1 minute, and then supplement to 50 mL with RPMI1640 10% FBS. MOLP-8 cells expressing CD38 were stained with PKH67 fluorescent dye, and macrophages obtained from purified monocytes were stained with PKH26, so that they could be identified by flow cytometry. MOLP-8 cells and macrophages were then placed in culture in the presence of
如通過在所有實驗中獲得的較高總吞噬作用百分比(%)和較低EC50(相對)值(EC50rel)所證明,與mAb 1相比,mAb 3展現出提高的針對MOLP-8細胞株的ADCP活性。總吞噬作用%的幾何平均值對於mAb 3是40.85%,而對於mAb 1是18.57%,並且EC50rel的幾何平均值對於mAb 3是31.87 ng/mL(或212.57 pM),而對於mAb 1,所述值超過64.86 ng/mL(或超過432.62 pM)。基於EC50rel值,mAb 3在ADCP活性方面比mAb 1要好至少2.035倍。活體內功效 As demonstrated by the higher percentage of total phagocytosis (%) and lower EC50 (relative) value (EC50rel) obtained in all experiments, compared to
評價mAb 3的活體內功效。在第0天向人類化FcγR C57BL/6小鼠(Smith P, DiLillo DJ, Bournazos S, Li F, Ravetch JV; Proc Natl Acad Sci USA. 2012; 109(16):6181-6)靜脈內注射500,000個EL4-huCD38腫瘤細胞。使人類化FcγR C57BL/6小鼠的所有編碼FcγR的鼠基因缺失並將編碼為轉基因的人類FcγR插入小鼠基因組中。人類化FcγR C57BL/6小鼠概括了huFcγR表現模式和表現水準並且在發炎、細胞毒性和腫瘤清除的多個huIgG介導的模型中是功能性的。關於活化的FcγRIIIa和FcγRIIa受體,插入小鼠模型中的人類變異體分別是huFcγRIIIa 158F和huFcγRIIa 131R(受體的低親和力變異體)。人類FcγR表現模式和表現水準的特徵概括在小鼠中。Evaluate the efficacy of mAb 3 in vivo. Humanized FcγR C57BL/6 mice (Smith P, DiLillo DJ, Bournazos S, Li F, Ravetch JV; Proc Natl Acad Sci USA. 2012; 109(16):6181-6) were injected intravenously with 500,000 on day 0 One EL4-huCD38 tumor cell. All mouse genes encoding FcγR in the humanized FcγR C57BL/6 mice were deleted and the human FcγR encoded as a transgene was inserted into the mouse genome. The humanized FcγR C57BL/6 mouse recapitulates the expression pattern and level of huFcγR and is functional in multiple huIgG-mediated models of inflammation, cytotoxicity, and tumor clearance. Regarding the activated FcγRIIIa and FcγRIIa receptors, the human variants inserted into the mouse model are huFcγRIIIa 158F and huFcγRIIa 131R (low affinity variants of the receptor), respectively. The characteristics of human FcγR expression patterns and performance levels are summarized in mice.
