TW202130367A - Preparation and use of bispecific antibodies binding pd-1 and pd-l1 - Google Patents

Preparation and use of bispecific antibodies binding pd-1 and pd-l1 Download PDF

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TW202130367A
TW202130367A TW110101453A TW110101453A TW202130367A TW 202130367 A TW202130367 A TW 202130367A TW 110101453 A TW110101453 A TW 110101453A TW 110101453 A TW110101453 A TW 110101453A TW 202130367 A TW202130367 A TW 202130367A
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謝瑞霞
馬麗強
汪音爵
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大陸商信達生物製藥（蘇州）有限公司
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Abstract

The present invention relates to preparations containing anti-PD-1/PD-L1 antibodies, and more particularly to pharmaceutical preparations containing anti-PD-1/PD-L1 antibodies, buffers, stabilizers and surfactants. In addition, the present invention also relates to uses of these preparations for treating or prevention of diseases.

Description

結合PD-1和PD-L1的雙特異性抗體的製劑及其用途 Preparation and use of bispecific antibody combining PD-1 and PD-L1

本發明涉及抗體製劑領域。更具體而言，本發明涉及包含結合人程序性細胞死亡1(PD-1)和人PD-1配體1的雙特異性抗體(也稱為抗PD-1/PD-L1抗體)的穩定的製劑，以及所述製劑的治療和/或預防用途。 The present invention relates to the field of antibody preparations. More specifically, the present invention relates to the stabilization of bispecific antibodies (also known as anti-PD-1/PD-L1 antibodies) that bind human programmed cell death 1 (PD-1) and human PD-1 ligand 1 Preparations, and the therapeutic and/or preventive uses of the preparations.

免疫檢查點路徑用於維持自身耐受性及控制T細胞活化，但癌細胞可使用該等路徑來抑制抗腫瘤反應並防止其破壞。PD-1/PD-L1路徑是一種該免疫檢查點。在T細胞上發現人類PD-1，且人類PD-L1由多種腫瘤類型異常表達；PD-L1與PD-1的結合抑制T細胞增殖及細胞介素產生。PD-1/PD-L1抑制性軸已經被腫瘤征服，作為在抗腫瘤免疫反應的背景下使腫瘤進化成形的自然選擇過程的一部分。 Immune checkpoint pathways are used to maintain self-tolerance and control T cell activation, but cancer cells can use these pathways to inhibit anti-tumor responses and prevent their destruction. The PD-1/PD-L1 pathway is one such immune checkpoint. Human PD-1 is found on T cells, and human PD-L1 is abnormally expressed by a variety of tumor types; the combination of PD-L1 and PD-1 inhibits T cell proliferation and cytokine production. The PD-1/PD-L1 inhibitory axis has been conquered by tumors as part of the natural selection process that shapes tumor evolution in the context of anti-tumor immune responses.

儘管靶向PD-1/PD-L1路徑的治療劑在臨床上得到了驗證且已經在治療癌症方面取得了顯著臨床進展，但僅一部分患者受益於該治療(參見，例如，Sharma,P.及Allison,J.P.,Immune checkpoint targeting in cancer therapy：toward combination strategies with curative potential. Cell.2015；161：2015-14)。舉例而言，僅約20%患有非小細胞肺癌(NSCLC)的患者對PD-1抗體治療有反應。 Although therapeutic agents targeting the PD-1/PD-L1 pathway have been clinically validated and have made significant clinical progress in the treatment of cancer, only a portion of patients have benefited from this treatment (see, for example, Sharma, P. and Allison,JP,Immune checkpoint targeting in cancer therapy:toward combination strategies with curative potential. Cell. 2015; 161: 2015-14). For example, only about 20% of patients with non-small cell lung cancer (NSCLC) respond to PD-1 antibody treatment.

儘管目前正在進行涉及共同給藥PD-L1抗體及PD-1抗體的臨床試驗(參見，例如，EUROPEAN SOCIETY FOR MEDICAL ONCOLOGY(ESMO)，摘要編號2130；2016年10月)，但該治療方案涉及輸注兩種單獨的抗體產品，每種抗體的劑量相對較高。此外，尚未知與單一療法相比，該組合療法是否會提供效能的改良而不會加劇不良事件特性。 Although clinical trials involving the co-administration of PD-L1 antibodies and PD-1 antibodies are currently underway (see, for example, EUROPEAN SOCIETY FOR MEDICAL ONCOLOGY (ESMO), Abstract No. 2130; October 2016), this treatment regimen involves infusion Two separate antibody products, the dosage of each antibody is relatively high. In addition, it is not known whether the combination therapy will provide an improvement in efficacy compared to monotherapy without exacerbating the characteristics of adverse events.

因此，需要以高親和性結合PD-L1及PD-1、有效地中和由所有PDx家族配體的PD-L1及PD-1活化及/或相對於已知靶向PD-1/PD-L1路徑的治療劑提供優異活性或甚至其組合的雙特異性抗體作為某些癌症的更有效的藥理學介入。例如，PCT申請號PCT/US2018/041205中公開了一種對人PD-1/PD-L1特異性的示例性的治療性抗體，其公開內容特此藉由引用整體併入。同時，本領域中需要能夠用來治療、預防或延緩各種癌症和免疫相關疾病的抗PD-1/PD-L1抗體製劑。 Therefore, it is necessary to bind PD-L1 and PD-1 with high affinity, effectively neutralize the activation of PD-L1 and PD-1 by all PDx family ligands, and/or relative to known targets PD-1/PD- The therapeutic agent of the L1 pathway provides bispecific antibodies with excellent activity or even a combination thereof as a more effective pharmacological intervention for certain cancers. For example, PCT application number PCT/US2018/041205 discloses an exemplary therapeutic antibody specific for human PD-1/PD-L1, the disclosure of which is hereby incorporated by reference in its entirety. At the same time, there is a need in the art for anti-PD-1/PD-L1 antibody preparations that can be used to treat, prevent or delay various cancers and immune-related diseases.

用於人類受試者的抗體在使用之前必須儲存和運輸至施用地點。在受試者中可再現地獲得期望的抗體藥物水平要求所述藥物儲存在維持藥物的生物活性的製劑中。同時，相較於靜脈輸注，高濃度製劑皮下給藥更方便安全，能實現病人在家自主給藥，便於慢性病的長期給藥治療；同時高濃度製劑可以滿足高劑量的給藥要求，能夠降低生產成本。開發高濃度製劑已然成為單抗類製劑的發展趨勢。同時，凍乾製劑能夠帶來更優越的製劑穩定性，且藉由複溶時加入更少的液體能夠製備更高濃度的液體製劑。 Antibodies used in human subjects must be stored and transported to the site of administration before use. Reproducibly obtaining the desired antibody drug level in the subject requires that the drug be stored in a formulation that maintains the biological activity of the drug. At the same time, compared to intravenous infusion, subcutaneous administration of high-concentration preparations is more convenient and safer, enabling patients to self-administer at home, which is convenient for long-term treatment of chronic diseases; at the same time, high-concentration preparations can meet the requirements of high-dose administration and reduce production. cost. The development of high-concentration preparations has become the development trend of monoclonal antibody preparations. At the same time, lyophilized formulations can bring about superior formulation stability, and higher concentration liquid formulations can be prepared by adding less liquid during reconstitution.

因此，在本領域中對於含有足夠穩定且適於以各種形式施用給受試者的抗PD-1/PD-L1抗體的新穎藥物製劑仍存在需要。特別需要這樣的製劑，其能夠表現出長的貯存期限，在儲存和運輸時是穩定的，並且適合以高濃度(例如用於皮下施用)和低濃度(例如用於靜脈內施用)施用。 Therefore, there is still a need in the art for novel pharmaceutical formulations containing anti-PD-1/PD-L1 antibodies that are sufficiently stable and suitable for administration to subjects in various forms. There is a particular need for formulations that can exhibit a long shelf life, are stable during storage and transportation, and are suitable for administration at high concentrations (for example, for subcutaneous administration) and low concentrations (for example, for intravenous administration).

發明概述Summary of the invention

本發明藉由提供含有特異結合至PD-1/PD-L1的抗體的穩定製劑來滿足上述需求。 The present invention meets the above needs by providing a stable formulation containing antibodies that specifically bind to PD-1/PD-L1.

在第一方面中，本發明涉及抗PD-1/PD-L1抗體或其抗原結合片段的凍乾製劑，其包含：(i)抗PD-1/PD-L1抗體或其抗原結合片段；(ii)緩衝體系；(iii)穩定劑，該穩定劑包括多元醇和/或胺基酸；和(iv)表面活性劑，其中該製劑當重構時具有5.5-6.5之間的pH，例如，pH約為5.5、6.0或6.5。 In the first aspect, the present invention relates to a lyophilized preparation of an anti-PD-1/PD-L1 antibody or an antigen-binding fragment thereof, which comprises: (i) an anti-PD-1/PD-L1 antibody or an antigen-binding fragment thereof; ii) a buffer system; (iii) a stabilizer, the stabilizer includes a polyol and/or an amino acid; and (iv) a surfactant, wherein the formulation has a pH between 5.5-6.5 when reconstituted, for example, pH Approximately 5.5, 6.0 or 6.5.

在某些實施方案中，該凍乾製劑能夠在約1mg/mL至約200mg/mL之間的濃度重構該抗體或其抗原結合片段。 In certain embodiments, the lyophilized formulation can reconstitute the antibody or antigen-binding fragment thereof at a concentration between about 1 mg/mL to about 200 mg/mL.

在某些實施方案中，該表面活性劑選自聚山梨酯80、聚山梨酯20、泊洛沙姆、聚乙二醇聚山梨酯80或其組合，且以約0.01%(w/v)-1%(w/v)的重量比存在。 In certain embodiments, the surfactant is selected from polysorbate 80, polysorbate 20, poloxamer, polyethylene glycol polysorbate 80, or a combination thereof, and is at about 0.01% (w/v) -1% (w/v) weight ratio exists.

在某些實施方案中，該緩衝體系選自組胺酸緩衝體系、組胺酸和鹽酸組胺酸緩衝體系、枸櫞酸和枸櫞酸鈉緩衝體系、醋酸醋酸鈉緩衝體系、磷酸鹽緩衝體系，且以約0.01%(w/v)-1%(w/v)的重量比存在。 In some embodiments, the buffer system is selected from the group consisting of histidine buffer system, histidine and hydrochloride histidine buffer system, citric acid and sodium citrate buffer system, sodium acetate buffer system, phosphate buffer system , And exist in a weight ratio of about 0.01%(w/v)-1%(w/v).

在某些實施方案中，該多元醇選自山梨醇、甘露醇或其組合，該胺基酸選自精胺酸、鹽酸精胺酸、甲硫胺酸、甘胺酸、脯胺酸或其組合； In certain embodiments, the polyol is selected from sorbitol, mannitol or a combination thereof, and the amino acid is selected from arginine, arginine hydrochloride, methionine, glycine, proline or combination;

較佳地，該穩定劑包括山梨醇和精胺酸；更佳地，該山梨醇以約1-10%(w/v)，例如約1、2、3、4、5、6、7、8、9、10%(w/v)的重量比存在，該精胺酸以約1-10%(w/v)，例如約1、2、3、4、5、6、7、8、9、10%(w/v)的重量比存在； Preferably, the stabilizer includes sorbitol and arginine; more preferably, the sorbitol contains about 1-10% (w/v), such as about 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10% (w/v) by weight ratio, the arginine is about 1-10% (w/v), for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10% (w/v) weight ratio exists;

較佳地，該穩定劑包括山梨醇和鹽酸精胺酸；更佳地，該山梨醇以約1-10%(w/v)，例如約1、2、3、4、5、6、7、8、9、10%(w/v)的重量比存在，以約1-10%(w/v)，例如約1、2、3、4、5、6、7、8、9、10%(w/v)的重量比存在。 Preferably, the stabilizer includes sorbitol and arginine hydrochloride; more preferably, the sorbitol is about 1-10% (w/v), such as about 1, 2, 3, 4, 5, 6, 7, The weight ratio of 8, 9, 10% (w/v) is present, with about 1-10% (w/v), for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10% The weight ratio of (w/v) exists.

在某些實施方案中，該穩定劑還包括甲硫胺酸；更佳地，該甲硫胺酸以約0.1-10%(w/v)的重量比存在。 In certain embodiments, the stabilizer further includes methionine; more preferably, the methionine is present in a weight ratio of about 0.1-10% (w/v).

在第二方面中，本發明涉及抗PD-1/PD-L1抗體或其抗原結合片段的凍乾製劑，其藉由凍乾水溶液製成，該水溶液包含：(i)抗PD-1/PD-L1抗體或其抗原結合片段；(ii)緩衝體系；(iii)穩定劑，該穩定劑包括多元醇和/或胺基酸；和(iv)表面活性劑，該液體製劑具有約5.5-6.5的pH，例如，pH約為5.5、6.0或6.5；較佳地，該液體製劑具有約6.0的pH。 In the second aspect, the present invention relates to a freeze-dried preparation of an anti-PD-1/PD-L1 antibody or an antigen-binding fragment thereof, which is prepared by a freeze-dried aqueous solution, the aqueous solution comprising: (i) anti-PD-1/PD -L1 antibody or its antigen-binding fragment; (ii) buffer system; (iii) stabilizer, the stabilizer includes polyol and/or amino acid; and (iv) surfactant, the liquid preparation has a concentration of about 5.5-6.5 The pH, for example, the pH is about 5.5, 6.0 or 6.5; preferably, the liquid formulation has a pH of about 6.0.

在第三方面中，本發明涉及抗PD-1/PD-L1抗體或其抗原結合片段的液體藥物製劑，其包含水溶液，該水溶液包含：(i)抗PD-1/PD-L1抗體或其抗原結合片段；(ii)緩衝體系；(iii)穩定劑，該穩定劑包括多元醇和/或胺基酸；和(iv)表面活性劑，該液體製劑具有約5.5-6.5的pH，例如，pH約為5.5、6.0或6.5；較佳地，該液體製劑具有約6.0的pH。 In the third aspect, the present invention relates to a liquid pharmaceutical preparation of an anti-PD-1/PD-L1 antibody or an antigen-binding fragment thereof, which comprises an aqueous solution comprising: (i) an anti-PD-1/PD-L1 antibody or its Antigen-binding fragment; (ii) buffer system; (iii) stabilizer, the stabilizer includes polyol and/or amino acid; and (iv) surfactant, the liquid formulation has a pH of about 5.5-6.5, for example, pH It is about 5.5, 6.0 or 6.5; preferably, the liquid formulation has a pH of about 6.0.

在某些實施方案中，上述的凍乾製劑或液體製劑中，該抗PD-1/PD-L1抗體或其抗原結合片段以約1mg/mL至約200mg/mL的濃度存在於水溶液中，例如以約1、5、10、20、25、30、35、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190或200mg/ml的濃度存在於水溶液中。 In certain embodiments, in the above-mentioned lyophilized formulation or liquid formulation, the anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof is present in an aqueous solution at a concentration of about 1 mg/mL to about 200 mg/mL, for example With about 1, 5, 10, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200mg/ The concentration of ml exists in the aqueous solution.

在某些實施方案中，上述的凍乾製劑或液體製劑中，該表面活性劑選自聚山梨酯80、聚山梨酯20、泊洛沙姆、聚乙二醇聚山梨酯80或其組合，且以約0.1-10mg/ml，例如約0.1、0.5、1、2、3、4、5、6、7、8、9、10mg/ml的濃度存在。 In certain embodiments, in the above-mentioned lyophilized formulation or liquid formulation, the surfactant is selected from polysorbate 80, polysorbate 20, poloxamer, polyethylene glycol polysorbate 80 or a combination thereof, And it exists at a concentration of about 0.1-10 mg/ml, for example, about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/ml.

在某些實施方案中，上述的凍乾製劑或液體製劑中，該緩衝體系選自組胺酸緩衝體系、組胺酸和鹽酸組胺酸緩衝體系、枸櫞酸和枸櫞酸鈉緩衝體系、醋酸醋酸鈉緩衝體系、磷酸鹽緩衝體系，且以約1-100mM，例如，1、5、10、15、20、30、40、50、60、70、80、90、100mM的濃度存在。 In some embodiments, in the above-mentioned freeze-dried preparation or liquid preparation, the buffer system is selected from the group consisting of histidine buffer system, histidine and histidine hydrochloride buffer system, citric acid and sodium citrate buffer system, Sodium acetate acetate buffer system, phosphate buffer system, and exist in a concentration of about 1-100 mM, for example, 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 mM.

較佳地，該緩衝體系為組胺酸緩衝體系；更佳地，該組胺酸以約0.1-10mg/mL，例如約0.1、0.5、1、2、3、4、5、6、7、8、9、10mg/ml的濃度存在； Preferably, the buffer system is a histidine buffer system; more preferably, the histidine is about 0.1-10 mg/mL, such as about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, Exist at the concentration of 8, 9, 10mg/ml;

較佳地，該緩衝體系為組胺酸和鹽酸組胺酸緩衝體系；更佳地，該組胺酸和該鹽酸組胺酸分別以約0.1-10mg/mL，例如約0.1、0.5、1、2、3、4、5、6、7、8、9、10mg/ml的濃度存在。 Preferably, the buffer system is a histidine and histidine hydrochloride buffer system; more preferably, the histidine and the histidine hydrochloride are respectively about 0.1-10 mg/mL, such as about 0.1, 0.5, 1, Concentrations of 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/ml exist.

在某些實施方案中，上述的凍乾製劑或液體製劑中，該多元醇選自山梨醇、甘露醇或其組合，該胺基酸選自精胺酸、鹽酸精胺酸、甲硫胺酸、甘胺酸、脯胺酸或其組合；較佳地，該穩定劑包括山梨醇和精胺酸；更佳地，該山梨醇以約10-100mg/mL，例如約10、20、30、40、50、60、70、80、90、100mg/mL的濃度存在，該精胺酸以例如約10、20、30、40、50、60、70、80、90、100mg/mL存在； In some embodiments, in the above-mentioned freeze-dried formulation or liquid formulation, the polyol is selected from sorbitol, mannitol or a combination thereof, and the amino acid is selected from arginine, arginine hydrochloride, and methionine , Glycine, proline or a combination thereof; preferably, the stabilizer includes sorbitol and arginine; more preferably, the sorbitol is about 10-100 mg/mL, such as about 10, 20, 30, 40 , 50, 60, 70, 80, 90, 100 mg/mL, and the arginine is present at, for example, about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg/mL;

較佳地，該穩定劑包括山梨醇和鹽酸精胺酸；更佳地，該山梨醇和該鹽酸精胺酸分別以約10-100mg/mL，例如約10、20、30、40、50、60、70、80、90、100mg/mL的濃度存在。 Preferably, the stabilizer includes sorbitol and arginine hydrochloride; more preferably, the sorbitol and the arginine hydrochloride are respectively about 10-100 mg/mL, such as about 10, 20, 30, 40, 50, 60, Concentrations of 70, 80, 90, 100 mg/mL exist.

在某些實施方案中，上述的凍乾製劑或液體製劑中，該穩定劑還包括甲硫胺酸；較佳地，該甲硫胺酸以約1-100mg/mL，例如約1、5、10、20、30、40、50、60、70、80、90、100mg/mL的濃度存在。 In some embodiments, in the above-mentioned freeze-dried formulations or liquid formulations, the stabilizer further includes methionine; preferably, the methionine is at a concentration of about 1-100 mg/mL, such as about 1, 5, There are concentrations of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg/mL.

在本文中描述的任意製劑中，該抗PD-1/PD-L1抗體或其抗原結合片段結合人類PD-L1(SEQ ID NO：1)及人類PD-1(SEQ ID NO：2)。較佳地，該抗PD-1/PD-L1抗體或其抗原結合片段包含：第一重鏈(HC1)，其包含第一重鏈可變區(HCVR1)和恆定區；第一輕鏈(LC1)，其包含第一輕鏈可變區(LCVR1)和恆定區；第二重鏈(HC2)，其包含第二重鏈可變區(HCVR2)和恆定區；以及第二輕鏈(LC2)，其包含第二輕鏈可變區(LCVR2)和恆定區，其中： In any of the formulations described herein, the anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof binds human PD-L1 (SEQ ID NO: 1) and human PD-1 (SEQ ID NO: 2). Preferably, the anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof comprises: a first heavy chain (HC1), which comprises a first heavy chain variable region (HCVR1) and a constant region; a first light chain ( LC1), which includes a first light chain variable region (LCVR1) and a constant region; a second heavy chain (HC2), which includes a second heavy chain variable region (HCVR2) and a constant region; and a second light chain (LC2 ), which contains the second light chain variable region (LCVR2) and the constant region, where:

該HCVR1包含SEQ ID NO：3所示的重鏈可變區所含的三個互補決定區域HCDR1、HCDR2和HCDR3，並且該LCVR1包含SEQ ID NO：4所示的輕鏈可變區所含的LCDR1、LCDR2和LCDR3；以及 The HCVR1 includes the three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 3, and the LCVR1 includes the three complementary determining regions contained in the light chain variable region shown in SEQ ID NO: 4 LCDR1, LCDR2 and LCDR3; and

該HCVR2包含SEQ ID NO：5所示的重鏈可變區中所含的三個互補決定區域(CDR)HCDR1、HCDR2和HCDR3，並且該LCVR2包含SEQ ID NO：8所示的輕鏈可變區所含的三個互補決定區域LCDR1、LCDR2和LCDR3。 The HCVR2 includes the three complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 5, and the LCVR2 includes the light chain variable shown in SEQ ID NO: 8 The three complementary determining areas LCDR1, LCDR2 and LCDR3 contained in the area.

在一些實施方案中，本發明抗體製劑中的抗PD-1/PD-L1抗體可以包含：第一重鏈(HC1)，其包含第一重鏈可變區(HCVR1)和恆定區；第一輕鏈(LC1)，其包含第一輕鏈可變區(LCVR1)和恆定區；第二重鏈(HC2)，其包含第二重鏈可變區(HCVR2)和恆定區；以及第二輕鏈(LC2)，其包含第二輕鏈可變區(LCVR2)和恆定區，其中： In some embodiments, the anti-PD-1/PD-L1 antibody in the antibody preparation of the present invention may include: a first heavy chain (HC1), which includes a first heavy chain variable region (HCVR1) and a constant region; The light chain (LC1), which includes the first light chain variable region (LCVR1) and the constant region; the second heavy chain (HC2), which includes the second heavy chain variable region (HCVR2) and the constant region; and the second light chain Chain (LC2), which includes the second light chain variable region (LCVR2) and constant region, where:

a)該HCVR1包含具有SEQ ID NO：16所示的胺基酸序列的互補決定區域CDR1、具有SEQ ID NO：17所示的胺基酸序列的互補決定區域CDR2及具有SEQ ID NO：18所示的胺基酸序列的互補決定區域CDR3； a) The HCVR1 includes the complementarity determining region CDR1 having the amino acid sequence shown in SEQ ID NO: 16, the complementarity determining region CDR2 having the amino acid sequence shown in SEQ ID NO: 17, and the complementarity determining region CDR2 having the amino acid sequence shown in SEQ ID NO: 18; The complementarity determining region CDR3 of the amino acid sequence shown;

b)該LCVR1包含具有SEQ ID NO：19所示的胺基酸序列的互補決定區域CDR1、具有SEQ ID NO：20所示的胺基酸序列的互補決定區域CDR2及具有SEQ ID NO：21所示的胺基酸序列的互補決定區域CDR3； b) The LCVR1 includes the complementarity determining region CDR1 having the amino acid sequence shown in SEQ ID NO: 19, the complementarity determining region CDR2 having the amino acid sequence shown in SEQ ID NO: 20, and the complementarity determining region CDR2 having the amino acid sequence shown in SEQ ID NO: 21. The complementarity determining region CDR3 of the amino acid sequence shown;

c)該HCVR2包含具有SEQ ID NO：22所示的胺基酸序列的互補決定區域CDR1、具有SEQ ID NO：23或SEQ ID NO：24所示的胺基酸序列的互補決定區域CDR2及具有SEQ ID NO：25所示的胺基酸序列的互補決定區域CDR3；以及 c) The HCVR2 includes the complementarity determining region CDR1 having the amino acid sequence shown in SEQ ID NO: 22, the complementarity determining region CDR2 having the amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 24, and The complementarity determining region CDR3 of the amino acid sequence shown in SEQ ID NO: 25; and

d)該LCVR2包含具有SEQ ID NO：26所示的胺基酸序列的互補決定區域CDR1、具有SEQ ID NO：27所示的胺基酸序列的互補決定區域CDR2及具有SEQ ID NO：28所示的胺基酸序列的互補決定區域CDR3。 d) The LCVR2 includes the complementarity determining region CDR1 having the amino acid sequence shown in SEQ ID NO: 26, the complementarity determining region CDR2 having the amino acid sequence shown in SEQ ID NO: 27, and the complementarity determining region CDR2 having the amino acid sequence shown in SEQ ID NO: 28. The complementarity determining region of the amino acid sequence shown is CDR3.