從注射腫瘤細胞後第1天開始,在第1、4、7和14天腹膜內投予測試或對照mAb。同型對照mAb是以10 mg/kg投予。mAb 1、3和10是以10和1.25 mg/kg投予。在此生存期模型中,主要功效終點是中位生存時間(MST)、延長壽命百分比(ILS%)和長期存活者(定義為優於或等於對照組MST的2倍的生存持續時間)。用Cox模型評價各組之間的生存期差異。Starting from
如表17所示,同型對照組具有39天的MST。百分之二十的同型對照組在此模型下展現出長期生存。用10 mg/kg的mAb 1治療的組具有超過82.5天的MST、大於112%的延長壽命。用10 mg/kg的mAb 1治療的組中有百分之五十是長期存活者。用1.25 mg/kg的mAb 1治療的小鼠具有大於46.5天的MST和大於19%的延長壽命。用1.25 mg/kg的mAb 1治療的組中有百分之三十是長期存活者。As shown in Table 17, the isotype control group had an MST of 39 days. Twenty percent of the isotype control group exhibited long-term survival under this model. The group treated with
用10 mg/kg的mAb 10治療的組具有大於70天的MST、70%的延長壽命。用10 mg/kg的mAb 10治療的組中有百分之五十是長期存活者。用1.25 mg/kg的mAb 10治療的小鼠具有大於42天的MST和8%的延長壽命。用1.25 mg/kg的mAb 10治療的組中僅10%是長期存活者。The group treated with
用10 mg/kg的mAb 3治療的組具有超過90天的MST、大於131%的延長壽命。顯著地,用10 mg/kg的mAb 3治療的組中有90%是長期存活者。與同型對照組相比,10 mg/kg的mAb 3在統計上顯著地更有活性(p = 0.0351)。甚至更驚人地,用1.25 mg/kg的mAb 3治療的小鼠也具有大於90天的MST和大於131%的延長壽命。此外,用1.25 mg/kg的mAb 3治療的組中有90%是長期存活者。與同型對照組相比,1.25 mg/kg的mAb 3在統計上顯著地更有活性(p = 0.0380),並且在統計上顯著地優於相同劑量下的mAb 10(p = 0.0380)。The group treated with mAb 3 at 10 mg/kg had an MST over 90 days and an extended life span of greater than 131%. Significantly, 90% of the group treated with mAb 3 at 10 mg/kg were long-term survivors. Compared with the isotype control group, mAb 3 at 10 mg/kg was statistically significantly more active (p = 0.0351). Even more surprisingly, mice treated with 1.25 mg/kg of mAb 3 also had an MST greater than 90 days and an extended life span of greater than 131%. In addition, 90% of the group treated with 1.25 mg/kg mAb 3 were long-term survivors. Compared with the isotype control group, mAb 3 at 1.25 mg/kg was statistically significantly more active (p = 0.0380), and statistically significantly better than
用10 mg/kg劑量下的mAb 1、mAb 3和mAb 10治療攜帶低親和力huFcγRIIIa(F158/F158)的小鼠中的該腫瘤模型導致壽命和長期存活者數量的統計上顯著的改善。然而,驚人的是,在1.25 mg/kg劑量下,mAb 3仍展現出壽命和長期存活者數量的統計上顯著的改善,而mAb 1和mAb 10則無活性。表 17
使用Malvern MicroCal VP熱量計,通過差示掃描量熱法(DSC)量測mAb 1、3、5、6、7和8的熱穩定性。在包含10 mM組胺酸HCl的緩衝液(pH 6.0)中測試樣品。將所製備的樣品連同作為參照的緩衝液一起載入到Wheaton 96孔圓底盤的各孔中,一式三份。使用1ºC/min的溫度斜率和20ºC至120ºC的溫度範圍,收集熱掃描並使用origin軟體2.0進行處理。Using a Malvern MicroCal VP calorimeter, the thermal stability of
如表18所示,mAb 3、5、6、7和8在約40ºC下顯示出現蛋白質構象變化,此溫度比mAb 1的出現溫度低約17ºC,並且比mAb的典型的溫度低約15ºC-20ºC(圖1)。此外,在約50ºC下相對於70ºC下,mAb 3、5、6、7和8的CH2結構域的去折疊比mAb 1開始得要早得多。表 18 熱穩定性( ºC )
通過毛細管等電聚焦(cIEF)量測mAb 1、3、5、6、7和8中的每一個的等電點(pI)。理論pI是使用分析平臺SEDNTERP並假設以下胺基酸pKa值:Arg = 12,Asp = 4.5,Glu = 4.6,His = 6.2,Lys = 10.4且Tyr = 9.7,由組合物確定的。假設在Cys殘基中的所有巰基側鏈是雙硫鍵鍵結的,沒有貢獻pKa(Laue TM, Shah BD, Ridgeway TM和Pelletier SL.Computer-aided interpretation of analytical sedimentation data for protein.In Analytical Ultracentrifugation in Biochemistry and Polymer Science.Harding SE, Rowe AJ和Horton Jc編輯 第90-124頁. Royal Society of Chemistry, 1991)。The isoelectric point (pI) of each of
驚人的是,如表19所示,基於序列,mAb 3的pI量測值低於pI計算值。表 19