在某些實施方案中，該抗體或其抗原結合片段包含與SEQ ID NO：3具有至少90%,95%,98%或99%或更高同一性的HCVR1；和/或與SEQ ID NO：4具有至少90%,95%,98%或99%或更高同一性的LCVR1；和/或與SEQ ID NO：5具有至少90%,95%,98%或99%或更高同一性的HCVR2；和/或與SEQ ID NO：8具有至少90%,95%,98%或99%或更高同一性的LCVR2； In certain embodiments, the antibody or antigen-binding fragment thereof comprises HCVR1 having at least 90%, 95%, 98%, or 99% or more identity with SEQ ID NO: 3; and/or with SEQ ID NO: 4 LCVR1 with at least 90%, 95%, 98% or 99% or higher identity; and/or at least 90%, 95%, 98% or 99% or higher identity with SEQ ID NO: 5 HCVR2; and/or LCVR2 with at least 90%, 95%, 98% or 99% or higher identity with SEQ ID NO: 8;

較佳地，在上述抗PD-1/PD-L1抗體或其抗原結合片段中，a)該HCVR1包含具有SEQ ID NO：3所示的胺基酸序列；b)該LCVR2包含具有SEQ ID NO：4所示的胺基酸序列；c)該HCVR2包含具有SEQ ID NO：5所示的胺基酸序列；及d)該LCVR2包含具有SEQ ID NO：8所示的胺基酸序列。 Preferably, in the aforementioned anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof, a) the HCVR1 contains the amino acid sequence shown in SEQ ID NO: 3; b) the LCVR2 contains the amino acid sequence shown in SEQ ID NO: : The amino acid sequence shown in 4; c) the HCVR2 includes the amino acid sequence shown in SEQ ID NO: 5; and d) the LCVR2 includes the amino acid sequence shown in SEQ ID NO: 8.

在某些實施方案中，該抗體或其抗原結合片段包含與SEQ ID NO：49或SEQ ID NO：29具有至少90%,95%,98%或99%或更高同一性的HC1；和/或與SEQ ID NO：30具有至少90%,95%,98%或99%或更高同一性的LC1；和/或與SEQ ID NO：33或SEQ ID NO：31或SEQ ID NO：32具有至少90%,95%,98%或99%或更高同一性的HC2；和/或與SEQ ID NO：34具有至少90%,95%,98%或99%或更高同一性的LC2； In certain embodiments, the antibody or antigen-binding fragment thereof comprises HC1 that is at least 90%, 95%, 98%, or 99% or more identical to SEQ ID NO: 49 or SEQ ID NO: 29; and/ Or LC1 with at least 90%, 95%, 98% or 99% or higher identity with SEQ ID NO: 30; and/or with SEQ ID NO: 33 or SEQ ID NO: 31 or SEQ ID NO: 32 HC2 with at least 90%, 95%, 98% or 99% or higher identity; and/or LC2 with at least 90%, 95%, 98% or 99% or higher identity with SEQ ID NO: 34;

較佳地，在上述抗PD-1/PD-L1抗體或其抗原結合片段中，該HC1、該LC1、該HC2及該LC2分別包含SEQ ID NO：49、SEQ ID NO：30、 SEQ ID NO：31及SEQ ID NO：34所示的胺基酸序列；或者分別包含SEQ ID NO：49、SEQ ID NO：30、SEQ ID NO：32及SEQ ID NO：34所示的胺基酸序列；或者分別包含SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：33及SEQ ID NO：34所示的胺基酸序列。 Preferably, in the aforementioned anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof, the HC1, the LC1, the HC2 and the LC2 respectively comprise SEQ ID NO: 49, SEQ ID NO: 30, The amino acid sequence shown in SEQ ID NO: 31 and SEQ ID NO: 34; or the amino acid sequence shown in SEQ ID NO: 49, SEQ ID NO: 30, SEQ ID NO: 32 and SEQ ID NO: 34, respectively Acid sequence; or respectively comprise the amino acid sequence shown in SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 33 and SEQ ID NO: 34.

在某些實施方案中，該抗PD-1/PD-L1抗體為PCT申請號PCT/US2018/041205中公開的抗PD-1/PD-L1抗體。為了本申請的目的，該PCT申請的全部內容特此併入本文作為參考。較佳地，該抗體選自：PD-1/PD-L1雙特異性Ab v13844、Ab v3.0或Ab v3.2。 In certain embodiments, the anti-PD-1/PD-L1 antibody is the anti-PD-1/PD-L1 antibody disclosed in PCT application number PCT/US2018/041205. For the purposes of this application, the entire content of the PCT application is hereby incorporated herein by reference. Preferably, the antibody is selected from: PD-1/PD-L1 bispecific Ab v13844, Ab v3.0 or Ab v3.2.

在某些實施方案中，本發明所述的製劑中該抗體在至少約12個月內表現出穩定性，較佳地，該抗體在至少約24個月內表現出穩定性，更佳地，該抗體在至少約36個月內表現出穩定性。 In certain embodiments, the antibody in the formulation of the present invention exhibits stability for at least about 12 months, preferably, the antibody exhibits stability for at least about 24 months, more preferably, The antibody exhibits stability for at least about 36 months.

在某些實施方案中，製劑在儲存後，例如在2-8℃儲存至少24個月後，或在室溫儲存至少6個月後，或在40℃±2℃儲存1個月後，是穩定的，較佳地具有如下特徵之一或多項： In certain embodiments, the formulation after storage, for example, after storage at 2-8°C for at least 24 months, or after storage at room temperature for at least 6 months, or after storage at 40°C ± 2°C for 1 month, is Stable, preferably having one or more of the following characteristics:

(i)藉由SEC-HPLC法測量，製劑具有大於90%的純度，較佳大於92%、94%、96%、98%、99%的純度； (i) Measured by the SEC-HPLC method, the preparation has a purity greater than 90%, preferably greater than 92%, 94%, 96%, 98%, 99% purity;

(ii)藉由還原型或非還原型CE-SDS法測量，製劑具有大於90%的純度，較佳大於92%、94%、96%、98%的純度； (ii) Measured by the reduced or non-reduced CE-SDS method, the preparation has a purity greater than 90%, preferably greater than 92%, 94%, 96%, 98% purity;

(iii)藉由iCIEF或CEX-HPLC法測量，主成分

Figure 110101453-A0101-12-0009-41
35%，較佳大於或等於35%、40%、45%或50%。 (iii) Measured by iCIEF or CEX-HPLC method, the main component
Figure 110101453-A0101-12-0009-41
35%, preferably greater than or equal to 35%, 40%, 45% or 50%.

在一個方面，本發明提供了一種製備本發明的製劑的方法，包括以下步驟： In one aspect, the present invention provides a method for preparing the formulation of the present invention, including the following steps:

(1)將除表面活性劑之外的成分配製成溶液； (1) Make a solution with ingredients other than surfactants;

(2)藉由超濾，採用步驟(1)製備的溶液對該PD-1/PD-L1雙特異性抗體或其抗原結合片段進行換液，然後濃縮至目標濃度； (2) Use the solution prepared in step (1) to exchange the PD-1/PD-L1 bispecific antibody or its antigen-binding fragment by ultrafiltration, and then concentrate to the target concentration;

(3)添加該表面活性劑到步驟(2)製備的液體中。 (3) Add the surfactant to the liquid prepared in step (2).

在一個方面，本發明提供了一種遞送裝置，其包含有效量的本發明的液體抗體製劑或凍乾製劑。在一個實施方案中，本發明的遞送裝置以包含有效量的本發明的液體抗體製劑或凍乾製劑的預填裝注射器形式提供，例如用於靜脈內、皮下、皮內或者肌內注射，或靜脈內輸注。 In one aspect, the invention provides a delivery device comprising an effective amount of the liquid antibody preparation or lyophilized preparation of the invention. In one embodiment, the delivery device of the present invention is provided in the form of a pre-filled syringe containing an effective amount of the liquid antibody formulation or lyophilized formulation of the present invention, for example for intravenous, subcutaneous, intradermal or intramuscular injection, or Intravenous infusion.

在又一方面，本發明提供向受試者，例如哺乳動物遞送抗PD-1/PD-L1抗體蛋白的方法，包括給予該受試者有效量的本發明的液體抗體製劑或凍乾製劑的步驟，該遞送是例如藉由使用預填裝注射器的遞送裝置實施的。 In yet another aspect, the present invention provides a method for delivering anti-PD-1/PD-L1 antibody protein to a subject, such as a mammal, comprising administering to the subject an effective amount of the liquid antibody preparation or lyophilized preparation of the present invention Step, the delivery is performed by, for example, a delivery device using a pre-filled syringe.

在又一方面，本發明提供本發明的液體抗體製劑或凍乾製劑的用途，用於製備在受試者中治療或預防腫瘤的遞送裝置(如，預填裝注射器)或藥物，該腫瘤為例如癌症，包括但不限於實體瘤、血液學癌、軟組織腫瘤和轉移性病灶。在一些實施方案中，本文所述的癌症包括但不限於霍奇金氏或非霍奇金氏淋巴瘤、黑色素瘤、腎細胞癌、腎癌、肺癌、膀胱癌、胃及食道癌、結腸直腸癌、肝癌、肝細胞癌、膽管癌、胰臟癌、乳癌、三陰性乳癌、卵巢癌、子宮內膜癌、***癌、小細胞肺癌(SCLC)、非小細胞肺(NSCLC)、間皮瘤、頭頸鱗狀細胞癌(SCCHN)、軟組織肉瘤或多形性神經膠母細胞瘤。較佳地，該肺癌為NSCLC(鱗狀及非鱗狀)、小細胞肺癌或間皮瘤。 In yet another aspect, the present invention provides the use of the liquid antibody preparation or lyophilized preparation of the present invention to prepare a delivery device (eg, a pre-filled syringe) or a drug for treating or preventing tumors in a subject, and the tumor is For example, cancers include, but are not limited to, solid tumors, hematological cancers, soft tissue tumors, and metastatic lesions. In some embodiments, the cancers described herein include, but are not limited to, Hodgkin's or non-Hodgkin's lymphoma, melanoma, renal cell carcinoma, kidney cancer, lung cancer, bladder cancer, gastric and esophageal cancer, colorectal cancer Cancer, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, pancreatic cancer, breast cancer, triple negative breast cancer, ovarian cancer, endometrial cancer, prostate cancer, small cell lung cancer (SCLC), non-small cell lung (NSCLC), mesothelioma , Squamous cell carcinoma of the head and neck (SCCHN), soft tissue sarcoma or glioblastoma multiforme. Preferably, the lung cancer is NSCLC (squamous and non-squamous), small cell lung cancer or mesothelioma.

本發明還提供了一種藉由向受試者施用本發明的液體抗體製劑或凍乾製劑或包含該液體抗體製劑或凍乾製劑的遞送裝置(如，預填裝注射器)或藥物，在受試者中增強免疫效應細胞反應和/或減少免疫抑制的方法。 The present invention also provides a liquid antibody preparation or lyophilized preparation of the present invention or a delivery device (such as a pre-filled syringe) or a drug containing the liquid antibody preparation or lyophilized preparation to a subject. Methods of enhancing immune effector cell response and/or reducing immunosuppression.

本發明還提供了一種藉由向受試者施用本發明的液體抗體製劑或凍乾製劑或包含該液體抗體製劑或凍乾製劑的遞送裝置(如，預填裝注射器)或藥物，預防和/或治療受試者的疾病，例如上述腫瘤的方法。 The present invention also provides a preventive and/ Or a method for treating a subject's disease, such as the above-mentioned tumor.

本發明製劑的有益效果： The beneficial effects of the preparation of the present invention:

1)本發明提供的凍乾及液體抗體製劑性質穩定，在5℃±3℃條件下可穩定至少24個月。 1) The freeze-dried and liquid antibody preparations provided by the present invention are stable in nature, and can be stable for at least 24 months at 5°C±3°C.

2)本發明提供的處方不僅可以保證低濃度液體製劑的穩定，還可以保證高濃度液體製劑的穩定而不額外添加輔料，為高濃度液體製劑皮下給藥提供可能。在實施案例中，本發明高濃度液體製劑能夠直接製備成凍乾製劑無需優化處方，凍乾後能保持其結構與功能的完整性。 2) The prescription provided by the present invention can not only ensure the stability of low-concentration liquid preparations, but also can ensure the stability of high-concentration liquid preparations without adding auxiliary materials, providing the possibility of subcutaneous administration of high-concentration liquid preparations. In an implementation case, the high-concentration liquid preparation of the present invention can be directly prepared into a freeze-dried preparation without optimizing the prescription, and can maintain its structural and functional integrity after freeze-drying.

3)本發明提供的凍乾製劑易複溶，在液體製劑可接受的注射黏度範圍內(

Figure 110101453-A0101-12-0011-38
10cP)，且在常溫下放置一天后各項檢測指標均未發生變化。藉由減少複溶液體積可以製備更高濃度(例如170mg/ml)液體製劑。相同的給藥量，減少給藥體積，提高臨床給藥的順應性。 3) The freeze-dried preparation provided by the present invention is easy to reconstitute and is within the acceptable injection viscosity range of the liquid preparation (
Figure 110101453-A0101-12-0011-38
10cP), and there is no change in all detection indicators after being placed at room temperature for one day. By reducing the volume of the reconstituted solution, a higher concentration (for example, 170 mg/ml) liquid preparation can be prepared. The same amount of administration reduces the volume of administration and improves the compliance of clinical administration.

本發明的其它實施方案將藉由參閱此後的詳細說明而清楚明瞭。 Other embodiments of the present invention will be made clear by referring to the detailed description hereinafter.

結合以下附圖一起閱讀時，將更好地理解以下詳細描述的本發明的較佳實施方案。出於說明本發明的目的，圖中顯示了目前較佳的實施方案。然而，應當理解本發明不限於圖中所示實施方案的精確安排和手段。 When read in conjunction with the following drawings, the preferred embodiments of the present invention described in detail below will be better understood. For the purpose of illustrating the present invention, the figure shows the presently preferred embodiment. However, it should be understood that the present invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

圖1顯示了處方前試驗中，藉由SEC-HPLC法測定的各樣品中蛋白純度隨時間變化圖。圖中Met表示甲硫胺酸。 Figure 1 shows a graph of the protein purity in each sample measured by the SEC-HPLC method over time in the pre-prescription test. Met in the figure represents methionine.

圖2顯示了處方前試驗中，藉由iCIEF法測定的各樣品中電荷變異體主成分和酸性組分隨時間變化圖。 Figure 2 shows the changes in the main components and acidic components of the charge variants in each sample measured by the iCIEF method over time in the pre-prescription test.

圖3顯示了處方篩選試驗中，在40℃±2℃條件下，藉由SEC-HPLC法測定的各樣品中蛋白純度隨時間變化圖 Figure 3 shows the variation of protein purity in each sample measured by SEC-HPLC method over time under the condition of 40℃±2℃ in the prescription screening test

圖4顯示了處方篩選試驗中，在25℃±2℃條件下，藉由SEC-HPLC法測定的各樣品中蛋白純度隨時間變化圖。 Figure 4 shows a graph of the protein purity in each sample measured by the SEC-HPLC method over time under the condition of 25°C±2°C in the prescription screening test.

圖5顯示了處方篩選試驗中，在40℃±2℃條件下，藉由非還原型CE-SDS法測定的各樣品中蛋白純度隨時間變化圖。 Figure 5 shows the time-varying graph of the protein purity in each sample measured by the non-reducing CE-SDS method under the condition of 40℃±2℃ in the prescription screening test.

圖6顯示了處方篩選試驗中，在40℃±2℃條件下，藉由CEX-HPLC法測定的各樣品中電荷變異體主成分和酸性組分隨時間變化圖。 Figure 6 shows a graph of the main components and acidic components of the charge variants in each sample measured by the CEX-HPLC method at 40°C ± 2°C in the prescription screening test over time.

圖7顯示了處方篩選試驗中，在25℃±2℃條件下，藉由CEX-HPLC法測定的各樣品中電荷變異體主成分和酸性組分隨時間變化圖。 Figure 7 shows a graph of the main components and acidic components of the charge variants in each sample determined by the CEX-HPLC method at 25°C ± 2°C in the prescription screening test over time.

發明詳述Detailed description of the invention

在詳細描述本發明之前，應瞭解，本發明不受限於本說明書中的特定方法及實驗條件，因為該方法以及條件是可以改變的。另外，本文所用術語僅是供說明特定實施方案之用，而不意欲為限制性的。 Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific methods and experimental conditions in this specification, because the methods and conditions can be changed. In addition, the terms used herein are only for describing specific embodiments, and are not intended to be limiting.

定義definition

除非另有定義，否則本文中使用的所有技術和科學術語均具有與本領域一般技術人員通常所理解的含義相同的含義。為了本發明的目的，下文定義了以下術語。 Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. For the purpose of the present invention, the following terms are defined below.

術語“約”在與數字數值聯合使用時意為涵蓋具有比指定數字數值小5%的下限和比指定數字數值大5%的上限的範圍內的數字數值。 The term "about" when used in conjunction with a numerical value means to cover a numerical value within a range that has a lower limit that is 5% smaller than the specified numerical value and an upper limit that is 5% larger than the specified numerical value.

術語“和/或”當用於連接兩個或多個可選項時，應理解為意指可選項中的任一項或可選項中的任意兩項或多項。 When the term "and/or" is used to connect two or more alternatives, it should be understood to mean any one of the alternatives or any two or more of the alternatives.

如本文中所用，術語“包含”或“包括”意指包括所述的要素、整數或步驟，但是不排除任意其他要素、整數或步驟。在本文中，當使用術語“包含”或“包括”時，除非另有指明，否則也涵蓋由所述及的要素、整數或步驟組成的情形。例如，當提及“包含”某個具體序列的抗體可變區時，也旨在涵蓋由該具體序列組成的抗體可變區。 As used herein, the term "comprising" or "including" means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps. In this document, when the term "comprises" or "includes" is used, unless otherwise specified, it also encompasses the situation consisting of the stated elements, integers or steps. For example, when referring to an antibody variable region that "comprises" a specific sequence, it is also intended to encompass the antibody variable region composed of the specific sequence.

術語“免疫檢查點分子”意指免疫系統中存在的一類抑制性信號分子，藉由調節外周組織中免疫反應的持續性和強度避免組織損傷，並參與維持對於自身抗原的耐受(Pardoll DM.,The blockade of immune checkpoints in cancer immunotherapy.Nat Rev Cancer,2012,12(4)：252-264)。研究發現，腫瘤細胞能夠逃避體內免疫系統而失控增殖的原因之一是利用了免疫檢查點分子的抑制性信號通路，由此抑制了T淋巴細胞活性， 使得T淋巴細胞不能有效發揮對腫瘤的殺傷效應(Yao S,Zhu Y和Chen L.,Advances in targeting cell surface signaling molecules for immune modulation.Nat Rev Drug Discov,2013,12(2)：130-146)。免疫檢查點分子包括但不限於程序性死亡1(PD-1)、PD-L1、PD-L2、細胞毒T淋巴細胞抗原4(CTLA-4)、LAG-3和TIM-3。 The term "immune checkpoint molecule" refers to a type of inhibitory signal molecule present in the immune system, which prevents tissue damage by regulating the persistence and intensity of immune response in peripheral tissues, and participates in maintaining tolerance to self-antigens (Pardoll DM. , The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer, 2012, 12(4): 252-264). Studies have found that one of the reasons why tumor cells can evade the immune system in the body and proliferate out of control is the use of the inhibitory signaling pathway of immune checkpoint molecules, thereby inhibiting the activity of T lymphocytes. Make T lymphocytes unable to effectively exert the killing effect on tumors (Yao S, Zhu Y and Chen L., Advances in targeting cell surface signaling molecules for immune modulation. Nat Rev Drug Discov, 2013, 12(2): 130-146) . Immune checkpoint molecules include but are not limited to programmed death 1 (PD-1), PD-L1, PD-L2, cytotoxic T lymphocyte antigen 4 (CTLA-4), LAG-3 and TIM-3.

如本文所用的術語“程序性細胞死亡配體1”、“PD-L1”、“程序性死亡配體1”、“分化簇274”、“CD274”或“B7同系物1”是指來自任何脊椎動物來源的任何天然PD-L1，該任何脊椎動物來源包括哺乳動物，諸如靈長類(例如，人)和齧齒類(例如，小鼠和大鼠)。該術語涵蓋“全長”、未加工的PD-L1以及由細胞中的加工所產生的任何形式的PD-L1。PD-L1可作為跨膜蛋白或作為可溶性蛋白存在。該術語還涵蓋天然存在的PD-L1的變體，例如剪接變體或等位基因變體。PD-L1的基本結構包括4個結構域：胞外Ig樣V型結構域和Ig樣C2型結構域、跨膜結構域以及細胞質結構域。可在NCBI Gene ID No.29126下找到關於人PD-L1基因(包括基因組DNA序列)的另外信息。可在NCBI Gene ID No.60533下找到關於小鼠PD-L1基因(包括基因組DNA序列)的另外信息。示例性全長人PD-L1蛋白的胺基酸序列可例如在NCBI登錄號NP_001254653或UniProt登錄號Q9NZQ7下找到，而可例如在NCBI登錄號NP_068693或Uniprot登錄號Q9EP73下找到示例性全長小鼠PD-L1蛋白序列。在本文中，術語“抗體”以最廣意義使用，指包含抗原結合位點的蛋白質，涵蓋各種結構的天然抗體和人工抗體，包括但不限於完整抗體和抗體的抗原結合片段。 As used herein, the terms "programmed cell death ligand 1", "PD-L1", "programmed cell death ligand 1", "cluster of differentiation 274", "CD274" or "B7 homolog 1" refer to any Any natural PD-L1 of vertebrate origin, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full length", unprocessed PD-L1 and any form of PD-L1 produced by processing in the cell. PD-L1 can exist as a transmembrane protein or as a soluble protein. The term also encompasses naturally occurring variants of PD-L1, such as splice variants or allelic variants. The basic structure of PD-L1 includes 4 domains: extracellular Ig-like V-type domain and Ig-like C2-type domain, transmembrane domain and cytoplasmic domain. Additional information about the human PD-L1 gene (including genomic DNA sequence) can be found under NCBI Gene ID No. 29126. Additional information about the mouse PD-L1 gene (including genomic DNA sequence) can be found under NCBI Gene ID No. 60533. The amino acid sequence of an exemplary full-length human PD-L1 protein can be found, for example, under NCBI accession number NP_001254653 or UniProt accession number Q9NZQ7, and an exemplary full-length mouse PD-L1 protein can be found, for example, under NCBI accession number NP_068693 or Uniprot accession number Q9EP73. L1 protein sequence. As used herein, the term "antibody" is used in the broadest sense and refers to a protein containing an antigen binding site, encompassing natural antibodies and artificial antibodies of various structures, including but not limited to intact antibodies and antigen-binding fragments of antibodies.

術語“全抗體”、“全長抗體”、“完全抗體”和“完整抗體”在本文中可互換地用來指包含由二硫鍵相互連接的至少兩條重鏈(H)和兩條輕鏈(L)的糖蛋白。每條重鏈由重鏈可變區(本文中縮寫為VH)和重鏈恆定區組成。重鏈恆定區由3個結構域CH1、CH2和CH3組成。每條輕鏈由輕鏈可變區(本文中縮寫為VL)和輕鏈恆定區組成。輕鏈恆定區由一個結構域CL組成。VH區和VL區可以進一步再劃分為超變區(為互補決定區(CDR)，其間插有較保守的區域(為構架區(FR))。每個VH和VL由三個CDR和4個FR組成，從胺基端到羧基端以如下順序排列：FR1,CDR1,FR2,CDR2,FR3,CDR3,FR4。恆定區不直接參與抗體與抗原的結合，但是顯示出多種效應子功能。 The terms "whole antibody", "full-length antibody", "full antibody" and "whole antibody" are used interchangeably herein to refer to at least two heavy chains (H) and two light chains interconnected by disulfide bonds. (L) Glycoprotein. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region. The heavy chain constant region is composed of three structural domains CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated as VL herein) and a light chain constant region. The light chain constant region consists of a domain CL. The VH and VL regions can be further divided into hypervariable regions (complementarity determining regions (CDR)), with more conservative regions (framework regions (FR)) interposed between them. Each VH and VL consists of three CDRs and four The FR composition is arranged in the following order from the amino end to the carboxyl end: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region does not directly participate in the binding of the antibody to the antigen, but displays a variety of effector functions.

如本文所用，術語“多特異性”抗體指具有至少兩個抗原結合位點的抗體，該至少兩個抗原結合位點中的每一個抗原結合位點與相同抗原的不同表位或與不同抗原的不同表位結合。本文提供的抗體通常是多特異性抗體，例如雙特異性抗體。多特異性抗體是對至少兩個不同抗原表位具有結合特異性的抗體。在一個實施方案中，本文提供了這樣的雙特異性抗體，其具有針對第一抗原和第二抗原的結合特異性。例如第一抗原為PD-L1，第二抗原為PD-1。 As used herein, the term "multispecific" antibody refers to an antibody having at least two antigen binding sites, each of which is associated with a different epitope of the same antigen or with a different antigen. Binding of different epitopes. The antibodies provided herein are generally multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are antibodies that have binding specificities for at least two different epitopes. In one embodiment, provided herein are bispecific antibodies that have binding specificities for a first antigen and a second antigen. For example, the first antigen is PD-L1, and the second antigen is PD-1.

術語“人源化”抗體指包含來自非人類HVR的胺基酸殘基和來自人FR的胺基酸殘基的嵌合抗體。在一些實施方案中，人源化抗體包含全部或基本上全部的HVR(例如，CDR)與非人抗體的那些HVR對應並且全部或基本上全部的FR區與人抗體的那些FR對應。人源化抗體任選地 可以包含從人抗體衍生的抗體恆定區的至少一部分。抗體(例如非人抗體)的“人源化形式”指已經歷過人源化的抗體。 The term "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVR and amino acid residues from human FR. In some embodiments, the humanized antibody comprises all or substantially all of the HVR (eg, CDR) corresponding to those of the non-human antibody and all or substantially all of the FR regions corresponding to those of the human antibody. Humanized antibody optionally It may comprise at least a part of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

術語“Fc區”在本文中用於定義免疫球蛋白重鏈的C端區域，該區域包含至少一部分的恆定區。該術語包括天然序列Fc區和變體Fc區。在某些實施方案中，人IgG重鏈Fc區從Cys226或Pro230延伸至重鏈的羰基端。然而，Fc區的C端賴胺酸(Lys447)可以存在或者可以不存在。除非另外說明，Fc區或恆定區中的胺基酸殘基的編號是根據EU編號系統，其也被稱為EU索引，如在Kabat等，Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,MD,1991中所述。 The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In certain embodiments, the Fc region of a human IgG heavy chain extends from Cys226 or Pro230 to the carbonyl end of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified, the numbering of amino acid residues in the Fc region or constant region is based on the EU numbering system, which is also called the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service , National Institutes of Health, Bethesda, MD, 1991.

術語“可變區”或“可變結構域”是指參與抗體與抗原結合的抗體重或輕鏈的結構域。天然抗體的重鏈和輕鏈的可變結構域通常具有相似的結構，其中每個結構域包含四個保守的框架區(FR)和三個互補決定區(參見，例如，Kindt等Kuby Immunology，6th ed.,W.H.Freeman and Co.91頁(2007))。單個VH或VL結構域可以足以給予抗原結合特異性。此外，可以使用來自與特定抗原結合的抗體的VH或VL結構域來分離結合該抗原的抗體，以分別篩選互補VL或VH結構域的文庫。參見，例如，Portolano等，J.Immunol.150：880-887(1993)；Clarkson等，Nature 352：624-628(1991)。 The term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that participates in the binding of an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies usually have similar structures, where each domain contains four conserved framework regions (FR) and three complementarity determining regions (see, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co. 91 pages (2007)). A single VH or VL domain may be sufficient to give antigen binding specificity. In addition, VH or VL domains from antibodies that bind to a specific antigen can be used to isolate antibodies that bind to that antigen to screen libraries of complementary VL or VH domains, respectively. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

可變區通常表現出由三個高變區連接的相對保守的構架區(FR)的相同的一般結構，該高變區也被稱為互補決定區或CDR。通常藉由構架區定位(align)來自每對的兩條鏈的CDR，該CDR使得可結合特異性 表位。兩條輕鏈和重鏈可變區從N-末端到C-末端通常包含結構域FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。 Variable regions usually exhibit the same general structure of relatively conserved framework regions (FR) connected by three hypervariable regions, which are also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are usually aligned by the framework regions, which allow specific binding gauge. The variable regions of the two light and heavy chains usually comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from N-terminus to C-terminus.

“互補決定區”或“CDR區”或“CDR”或“高變區”(在本文中與超變區“HVR”可以互換使用)，是抗體可變結構域中在序列上高變並且形成在結構上確定的環(“超變環”)和/或含有抗原接觸殘基(“抗原接觸點”)的區域。CDR主要負責與抗原表位結合。重鏈和輕鏈的CDR通常被稱作CDR1、CDR2和CDR3，從N-端開始順序編號。位於抗體重鏈可變結構域內的CDR被稱作HCDR1、HCDR2和HCDR3，而位於抗體輕鏈可變結構域內的CDR被稱作LCDR1、LCDR2和LCDR3。在一個給定的輕鏈可變區或重鏈可變區胺基酸序列中，各CDR的精確胺基酸序列邊界可以使用許多公知的抗體CDR指派系統的任一種或其組合確定，該指派系統包括例如：基於抗體的三維結構和CDR環的拓撲學的Chothia(Chothia等人.(1989)Nature 342：877-883，Al-Lazikani等人，“Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))，基於抗體序列可變性的Kabat(Kabat等人，Sequences of Proteins of Immunological Interest，第4版，U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath)，Contact(University College London)，國際ImMunoGeneTics database(IMGT)(萬維網imgt.cines.fr/)，以及基於利用大量晶體結構的近鄰傳播聚類(affinity propagation clustering)的North CDR定義(North等，“A New Clustering of Antibody CDR Loop Conformations”，Journal of Molecular Biology，406，228-256(2011))。 "Complementarity determining region" or "CDR region" or "CDR" or "hypervariable region" (herein can be used interchangeably with hypervariable region "HVR") is an antibody variable domain that is hypervariable in sequence and forms Structurally defined loops ("hypervariable loops") and/or regions containing antigen contact residues ("antigen contact points"). CDR is mainly responsible for binding to antigen epitopes. The CDRs of the heavy chain and light chain are usually referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. The CDRs located in the variable domain of the antibody heavy chain are called HCDR1, HCDR2, and HCDR3, and the CDRs located in the variable domain of the antibody light chain are called LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems. The system includes, for example: Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal based on the three-dimensional structure of antibodies and the topology of CDR loops of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDepartment of Health and Human Services, National Institutes of Health (1987) ), AbM (University of Bath), Contact (University College London), International ImmunoGeneTics database (IMGT) (World Wide Web imgt.cines.fr/), and North based on affinity propagation clustering using a large number of crystal structures CDR definition (North et al., "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256 (2011)).

例如，使用Kabat和Chothia編號的CDR區域的不同定義範圍。 For example, the different definition ranges of CDR regions numbered by Kabat and Chothia are used.

Figure 110101453-A0101-12-0018-1
Figure 110101453-A0101-12-0018-1

CDR也可以基於與參考CDR序列(例如本發明示例性CDR之任一)具有相同的Kabat編號位置而確定。 The CDR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (for example, any of the exemplary CDRs of the present invention).

除非另有說明，否則在本發明中，術語“CDR”或“CDR序列”涵蓋以上述任一種方式確定的CDR序列。 Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-mentioned ways.

除非另有說明，否則在本發明中，當提及抗體可變區中的殘基位置(包括重鏈可變區殘基和輕鏈可變區殘基)時，是指根據Kabat編號系統(Kabat等人，Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.(1991))的編號位置。 Unless otherwise specified, in the present invention, when referring to residue positions in the variable region of an antibody (including heavy chain variable region residues and light chain variable region residues), it refers to the Kabat numbering system ( Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

在一個實施方案中，本發明抗體的CDR藉由North定義規則確定邊界，例如下文表1b和表1c所示。 In one embodiment, the CDR of the antibody of the present invention is bounded by North definition rules, for example, as shown in Table 1b and Table 1c below.

然而，應該注意，基於不同的指派系統獲得的同一抗體的可變區的CDR的邊界可能有所差異。即不同指派系統下定義的同一抗體可變區的CDR序列有所不同。因此，在涉及用本發明定義的具體CDR序列限定抗體時，該抗體的範圍還涵蓋了這樣的抗體，其可變區序列包含該具體CDR序列，但是由於應用了不同的方案(例如不同的指派系統規則或組合)而導致其所聲稱的CDR邊界與本發明所定義的具體CDR邊界不同。 However, it should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different. Therefore, when it comes to defining an antibody with a specific CDR sequence defined in the present invention, the scope of the antibody also covers antibodies whose variable region sequence contains the specific CDR sequence, but due to the application of different schemes (such as different assignments). System rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined in the present invention.

具有不同特異性(即，針對不同抗原的不同結合位點)的抗體具有不同的CDR。然而，儘管CDR在抗體與抗體之間是不同的，但是CDR內只有有限數量的胺基酸位置直接參與抗原結合。使用Kabat,Chothia,AbM、Contact和North方法中的至少兩種，可以確定最小重疊區域，從而提供用於抗原結合的“最小結合單位”。最小結合單位可以是CDR的一個子部分。正如本領域技術人員明瞭，藉由抗體的結構和蛋白折疊，可以確定CDR序列其餘部分的殘基。因此，本發明也考慮本文所給出的任何CDR的變體。例如，在一個CDR的變體中，最小結合單位的胺基酸殘基可以保 持不變，而根據Kabat或Chothia定義的其餘CDR殘基可以被保守胺基酸殘基替代。 Antibodies with different specificities (ie, different binding sites for different antigens) have different CDRs. However, although CDRs are different from antibody to antibody, there are only a limited number of amino acid positions within the CDR that directly participate in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact, and North methods, the minimum overlap area can be determined, thereby providing the "minimum binding unit" for antigen binding. The minimum binding unit can be a sub-portion of the CDR. As those skilled in the art know, the structure of the antibody and protein folding can determine the residues of the rest of the CDR sequence. Therefore, the present invention also considers any CDR variants given herein. For example, in a CDR variant, the amino acid residue of the smallest binding unit can preserve The remaining CDR residues defined by Kabat or Chothia can be replaced by conservative amino acid residues.

術語“抗體製劑”指一種製備物，該製備物處於允許作為活性成分的抗體的生物活性可以有效發揮的形式，並且不含有對於待施用該製劑的受試者而言具有不可接受毒性的其它組分。這類抗體製劑通常是無菌的。通常，抗體製劑中包含可藥用賦形劑。“可藥用”賦形劑是可以合理地施用至受試哺乳動物以便製劑中所用活性成分的有效劑量可以遞送至受試者的試劑。賦形劑的濃度與施用模式相適應，例如可以是注射可接受的。 The term "antibody preparation" refers to a preparation that is in a form that allows the biological activity of the antibody as an active ingredient to be effectively exhibited, and does not contain other groups that have unacceptable toxicity to the subject to which the preparation is to be administered. point. Such antibody preparations are usually sterile. Generally, pharmaceutically acceptable excipients are included in antibody preparations. A "pharmaceutically acceptable" excipient is an agent that can be reasonably administered to a tested mammal so that an effective dose of the active ingredient used in the formulation can be delivered to the subject. The concentration of the excipient is adapted to the mode of administration, for example, it may be acceptable for injection.

術語“抗PD-1/PD-L1抗體製劑”在本文中也簡稱為“本發明的抗體製劑”，意指包含抗PD-1/PD-L1抗體蛋白作為活性成分並包含可藥用賦形劑的製備物。將抗PD-1/PD-L1抗體蛋白與可藥用賦形劑組合後，作為活性成分的抗PD-1/PD-L1抗體蛋白適於治療性或預防性施與人類或非人類動物。本發明的抗體製劑可以例如製備成水性形式的液體製劑，例如，即用式預填裝注射器，或者製備成凍乾製劑，在即將使用前藉由溶解和/或懸浮於生理可接受的溶液中進行重構(即，複溶)。在一些實施方案中，抗PD-1/PD-L1抗體蛋白製劑是液體製劑形式。 The term "anti-PD-1/PD-L1 antibody preparation" is also referred to as "antibody preparation of the present invention" herein, meaning that it contains anti-PD-1/PD-L1 antibody protein as an active ingredient and contains pharmaceutically acceptable excipients. Preparation of the agent. After the anti-PD-1/PD-L1 antibody protein is combined with a pharmaceutically acceptable excipient, the anti-PD-1/PD-L1 antibody protein as an active ingredient is suitable for therapeutic or preventive administration to human or non-human animals. The antibody preparation of the present invention can be prepared, for example, as a liquid preparation in an aqueous form, for example, a ready-to-use pre-filled syringe, or prepared as a lyophilized preparation, by dissolving and/or suspending in a physiologically acceptable solution immediately before use Perform reconstitution (ie, reconstitution). In some embodiments, the anti-PD-1/PD-L1 antibody protein preparation is in the form of a liquid preparation.

“穩定的”抗體製劑是製劑中的抗體在儲存於特定條件下之後保有可接受程度的物理穩定性和/或化學穩定性。儘管抗體製劑中所含的抗體在儲存特定時間之後可能不會100%維持其化學結構，但通常在儲存特定時間之後維持約90%、約95%、約96%、約97%、約98%或約99%的抗體結構或功能，則認為抗體製劑是“穩定的”。在一些具體的實施方案中，本發明的抗PD-1/PD-L1抗體蛋白製劑在製造、製備、運輸和長期儲存過程中表現出低至檢測不到的抗體聚集或降解或化學修飾，從而極少或 甚至是沒有抗PD-1/PD-L1抗體蛋白的生物活性損失，表現出高度穩定性。在一些實施方案中，本發明的抗PD-1/PD-L1抗體蛋白製劑在儲存後，基本上保留其物理和化學穩定性。較佳地，本發明液體製劑可以在室溫穩定至少6個月或在40℃±2℃儲存1個月，和/或在25℃±2℃穩定至少3個月，和/或在2-8℃穩定至少24個月。 A "stable" antibody preparation is when the antibody in the preparation retains an acceptable degree of physical and/or chemical stability after storage under specific conditions. Although the antibody contained in the antibody preparation may not maintain 100% of its chemical structure after storage for a specific time, it usually maintains about 90%, about 95%, about 96%, about 97%, about 98% after storage for a specific time. Or about 99% of the structure or function of the antibody, the antibody preparation is considered "stable." In some specific embodiments, the anti-PD-1/PD-L1 antibody protein preparations of the present invention exhibit undetectable antibody aggregation or degradation or chemical modification during manufacturing, preparation, transportation and long-term storage, thereby Rarely or Even there is no loss of the biological activity of the anti-PD-1/PD-L1 antibody protein, showing a high degree of stability. In some embodiments, the anti-PD-1/PD-L1 antibody protein formulation of the present invention substantially retains its physical and chemical stability after storage. Preferably, the liquid formulation of the present invention can be stable at room temperature for at least 6 months or stored at 40°C±2°C for 1 month, and/or stable at 25°C±2°C for at least 3 months, and/or at 2- Stable at 8°C for at least 24 months.

本領域已知多種分析技術可以用於測定蛋白質的穩定性，參見例如Peptide and Protein Drug Delivery,247-301,Vincent Lee Ed.,Marcel Dekker,Inc.,New York,N.Y.,Pubs(1991)and Jones,A.Adv.Drug Delivery Rev.10：29-90(1993)。可以在選定的溫度和選定的儲存時間測量穩定性。例如，可以基於預期的製劑貨架期來選擇儲存時間。備選地，可以使用加速穩定性試驗。在一些實施方案中，藉由對抗體製劑進行各種脅迫測試來進行穩定性測試。這些測試可以代表調配的抗體製劑在製造、儲存或運輸期間可能遭遇到的極端條件，也可以代表在非製造、儲存或運輸期間可能使抗體製劑中的抗體的不穩定性加速的條件。例如，可以將經調配的抗PD-1/PD-L1抗體蛋白製劑充填至玻璃小瓶中以檢驗在高溫脅迫下的抗體穩定性。 A variety of analytical techniques known in the art can be used to determine the stability of proteins, see, for example, Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs (1991) and Jones , A. Adv. Drug Delivery Rev. 10: 29-90 (1993). The stability can be measured at a selected temperature and selected storage time. For example, the storage time can be selected based on the expected shelf life of the formulation. Alternatively, an accelerated stability test can be used. In some embodiments, the stability test is performed by performing various stress tests on the antibody preparation. These tests can represent extreme conditions that may be encountered during manufacture, storage, or transportation of the formulated antibody preparation, or conditions that may accelerate the instability of the antibody in the antibody preparation during non-manufacture, storage, or transportation. For example, the formulated anti-PD-1/PD-L1 antibody protein preparation can be filled into a glass vial to test the antibody stability under high temperature stress.

經一段儲存時間後，製劑不顯示聚集、沉澱、混濁和/或變性；或顯示非常少的聚集、沉澱、混濁和/或變性，則可以認為抗體在製劑中“保持其物理穩定性”。由於製劑中抗體的聚集可以潛在地導致患者增加的免疫反應，從而導致安全性問題。因此，需要使在製劑中的抗體聚集最小化或防止聚集。光散射法可以用於測定製劑中的可見聚集物。SEC可以用於測定製劑中的可溶性聚集物。此外，可以藉由目視檢查製劑的外觀、顏色和/或澄清度、或者藉由OD350nm法檢測製劑的濁度、或者藉由非還原型CE-SDS法測定製劑的純度，來指示製劑的穩定性。在一個實施方案中， 藉由測定在特定溫度下儲存特定時間之後製劑中的抗體單體的百分比來測量製劑的穩定性，其中製劑中的抗體單體的百分比越高，則製劑的穩定性越高。 After a period of storage time, the preparation does not show aggregation, precipitation, turbidity and/or denaturation; or shows very little aggregation, precipitation, turbidity and/or denaturation, it can be considered that the antibody "maintains its physical stability" in the preparation. The accumulation of antibodies in the preparation can potentially lead to an increased immune response in patients, leading to safety issues. Therefore, there is a need to minimize or prevent aggregation of the antibody in the formulation. The light scattering method can be used to determine the visible aggregates in the formulation. SEC can be used to determine soluble aggregates in the formulation. In addition, the stability of the preparation can be indicated by visually inspecting the appearance, color and/or clarity of the preparation, or by detecting the turbidity of the preparation by the OD 350nm method, or by measuring the purity of the preparation by the non-reducing CE-SDS method sex. In one embodiment, the stability of the formulation is measured by determining the percentage of antibody monomers in the formulation after storage at a specific temperature for a specific time, where the higher the percentage of antibody monomers in the formulation, the more stable the formulation. high.

“可接受程度的”物理穩定性可以表示於特定溫度下儲存特定時間之後，在製劑中檢測到至少約90%的抗PD-1/PD-L1抗體蛋白單體。在一些實施方案中，在特定溫度儲存至少2周、至少28天、至少1個月、至少2個月、至少3個月、至少4個月、至少5個月、至少6個月、至少7個月、至少8個月、至少9個月、至少10個月、至少11個月、至少12個月、至少18個月、至少24個月或更久後，可接受程度的物理穩定性表示至少約90%、91%、92%、93%、94%、95%、96%、97%、98%、99%的抗PD-1/PD-L1抗體蛋白單體。當評估物理穩定性時，藥物製劑儲存的特定溫度可為約-80℃至約45℃的任一溫度，例如儲存於約-80℃、約-30℃、約-20℃、約0℃、約4℃-8℃、約5℃、約25℃、約35℃、約37℃、約40℃、約42℃或約45℃。例如，若儲存於約40℃±2℃ 1個月之後，檢測到至少約90%、91%、92%、93%、94%、95%、96%、97%、98%、99%的抗PD-1/PD-L1抗體蛋白單體，則藥物製劑視為是穩定的；若儲存於約25℃ 2個月之後，檢測到至少約90%、91%、92%、93%、94%、95%、96%、97%、98%、99%的抗PD-1/PD-L1抗體蛋白單體，則藥物製劑視為是穩定的；若儲存於約5℃ 9個月之後，檢測到至少約90%、91%、92%、93%、94%、95%、96%、97%、98%、99%的抗PD-1/PD-L1抗體蛋白單體，則藥物製劑視為是穩定的。 An "acceptable degree" of physical stability can mean that at least about 90% of the anti-PD-1/PD-L1 antibody protein monomer is detected in the preparation after storage at a specific temperature for a specific time. In some embodiments, storage at a specific temperature for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months After months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months or more, an acceptable degree of physical stability indicates At least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the anti-PD-1/PD-L1 antibody protein monomer. When evaluating physical stability, the specific temperature at which the pharmaceutical preparation is stored may be any temperature from about -80°C to about 45°C, for example, when stored at about -80°C, about -30°C, about -20°C, about 0°C, About 4°C-8°C, about 5°C, about 25°C, about 35°C, about 37°C, about 40°C, about 42°C, or about 45°C. For example, if stored at about 40℃±2℃ for 1 month, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of Anti-PD-1/PD-L1 antibody protein monomer, the pharmaceutical preparation is considered stable; if stored at about 25℃ for 2 months, at least about 90%, 91%, 92%, 93%, 94% are detected %, 95%, 96%, 97%, 98%, 99% of the anti-PD-1/PD-L1 antibody protein monomer, the pharmaceutical preparation is considered stable; if stored at about 5℃ for 9 months, At least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the anti-PD-1/PD-L1 antibody protein monomer is detected, then the pharmaceutical preparation Considered to be stable.

經一段儲存時間後，如果製劑中的抗體不顯示顯著的化學改變，則可以認為抗體在製劑中“保持其化學穩定性”。大多數化學不穩定性源自於形成了抗體的共價修飾形式(例如，抗體的電荷變異體)。例如由天冬 胺酸異構化、N和C末端修飾，可以形成鹼性變異體；由脫醯胺化、唾液酸化和糖化，可以產生酸性變異體。化學穩定性可以藉由檢測和/或定量抗體的化學改變形式來評估。例如，可以藉由陽離子交換色譜(CEX)或成像毛細管等電聚焦電泳(iCIEF)檢測製劑中抗體的電荷變異體。在一個實施方案中，藉由測定在特定溫度下儲存特定時間之後製劑中抗體的電荷變異體百分比變化值來測量製劑的穩定性，其中該變化值越小，則製劑的穩定性越高。 After a period of storage, if the antibody in the preparation does not show a significant chemical change, it can be considered that the antibody "maintains its chemical stability" in the preparation. Most chemical instabilities result from the formation of covalently modified forms of antibodies (for example, charge variants of antibodies). For example by Tiandong Amino acid isomerization, N- and C-terminal modification can form basic variants; deamidation, sialylation and saccharification can produce acidic variants. Chemical stability can be assessed by detecting and/or quantifying the chemically modified form of the antibody. For example, the charge variant of the antibody in the preparation can be detected by cation exchange chromatography (CEX) or imaging capillary isoelectric focusing electrophoresis (iCIEF). In one embodiment, the stability of the formulation is measured by determining the percentage change in the charge variant of the antibody in the formulation after storage at a specific temperature for a specific time, wherein the smaller the change, the higher the stability of the formulation.