基於熱穩定性結果,開發凍乾的調配物以確保mAb 3的足夠穩定性以適合於mAb在 ≤ -30ºC下儲存和凍乾的mAb在2ºC-8ºC下儲存。在穩定性研究中測試使用不同緩衝系統且在不同pH下的mAb 3調配物,所述穩定性研究包括凍融循環、攪動研究以及在40ºC、25ºC、5ºC、-30ºC和-80ºC的應力、加速和預期儲存條件下的短期(12周)培育。在這些實驗中,除了粒子形成(目測檢查、基於光模糊的粒子計數和微流成像)之外,還評價了蛋白質的聚集和化學降解。Based on the thermal stability results, a lyophilized formulation was developed to ensure sufficient stability of mAb 3 to be suitable for storage of mAb at ≤ -30ºC and lyophilized mAb for storage at 2ºC-8ºC. Test mAb 3 formulations using different buffer systems and at different pHs in stability studies. The stability studies include freeze-thaw cycles, agitation studies, and stress and acceleration at 40ºC, 25ºC, 5ºC, -30ºC and -80ºC And short-term (12 weeks) cultivation under expected storage conditions. In these experiments, in addition to particle formation (visual inspection, particle counting based on light blur, and microfluidic imaging), protein aggregation and chemical degradation were also evaluated.
基於來自調配物開發研究的結果,選擇含有10 mM L-組胺酸-HCl、8% w/v蔗糖和0.05% w/v 聚山梨酯80的調配物(pH 6.2)。凍乾程序開發 Based on the results from the formulation development study, a formulation (pH 6.2) containing 10 mM L-histidine-HCl, 8% w/v sucrose and 0.05% w/v polysorbate 80 was selected. Freeze-drying program development
開發mAb 3的凍乾程序以確保經調配抗體在凍乾之後具有可接受的穩定性和餅屬性。使用實驗室規模的凍乾器(由SP Scientific製造的Genesis EL35),基於多次開發運行確定凍乾循環的程序參數(乾燥溫度、腔室壓力等)。循環的穩健性是通過在冷凍速率、乾燥溫度和腔室真空壓力的受控偏移下進行一系列凍乾運行來建立。對凍乾的經調配抗體的穩定性研究包括在40ºC、25ºC和5ºC的應力、加速和預期儲存條件下的短期(12周)孵育。在這些實驗中,除了聚集、化學降解和粒子形成之外,還評價了凍乾粉末屬性(餅外觀、重構時間、氧氣頂部空間、水分含量)。基於調配物和凍乾開發研究,mAb 3的組合物被選擇為50 mg/mL mAb 3、10 mM L-組胺酸-HCl、8% w/v蔗糖、0.05% w/v聚山梨酯80並且pH為6.2。將經調配抗體在 ≤ -30ºC下儲存並將凍乾的經調配抗體在2ºC至8ºC下儲存。A lyophilization procedure for mAb 3 was developed to ensure that the formulated antibody has acceptable stability and cake properties after lyophilization. Using a laboratory-scale lyophilizer (Genesis EL35 manufactured by SP Scientific), the program parameters (drying temperature, chamber pressure, etc.) of the lyophilization cycle are determined based on multiple development runs. The robustness of the cycle is established by performing a series of freeze-drying runs under controlled deviations of the freezing rate, drying temperature, and chamber vacuum pressure. Stability studies of lyophilized formulated antibodies include short-term (12 weeks) incubation under stress, acceleration, and expected storage conditions at 40ºC, 25ºC, and 5ºC. In these experiments, in addition to aggregation, chemical degradation, and particle formation, freeze-dried powder properties (cake appearance, reconstitution time, oxygen headspace, moisture content) were also evaluated. Based on the formulation and lyophilization development research, the composition of mAb 3 was selected as 50 mg/
無no
圖1是顯示mAb 1、2、3、5、6、7和8出現去折疊的圖。Figure 1 is a graph showing the unfolding of
圖2是顯示mAb 1、3、5、6、7和8的等電點(PI)的圖。Figure 2 is a graph showing the isoelectric points (PI) of
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