“可接受程度”的化學穩定性可以表示於特定溫度下儲存特定時間之後製劑中電荷變異體(例如主成分或酸性組分或鹼性組分)的百分比變化值不超過50%，例如不超過30%、不超過20%。在一些實施方案中，在特定溫度儲存至少2周、至少28天、至少1個月、至少2個月、至少3個月、至少4個月、至少5個月、至少6個月、至少7個月、至少8個月、至少9個月、至少10個月、至少11個月、至少12個月、至少18個月、至少24個月或更久後，可接受程度的化學穩定性可以表現為主成分電荷變異體的百分比變化值不超過約50%、40%、30%、20%、15%。當評估化學穩定性時，儲存藥物製劑的溫度可為約-80℃至約45℃的任一溫度，例如儲存於約-80℃、約-30℃、約-20℃、約0℃、約4℃-8℃、約5℃、約25℃或約45℃。例如，若在儲存於5℃ 24個月之後，主成分電荷變異體的百分比變化值少於約25%、24%、23%、22%、21%、20%、19%、18%、17%、16%、15%、14%、13%、12%、10%、9%、8%、7%、6%、5%、4%、3%、2%、1%、0.5%或0.1%，則藥物製劑可被視為是穩定的；若在儲存於25℃ 2個月後，主成分電荷變異體的百分比變化值少於約20%、19%、18%、17%、16%、15%、14%、13%、12%、10%、9%、8%、7%、6%、5%、4%、3%、2%、1%、0.5%或0.1%，則藥物製劑亦可被視為是穩 定的；若在儲存於40℃ 1個月之後，主成分電荷變異體的百分比變化值少於約50%、40%、30%、20%、10%、5%或4%，則藥物製劑亦可被視為是穩定的。 The "acceptable degree" of chemical stability can mean that the percentage change of the charge variant (such as the main component or acidic component or alkaline component) in the preparation after storage at a specific temperature for a specific time does not exceed 50%, for example, does not exceed 30%, not more than 20%. In some embodiments, storage at a specific temperature for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months After months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months or more, an acceptable degree of chemical stability can be The percentage change value of the main component charge variant does not exceed about 50%, 40%, 30%, 20%, 15%. When evaluating chemical stability, the storage temperature of the pharmaceutical preparation can be any temperature from about -80°C to about 45°C, for example, when stored at about -80°C, about -30°C, about -20°C, about 0°C, about 4°C-8°C, about 5°C, about 25°C, or about 45°C. For example, if stored at 5℃ for 24 months, the percentage change value of the principal component charge variant is less than about 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17 %, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1%, the pharmaceutical preparation can be considered stable; if the percentage change value of the principal component charge variant is less than about 20%, 19%, 18%, 17%, 16% after storage at 25°C for 2 months %, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1%, The pharmaceutical preparations can also be considered as stable If the percentage change value of the charge variant of the principal component is less than about 50%, 40%, 30%, 20%, 10%, 5% or 4% after storage at 40℃ for 1 month, then the pharmaceutical preparation It can also be considered stable.

術語“凍乾製劑”是指藉由液體製劑的冷凍乾燥處理得到或能夠得到的組合物。較佳地，其為具有少於5%、較佳少於3%水含量的固體組合物。 The term "lyophilized preparation" refers to a composition obtained or obtainable by freeze-drying a liquid preparation. Preferably, it is a solid composition having a water content of less than 5%, preferably less than 3%.

術語“重構製劑”是指將固體製劑(例如凍乾製劑)溶解和/或懸浮於生理可接受的溶液中得到的液體製劑。 The term "reconstituted preparation" refers to a liquid preparation obtained by dissolving and/or suspending a solid preparation (for example, a lyophilized preparation) in a physiologically acceptable solution.

文中使用的術語“室溫”是指15℃至30℃、較佳20℃至27℃、更佳25℃的溫度。 The term "room temperature" as used herein refers to a temperature of 15°C to 30°C, preferably 20°C to 27°C, more preferably 25°C.

“脅迫條件”是指在化學和/或物理上不利於抗體蛋白的環境，該環境可以導致不可接受的抗體蛋白失穩定，例如，高溫、振盪、凍融、光照。“高溫脅迫”是指，將抗體製劑置於室溫或甚至於更高溫度(例如40℃±2℃)儲存一段時間。藉由高溫脅迫加速試驗，可以檢查抗體製劑的穩定性。 "Stress conditions" refer to environments that are chemically and/or physically unfavorable to the antibody protein, which can lead to unacceptable instability of the antibody protein, such as high temperature, shaking, freezing and thawing, and light. "High temperature stress" refers to storing the antibody preparation at room temperature or even at a higher temperature (for example, 40°C±2°C) for a period of time. Through the accelerated test of high temperature stress, the stability of the antibody preparation can be checked.

如本文所使用，術語“腸胃外施用”意指腸內和局部給藥以外的給藥方式，通常藉由注射或輸注方式，並且包括但不限於，靜脈內、肌內、動脈內、鞘內、囊內、眶內、心內、皮內、腹膜內、經氣管、皮下、表皮下(subcuticular)、關節內、囊下、蛛網膜下、脊管柱內、硬膜外和胸骨內注射以及輸注。在一些實施方案中，本發明的穩定抗PD-1/PD-L1抗體蛋白製劑腸胃外施用於受試者。在一個實施方案中，本發明的抗PD-1/PD-L1抗體蛋白製劑以皮下、皮內、肌內或靜脈內注射方式施用於受試者。 As used herein, the term "parenteral administration" means administration methods other than enteral and local administration, usually by injection or infusion, and includes, but is not limited to, intravenous, intramuscular, intraarterial, and intrathecal , Intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injections and Infusion. In some embodiments, the stabilized anti-PD-1/PD-L1 antibody protein formulation of the present invention is administered to a subject parenterally. In one embodiment, the anti-PD-1/PD-L1 antibody protein formulation of the present invention is administered to a subject by subcutaneous, intradermal, intramuscular or intravenous injection.

I.抗體製劑I. Antibody preparations

本發明的一方面提供穩定的凍乾製劑，其包含(i)抗PD-1/PD-L1抗體或其抗原結合片段，(ii)緩衝體系，(iii)穩定劑，該穩定劑包括多元醇和/或胺基酸，和(iv)表面活性劑，其中該製劑當重構時具有5.5-6.5之間的pH，例如，pH約為5.5、6.0或6.5。 One aspect of the present invention provides a stable lyophilized formulation, which comprises (i) an anti-PD-1/PD-L1 antibody or an antigen-binding fragment thereof, (ii) a buffer system, (iii) a stabilizer, the stabilizer includes a polyol and /Or an amino acid, and (iv) a surfactant, wherein the formulation has a pH between 5.5 and 6.5 when reconstituted, for example, the pH is about 5.5, 6.0, or 6.5.

本發明的另一方面提供穩定的凍乾製劑，其藉由凍乾水溶液製成，該水溶液包含：(i)抗PD-1/PD-L1抗體或其抗原結合片段，(ii)緩衝體系，(iii)穩定劑，該穩定劑包括多元醇和/或胺基酸，和(iv)表面活性劑，該液體製劑具有約5.5-6.5的pH，例如，pH約為5.5、6.0或6.5；較佳地，該液體製劑具有約6.0的pH。 Another aspect of the present invention provides a stable freeze-dried formulation, which is prepared by a freeze-dried aqueous solution, the aqueous solution comprising: (i) anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof, (ii) a buffer system, (iii) A stabilizer, the stabilizer includes a polyol and/or amino acid, and (iv) a surfactant, the liquid formulation has a pH of about 5.5-6.5, for example, the pH is about 5.5, 6.0 or 6.5; preferably Specifically, the liquid formulation has a pH of about 6.0.

本發明的再一方面提供穩定的液體抗體製劑，其包含水溶液，該水溶液包含(i)抗PD-1/PD-L1抗體或其抗原結合片段，(ii)緩衝體系，(iii)穩定劑，該穩定劑包括多元醇和/或胺基酸，和(iv)表面活性劑，該液體製劑具有約5.5-6.5的pH，例如，pH約為5.5、6.0或6.5；較佳地，該液體製劑具有約6.0的pH。 Another aspect of the present invention provides a stable liquid antibody preparation, which comprises an aqueous solution comprising (i) an anti-PD-1/PD-L1 antibody or an antigen-binding fragment thereof, (ii) a buffer system, and (iii) a stabilizer, The stabilizer includes a polyol and/or amino acid, and (iv) a surfactant. The liquid formulation has a pH of about 5.5-6.5, for example, the pH is about 5.5, 6.0 or 6.5; preferably, the liquid formulation has PH of about 6.0.

在一個較佳方案中，本發明的液體抗體製劑是注射製劑形式。 In a preferred embodiment, the liquid antibody preparation of the present invention is in the form of an injection preparation.

(i)抗PD-1/PD-L1抗體 (i) Anti-PD-1/PD-L1 antibody

“抗PD-1/PD-L1抗體”或“結合PD-1和PD-L1的雙特異性抗體”或“結合PD-1和PD-L1的抗體”是指這樣的抗體，該抗體能夠以足夠的親和力結合PD-1以及PD-L1分子，以致該抗體可以用作同時靶向PD-1和PD-L1分子的治療劑和/或預防劑。 "Anti-PD-1/PD-L1 antibody" or "bispecific antibody that binds PD-1 and PD-L1" or "antibody that binds PD-1 and PD-L1" refers to an antibody that can Sufficient affinity binds PD-1 and PD-L1 molecules so that the antibody can be used as a therapeutic and/or preventive agent that simultaneously targets PD-1 and PD-L1 molecules.

如本文所用，術語“結合”或“特異性結合”意指結合作用對抗原是選擇性的，並且可以與不想要的或非特異的相互作用區別開。抗體與特定抗原結合的能力可以藉由酶聯免疫吸附測定法(ELISA)、表面等離子共振法(SPR)或生物膜層光學干涉技術(ForteBio)或本領域已知的其它常規結合測定法測定。作為本發明的實施例，指抗體或抗原結合片段在體外測定法中，較佳地在採用純化的野生型抗原的生物膜層光學干涉測量中與抗原表位結合。在某些實施方案中，在抗體或抗原結合片段較佳地識別蛋白質和/或大分子的複雜混合物中的其靶抗原時，將抗體或抗原結合片段稱作特異性結合抗原。 As used herein, the term "binding" or "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antibody to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) or biofilm optical interference technology (ForteBio) or other conventional binding assays known in the art. As an embodiment of the present invention, it refers to that the antibody or antigen-binding fragment binds to the epitope in the in vitro assay, preferably in the optical interferometry of the biofilm layer using purified wild-type antigen. In certain embodiments, when an antibody or antigen-binding fragment preferably recognizes its target antigen in a complex mixture of proteins and/or macromolecules, the antibody or antigen-binding fragment is referred to as a specific binding antigen.

如本文所用“結合”或“特異性結合”在提及抗體對人類PD-L1(SEQ ID NO：1)、人類PD-1(SEQ ID NO：2)或二者的親和性時，除非另有指示，否則指KD小於約1×10-6M、較佳小於約1×10-9M，如借由上述業內已知的常見方法於37℃下所測定。 As used herein, "binding" or "specific binding" refers to the affinity of an antibody to human PD-L1 (SEQ ID NO: 1), human PD-1 (SEQ ID NO: 2), or both, unless otherwise stated. If indicated, otherwise it means that K D is less than about 1×10 -6 M, preferably less than about 1×10 -9 M, as determined by the above-mentioned common method known in the industry at 37°C.

本發明中的抗體為同時靶向PD-L1及PD-1的異二聚體雙特異性抗體，經由使兩條不同重鏈及兩條不同輕鏈配對成一個IgG样抗體而成。此外，本發明中的抗體提供具有以下特徵中之一或多個的抗人類PD-L1及抗人類PD-1異二聚體雙特異性抗體：阻斷PD軸的所有三種相互作用(PD-L1結合至PD-1，PD-L2結合至PD-1及PD-L1結合至CD80)，橋接PD-L1及PD-1過表達細胞，由於靠近結合的T細胞及腫瘤細胞而增加T細胞活化及腫瘤細胞殺死，在具有中等至高PD配體表達程度的腫瘤細胞中於低劑量下展現顯著抗腫瘤活性，並展現物理及化學穩定性，包括但不限於活體內穩定性、熱穩定性、溶解性、低自締合及藥物動力學特徵。 The antibody in the present invention is a heterodimeric bispecific antibody that simultaneously targets PD-L1 and PD-1, and is formed by pairing two different heavy chains and two different light chains to form an IgG-like antibody. In addition, the antibodies of the present invention provide anti-human PD-L1 and anti-human PD-1 heterodimer bispecific antibodies having one or more of the following characteristics: blocking all three interactions of the PD axis (PD- L1 binds to PD-1, PD-L2 binds to PD-1 and PD-L1 binds to CD80), bridges PD-L1 and PD-1 overexpressing cells, and increases T cell activation due to proximity to bound T cells and tumor cells And tumor cell killing. It exhibits significant anti-tumor activity at low doses in tumor cells with moderate to high PD ligand expression levels, and exhibits physical and chemical stability, including but not limited to in vivo stability, thermal stability, Solubility, low self-association and pharmacokinetic characteristics.

因此，在一些實施方案中，本發明抗體製劑中的抗PD-1/PD-L1抗體包含：a)包含含有SEQ ID NO：3的胺基酸序列的重鏈可變區 (HCVR1)的第一重鏈(HC1)；b)包含含有SEQ ID NO：4的胺基酸序列的輕鏈可變區(LCVR1)的第一輕鏈(LC1)；c)包含含有SEQ ID NO：5的胺基酸序列的重鏈可變區(HCVR2)的第二重鏈(HC2)；及d)包含含有SEQ ID NO：8的胺基酸序列的輕鏈可變區(LCVR2)的第二輕鏈(LC2)。 Therefore, in some embodiments, the anti-PD-1/PD-L1 antibody in the antibody preparation of the present invention comprises: a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 (HCVR1) the first heavy chain (HC1); b) comprises the first light chain (LC1) of the light chain variable region (LCVR1) containing the amino acid sequence of SEQ ID NO: 4; c) comprises the first light chain (LC1) containing the amino acid sequence of SEQ ID NO: 4 The second heavy chain (HC2) of the heavy chain variable region (HCVR2) of the amino acid sequence of NO: 5; and d) the light chain variable region (LCVR2) comprising the amino acid sequence of SEQ ID NO: 8 The second light chain (LC2).

在一些實施方案中，本發明抗體製劑中的抗PD-1/PD-L1抗體包含HC1、LC1、HC2及LC2，其中：a)該HC1在HCVR中包含具有SEQ ID NO：16的胺基酸序列的CDR1、具有SEQ ID NO：17的胺基酸序列的CDR2及具有SEQ ID NO：18的胺基酸序列的CDR3；b)該LC1在LCVR中包含具有SEQ ID NO：19的胺基酸序列的CDR1、具有SEQ ID NO：20的胺基酸序列的CDR2及具有SEQ ID NO：21的胺基酸序列的CDR3；c)該HC2包含在HCVR中具有SEQ ID NO：22的胺基酸序列的CDR1、具有SEQ ID NO：23或SEQ ID NO：24的胺基酸序列的CDR2及具有SEQ ID NO：25的胺基酸序列的CDR3；d)該LC2在LCVR中包含具有SEQ ID NO：26的胺基酸序列的CDR1、具有SEQ ID NO：27的胺基酸序列的CDR2及具有SEQ ID NO：28的胺基酸序列的CDR3；e)HC1的CH1域包含S183K的胺基酸取代；f)LC1的恆定區包含S176E及Y178E的胺基酸取代的人類λ变體；g)HC2的CH1域包含L128E、K147T及Q175E的胺基酸取代；h)LC2的恆定區包含S131R、V133G及S176R的胺基酸取代的人類κ变體；且i)HC1的CH3域包含T350V、L351Y、F405A及Y407V的胺基酸取代，且HC2的CH3域包含T350V、T366L、K392L及T394W的胺基酸取代；或HC1的CH3域包含T350V、T366L、K392L及T394W的胺基酸取代，且HC2的CH3域包含T350V、L351Y、F405A及Y407V的胺基酸取代；且j)其中HC1及HC2是免疫效應功能 無效的人類IgG1 Fc變體，其中編號是根據EU索引。較佳地，HC1及HC2包含L234A、L235A及D265S的胺基酸取代。 In some embodiments, the anti-PD-1/PD-L1 antibody in the antibody preparation of the present invention comprises HC1, LC1, HC2, and LC2, wherein: a) the HC1 comprises an amino acid having SEQ ID NO: 16 in HCVR CDR1 of the sequence, CDR2 having the amino acid sequence of SEQ ID NO: 17, and CDR3 having the amino acid sequence of SEQ ID NO: 18; b) the LC1 includes the amino acid of SEQ ID NO: 19 in the LCVR CDR1 of the sequence, CDR2 having the amino acid sequence of SEQ ID NO: 20, and CDR3 having the amino acid sequence of SEQ ID NO: 21; c) the HC2 contains the amino acid of SEQ ID NO: 22 in HCVR CDR1 of the sequence, CDR2 having the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 24, and CDR3 having the amino acid sequence of SEQ ID NO: 25; d) The LC2 includes the amino acid sequence of SEQ ID NO: : CDR1 with the amino acid sequence of 26, CDR2 with the amino acid sequence of SEQ ID NO: 27, and CDR3 with the amino acid sequence of SEQ ID NO: 28; e) CH1 domain of HC1 contains the amino acid of S183K Substitution; f) The constant region of LC1 contains the amino acid-substituted human lambda variants of S176E and Y178E; g) The CH1 domain of HC2 contains the amino acid substitutions of L128E, K147T and Q175E; h) The constant region of LC2 contains S131R, The amino acid-substituted human κ variants of V133G and S176R; and i) the CH3 domain of HC1 includes the amino acid substitutions of T350V, L351Y, F405A, and Y407V, and the CH3 domain of HC2 includes the amines of T350V, T366L, K392L, and T394W Or the CH3 domain of HC1 includes amino acid substitutions of T350V, T366L, K392L and T394W, and the CH3 domain of HC2 includes amino acid substitutions of T350V, L351Y, F405A and Y407V; and j) where HC1 and HC2 are Immune effector function Invalid human IgG1 Fc variant, where the numbering is based on the EU index. Preferably, HC1 and HC2 include amino acid substitutions of L234A, L235A, and D265S.

在一些實施方案中，本發明抗體製劑中的抗PD-1/PD-L1抗體包含：a)HC1，其自N端至C端依序包含含有SEQ ID NO：3的胺基酸序列的HCVR、CH1域中SEQ ID NO：9的胺基酸序列、CH2域中SEQ ID NO：10的胺基酸序列及CH3域中SEQ ID NO：11或SEQ ID NO：12的胺基酸序列；b)LC1，其自N端至C端依序包含含有SEQ ID NO：4的胺基酸序列的LCVR及恆定區中SEQ ID NO：14的胺基酸序列；c)HC2，其自N端至C端依序包含含有SEQ ID NO：5的胺基酸序列的HCVR、CH1域中SEQ ID NO：13的胺基酸序列、CH2域中SEQ ID NO：10的胺基酸序列及CH3域中SEQ ID NO：12的胺基酸序列或SEQ ID NO：11的胺基酸序列；及d)LC2，其自N端依序包含含有SEQ ID NO：8的胺基酸序列的LCVR及恆定區中SEQ ID NO：15的胺基酸序列，條件是當SEQ ID NO：11的胺基酸序列存於該HC1的CH3域中時，SEQ ID NO：12的胺基酸序列存於該HC2的CH3域中；或當SEQ ID NO：12的胺基酸序列存於該HC1的CH3域中時，SEQ ID NO：11的胺基酸序列存於該HC2的CH3域中。 In some embodiments, the anti-PD-1/PD-L1 antibody in the antibody preparation of the present invention comprises: a) HC1, which sequentially comprises HCVR containing the amino acid sequence of SEQ ID NO: 3 from N-terminus to C-terminus The amino acid sequence of SEQ ID NO: 9 in the CH1 domain, the amino acid sequence of SEQ ID NO: 10 in the CH2 domain, and the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12 in the CH3 domain; b ) LC1, which sequentially includes the LCVR containing the amino acid sequence of SEQ ID NO: 4 and the amino acid sequence of SEQ ID NO: 14 in the constant region from N-terminus to C-terminus; c) HC2, which is from N-terminus to The C-terminus contains the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 13 in the CH1 domain, the amino acid sequence of SEQ ID NO: 10 in the CH2 domain, and the amino acid sequence of SEQ ID NO: 10 in the CH3 domain in sequence. The amino acid sequence of SEQ ID NO: 12 or the amino acid sequence of SEQ ID NO: 11; and d) LC2, which sequentially includes the LCVR and constant regions containing the amino acid sequence of SEQ ID NO: 8 from the N-terminus The amino acid sequence of SEQ ID NO: 15, provided that when the amino acid sequence of SEQ ID NO: 11 is stored in the CH3 domain of HC1, the amino acid sequence of SEQ ID NO: 12 is stored in the HC2 In the CH3 domain; or when the amino acid sequence of SEQ ID NO: 12 is stored in the CH3 domain of HC1, the amino acid sequence of SEQ ID NO: 11 is stored in the CH3 domain of HC2.

在一些實施方案中，本發明抗體製劑中的抗PD-1/PD-L1抗體中：a)該HC1自N端至C端依序包含含有SEQ ID NO：3的胺基酸序列的HCVR1、CH1域中SEQ ID NO：9的胺基酸序列、CH2域中SEQ ID NO：10的胺基酸序列及CH3域中SEQ ID NO：11的胺基酸序列；b)該LC1，其自N端至C端依序包含含有SEQ ID NO：4的胺基酸序列的LCVR1及恆定區中SEQ ID NO：14的胺基酸序列；及c)該HC2，其自N端至C端依序包含含有SEQ ID NO：6的胺基酸序列的HCVR2、CH1域中SEQ ID NO：13的胺基酸序列、CH2域的區中SEQ ID NO：10的胺基酸序列及CH3域中SEQ ID NO：12的胺基酸序列；及d)該LC2，其自N端依序包含含有SEQ ID NO：8的胺基酸序列的LCVR2及恆定區中SEQ ID NO：15的胺基酸序列。 In some embodiments, in the anti-PD-1/PD-L1 antibody in the antibody preparation of the present invention: a) The HC1 contains HCVR1 containing the amino acid sequence of SEQ ID NO: 3 sequentially from N-terminus to C-terminus The amino acid sequence of SEQ ID NO: 9 in the CH1 domain, the amino acid sequence of SEQ ID NO: 10 in the CH2 domain, and the amino acid sequence of SEQ ID NO: 11 in the CH3 domain; b) the LC1, which is from N The end to the C-terminus includes the LCVR1 containing the amino acid sequence of SEQ ID NO: 4 and the amino acid sequence of SEQ ID NO: 14 in the constant region in sequence; and c) the HC2, which is sequentially from the N-terminus to the C-terminus Contains SEQ ID NO: 6 amino acid sequence in the HCVR2, CH1 domain of SEQ The amino acid sequence of ID NO: 13, the amino acid sequence of SEQ ID NO: 10 in the region of the CH2 domain, and the amino acid sequence of SEQ ID NO: 12 in the CH3 domain; and d) the LC2, which is from the N-terminus The sequence includes LCVR2 containing the amino acid sequence of SEQ ID NO: 8 and the amino acid sequence of SEQ ID NO: 15 in the constant region.

在一些實施方案中，本發明抗體製劑中的抗PD-1/PD-L1抗體中：a)包含SEQ ID NO：49的胺基酸序列的HC1；b)包含SEQ ID NO：30的胺基酸序列的LC1；c)包含SEQ ID NO31的胺基酸序列的HC2；及d)包含SEQ ID NO：34的胺基酸序列的LC2。 In some embodiments, the anti-PD-1/PD-L1 antibody in the antibody preparation of the present invention: a) HC1 comprising the amino acid sequence of SEQ ID NO: 49; b) comprising the amino acid sequence of SEQ ID NO: 30 Acid sequence LC1; c) HC2 comprising the amino acid sequence of SEQ ID NO31; and d) LC2 comprising the amino acid sequence of SEQ ID NO:34.

本發明提供結合人類PD-L1(SEQ ID NO：1)及人類PD-1(SEQ ID NO：2)的抗體，其包含：a)包含SEQ ID NO：49的胺基酸序列的HC1；b)包含SEQ ID NO：30的胺基酸序列的LC1；c)包含SEQ ID NO32的胺基酸序列的HC2；及d)包含SEQ ID NO：34的胺基酸序列的LC2。 The present invention provides an antibody that binds to human PD-L1 (SEQ ID NO: 1) and human PD-1 (SEQ ID NO: 2), which comprises: a) HC1 comprising the amino acid sequence of SEQ ID NO: 49; b ) LC1 comprising the amino acid sequence of SEQ ID NO: 30; c) HC2 comprising the amino acid sequence of SEQ ID NO: 32; and d) LC2 comprising the amino acid sequence of SEQ ID NO: 34.

在一些實施方案中，本發明抗體製劑中的抗PD-1/PD-L1抗體中：a)包含SEQ ID NO：29的胺基酸序列的HC1；b)包含SEQ ID NO：30的胺基酸序列的LC1；c)包含SEQ ID NO：33的胺基酸序列的HC2；及d)包含SEQ ID NO：34的胺基酸序列的LC2。 In some embodiments, the anti-PD-1/PD-L1 antibody in the antibody preparation of the present invention: a) HC1 comprising the amino acid sequence of SEQ ID NO: 29; b) comprising the amino group of SEQ ID NO: 30 Acid sequence LC1; c) HC2 comprising the amino acid sequence of SEQ ID NO: 33; and d) LC2 comprising the amino acid sequence of SEQ ID NO: 34.

在一些實施方案中，本發明抗體製劑中的抗PD-1/PD-L1抗體可以包含與SEQ ID NO：3具有至少90%,95%,98%或99%或更高同一性的HCVR1；和/或與SEQ ID NO：4具有至少90%,95%,98%或99%或更高同一性的LCVR1；和/或與SEQ ID NO：5(即SEQ ID NO：6或SEQ ID NO：7)具有至少90%,95%,98%或99%或更高同一性的HCVR2；和/或與SEQ ID NO：8具有至少90%,95%,98%或99%或更高同一性的LCVR2。在本文中，“序列同一性”是指在比較窗中以逐個核苷酸或逐個胺基酸為基礎的序列相同的程度。可以藉由以下方式計算“序列同一性百分 比”：將兩條最佳比對的序列在比較窗中進行比較，確定兩條序列中存在相同核酸鹼基(例如，A、T、C、G、I)或相同胺基酸殘基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的數目以得到匹配位置的數目，將匹配位置的數目除以比較窗中的總位置數(即，窗大小)，並且將結果乘以100，以產生序列同一性百分比。為了確定序列同一性百分數而進行的最佳比對，可以按本領域已知的多種方式實現，例如，使用可公開獲得的計算機軟體如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)軟體。本領域技術人員可以確定用於比對序列的適宜參數，包括為實現正在比較的全長序列範圍內或目標序列區域內最大比對所需要的任何算法。 In some embodiments, the anti-PD-1/PD-L1 antibody in the antibody preparation of the present invention may comprise HCVR1 having at least 90%, 95%, 98% or 99% or higher identity with SEQ ID NO: 3; And/or LCVR1 having at least 90%, 95%, 98% or 99% or more identity with SEQ ID NO: 4; and/or with SEQ ID NO: 5 (ie SEQ ID NO: 6 or SEQ ID NO : 7) HCVR2 having at least 90%, 95%, 98% or 99% or higher identity; and/or at least 90%, 95%, 98% or 99% or higher identity with SEQ ID NO: 8 Sexual LCVR2. As used herein, "sequence identity" refers to the degree of sequence identity on a nucleotide-by-nucleotide or amino acid-by-amino acid basis in the comparison window. The "percentage of sequence identity" can be calculated by the following method "Comparison": Compare the two optimally aligned sequences in the comparison window to determine the presence of the same nucleic acid base (for example, A, T, C, G, I) or the same amino acid residue ( For example, the number of positions of Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) to get the matching position Divide the number of matching positions by the total number of positions in the comparison window (ie, the window size), and multiply the result by 100 to produce the percent sequence identity. The best comparison to determine the percent sequence identity Yes, it can be implemented in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate sequence for alignment Parameters, including any algorithms needed to achieve the maximum alignment within the full-length sequence being compared or within the target sequence region.

在一些實施方案中，本發明製劑中的抗PD-1/PD-L1抗體的HCVR1與SEQ ID NO：3相比具有不超過10個，較佳地不超過5個、4個或3個不同殘基，較佳地該不同殘基為保守胺基酸替代。在一些實施方案中，本發明製劑中的抗PD-1/PD-L1抗體的LCVR1與SEQ ID NO：4相比具有不超過10個，較佳地不超過5個、4個或3個不同殘基，較佳地該不同殘基為保守胺基酸替代。在一些實施方案中，本發明製劑中的抗PD-1/PD-L1抗體的HCVR2與SEQ ID NO：5(即SEQ ID NO：6或SEQ ID NO：7)相比具有不超過10個，較佳地不超過5個、4個或3個不同殘基，較佳地該不同殘基為保守胺基酸替代。在一些實施方案中，本發明製劑中的抗PD-1/PD-L1抗體的LCVR2與SEQ ID NO：8相比具有不超過10個，較佳地不超過5個、4個或3個不同殘基，較佳地該不同殘基為保守胺基酸替代。“保守性取代”是指導致某個胺基酸置換為化學上相似的胺基酸的胺基酸改變。提供功能上相似胺基酸的保守性置換表是本領域熟知的。 在本發明任一實施方案中，在一個較佳的方面，保守取代殘基來自以下的保守替代表A，較佳地為表A中所示較佳置換殘基。 In some embodiments, the HCVR1 of the anti-PD-1/PD-L1 antibody in the formulation of the present invention has no more than 10, preferably no more than 5, 4, or 3 differences compared to SEQ ID NO: 3. Residues, preferably the different residues are conservative amino acid substitutions. In some embodiments, the LCVR1 of the anti-PD-1/PD-L1 antibody in the formulation of the present invention has no more than 10, preferably no more than 5, 4, or 3 differences compared to SEQ ID NO: 4. Residues, preferably the different residues are conservative amino acid substitutions. In some embodiments, the anti-PD-1/PD-L1 antibody in the formulation of the present invention has no more than 10 HCVR2 compared to SEQ ID NO: 5 (ie, SEQ ID NO: 6 or SEQ ID NO: 7), Preferably no more than 5, 4 or 3 different residues, preferably the different residues are conservative amino acid substitutions. In some embodiments, the LCVR2 of the anti-PD-1/PD-L1 antibody in the formulation of the present invention has no more than 10, preferably no more than 5, 4, or 3 differences compared to SEQ ID NO: 8. Residues, preferably the different residues are conservative amino acid substitutions. "Conservative substitution" refers to an amino acid change that causes a certain amino acid to be replaced with a chemically similar amino acid. It is well known in the art to provide conservative substitution tables for functionally similar amino acids. In any embodiment of the invention, in a preferred aspect, the conservatively substituted residues are derived from the conservative substitution table A below, preferably the preferred substituted residues shown in Table A.

Figure 110101453-A0101-12-0031-2
Figure 110101453-A0101-12-0031-2

Figure 110101453-A0101-12-0032-3
Figure 110101453-A0101-12-0032-3

本發明的抗體是工程化的非天然存在的多肽複合物。本發明的DNA分子是非天然存在的DNA分子，其包含編碼具有本發明抗體中一種多肽的胺基酸序列的多肽的多核苷酸序列。 The antibodies of the present invention are engineered non-naturally occurring polypeptide complexes. The DNA molecule of the present invention is a non-naturally occurring DNA molecule that contains a polynucleotide sequence encoding a polypeptide having the amino acid sequence of a polypeptide in the antibody of the present invention.

本發明的抗體是IgG型抗體，具有重鏈和輕鏈，它們藉由鏈內和鏈間二硫鍵交聯。每條重鏈由N-末端HCVR和重鏈恆定區(“HCCR”)組成。每條輕鏈由N末端LCVR和輕鏈恆定區(“LCCR”)組成。輕鏈各自與重鏈形成二硫鍵，並且兩條重鏈在彼此之間形成兩個二硫鍵。 The antibody of the present invention is an IgG type antibody, which has a heavy chain and a light chain, which are cross-linked by intra-chain and inter-chain disulfide bonds. Each heavy chain consists of an N-terminal HCVR and a heavy chain constant region ("HCCR"). Each light chain consists of an N-terminal LCVR and a light chain constant region ("LCCR"). The light chains each form a disulfide bond with the heavy chain, and the two heavy chains form two disulfide bonds between each other.

重鏈的恆定區含有CH1，CH2和CH3結構域。CH1來自HCVR；CH1和HCVR形成Fab的重鏈部分。CH2位於鉸鏈區之後和CH3之前。CH3位於CH2之後，位於重鏈的羧基末端。輕鏈的恆定區含有一個結構域CL。CL來自LCVR；CL和LCVR形成Fab的輕鏈部分。 The constant region of the heavy chain contains CH1, CH2 and CH3 domains. CH1 is derived from HCVR; CH1 and HCVR form the heavy chain part of the Fab. CH2 is located after the hinge area and before CH3. CH3 is located after CH2, at the carboxyl end of the heavy chain. The constant region of the light chain contains a domain CL. CL is derived from LCVR; CL and LCVR form the light chain portion of the Fab.

在一個較佳的實施方案中，本發明抗體製劑中的抗PD-1/PD-L1抗體是PCT申請號PCT/US2018/041205(國際申請日：2018年7月9日)中公開的抗PD-1/PD-L1抗體，藉由引用將全部內容併入本文。該抗PD-1/PD-L1抗體的序列延用該專利申請中的序列號，沒有重新進行編號。較佳地，該抗體選自：PD-1/PD-L1雙特異性Ab v13844、Ab v3.0或Ab v3.2。更較佳地，該抗體為PD-1/PD-L1雙特異性Ab v3.2。在一個實施方案中，該抗PD-1/PD-L1抗體是由例如293細胞或CHO細胞等重組表達產生並經純化的抗體，其含有衍生自人IgG1的Fc部分變體。在本發明液體製劑中的該抗體表現出顯著的抗腫瘤活性。例如，在植入NCI- H292腫瘤細胞與PBMC的混合物的來自Jackson Laboratories的NSG小鼠中，施用本發明的抗體例如PD-1/PD-L1雙特異性Ab(v13844、v3.0或v3.2)以10mg/kg qw進行治療，完全緩解(CR)分別為7/8、5/8及5/8，比組合療法(親代PD-L1抗體+親代PD-1抗體)(CR為2/8)更有效。 In a preferred embodiment, the anti-PD-1/PD-L1 antibody in the antibody preparation of the present invention is the anti-PD disclosed in PCT Application No. PCT/US2018/041205 (International Filing Date: July 9, 2018). -1/PD-L1 antibody, the entire content is incorporated herein by reference. The sequence of the anti-PD-1/PD-L1 antibody continued to use the serial number in the patent application without renumbering. Preferably, the antibody is selected from: PD-1/PD-L1 bispecific Ab v13844, Ab v3.0 or Ab v3.2. More preferably, the antibody is PD-1/PD-L1 bispecific Ab v3.2. In one embodiment, the anti-PD-1/PD-L1 antibody is a purified antibody produced by recombinant expression such as 293 cells or CHO cells, which contains a variant of the Fc portion derived from human IgG1. The antibody in the liquid preparation of the present invention exhibits significant anti-tumor activity. For example, in the implantation of NCI- In NSG mice from Jackson Laboratories with a mixture of H292 tumor cells and PBMC, the antibody of the present invention such as PD-1/PD-L1 bispecific Ab (v13844, v3.0 or v3.2) was administered at 10 mg/kg qw After treatment, complete remission (CR) was 7/8, 5/8, and 5/8, respectively, which was more effective than combination therapy (parental PD-L1 antibody + parental PD-1 antibody) (CR 2/8).

本發明的抗體是異二聚體，因為抗體的每個臂由於形成抗體的兩條不同的重鏈和兩條不同的輕鏈而表現出與其同源抗原的選擇性單價結合。在本發明中，抗體的一個臂結合人PD-L1(SEQ ID NO：1)，另一個臂結合人PD-1(SEQ ID NO：2)。這些多肽鏈的CH2和/或CH3結構域在序列上不需要相同，並且有利地被修飾以促進兩條多肽鏈之間的複合。例如，可以將胺基酸取代(較佳用包含形成“旋鈕”的龐大側基的胺基酸取代，例如色胺酸)引入CH2或CH3結構域，使得空間干擾將阻止與類似突變的相互作用。結構域將強制突變結構域與已經設計了互補或調節突變的結構域配對，即“空穴”(例如，用甘胺酸取代)。可以將這些突變組工程化為包含形成Fc區的CH2-CH3結構域的任何多肽對。有利於異二聚化而不是同型二聚化的蛋白質工程方法是本領域熟知的，特別是關於免疫球蛋白樣分子的工程化(參見例如WO96/27011，WO 98/050431，EP 1870459，WO 2007/110205，WO 2007/147901，WO 2009/089004，WO 2010/129304，WO 2011/90754，WO 2011/143545，WO2012/058768，WO2013/157954，WO2013/096291，EP1870459A1，以及Ridgway等。(1996)“'Knobs-Into-Holes'工程抗體CH3重鏈結構域異二聚化，“蛋白質工程9：617-621，Atwell等人(1997)”使用噬菌體展示文庫改造Homodimer的結構域的穩定的異二聚體，“J.Mol.Biol.270：26-35，和Xie等人(2005)“雙特異性抗體的新形式：高效異源二聚化，表達和腫瘤細胞裂解”，J.Immunol.Methods 296：95-101)。通常，在本領域已知的方法中，CH3 第一重鏈的結構域和第二重鏈的CH3結構域均以互補的方式工程化，使得包含一個工程化的CH3結構域的重鏈不再能夠與另一個相同結構的重鏈同源二聚體化(例如CH3-工程化的第一條重鏈不能再與另一條CH3工程化的第一條重鏈同源二聚體化；而CH3-工程化的第二條重鏈不能再與另一條CH3-工程化的第二條重鏈同源二聚體化。由此迫使包含一個工程化CH3結構域的重鏈與包含CH3結構域的另一條重鏈異二聚化，該CH3結構域以互補方式工程化。對於本發明的該實施方案，第一重鏈的CH3結構域和第二重鏈的CH3結構域藉由胺基酸取代以互補方式工程化，使得第一重鏈和第二重鏈被迫異二聚化。而第一條重鏈和第二條重鏈不能再同源二聚化(例如出於空間原因)。 The antibody of the present invention is a heterodimer because each arm of the antibody exhibits selective monovalent binding to its homologous antigen due to two different heavy chains and two different light chains that form the antibody. In the present invention, one arm of the antibody binds human PD-L1 (SEQ ID NO: 1), and the other arm binds human PD-1 (SEQ ID NO: 2). The CH2 and/or CH3 domains of these polypeptide chains do not need to be identical in sequence, and are advantageously modified to promote the complexation between the two polypeptide chains. For example, amino acid substitutions (preferably with amino acids containing bulky side groups forming the "knob", such as tryptophan) can be introduced into the CH2 or CH3 domain, so that steric interference will prevent interaction with similar mutations . The domain will force the mutation domain to pair with the domain for which complementary or regulatory mutations have been designed, that is, "holes" (for example, substitution with glycine). These mutant sets can be engineered into any polypeptide pair comprising the CH2-CH3 domain that forms the Fc region. Protein engineering methods that favor heterodimerization rather than homodimerization are well known in the art, especially regarding the engineering of immunoglobulin-like molecules (see, for example, WO96/27011, WO 98/050431, EP 1870459, WO 2007 /110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO2012/058768, WO2013/157954, WO2013/096291, EP1870459A1, and Ridgway et al. (1996)" 'Knobs-Into-Holes' engineered antibody CH3 heavy chain domain heterodimerization, "Protein Engineering 9: 617-621, Atwell et al. (1997)" Use phage display library to transform the stable heterodimerization of the homodimer domain Biol. 270: 26-35, and Xie et al. (2005) "A new form of bispecific antibodies: high-efficiency heterodimerization, expression and tumor cell lysis", J. Immunol. Methods 296: 95-101). Generally, in methods known in the art, CH3 The domain of the first heavy chain and the CH3 domain of the second heavy chain are both engineered in a complementary manner, so that the heavy chain containing one engineered CH3 domain can no longer be homologous to another heavy chain of the same structure. Polymerization (for example, the first heavy chain of CH3-engineering can no longer be homodimerized with the first heavy chain of another CH3 engineering; and the second heavy chain of CH3-engineering can no longer be homodimerized with another CH3-engineered heavy chain. Another CH3-engineered second heavy chain is homodimerized. This forces the heavy chain containing one engineered CH3 domain to heterodimerize with the other heavy chain containing CH3 domain, and the CH3 structure The domains are engineered in a complementary manner. For this embodiment of the invention, the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain are engineered in a complementary manner by amino acid substitution, so that the first heavy chain and The second heavy chain is forced to heterodimerize, and the first heavy chain and the second heavy chain can no longer homodimerize (for example for spatial reasons).

支持本領域已知的用於支持重鏈異二聚化的不同方法，作為在根據本發明的雙特異性抗體中使用的不同替代方案。在本發明的一些實施方案中，突變摻入CH1和CH3區內的重鏈序列和輕鏈恆定區內的輕鏈序列中。CH1和LC突變有利於必需的輕鏈和重鏈對的天然配對並且不利於輕鏈錯配。使CH3突變有利於兩個不同重鏈的異二聚體配對並且不利於同型二聚體的形成。 The different methods known in the art for supporting heavy chain heterodimerization are supported as different alternatives used in the bispecific antibodies according to the present invention. In some embodiments of the invention, mutations are incorporated into the heavy chain sequence in the CH1 and CH3 regions and the light chain sequence in the light chain constant region. CH1 and LC mutations facilitate the natural pairing of the necessary light and heavy chain pairs and are not conducive to light chain mismatches. Mutation of CH3 is conducive to the pairing of heterodimers of two different heavy chains and is not conducive to the formation of homodimers.

在一些實施方案中，本發明的抗體含有衍生自人IgG1的Fc部分變體。眾所周知，IgG1與Fc-γ受體家族(FcγR)以及C1q結合。與這些受體的相互作用可以誘導抗體依賴性細胞毒性(ADCC)和補體依賴性細胞毒性(CDC)。因此，對於本發明的抗體，某些胺基酸取代被引入人IgG1 Fc區以消除免疫效應子功能。抗體重鏈的CH2區域中的突變可以包括EU編號中的位置234,235和265，以減少或消除免疫效應子功能。 In some embodiments, the antibodies of the invention contain variants of the Fc portion derived from human IgG1. It is well known that IgG1 binds to the Fc-γ receptor family (FcγR) and C1q. Interactions with these receptors can induce antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Therefore, for the antibodies of the present invention, certain amino acid substitutions are introduced into the Fc region of human IgG1 to eliminate immune effector functions. Mutations in the CH2 region of the antibody heavy chain can include positions 234, 235, and 265 in the EU numbering to reduce or eliminate immune effector functions.

藉由將編碼HCVR的DNA可操作地連接到編碼重鏈恆定區或其變體的另一DNA分子，可以將編碼HCVR區的分離的DNA轉化 為全長重鏈基因。人類以及其他哺乳動物重鏈恆定區基因的序列是本領域已知的。包含這些區域的DNA片段可以例如藉由標準PCR擴增獲得。較佳地，對於本發明的抗體，重鏈的重鏈恆定區是人IgG1的變體。 By operably linking the DNA encoding HCVR to another DNA molecule encoding the heavy chain constant region or a variant thereof, the isolated DNA encoding the HCVR region can be transformed It is a full-length heavy chain gene. The sequences of human and other mammalian heavy chain constant region genes are known in the art. DNA fragments containing these regions can be obtained, for example, by standard PCR amplification. Preferably, for the antibody of the present invention, the heavy chain constant region of the heavy chain is a variant of human IgG1.

藉由將編碼LCVR的DNA可操作地連接到編碼輕鏈恆定區或其變體的另一DNA分子，可以將編碼LCVR區的分離的DNA轉化為全長輕鏈基因。人類以及其他哺乳動物輕鏈恆定區基因的序列是本領域已知的。包含這些區域的DNA片段可藉由標準PCR擴增獲得。輕鏈恆定區可以是κ或λ恆定區。較佳地，對於本發明的抗體，抗體的抗PD-L1部分的輕鏈恆定區是λ輕鏈的變體，並且抗體的抗PD-1部分的輕鏈恆定區是κ輕鏈的變體。 By operably linking the DNA encoding the LCVR to another DNA molecule encoding the light chain constant region or a variant thereof, the isolated DNA encoding the LCVR region can be converted into a full-length light chain gene. The sequences of human and other mammalian light chain constant region genes are known in the art. DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region. Preferably, for the antibody of the present invention, the light chain constant region of the anti-PD-L1 portion of the antibody is a variant of the lambda light chain, and the light chain constant region of the anti-PD-1 portion of the antibody is a variant of the kappa light chain .

在序列與表達控制序列可操作地連接後，本發明的多核苷酸可以在宿主細胞中表達。表達載體通常可以作為附加體或作為附著體在宿主生物中複製宿主染色體DNA的組成部分。通常，表達載體含有選擇標記，例如四環素、新黴素、穀胺醯胺合成酶和二氫葉酸還原酶，以允許檢測用所需DNA序列轉化的那些細胞。 After the sequence is operably linked to the expression control sequence, the polynucleotide of the present invention can be expressed in the host cell. An expression vector can usually be used as an episome or as an attachment to replicate a component of the host's chromosomal DNA in the host organism. Typically, expression vectors contain selectable markers, such as tetracycline, neomycin, glutamine synthetase, and dihydrofolate reductase, to allow detection of those cells transformed with the desired DNA sequence.

本發明的抗體製劑中所包含的抗體或其抗原結合片段的量可隨著製劑的特定目的特性、特定環境、和使用製劑的特定目的而改變。在一些實施方案中，抗體製劑為液體製劑，其可含有約1-200mg/ml，例如約10、15、20、25、30、35、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190或200mg/ml，較佳地約10-100mg/mL，例如約10、15、20、25、30、35、40、50、60、70、80、90或100mg/ml的抗PD-1/PD-L1抗體。在一些實施方案中，抗體製劑為凍乾製劑，該凍乾製劑能夠在約1mg/mL至約200mg/mL之間的濃度，例如約10、15、20、25、30、35、40、50、60、70、80、90、100、110、120、 130、140、150、160、170、180、190或200mg/ml，較佳地約10-100mg/mL，例如約10、15、20、25、30、35、40、50、60、70、80、90或100mg/ml的濃度來重構該抗體或其抗原結合片段。 The amount of the antibody or antigen-binding fragment thereof contained in the antibody preparation of the present invention may vary according to the specific purpose characteristics of the preparation, the specific environment, and the specific purpose for which the preparation is used. In some embodiments, the antibody preparation is a liquid preparation, which may contain about 1-200 mg/ml, such as about 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg/ml, preferably about 10-100 mg/mL, such as about 10, 15, 20, 25, 30, 35, 40, 50, Anti-PD-1/PD-L1 antibody at 60, 70, 80, 90 or 100 mg/ml. In some embodiments, the antibody formulation is a lyophilized formulation that can be at a concentration between about 1 mg/mL to about 200 mg/mL, such as about 10, 15, 20, 25, 30, 35, 40, 50 , 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mg/ml, preferably about 10-100 mg/mL, such as about 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, Concentrations of 80, 90, or 100 mg/ml are used to reconstitute the antibody or antigen-binding fragment thereof.

(ii)緩衝體系 (ii) Buffer system

緩衝體系是可以將溶液的pH維持在可接受範圍的體系。在一些實施方案中，用於本發明製劑中的緩衝體系可以將本發明製劑的pH控制在大約5.5-6.5的pH範圍，例如約5.5、6.0或6.5的pH。在一些具體的實施方案中，本發明的抗體製劑具有約6的pH。 The buffer system is a system that can maintain the pH of the solution in an acceptable range. In some embodiments, the buffer system used in the formulation of the present invention can control the pH of the formulation of the present invention in a pH range of about 5.5-6.5, such as a pH of about 5.5, 6.0, or 6.5. In some specific embodiments, the antibody preparations of the invention have a pH of about 6.

在一些實施方案中，在本發明所述的凍乾製劑中，該緩衝體系選自組胺酸緩衝體系、組胺酸和鹽酸組胺酸緩衝體系、枸櫞酸和枸櫞酸鈉緩衝體系、醋酸醋酸鈉緩衝體系、磷酸鹽緩衝體系，且以約0.01%(w/v)-1%(w/v)的重量比，例如約0.01、0.05、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1%(w/v)的重量比存在；較佳地，該緩衝體系以約0.01%(w/v)-0.2%(w/v)的重量比存在； In some embodiments, in the lyophilized formulation of the present invention, the buffer system is selected from the group consisting of histidine buffer system, histidine and histidine hydrochloride buffer system, citric acid and sodium citrate buffer system, Sodium acetate acetate buffer system, phosphate buffer system, and in a weight ratio of about 0.01% (w/v) to 1% (w/v), such as about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 , 0.7, 0.8, 0.9, 1% (w/v) by weight ratio; preferably, the buffer system is present at a weight ratio of about 0.01% (w/v)-0.2% (w/v);

在一些實施方案中，該緩衝體系為組胺酸緩衝體系。較佳地，該組胺酸以約0.01-1%(w/v)，例如0.01、0.05、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1%(w/v)的重量比存在。在一些實施方案中，該組胺酸以約0.155%(w/v)的重量比存在； In some embodiments, the buffer system is a histidine buffer system. Preferably, the histidine content is about 0.01-1% (w/v), such as 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1% (w/v) The weight ratio exists. In some embodiments, the histidine is present in a weight ratio of about 0.155% (w/v);

在一些實施方案中，該緩衝體系為組胺酸和鹽酸組胺酸緩衝體系。較佳地，該組胺酸和該鹽酸組胺酸分別以約0.01-1%(w/v)，例如約0.01、0.05、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1%(w/v)的重量比存 在。在一些實施方案中，該組胺酸和鹽酸組胺酸分別以約0.085%(w/v)和0.1%(w/v)的重量比存在。 In some embodiments, the buffer system is a histidine and histidine hydrochloride buffer system. Preferably, the histidine and the hydrochloric acid histidine are respectively about 0.01-1% (w/v), such as about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 , 1% (w/v) weight ratio exist. In some embodiments, the histidine and hydrochloric acid histidine are present in a weight ratio of about 0.085% (w/v) and 0.1% (w/v), respectively.

在一些實施方案中，本發明的液體抗體製劑中該緩衝體系選自組胺酸緩衝體系、組胺酸和鹽酸組胺酸緩衝體系、枸櫞酸和枸櫞酸鈉緩衝體系、醋酸醋酸鈉緩衝體系、磷酸鹽緩衝體系，且以約1-100mM，例如，1、5、10、15、20、30、40、50、60、70、80、90、100mM的濃度存在。較佳地，該緩衝體系以約1-20mM，例如，1、5、10、15、20mM的濃度存在；更佳地，以約10mM的濃度存在。 In some embodiments, the buffer system in the liquid antibody preparation of the present invention is selected from the group consisting of histidine buffer system, histidine and hydrochloride histidine buffer system, citric acid and sodium citrate buffer system, and sodium acetate buffer system. System, phosphate buffer system, and exist at a concentration of about 1-100 mM, for example, 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 mM. Preferably, the buffer system is present at a concentration of about 1-20 mM, for example, 1, 5, 10, 15, 20 mM; more preferably, it is present at a concentration of about 10 mM.

在一個實施方案中，本發明的液體抗體製劑中的該緩衝體系為組胺酸緩衝體系。選地，該組胺酸以約0.1-10mg/mL，例如約0.1、0.5、1、2、3、4、5、6、7、8、9、10mg/ml的濃度存在。在更佳的實施方案中，該組胺酸以約1.55mg/mL的濃度存在； In one embodiment, the buffer system in the liquid antibody preparation of the present invention is a histidine buffer system. Optionally, the histidine is present at a concentration of about 0.1-10 mg/mL, for example about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/ml. In a more preferred embodiment, the histidine is present at a concentration of about 1.55 mg/mL;

本發明的液體抗體製劑中的該緩衝體系為組胺酸和鹽酸組胺酸緩衝體系。較佳地，該組胺酸和該鹽酸組胺酸分別以約0.1-10mg/mL，例如約0.1、0.5、1、2、3、4、5、6、7、8、9、10mg/ml的濃度存在。在更佳的實施方案中，用於本發明製劑中的緩衝劑是總濃度約10mM的組胺酸和鹽酸組胺酸，例如，該組胺酸和該鹽酸組胺酸分別以約0.85mg/mL和約1.00mg/mL的濃度存在。 The buffer system in the liquid antibody preparation of the present invention is a histidine and histidine hydrochloride buffer system. Preferably, the histidine and the histidine hydrochloride are respectively about 0.1-10 mg/mL, for example about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/ml The concentration exists. In a more preferred embodiment, the buffer used in the formulation of the present invention is histidine and histidine hydrochloride at a total concentration of about 10 mM. mL and about 1.00 mg/mL are present.

(iii)穩定劑 (iii) Stabilizer

無論是用於凍乾製劑或液體製劑，用於本發明的合適的穩定劑可以選自多元醇、胺基酸和它們的任意組合。 Whether used in lyophilized formulations or liquid formulations, suitable stabilizers used in the present invention may be selected from polyols, amino acids, and any combination thereof.

作為穩定劑的多元醇可以選自但不限於：甘露醇、山梨醇或其組合。在一個實施方案中，作為穩定劑的多元醇是山梨醇。 The polyol as a stabilizer can be selected from but not limited to: mannitol, sorbitol or a combination thereof. In one embodiment, the polyol used as a stabilizer is sorbitol.

作為穩定劑的胺基酸可以選自但不限於：精胺酸、鹽酸精胺酸、甲硫胺酸、甘胺酸、脯胺酸或其組合。在一些實施方案中，作為穩定劑的胺基酸是精胺酸和/或鹽酸精胺酸。 The amino acid as the stabilizer can be selected from but not limited to: arginine, arginine hydrochloride, methionine, glycine, proline or a combination thereof. In some embodiments, the amino acid as a stabilizer is arginine and/or arginine hydrochloride.

在一些實施方案中，在本發明所述的凍乾製劑中，該穩定劑包括山梨醇和精胺酸。較佳地，該山梨醇以約1-10%(w/v)，例如約1、2、3、4、5、6、7、8、9、10%(w/v)的重量比存在，該精胺酸以約1-10%(w/v)，例如約1、2、3、4、5、6、7、8、9、10%(w/v)的重量比存在。在一個更佳的實施方案中，該山梨醇和該精胺酸分別以約3%(w/v)和約1.742%(w/v)的重量比存在。 In some embodiments, in the lyophilized formulation of the present invention, the stabilizer includes sorbitol and arginine. Preferably, the sorbitol is present in a weight ratio of about 1-10% (w/v), such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10% (w/v) The arginine is present in a weight ratio of about 1-10% (w/v), for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10% (w/v). In a more preferred embodiment, the sorbitol and the arginine are present in a weight ratio of about 3% (w/v) and about 1.742% (w/v), respectively.

在一些實施方案中，在本發明所述的凍乾製劑中，該穩定劑包括山梨醇和鹽酸精胺酸。較佳地，該山梨醇以約1-10%(w/v)，例如約1、2、3、4、5、6、7、8、9、10%(w/v)的重量比存在，以約1-10%(w/v)，例如約1、2、3、4、5、6、7、8、9、10%(w/v)的重量比存在。在一個更佳的實施方案中，該山梨醇和該鹽酸精胺酸分別以約3%(w/v)和約2.107%(w/v)的重量比存在。 In some embodiments, in the lyophilized formulation of the present invention, the stabilizer includes sorbitol and arginine hydrochloride. Preferably, the sorbitol is present in a weight ratio of about 1-10% (w/v), such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10% (w/v) , Present in a weight ratio of about 1-10% (w/v), for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10% (w/v). In a more preferred embodiment, the sorbitol and the arginine hydrochloride are present in a weight ratio of about 3% (w/v) and about 2.107% (w/v), respectively.

在一些實施方案中，在本發明該的凍乾製劑中，該穩定劑還包括甲硫胺酸。在本發明液體製劑中，該甲硫胺酸可以以約0.1-10%(w/v)的重量比存在，例如以約0.1、0.5、1、2、3、4、5、6、7、8、9、10%(w/v)的重量比存在。在一個實施方案中，該甲硫胺酸以約0.749%(w/v)的重量比存在。 In some embodiments, in the lyophilized formulation of the present invention, the stabilizer further includes methionine. In the liquid formulation of the present invention, the methionine may be present in a weight ratio of about 0.1-10% (w/v), such as about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, The weight ratio of 8, 9, 10% (w/v) exists. In one embodiment, the methionine is present in a weight ratio of about 0.749% (w/v).

在一些實施方案中，本發明液體製劑包含山梨醇和精胺酸作為穩定劑。在本發明液體製劑中的該山梨醇和該精胺酸的濃度分別可以是約10-100mg/mL，例如約10、20、30、40、50、60、70、80、90、100mg/mL。較佳地，在本發明液體製劑中的該山梨醇和該精胺酸分別可以以約10-50mg/mL，例如約10、20、30、40、50mg/mL的濃度存在。在一個實施方案中，該山梨醇和該精胺酸分別以約30mg/mL和約17.42mg/mL的濃度存在； In some embodiments, the liquid formulation of the present invention contains sorbitol and arginine as stabilizers. The concentration of the sorbitol and the arginine in the liquid preparation of the present invention may be about 10-100 mg/mL, for example, about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg/mL, respectively. Preferably, the sorbitol and the arginine in the liquid preparation of the present invention may be present at a concentration of about 10-50 mg/mL, for example, about 10, 20, 30, 40, 50 mg/mL, respectively. In one embodiment, the sorbitol and the arginine are present at a concentration of about 30 mg/mL and about 17.42 mg/mL, respectively;

在一些實施方案中，本發明液體製劑包含山梨醇和鹽酸精胺酸作為穩定劑。在本發明液體製劑中的該山梨醇和該鹽酸精胺酸分別可以以約10-100mg/mL，例如約10、20、30、40、50、60、70、80、90、100mg/mL的濃度存在。較佳地，在本發明液體製劑中的該山梨醇和該鹽酸精胺酸分別可以以約10-50mg/mL，例如約10、20、30、40、50mg/mL的濃度存在。在一個實施方案中，山梨醇和鹽酸精胺酸分別以約30mg/mL和約21.07mg/mL的濃度存在。 In some embodiments, the liquid formulation of the present invention contains sorbitol and arginine hydrochloride as stabilizers. The sorbitol and the arginine hydrochloride in the liquid preparation of the present invention may be in a concentration of about 10-100 mg/mL, for example, about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg/mL, respectively. exist. Preferably, the sorbitol and the arginine hydrochloride in the liquid preparation of the present invention may be present at a concentration of about 10-50 mg/mL, for example, about 10, 20, 30, 40, 50 mg/mL, respectively. In one embodiment, sorbitol and arginine hydrochloride are present at a concentration of about 30 mg/mL and about 21.07 mg/mL, respectively.

在一些實施方案中，本發明液體製劑中該穩定劑還包括甲硫胺酸。在本發明液體製劑中，該甲硫胺酸可以以約1-100mg/mL，例如約1、5、10、20、30、40、50、60、70、80、90、100mg/mL的濃度存在。更佳地，該甲硫胺酸以約1-50mg/mL。在一個實施方案中，該甲硫胺酸以約7.49mg/mL的濃度存在。 In some embodiments, the stabilizer in the liquid formulation of the present invention further includes methionine. In the liquid formulation of the present invention, the methionine may be at a concentration of about 1-100 mg/mL, for example, about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg/mL exist. More preferably, the methionine is about 1-50 mg/mL. In one embodiment, the methionine is present at a concentration of about 7.49 mg/mL.

(iv)表面活性劑 (iv) Surfactant

如本文所使用的，術語“表面活性劑”是指具有兩親結構的有機物質；即，它們由相反的溶解性傾向的基團所組成，通常是油溶性的烴鏈和水溶性的離子基團。 As used herein, the term "surfactant" refers to an organic substance with an amphiphilic structure; that is, they are composed of groups with opposite solubility tendencies, usually oil-soluble hydrocarbon chains and water-soluble ionic groups. group.

無論是用於凍乾製劑或液體製劑，用於本發明的合適的穩定劑可以是非離子型表面活性劑，例如，烷基聚(環氧乙烯)。可包括在本發明製劑中的特定非離子型表面活性劑包括，例如聚山梨酯80、聚山梨酯20、泊洛沙姆、聚乙二醇聚山梨酯80等。在一個較佳實施方案中，本發明的製劑中包含聚山梨酯-80作為表面活性劑。 Whether used in lyophilized formulations or liquid formulations, suitable stabilizers for use in the present invention may be non-ionic surfactants, for example, alkyl poly(ethylene oxide). Specific nonionic surfactants that can be included in the formulation of the present invention include, for example, polysorbate 80, polysorbate 20, poloxamer, polyethylene glycol polysorbate 80, and the like. In a preferred embodiment, the formulation of the present invention contains polysorbate-80 as a surfactant.

本發明抗體製劑中所含的表面活性劑的量可隨製劑的特定目的特性、特定環境、和使用製劑的特定目的而改變。 The amount of the surfactant contained in the antibody preparation of the present invention can be changed according to the specific purpose characteristics of the preparation, the specific environment, and the specific purpose for which the preparation is used.

在一些實施方案中，在本發明的所述的凍乾製劑中，該表面活性劑以約0.01%(w/v)-1%(w/v)，例如0.01、0.05、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1%(w/v)的重量比存在。較佳地，以約0.01%(w/v)-0.5%(w/v)，例如0.01、0.05、0.1、0.2、0.3、0.4、0.5%(w/v)的重量比存在。 In some embodiments, in the lyophilized formulation of the present invention, the surfactant is at a concentration of about 0.01% (w/v) to 1% (w/v), such as 0.01, 0.05, 0.1, 0.2, 0.3 , 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1% (w/v) weight ratio exists. Preferably, it is present in a weight ratio of about 0.01% (w/v)-0.5% (w/v), for example 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5% (w/v).

在一些實施方案中，在本發明的所述的凍乾製劑中，該表面活性劑為聚山梨酯80，更佳地，該聚山梨酯80以約0.02%(w/v)的重量比存在。 In some embodiments, in the lyophilized formulation of the present invention, the surfactant is polysorbate 80, more preferably, the polysorbate 80 is present in a weight ratio of about 0.02% (w/v) .

在一些實施方案中，在本發明的所述的液體製劑中，該表面活性劑以約0.1-10mg/ml，例如約0.1、0.5、1、2、3、4、5、6、7、8、9、10mg/ml的濃度存在。較佳地，約0.1-5mg/ml，例如約0.1、0.5、1、2、3、4、5mg/ml的濃度存在。 In some embodiments, in the liquid formulation of the present invention, the surfactant is at a concentration of about 0.1-10 mg/ml, for example about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8. , 9, 10mg/ml concentration exists. Preferably, it is present at a concentration of about 0.1-5 mg/ml, for example, about 0.1, 0.5, 1, 2, 3, 4, 5 mg/ml.

在較佳的一些實施方案中，製劑可含有約0.1-10mg/ml，例如約0.1、0.5、1、2、3、4、5、6、7、8、9、10mg/ml的聚山梨酯類表面活性劑(例如，聚山梨酯-80)。更佳地，該聚山梨酯80以約0.2mg/ml的濃度存在。 In some preferred embodiments, the formulation may contain about 0.1-10 mg/ml, for example about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/ml of polysorbate Class surfactants (for example, polysorbate-80). More preferably, the polysorbate 80 is present at a concentration of about 0.2 mg/ml.

(v)其它賦形劑 (v) Other excipients

在一些實施方案中，本發明的抗體製劑中任選地包含其它賦形劑。該其它賦形劑包括，例如，抗微生物劑、抗靜電劑、抗氧化劑、螯合劑、明膠等等。這些和另外已知的藥物賦形劑和/或適用於本發明製劑的添加劑是本領域公知的，例如，列出於“The Handbook of Pharmaceutical Excipients，第4版，Rowe等人編，American Pharmaceuticals Association(2003)；和Remington：the Science and Practice of Pharmacy，第21版，Gennaro編，Lippincott Williams & Wilkins(2005)”。 In some embodiments, other excipients are optionally included in the antibody formulations of the present invention. The other excipients include, for example, antimicrobial agents, antistatic agents, antioxidants, chelating agents, gelatin, and the like. These and other known pharmaceutical excipients and/or additives suitable for the formulation of the present invention are well known in the art, for example, listed in "The Handbook of Pharmaceutical Excipients, 4th Edition, Rowe et al., eds., American Pharmaceuticals Association (2003); and Remington: the Science and Practice of Pharmacy, 21st edition, edited by Gennaro, Lippincott Williams & Wilkins (2005)".

在一些實施方案中，本發明的抗體凍乾製劑包含：約2-17%(w/v)重組全人源抗程序性死亡受體1(PD-1)和抗程序性死亡配體1(PD-L1)雙特異性抗體、約0.294%(w/v)、約50mg/ml山梨醇、約0.20mg/ml聚山梨酯80，該水溶液具有約5.5的pH。 In some embodiments, the antibody lyophilized preparation of the present invention comprises: about 2-17% (w/v) recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 ( PD-L1) bispecific antibody, about 0.294% (w/v), about 50 mg/ml sorbitol, about 0.20 mg/ml polysorbate 80, the aqueous solution has a pH of about 5.5.

在一些較佳的實施方案中，本發明的抗體凍乾製劑包含：約2-17%(w/v)重組全人源抗程序性死亡受體1(PD-1)和抗程序性死亡配體1(PD-L1)雙特異性抗體、約0.155%(w/v)組胺酸、約3%(w/v)山梨醇、約0.749%(w/v)甲硫胺酸、約1.742%(w/v)精胺酸、約0.20mg/ml聚山梨酯80，該製劑當重構時具有6.0的pH。 In some preferred embodiments, the antibody lyophilized preparation of the present invention contains: about 2-17% (w/v) recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death compound Body 1 (PD-L1) bispecific antibody, about 0.155% (w/v) histidine, about 3% (w/v) sorbitol, about 0.749% (w/v) methionine, about 1.742 %(w/v) Arginine, about 0.20 mg/ml Polysorbate 80, this formulation has a pH of 6.0 when reconstituted.

在一些較佳的實施方案中，本發明的抗體凍乾製劑包含：約2-17%(w/v)重組全人源抗程序性死亡受體1(PD-1)和抗程序性死亡配體1(PD-L1)雙特異性抗體、約0.085%(w/v)組胺酸、約0.1%(w/v)鹽酸組胺酸、約3.00%(w/v)山梨醇、約0.749%(w/v)甲硫胺酸、約2.107%(w/v)鹽酸精胺酸、約0.02%(w/v)聚山梨酯80；該製劑當重構時具有6.0的pH。 In some preferred embodiments, the antibody lyophilized preparation of the present invention contains: about 2-17% (w/v) recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death compound Body 1 (PD-L1) bispecific antibody, about 0.085% (w/v) histidine, about 0.1% (w/v) histidine hydrochloride, about 3.00% (w/v) sorbitol, about 0.749 % (w/v) methionine, about 2.107% (w/v) arginine hydrochloride, about 0.02% (w/v) polysorbate 80; this formulation has a pH of 6.0 when reconstituted.

在一些實施方案中，本發明的抗體液體製劑中該水溶液包含：約20-170mg/ml重組全人源抗程序性死亡受體1(PD-1)和抗程序性死亡配體1(PD-L1)雙特異性抗體、約2.94mg/ml枸櫞酸鈉、約50mg/ml山梨醇、約0.20mg/ml聚山梨酯80，該水溶液具有約5.5的pH。 In some embodiments, the aqueous solution in the antibody liquid formulation of the present invention contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD- L1) Bispecific antibody, about 2.94 mg/ml sodium citrate, about 50 mg/ml sorbitol, about 0.20 mg/ml polysorbate 80, the aqueous solution has a pH of about 5.5.

在一些實施方案中，本發明的抗體液體製劑中該水溶液包含：約20-170mg/ml重組全人源抗程序性死亡受體1(PD-1)和抗程序性死亡配體1(PD-L1)雙特異性抗體、約2.94mg/ml枸櫞酸鈉、約50mg/ml山梨醇、約0.20mg/ml聚山梨酯80，該水溶液具有約6.0的pH。 In some embodiments, the aqueous solution in the antibody liquid formulation of the present invention contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD- L1) Bispecific antibody, about 2.94 mg/ml sodium citrate, about 50 mg/ml sorbitol, about 0.20 mg/ml polysorbate 80, the aqueous solution has a pH of about 6.0.

在一些實施方案中，本發明的抗體液體製劑中該水溶液包含：約20-170mg/ml重組全人源抗程序性死亡受體1(PD-1)和抗程序性死亡配體1(PD-L1)雙特異性抗體、約2.94mg/ml枸櫞酸鈉、約50mg/ml山梨醇、約2.94mg/ml甲硫胺酸、約0.20mg/ml聚山梨酯80，該水溶液具有約6.0的pH。 In some embodiments, the aqueous solution in the antibody liquid formulation of the present invention contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD- L1) Bispecific antibody, about 2.94mg/ml sodium citrate, about 50mg/ml sorbitol, about 2.94mg/ml methionine, about 0.20mg/ml polysorbate 80, the aqueous solution has about 6.0 pH.

在一些實施方案中，本發明的抗體液體製劑中該水溶液包含：約20-170mg/ml重組全人源抗程序性死亡受體1(PD-1)和抗程序性 死亡配體1(PD-L1)雙特異性抗體、約2.94mg/ml枸櫞酸鈉、約50mg/ml山梨醇、約0.20mg/ml聚山梨酯80，該水溶液具有約6.5的pH。 In some embodiments, the aqueous solution in the antibody liquid formulation of the present invention contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed Death ligand 1 (PD-L1) bispecific antibody, about 2.94 mg/ml sodium citrate, about 50 mg/ml sorbitol, about 0.20 mg/ml polysorbate 80, the aqueous solution has a pH of about 6.5.

在一些實施方案中，本發明的抗體液體製劑中該水溶液包含：約20-170mg/ml重組全人源抗程序性死亡受體1(PD-1)和抗程序性死亡配體1(PD-L1)雙特異性抗體、約2.94mg/ml枸櫞酸鈉、約50mg/ml山梨醇、約0.20mg/ml聚山梨酯80，該水溶液具有約6.0的pH。 In some embodiments, the aqueous solution in the antibody liquid formulation of the present invention contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD- L1) Bispecific antibody, about 2.94 mg/ml sodium citrate, about 50 mg/ml sorbitol, about 0.20 mg/ml polysorbate 80, the aqueous solution has a pH of about 6.0.

在一些實施方案中，本發明的抗體液體製劑中該水溶液包含：約20-170mg/ml重組全人源抗程序性死亡受體1(PD-1)和抗程序性死亡配體1(PD-L1)雙特異性抗體、約1.55mg/ml組胺酸、約50mg/ml山梨醇、約0.20mg/ml聚山梨酯80，該水溶液具有約6.0的pH。 In some embodiments, the aqueous solution in the antibody liquid formulation of the present invention contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD- L1) Bispecific antibody, about 1.55 mg/ml histidine, about 50 mg/ml sorbitol, about 0.20 mg/ml polysorbate 80, the aqueous solution has a pH of about 6.0.

在一些實施方案中，本發明的抗體液體製劑中該水溶液包含：約20-170mg/ml重組全人源抗程序性死亡受體1(PD-1)和抗程序性死亡配體1(PD-L1)雙特異性抗體、約1.55mg/ml組胺酸、約50mg/ml山梨醇、約7.49mg/ml甲硫胺酸、約0.20mg/ml聚山梨酯80，該水溶液具有約6.0的pH。 In some embodiments, the aqueous solution in the antibody liquid formulation of the present invention contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD- L1) Bispecific antibody, about 1.55mg/ml histidine, about 50mg/ml sorbitol, about 7.49mg/ml methionine, about 0.20mg/ml polysorbate 80, the aqueous solution has a pH of about 6.0 .

在一些較佳的實施方案中，本發明的抗體液體製劑中該水溶液包含：20-170mg/ml重組全人源抗程序性死亡受體1(PD-1)和抗程序性死亡配體1(PD-L1)雙特異性抗體、約1.55mg/ml組胺酸、約30mg/ml山梨醇、約7.49mg/ml甲硫胺酸、約17.42mg/ml精胺酸、約0.20mg/ml聚山梨酯80，該水溶液具有約6.0的pH。 In some preferred embodiments, the aqueous solution in the antibody liquid formulation of the present invention contains: 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 ( PD-L1) bispecific antibody, about 1.55mg/ml histidine, about 30mg/ml sorbitol, about 7.49mg/ml methionine, about 17.42mg/ml arginine, about 0.20mg/ml poly Sorbate 80, the aqueous solution has a pH of about 6.0.

在一些較佳的實施方案中，本發明的抗體液體製劑中，該水溶液包含：約20-170mg/ml重組全人源抗程序性死亡受體1(PD-1)和抗 程序性死亡配體1(PD-L1)雙特異性抗體、約0.85mg/ml組胺酸、約1.00mg/ml鹽酸組胺酸、約30.00mg/ml山梨醇、約7.49mg/ml甲硫胺酸、約21.07mg/ml鹽酸精胺酸、約0.20mg/ml聚山梨酯80，該水溶液具有約6.0的pH。 In some preferred embodiments, in the antibody liquid formulation of the present invention, the aqueous solution contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti Programmed death ligand 1 (PD-L1) bispecific antibody, about 0.85mg/ml histidine, about 1.00mg/ml histidine hydrochloride, about 30.00mg/ml sorbitol, about 7.49mg/ml methyl sulfide Amino acid, about 21.07 mg/ml arginine hydrochloride, about 0.20 mg/ml polysorbate 80, the aqueous solution has a pH of about 6.0.

II.製劑的製備II. Preparation of the formulation

本發明提供了包含抗PD-1/PD-L1抗體蛋白的穩定製劑。在本發明製劑中使用的抗PD-1/PD-L1抗體蛋白可以使用本領域已知的用於生產抗體的技術進行製備。例如，可以重組製備抗體。在一些實施方案中，本發明的抗體可以在CHO,NSO,HEK293或COS細胞中製備。較佳地，本發明的抗體可以在CHO或HEK293細胞中製備。可借由熟知方法將含有所關注多核苷酸序列(例如編碼抗體的多肽的多核苷酸及表達控制序列)的載體轉移至宿主細胞中，該方法根據宿主細胞類型而有所變化。 The present invention provides a stable formulation containing anti-PD-1/PD-L1 antibody protein. The anti-PD-1/PD-L1 antibody protein used in the formulation of the present invention can be prepared using techniques known in the art for antibody production. For example, antibodies can be produced recombinantly. In some embodiments, the antibodies of the present invention can be produced in CHO, NSO, HEK293 or COS cells. Preferably, the antibody of the present invention can be prepared in CHO or HEK293 cells. The vector containing the polynucleotide sequence of interest (for example, the polynucleotide encoding the polypeptide of the antibody and the expression control sequence) can be transferred to the host cell by a well-known method, and the method varies according to the type of the host cell.

抗體作為藥物的活性成分的應用現在已經很廣泛。用於將治療性抗體純化至藥用級的技術是本領域公知的。例如，Tugcu等(Maximizing productivity of chromatography steps for purification of monoclonal antibodies,Biotechnology and Bioengineering 99(2008)599-613.)描述在蛋白A捕獲步驟後使用離子交換色譜(陰離子IEX和/或陽離子CEX色譜)的單株抗體三管柱純化方法。Kelley等(Weak partitioning chromatography for anion exchange purification of monoclonal antibodies,Biotechnology and Bioengineering 101(2008)553-566)描述了兩管柱純化法，其中在蛋白A親和色譜後使用弱分配陰離子交換樹脂。 The application of antibodies as active ingredients of medicines is now widespread. Techniques for purifying therapeutic antibodies to pharmaceutical grade are well known in the art. For example, Tugcu et al. (Maximizing productivity of chromatography steps for purification of monoclonal antibodies, Biotechnology and Bioengineering 99 (2008) 599-613.) describe the use of ion exchange chromatography (anion IEX and/or cation CEX chromatography) after the protein A capture step Monoclonal antibody three-column purification method. Kelley et al. (Weak partitioning chromatography for anion exchange purification of monoclonal antibodies, Biotechnology and Bioengineering 101 (2008) 553-566) describe a two-column purification method in which a weak partitioning anion exchange resin is used after protein A affinity chromatography.

一般，重組產生的單株抗體可以利用常規的純化方法純化，以提供具有足夠的可重複性和適度純度的藥物物質用於抗體製劑的配製。例如，在抗體從重組表達細胞分泌至培養基中後，可以使用商業可得的蛋白濃縮過濾器例如Amicon的超濾裝置，濃縮來自該表達系統的上清液。之後，可以使用例如色譜、透析和親和純化等方式進行抗體的純化。蛋白A適應於作為親和配體用於純化IgG1、IgG2和IgG4型抗體。也可以使用其它抗體純化方法，例如離子交換色譜。在獲得足夠純度的抗體後，可以按照本領域已知的方法，製備包含抗體的製劑。 Generally, recombinantly produced monoclonal antibodies can be purified by conventional purification methods to provide pharmaceutical substances with sufficient reproducibility and moderate purity for the preparation of antibody preparations. For example, after the antibody is secreted from the recombinant expression cell into the culture medium, a commercially available protein concentration filter such as Amicon's ultrafiltration device can be used to concentrate the supernatant from the expression system. After that, the antibody can be purified using methods such as chromatography, dialysis, and affinity purification. Protein A is suitable as an affinity ligand for the purification of IgG1, IgG2 and IgG4 type antibodies. Other antibody purification methods, such as ion exchange chromatography, can also be used. After obtaining the antibody of sufficient purity, a preparation containing the antibody can be prepared according to methods known in the art.

例如，可以採用如下步驟進行製備：(1)在發酵結束後將發酵液離心澄清去除細胞等雜質以獲得上清；(2)使用親和層析(例如對IgG1、IgG2和IgG4型抗體具有特異親和力的蛋白A管柱)捕獲抗體；(3)進行病毒滅活；(4)精製純化(一般可以採用CEX陽離子交換層析)，以去除蛋白中的雜質；(4)病毒過濾(使病毒滴度降低例如4 log10以上)；(5)超濾/滲濾(可以用於將蛋白置換於利於其穩定的製劑緩衝液中並濃縮至合適的濃度供注射用)。參見例如，B.Minow,P.Rogge,K.Thompson,BioProcess International,Vol.10,No.6,2012,pp.48-57。 For example, the following steps can be used for preparation: (1) After fermentation, the fermentation broth is centrifuged to clarify impurities such as cells to obtain the supernatant; (2) affinity chromatography (for example, specific affinity for IgG1, IgG2, and IgG4 antibodies) Protein A column) to capture antibodies; (3) virus inactivation; (4) purification and purification (usually CEX cation exchange chromatography can be used) to remove impurities in the protein; (4) virus filtration (to make the virus titer To reduce, for example, 4 log10 or more); (5) Ultrafiltration/diafiltration (which can be used to replace the protein in a formulation buffer that is conducive to its stability and concentrate it to a suitable concentration for injection). See, for example, B. Minow, P. Rogge, K. Thompson, BioProcess International, Vol. 10, No. 6, 2012, pp. 48-57.

III.製劑的分析方法III. Analytical method of preparation

在抗體製劑的儲存過程中，抗體可能會發生聚集、降解或化學修飾，導致抗體異質性(包括大小異質性和電荷異質性)以及聚集物和片段等，從而影響抗體製劑的質量。因此，有必要進行抗體製劑穩定性的監測。 During the storage of antibody preparations, antibodies may undergo aggregation, degradation or chemical modification, resulting in antibody heterogeneity (including size heterogeneity and charge heterogeneity), aggregates and fragments, etc., thereby affecting the quality of antibody preparations. Therefore, it is necessary to monitor the stability of antibody preparations.

在本領域中已知多種方法可以用於檢測抗體製劑的穩定性。例如，可以藉由還原型CE-SDS、非還原型CE-SDS和SEC-HPLC等方法，分析抗體製劑的純度和評估抗體的聚集水平；可以藉由毛細管等電聚焦電泳(cIEF)、成像毛細管等電聚焦電泳(iCIEF)和離子交換色譜(IEX)等，分析抗體製劑中的電荷變異體。此外，可以藉由目視檢測製劑外觀，快速地判斷製劑的穩定性。也可以使用OD350nm法檢測製劑的濁度改變，該方法可以給出有關可溶性和不溶性聚集物量的信息。此外，可以使用紫外分光光度法(UV法)檢測製劑中的蛋白質含量變化。 Various methods are known in the art that can be used to test the stability of antibody preparations. For example, methods such as reduced CE-SDS, non-reduced CE-SDS, and SEC-HPLC can be used to analyze the purity of antibody preparations and assess the level of antibody aggregation; can be achieved by capillary isoelectric focusing (cIEF), imaging capillary Isoelectric focusing electrophoresis (iCIEF) and ion exchange chromatography (IEX), etc., analyze charge variants in antibody preparations. In addition, the stability of the preparation can be quickly judged by visually inspecting the appearance of the preparation. The OD 350nm method can also be used to detect changes in the turbidity of the preparation, which can give information about the amount of soluble and insoluble aggregates. In addition, ultraviolet spectrophotometry (UV method) can be used to detect changes in protein content in the preparation.

非還原型CE-SDS法是一種以毛細管為分離通道進行的抗體純度測定方法。在CE-SDS中，蛋白遷移由SDS結合引起的表面電荷來驅動，而該表面電荷與蛋白質的分子量成正比。由於所有的SDS-蛋白質複合物都具有相似的質量-電荷比，故可以在毛細管的分子篩凝膠基質中，實現基於分子的大小或流體動力學半徑的電泳分離。該方法已經被廣泛地用於監測變性的完整抗體的純度。一般，在非還原型CE-SDS法中，供試樣品與SDS樣品緩衝液和碘乙醯胺混合。之後，混合物可以於68-72℃孵育約10-15分鐘，冷卻至室溫後離心的上清液用於分析。採用紫外檢測器檢測蛋白的遷移，獲得電泳譜圖。抗體製劑純度可以計算為IgG主峰的峰面積占所有峰面積之和的百分比。關於CE-SDS法的進一步描述，可以參見例如Richard R.等，Application of CE SDS gel in development of biopharmaceutical antibody-based products,Electrophoresis,2008,29,3612-3620。 The non-reduced CE-SDS method is a method of antibody purity determination using capillary as a separation channel. In CE-SDS, protein migration is driven by the surface charge caused by SDS binding, and the surface charge is proportional to the molecular weight of the protein. Since all SDS-protein complexes have similar mass-to-charge ratios, electrophoretic separation based on molecular size or hydrodynamic radius can be achieved in the molecular sieve gel matrix of the capillary. This method has been widely used to monitor the purity of denatured intact antibodies. Generally, in the non-reducing CE-SDS method, the test sample is mixed with SDS sample buffer and iodoacetamide. After that, the mixture can be incubated at 68-72°C for about 10-15 minutes, and the supernatant centrifuged after cooling to room temperature is used for analysis. A UV detector is used to detect the migration of the protein and obtain an electrophoresis spectrum. The purity of the antibody preparation can be calculated as the percentage of the peak area of the main IgG peak to the sum of all peak areas. For a further description of the CE-SDS method, see, for example, Richard R. et al., Application of CE SDS gel in development of biopharmaceutical antibody-based products, Electrophoresis, 2008, 29, 3612-3620.

尺寸排阻高效液相色譜法，即SEC-HPLC法，是用於抗體標準和質控的另一重要方法。該方法主要依據分子的尺寸大小或流體動力學半徑差異來進行分子的分離。藉由SEC-HPLC，抗體可以分離出三種主要形式：高分子量形式(HMMS)、主峰(主要是抗體單體)、和低分子量形式(LMMS)。抗體純度可以計算為色譜圖上主峰面積占所有峰面積之和的百分比。藉由SEC-HPLC法，可以測量製劑產品中抗體單體的百分數，給出可溶性聚集物和剪切物的含量信息。關於SEC-HPLC法的進一步描述，可以參見例如，J.Pharm.Scien.,83：1645-1650,(1994)；Pharm.Res.,11：485(1994)；J.Pharm.Bio.Anal.,15：1928(1997)；J.Pharm.Bio.Anal.,14：1133-1140(1986)。此外，也可以參見例如，R.Yang等，High resolution separation of recombinant monoclonal antibodies by size exclusion ultra-high performance liquid chromatography(SE-UHPLC),Journal of Pharmaceutical and Biomedical Analysis(2015),http：//dx.doi.org/10.1016/j.jpba.2015.02.032；和Alexandre Goyon等，Protocols for the analytical characterization of therapeutic monoclonal antibodies.I-Non-denaturing chromatographic techniques,Journal of Chromatography，http：//dx.doi.org/10.1016/j.jchromb.2017.05.010。 Size exclusion high performance liquid chromatography, or SEC-HPLC, is another important method for antibody standards and quality control. This method is mainly based on the size of the molecule or the difference in hydrodynamic radius to separate the molecules. By SEC-HPLC, antibodies can be separated into three main forms: high molecular weight form (HMMS), main peak (mainly antibody monomer), and low molecular weight form (LMMS). Antibody purity can be calculated as the percentage of the main peak area on the chromatogram to the sum of all peak areas. With the SEC-HPLC method, the percentage of antibody monomers in the preparation product can be measured, and information about the content of soluble aggregates and shears can be given. For a further description of the SEC-HPLC method, see, for example, J. Pharm. Scien., 83: 1645-1650, (1994); Pharm. Res., 11: 485 (1994); J. Pharm. Bio. Anal. , 15: 1928 (1997); J. Pharm. Bio. Anal., 14: 1133-1140 (1986). In addition, see, for example, R. Yang et al., High resolution separation of recombinant monoclonal antibodies by size exclusion ultra-high performance liquid chromatography (SE-UHPLC), Journal of Pharmaceutical and Biomedical Analysis (2015), http://dx. doi.org/10.1016/j.jpba.2015.02.032; and Alexandre Goyon et al., Protocols for the analytical characterization of therapeutic monoclonal antibodies. I-Non-denaturing chromatographic techniques, Journal of Chromatography, http://dx.doi.org /10.1016/j.jchromb.2017.05.010.

成像毛細管等電聚焦電泳(iCIEF)可以用於分析抗體的電荷異質性。該方法可以提供電荷變異體的定量分佈情況。iCIEF基於分子在pH梯度中的電荷差異(表觀pI值)來實現分子分離的目的。在iCIEF中，分離管柱通常是短毛細管(例如，5cm長，100μm內徑的二氧化矽毛細管)，蛋白質在高電壓下在毛細管管柱中聚焦，並藉由在280nM操作的全管柱成 像檢測系統對聚焦進行實時在線監測。該技術的一個優點是，可以藉由該全管柱檢測系統同時記錄抗體樣品的各種電荷變異體。一般而言，在icIEF中，將樣品與尿素和icIEF緩衝液混合，其中該緩衝液含有甲基纖維素、pI分子量標準和兩性電解質。然後，可以在iCIEF分析儀例如iCE280分析儀(Protein Simple,Santa Clara,CA)上，使用iCIEF管柱例如ProtionSimple組裝的iCIEF管柱，在樣品聚焦一定時間後，測定280nm的吸光度，獲得聚焦抗體電荷變異體的譜圖。在iCEIF譜圖中，在主峰(即主成分)之前沖提的蛋白相關峰被分類為酸性組分；相對地，在主峰之後沖提的蛋白相關峰被分類為鹼性組分。主成分、酸性組分和鹼性組分的相對量可以表示為占總峰面積的百分數。關於iCIEF的進一步描述，可以參見例如，Salas-Solano O等，Robustness of iCIEF methodology for the analysis of monoclonal antibodies：an interlaboratory study,J Sep Sci.2012 Nov；35(22)：3124-9.doi：10.1002/jssc.201200633.Epub 2012 Oct 15；和Dada OO等，Characterization of acidic and basic variants of IgG1 therapeutic monoclonal antibodies based on non-denaturing IEF fractionation,Electrophoresis.2015 Nov；36(21-22)：2695-2702.doi：10.1002/elps.201500219.Epub 2015 Sep 18. Imaging capillary isoelectric focusing electrophoresis (iCIEF) can be used to analyze the charge heterogeneity of antibodies. This method can provide a quantitative distribution of charge variants. iCIEF achieves molecular separation based on the charge difference (apparent pI value) of molecules in the pH gradient. In iCIEF, the separation column is usually a short capillary (for example, a silica capillary with a length of 5 cm and an inner diameter of 100 μm). The protein is focused in the capillary column at a high voltage and formed by a full-tube column operating at 280 nM. The image detection system performs real-time online monitoring of the focus. One advantage of this technology is that the full-column detection system can simultaneously record various charge variants of antibody samples. Generally speaking, in icIEF, the sample is mixed with urea and icIEF buffer, where the buffer contains methyl cellulose, pi molecular weight standards and amphoteric electrolytes. Then, you can use an iCIEF column such as an iCIEF column assembled by ProtionSimple on an iCIEF analyzer such as iCE280 analyzer (Protein Simple, Santa Clara, CA). After the sample is focused for a certain period of time, the absorbance at 280 nm is measured to obtain the focused antibody charge. Spectra of variants. In the iCEIF spectrum, the protein-related peaks extracted before the main peak (that is, the principal component) are classified as acidic components; in contrast, the protein-related peaks extracted after the main peak are classified as basic components. The relative amounts of the main components, acidic components, and basic components can be expressed as a percentage of the total peak area. For a further description of iCIEF, see, for example, Salas-Solano O et al., Robustness of iCIEF methodology for the analysis of monoclonal antibodies: an interlaboratory study, J Sep Sci. 2012 Nov; 35(22): 3124-9. doi: 10.1002 /jssc.201200633.Epub 2012 Oct 15; and Dada OO, etc., Characterization of acidic and basic variants of IgG1 therapeutic monoclonal antibodies based on non-denaturing IEF fractionation, Electrophoresis. 2015 Nov; 36(21-22): 2695-2702. doi: 10.1002/elps.201500219.Epub 2015 Sep 18.

也可以藉由陽離子交換高效液相色譜法(CEX-HPLC)測定抗體製劑中抗體的電荷變異體。在該測定法中，以比主峰的保留時間更早從CEX-HPLC管柱沖提出的峰被標記為“酸性峰”，而那些以比主峰的保留時間更晚從CEX-HPLC管柱沖提出的峰被標記為“鹼性峰”。 The charge variant of the antibody in the antibody preparation can also be determined by cation exchange high performance liquid chromatography (CEX-HPLC). In this assay, the peaks extracted from the CEX-HPLC column with a retention time earlier than the main peak are marked as "acidic peaks", and those extracted from the CEX-HPLC column with a retention time later than the main peak The peaks are labeled as "basic peaks".

加速穩定性研究可以用於檢查產品的穩定性性質，有利於篩選穩定藥物製劑形式。例如，可以將製劑樣品放置於升高的溫度，例如約40℃±2℃、25℃±2℃條件下進行加速穩定性研究。檢測指標可以包括外觀、可見異物、蛋白含量、濁度、純度(SEC-HPLC法、非還原型CE-SDS法)和電荷變異體(iCIEF法、CEX-HPLC法)。 Accelerated stability studies can be used to check the stability properties of products, which is conducive to the screening of stable pharmaceutical formulations. For example, a sample of the formulation can be placed at an elevated temperature, such as about 40°C ± 2°C, 25°C ± 2°C, for accelerated stability studies. Detection indicators can include appearance, visible foreign matter, protein content, turbidity, purity (SEC-HPLC method, non-reduced CE-SDS method) and charge variants (iCIEF method, CEX-HPLC method).

本發明的抗PD-1/PD-L1抗體蛋白製劑是穩定的。在一些實施方案中，本發明的抗PD-1/PD-L1抗體蛋白製劑在儲存後，例如在2-8℃儲存至少24個月後，或在室溫儲存至少6個月後，或在40℃±2℃儲存1個月後，是穩定的。在一些實施方案中，於約5℃、25℃、37℃、40℃、或45℃儲存至少1個月、2個月或3個月後，例如，在5℃±3℃儲存3個月後，本發明的抗體製劑中的抗PD-1/PD-L1抗體蛋白純度是至少90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%以上，如藉由尺寸排阻色譜法或藉由非還原型CE-SDS所測定。在一個實施方案中，於約5℃、25℃、37℃、40℃、或45℃儲存至少1個月、2個月或3個月後，例如，在5℃±3℃儲存3個月後，本發明的抗體製劑中抗PD-1/PD-L1抗體蛋白的至少55%，較佳至少60%是非鹼性及非酸性形式(亦即，主峰或主要電荷形式)，如藉由CEX-HPLC法所測定。 The anti-PD-1/PD-L1 antibody protein preparation of the present invention is stable. In some embodiments, the anti-PD-1/PD-L1 antibody protein formulation of the present invention is stored, for example, after storage at 2-8°C for at least 24 months, or after storage at room temperature for at least 6 months, or after It is stable after storage at 40℃±2℃ for 1 month. In some embodiments, after storage at about 5°C, 25°C, 37°C, 40°C, or 45°C for at least 1 month, 2 months, or 3 months, for example, storage at 5°C ± 3°C for 3 months Afterwards, the anti-PD-1/PD-L1 antibody protein purity in the antibody preparation of the present invention is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or More than 99%, as measured by size exclusion chromatography or by non-reduced CE-SDS. In one embodiment, after storage at about 5°C, 25°C, 37°C, 40°C, or 45°C for at least 1 month, 2 months, or 3 months, for example, storage at 5°C ± 3°C for 3 months Later, at least 55%, preferably at least 60%, of the anti-PD-1/PD-L1 antibody protein in the antibody preparation of the present invention is in the non-basic and non-acidic form (that is, the main peak or the main charge form), such as by CEX -Measured by HPLC method.

IV.製劑的用途IV. Use of the preparation

本發明的包含抗PD-1/PD-L1抗體蛋白的本發明抗體製劑能夠用於治療或預防腫瘤等。 The antibody preparation of the present invention containing the anti-PD-1/PD-L1 antibody protein of the present invention can be used to treat or prevent tumors and the like.

本發明進一步提供本發明抗體製劑的用途，其用於製造用於治療或預防癌症的藥物，其中該藥物可與一或多種選自由以下組成的群組 的抗腫瘤劑同時、分開或依序給藥：順鉑(cisplatin)、卡鉑(carboplatin)、達卡巴嗪(dacarbazine)、脂質體多柔比星(liposomal doxorubicin)、多西他賽(docetaxel)、環磷醯胺(cyclophosphamide)及多柔比星、諾維本(navelbine)、艾瑞布林(eribulin)、太平洋紫杉醇(paclitaxel)、用於可注射懸浮液的太平洋紫杉醇蛋白質結合顆粒、伊沙匹隆(ixabepilone)、卡培他濱(capecitabine)、FOLFOX(亞葉酸鈣(leucovorin)、氟尿嘧啶(fluorouracil)及奧沙利鉑(oxaliplatin))、FOLFIRI(亞葉酸鈣、氟尿嘧啶及伊立替康(irinotecan))、吉西他濱(gemcitabine)、托泊替康(topotecan)、脂質體伊立替康、培美曲塞(pemetrexed)、西妥昔單抗(cetuximab)、尼沃魯單抗(nivolumab)、伊匹單抗(ipilimumab)、匹利珠單抗(pidilizumab)、派姆單抗(pembrolizumab)、曲美木單抗(tremelimumab)、烏瑞魯單抗(urelumab)、利麗單抗(lirilumab)、阿替珠單抗(atezolizumab)、愛帕司他(epacadostat)及德瓦魯單抗(durvalumab)。 The present invention further provides the use of the antibody preparation of the present invention for the manufacture of a drug for the treatment or prevention of cancer, wherein the drug can be combined with one or more selected from the group consisting of Simultaneous, separate or sequential administration of antitumor agents: cisplatin, carboplatin, dacarbazine, liposomal doxorubicin, docetaxel , Cyclophosphamide and doxorubicin, navelbine, eribulin, paclitaxel, paclitaxel protein-binding particles for injectable suspension, Isa Ixabepilone, capecitabine, FOLFOX (leucovorin, fluorouracil and oxaliplatin), FOLFIRI (calcium leucovorin, fluorouracil, and irinotecan )), gemcitabine, topotecan, liposomal irinotecan, pemetrexed, cetuximab, nivolumab, ipi Monoclonal antibody (ipilimumab), pilizumab (pidilizumab), pembrolizumab (pembrolizumab), tremelimumab (tremelimumab), urelumab (urelumab), lililumab (lirilumab), a Tezolizumab, epacadostat and durvalumab.

本發明提供本發明抗體製劑的用途，其用於製造用於治療癌症的藥物，其中該藥物可與電離輻射同時、分開或依次施用。 The present invention provides the use of the antibody preparation of the present invention for the manufacture of a drug for the treatment of cancer, wherein the drug can be administered simultaneously, separately or sequentially with ionizing radiation.

在一些實施方案中，預防或治療的癌症包括但不限於霍奇金氏(Hodgkin’s)或非霍奇金氏淋巴瘤、黑色素瘤、腎細胞癌、腎癌、肺癌、膀胱癌、胃及食道癌、結腸直腸癌、肝癌、肝細胞癌、膽管癌、胰臟癌、乳癌、三陰性乳癌、卵巢癌、子宮內膜癌、***癌、小細胞肺癌(SCLC)、非小細胞肺癌(NSCLC)、間皮瘤、頭頸鱗狀細胞癌(SCCHN)、軟組織肉瘤或多形性神經膠母細胞瘤。在一些實施方案中，癌症為胃腸道癌症，例如胃癌、直腸癌、結腸癌、結腸直腸癌等。 In some embodiments, cancers to be prevented or treated include, but are not limited to, Hodgkin's or non-Hodgkin's lymphoma, melanoma, renal cell carcinoma, kidney cancer, lung cancer, bladder cancer, stomach and esophageal cancer , Colorectal cancer, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, pancreatic cancer, breast cancer, triple negative breast cancer, ovarian cancer, endometrial cancer, prostate cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), Mesothelioma, squamous cell carcinoma of the head and neck (SCCHN), soft tissue sarcoma, or glioblastoma multiforme. In some embodiments, the cancer is a gastrointestinal cancer, such as gastric cancer, rectal cancer, colon cancer, colorectal cancer, and the like.

本發明也提供了本發明的製劑在製備藥物中的用途，其中該藥物用於向哺乳動物遞送抗PD-1/PD-L1抗體蛋白。本發明還提供了本發明的製劑用於治療或預防一種或多種上述疾病和病症的方法。較佳地，哺乳動物是人。 The present invention also provides the use of the preparation of the present invention in preparing a medicine, wherein the medicine is used to deliver anti-PD-1/PD-L1 antibody protein to mammals. The present invention also provides a method for the preparation of the present invention to treat or prevent one or more of the above-mentioned diseases and disorders. Preferably, the mammal is a human.

可以以多種途徑將本發明的抗體製劑施用於受試者或患者。例如，施用可以藉由輸注或藉由注射器進行。因此，在一個方面，本發明提供了一種遞送裝置(例如注射器)，其包含本發明的抗體製劑(例如，預填裝注射器)。患者將接受有效量的抗PD-1/PD-L1抗體蛋白作為主要活性成分，即足以治療、改善或預防目的疾病或病症的量。 The antibody formulation of the present invention can be administered to a subject or patient in a variety of ways. For example, administration can be by infusion or by syringe. Therefore, in one aspect, the present invention provides a delivery device (e.g., a syringe) comprising the antibody preparation of the present invention (e.g., a pre-filled syringe). The patient will receive an effective amount of anti-PD-1/PD-L1 antibody protein as the main active ingredient, that is, an amount sufficient to treat, ameliorate or prevent the target disease or condition.

本發明進一步提供一種預防和/或治療腫瘤的方法，包括向受試者施用有效量的本發明該的製劑。例如，該腫瘤是癌症，較佳地，該癌症為霍奇金或非霍奇金淋巴瘤、黑色素瘤、腎細胞癌、腎癌、肺癌、膀胱癌、胃及食道癌、結腸直腸癌、肝癌、肝細胞癌、膽管癌、胰臟癌、乳癌、三陰性乳癌、卵巢癌、子宮內膜癌、***癌、小細胞肺癌(SCLC)、非小細胞肺癌(NSCLC)、間皮瘤、頭頸的鱗狀細胞癌(SCCHN)、軟組織肉瘤或多形性神經膠母細胞瘤；更較佳地，該肺癌是NSCLC、小細胞肺癌或間皮瘤。 The present invention further provides a method for preventing and/or treating tumors, which comprises administering to a subject an effective amount of the preparation of the present invention. For example, the tumor is cancer, preferably, the cancer is Hodgkin's or non-Hodgkin's lymphoma, melanoma, renal cell carcinoma, kidney cancer, lung cancer, bladder cancer, gastric and esophageal cancer, colorectal cancer, liver cancer , Hepatocellular carcinoma, cholangiocarcinoma, pancreatic cancer, breast cancer, triple-negative breast cancer, ovarian cancer, endometrial cancer, prostate cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), mesothelioma, head and neck cancer Squamous cell carcinoma (SCCHN), soft tissue sarcoma or glioblastoma multiforme; more preferably, the lung cancer is NSCLC, small cell lung cancer or mesothelioma.

在一些實施方案中，本發明該的方法，還包括同時，分開或依次給予一種或多種抗腫瘤劑，該抗腫瘤劑選自順鉑(cisplatin)、卡鉑(carboplatin)、達卡巴嗪(dacarbazine)、脂質體多柔比星(liposomal doxorubicin)、多西他賽(docetaxel)、環磷醯胺(cyclophosphamide)及多柔比星、諾維本(navelbine)、艾瑞布林(eribulin)、太平洋紫杉醇(paclitaxel)、用於可注射懸浮液的太平洋紫杉醇蛋白質結合顆粒、伊沙匹隆 (ixabepilone)、卡培他濱(capecitabine)、FOLFOX(亞葉酸鈣(leucovorin)、氟尿嘧啶(fluorouracil)及奥沙利鉑(oxaliplatin))、FOLFIRI(亞葉酸鈣、氟尿嘧啶及伊立替康(irinotecan))、吉西他濱(gemcitabine)、托泊替康(topotecan)、脂質體伊立替康、培美曲塞(pemetrexed)、西妥昔單抗(cetuximab)、尼沃鲁單抗(nivolumab)、伊匹單抗(ipilimumab)、匹利珠單抗(pidilizumab)、派姆單抗(pembrolizumab)、曲美木單抗(tremelimumab)、烏瑞鲁單抗(urelumab)、利麗單抗(lirilumab)、阿替珠單抗(atezolizumab)、愛帕司他(epacadostat)及德瓦鲁單抗(durvalumab)。 In some embodiments, the method of the present invention further includes simultaneous, separate or sequential administration of one or more anti-tumor agents selected from the group consisting of cisplatin, carboplatin, dacarbazine ), liposomal doxorubicin, docetaxel, cyclophosphamide and doxorubicin, navelbine, eribulin, Pacific Paclitaxel, paclitaxel protein-binding particles for injectable suspension, ixabepilone (ixabepilone), capecitabine, FOLFOX (leucovorin, fluorouracil and oxaliplatin), FOLFIRI (calcium leucovorin, fluorouracil and irinotecan) , Gemcitabine, topotecan, liposomal irinotecan, pemetrexed, cetuximab, nivolumab, ipilimumab (ipilimumab), pidilizumab, pembrolizumab, tremelimumab, urelumab, lirilumab, atezumab Monoclonal antibody (atezolizumab), epacadostat (epacadostat) and devalumab (durvalumab).

治療效果可包括減少生理症狀。用於任何特定受試者的抗體的最佳有效量和濃度將取決於多種因素，包括患者的年齡、體重、健康狀況和/或性別、疾病的性質和程度、特定抗體的活性，身體對其清除率，並且也包括與該抗體製劑組合施用的任何可能的其它治療。對於具體的情況，所遞送的有效量可以在臨床醫師的判斷範圍內來確定。在這方面，已知的基於抗體的藥物的應用可以提供一定的指導。劑量可以是單劑量方案或多劑量方案。 The therapeutic effect may include reducing physical symptoms. The optimal effective amount and concentration of antibodies for any particular subject will depend on many factors, including the age, weight, health and/or gender of the patient, the nature and extent of the disease, the activity of the particular antibody, and the body’s Clearance rate, and also includes any possible other treatments administered in combination with the antibody formulation. For specific situations, the effective amount delivered can be determined within the judgment of the clinician. In this regard, the application of known antibody-based drugs can provide certain guidance. The dosage can be a single-dose schedule or a multiple-dose schedule.

用於本文時，“治療”指減緩、中斷、阻滯、緩解、停止、降低、或逆轉已存在的症狀、病症、病況或疾病的進展或嚴重性。想要的治療效果包括但不限於防止疾病出現或復發、減輕症狀、減小疾病的任何直接或間接病理學後果、防止轉移、降低病情進展速率、改善或緩和疾病狀態，以及緩解或改善預後。在一些實施方案中，本發明的抗體分子用來延緩疾病發展或用來減慢疾病的進展。 As used herein, "treatment" refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. The desired therapeutic effects include, but are not limited to, preventing the appearance or recurrence of the disease, reducing symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving the prognosis. In some embodiments, the antibody molecules of the present invention are used to delay the progression of a disease or to slow the progression of a disease.

用於本文時，“預防”包括對疾病或病症或特定疾病或病症的症狀的發生或發展的抑制。在一些實施方式中，具有癌症家族病史的受試者是預防性方案的候選。通常，在癌症的背景中，術語“預防”是指在癌症的病徵或症狀發生前，特別是在具有癌症風險的受試者中發生前的藥物施用。 As used herein, "prevention" includes the inhibition of the occurrence or development of a disease or condition or the symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for prophylactic regimens. Generally, in the context of cancer, the term "prevention" refers to the administration of drugs before the onset of signs or symptoms of cancer, especially in subjects at risk of cancer.

“治療有效量”指以需要的劑量並持續需要的時間段，有效實現所需治療結果的量。抗體或抗體片段或其綴合物或組合物的治療有效量可以根據多種因素如疾病狀態、個體的年齡、性別和重量和抗體或抗體部分在個體中激發所需反應的能力而變動。治療有效量也是這樣的一個量，其中抗體或抗體片段或其綴合物或組合物的任何有毒或有害作用不及治療有益作用。相對於未治療的對象，“治療有效量”較佳地抑制可度量參數(例如腫瘤生長率)至少約20%、更佳地至少約40%、甚至更佳地至少約50%、60%或70%和仍更佳地至少約80%。可以在預示人腫瘤中的功效的動物模型系統中評價化合物抑制可度量參數(例如，癌症)的能力。可選地，可以藉由檢驗化合物抑制的能力評價組合物的這種特性，該抑制在體外藉由熟練技術人員已知的測定法。 "Therapeutically effective amount" refers to the amount that is effective to achieve the desired therapeutic result at the required dose and for the required period of time. The therapeutically effective amount of the antibody or antibody fragment or its conjugate or composition can vary according to various factors such as disease state, the age, sex and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or its conjugate or composition are not as good as the therapeutically beneficial effects. Relative to an untreated subject, a "therapeutically effective amount" preferably inhibits a measurable parameter (such as tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70% and still more preferably at least about 80%. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be evaluated in an animal model system that predicts efficacy in human tumors. Alternatively, this property of the composition can be evaluated by testing the compound's ability to inhibit, the inhibition in vitro by an assay known to the skilled artisan.

“預防有效量”指以需要的劑量並持續需要的時間段，有效實現所需預防結果的量。 "Prophylactically effective amount" refers to an amount that effectively achieves the desired preventive result at the required dose and for the required period of time.

V.本發明的示例性雙特異性抗體V. Exemplary bispecific antibodies of the invention

Figure 110101453-A0101-12-0054-4
Figure 110101453-A0101-12-0054-4

Figure 110101453-A0101-12-0054-5
Figure 110101453-A0101-12-0054-5

Figure 110101453-A0101-12-0055-6
Figure 110101453-A0101-12-0055-6

描述以下實施例以輔助對本發明的理解。不意在且不應當以任何方式將實施例解釋成限制本發明的保護範圍。 The following examples are described to assist the understanding of the present invention. It is not intended and should not be construed in any way to limit the scope of protection of the present invention.

縮略詞描述Abbreviation description

CEX-HPLC：陽離子交換高效液相色譜 CEX-HPLC: Cation Exchange High Performance Liquid Chromatography

CE-SDS：十二烷基硫酸鈉毛細管凝膠電泳 CE-SDS: Sodium Lauryl Sulfate Capillary Gel Electrophoresis

iCIEF：成像毛細管等電聚焦電泳 iCIEF: Imaging Capillary Isoelectric Focusing Electrophoresis

SEC-HPLC：尺寸排阻高效液相色譜法 SEC-HPLC: size exclusion high performance liquid chromatography

實施例Example

為了開發出抗PD-1/PD-L1抗體注射液以及凍乾製劑長期穩定儲存的製劑處方，確保產品在有效期內(至少24個月)的質量可控，設計了處方篩選試驗，考察了不同輔料對於抗PD-1/PD-L1抗體製劑穩定性的影響。試驗所用材料和方法如下： In order to develop formulations for anti-PD-1/PD-L1 antibody injections and freeze-dried formulations for long-term stable storage, and to ensure that the product quality is controllable within the validity period (at least 24 months), a prescription screening test was designed and different The effect of excipients on the stability of anti-PD-1/PD-L1 antibody preparations. The materials and methods used in the test are as follows:

材料和方法 Materials and Method

1.1 本發明的製劑研究中使用的材料 1.1 Materials used in the formulation research of the present invention

Figure 110101453-A0101-12-0056-7
Figure 110101453-A0101-12-0056-7

1.2. 本發明的製劑研究中使用的儀器設備 1.2. Instruments and equipment used in the formulation of the present invention

Figure 110101453-A0101-12-0057-9
Figure 110101453-A0101-12-0057-9

1.3 製劑穩定性的檢測項目和檢測方法 1.3 Test items and test methods for the stability of the preparation

對抗體製劑檢測了以下項目：(1)檢測外觀以及是否存在可見異物；(2)藉由紫外法(UV法)測定製劑中的蛋白質含量；(3)藉由檢測在350nm處的吸光度，測定濁度；(4)藉由尺寸排阻色譜法，例如，尺寸排阻高效液相色譜法(size-exclusion chromatography-HPLC；SEC-HPLC)測定抗體製劑的純度，表示為單體的面積占所有峰面積之和的百分數；(5)藉由還原型十二烷基硫酸鈉毛細管電泳(還原型CE-SDS)和/或 非還原型十二烷基硫酸鈉毛細管電泳(非還原型CE-SDS)測定抗體製劑的純度，表示為單體的面積占所有峰面積之和的百分數；(6)藉由成像毛細管等電聚焦電泳法(iCIEF法)和陽離子交換高效液相色譜法(CEX-HPLC)測定抗體製劑中電荷變異體，表示為主成分、酸性組分和鹼性組分的百分數。 The following items were tested for antibody preparations: (1) the appearance and presence of visible foreign matter was detected; (2) the protein content in the preparation was measured by the ultraviolet method (UV method); (3) the absorbance at 350nm was measured to determine Turbidity; (4) Determine the purity of the antibody preparation by size-exclusion chromatography, for example, size-exclusion chromatography-HPLC (SEC-HPLC), expressed as the area of the monomer occupies all The percentage of the sum of peak areas; (5) By reducing sodium dodecyl sulfate capillary electrophoresis (reduced CE-SDS) and/or Non-reduced sodium dodecyl sulfate capillary electrophoresis (non-reduced CE-SDS) determines the purity of the antibody preparation, expressed as the percentage of the monomer area to the sum of all peak areas; (6) by imaging capillary isoelectric focusing Electrophoresis (iCIEF method) and cation-exchange high performance liquid chromatography (CEX-HPLC) were used to determine charge variants in antibody preparations, which were expressed as the percentages of main components, acidic components, and basic components.

可見異物檢測 Visible foreign body detection

按照國家藥典委員會，中華人民共和國藥典(2015年版，四部通則0904“可見異物檢查法”，北京：中國醫藥科技出版社.2015)中所記載的方法，採用澄明度檢測儀(天津天大天發生產，型號YB-2)，檢查樣品中的可見異物。 According to the method described in the National Pharmacopoeia Commission, the Pharmacopoeia of the People’s Republic of China (2015 edition, Four General Principles 0904 "Visible Foreign Body Inspection Method", Beijing: China Medical Science and Technology Press. 2015), the clarity detector (Tianjin Tianda Tianfa) Production, model YB-2), check for visible foreign objects in the sample.

蛋白含量測定 Protein content determination

使用紫外分光光度計(日本島津生產，型號UV-1800)測定樣品中的蛋白質含量。 An ultraviolet spectrophotometer (produced by Shimadzu, Japan, model UV-1800) was used to determine the protein content in the sample.

濁度測定 Turbidity measurement

使用紫外分光光度計(日本島津生產，型號UV-1800)，測定樣品在350nm的吸光度，確定樣品濁度。 Using an ultraviolet spectrophotometer (produced by Shimadzu, Japan, model UV-1800), measure the absorbance of the sample at 350 nm to determine the turbidity of the sample.

純度(SEC-HPLC法) Purity (SEC-HPLC method)

使用體積排阻色譜管柱分離，流動相為磷酸鹽緩衝液(稱取3.12g二水合磷酸二氫鈉，8.77g氯化鈉和34.84g精胺酸，超純水溶解後用鹽酸調節pH至6.8並定容至1000ml)，色譜管柱保護液為0.05%(w/v)NaN3，進樣量50μl，流速0.5ml/分鐘，採集時間30分鐘，管柱溫25℃，檢測波長280nm。取待測樣品用超純水稀釋至2mg/ml，作為供試品溶液。 取製劑緩衝液用上述相同處理方式稀釋後做為空白溶液。取空白溶液、供試品溶液各50μl注入液相色譜儀，開始檢測。 Use size exclusion chromatography column to separate, the mobile phase is phosphate buffer (weigh 3.12g sodium dihydrogen phosphate dihydrate, 8.77g sodium chloride and 34.84g arginine, after dissolving in ultrapure water, adjust the pH to 6.8 and dilute to 1000ml), the chromatographic column protection solution is 0.05% (w/v) NaN 3 , the injection volume is 50μl, the flow rate is 0.5ml/min, the collection time is 30 minutes, the column temperature is 25°C, and the detection wavelength is 280nm. Take the sample to be tested and dilute it to 2mg/ml with ultrapure water as the test solution. Take the preparation buffer solution and dilute it with the same treatment method as above and use it as a blank solution. Take 50μl each of the blank solution and the test solution into the liquid chromatograph to start the test.

純度(還原型CE-SDS法) Purity (reduced CE-SDS method)

採用毛細管凝膠電泳法檢測。毛細管為無塗層毛細管，內徑50μm，總長30.2cm，有效長度20.2cm。電泳前分別使用0.1mol/L氫氧化鈉、0.1mol/L鹽酸、超純水、電泳膠70psi沖洗毛細管管柱。將待測樣品用適量超純水稀釋至2.0mg/ml，取以上稀釋後的樣品50μl於1.5ml離心管中，分別向其中加入45μl pH 6.5的樣品緩衝液(稱取一水檸檬酸0.32g，十二水合磷酸氫二鈉2.45g，溶於45ml超純水中，定容至50ml，製得檸檬酸-磷酸鹽緩衝液，精密量取該緩衝液200μl，加10%(w/v)十二烷基硫酸鈉溶液80μl，加水至1ml，混勻，即得)、1μl內標(10kDa蛋白質，5mg/mL)(Beckman Coulter，貨號：390953)和5μl β-巰基乙醇，充分混勻後70±2℃加熱10±2分鐘，冷卻至室溫後轉移至樣品瓶作為供試品溶液。取與供試品相同體積的製劑緩衝液，按上述方法同樣操作，製得空白溶液。樣品進樣條件：-5kV 20秒；分離電壓：-15kV 35分鐘。毛細管管柱溫控制在25℃，檢測波長為220nm。 Detected by capillary gel electrophoresis. The capillary is an uncoated capillary with an inner diameter of 50μm, a total length of 30.2cm, and an effective length of 20.2cm. Wash the capillary column with 0.1mol/L sodium hydroxide, 0.1mol/L hydrochloric acid, ultrapure water, and 70psi electrophoresis gel before electrophoresis. Dilute the sample to be tested with an appropriate amount of ultrapure water to 2.0mg/ml, take 50μl of the above diluted sample into a 1.5ml centrifuge tube, and add 45μl pH 6.5 sample buffer to it (weigh 0.32g monohydrate citric acid 0.32g) , 2.45g disodium hydrogen phosphate dodecahydrate, dissolve in 45ml ultrapure water, dilute to 50ml to prepare citric acid-phosphate buffer solution, accurately measure 200μl of the buffer solution, add 10%(w/v) Sodium lauryl sulfate solution 80μl, add water to 1ml, mix well, ready to get), 1μl internal standard (10kDa protein, 5mg/mL) (Beckman Coulter, catalog number: 390953) and 5μl β-mercaptoethanol, mix thoroughly Heat at 70±2°C for 10±2 minutes, cool to room temperature and transfer to a sample bottle as the test solution. Take the same volume of preparation buffer as the test product, and perform the same operation as described above to prepare a blank solution. Sample injection conditions: -5kV for 20 seconds; separation voltage: -15kV for 35 minutes. The capillary column temperature is controlled at 25°C, and the detection wavelength is 220nm.

純度(非還原型CE-SDS法) Purity (non-reduced CE-SDS method)

採用毛細管凝膠電泳法檢測。毛細管為無塗層毛細管，內徑50μm，總長30.2cm，有效長度20.2cm。電泳前分別使用0.1mol/L氫氧化鈉、0.1mol/L鹽酸、超純水、電泳膠70psi沖洗毛細管管柱。將待測樣品用適量超純水稀釋至2.0mg/ml，取以上稀釋後的樣品50μl於1.5ml離心管中，分別向其中加入45μl pH 6.5的樣品緩衝液(稱取一水檸檬酸 0.32g，十二水合磷酸氫二鈉2.45g，溶於45ml超純水中，定容至50ml，製得檸檬酸-磷酸鹽緩衝液，精密量取該緩衝液200μl，加10%(w/v)十二烷基硫酸鈉溶液80μl，加水至1ml，混勻，即得)、1μl內標(10kDa蛋白質，5mg/mL)(Beckman Coulter，貨號：390953)和5μl 250mmol/L NEM溶液(稱取N-乙基順丁稀二醯亞胺62mg，溶於2ml超純水中)，充分混勻後70±2℃加熱10±2分鐘，冷卻至室溫後轉移至樣品瓶作為供試品溶液。取與供試品相同體積的製劑緩衝液，按上述方法同樣操作，製得空白溶液。樣品進樣條件：-5kV 20秒；分離電壓：-15kV 35分鐘。毛細管管柱溫控制在25℃，檢測波長為220nm。 Detected by capillary gel electrophoresis. The capillary is an uncoated capillary with an inner diameter of 50μm, a total length of 30.2cm, and an effective length of 20.2cm. Wash the capillary column with 0.1mol/L sodium hydroxide, 0.1mol/L hydrochloric acid, ultrapure water, and 70psi electrophoresis gel before electrophoresis. Dilute the sample to be tested with an appropriate amount of ultrapure water to 2.0mg/ml, take 50μl of the above diluted sample into a 1.5ml centrifuge tube, and add 45μl pH 6.5 sample buffer to it (weigh monohydrate citric acid 0.32g, 2.45g disodium hydrogen phosphate dodecahydrate, dissolved in 45ml ultrapure water, dilute to 50ml to prepare citric acid-phosphate buffer solution, accurately measure 200μl of the buffer solution, add 10% (w/ v) 80μl of sodium lauryl sulfate solution, add water to 1ml, and mix well to obtain), 1μl internal standard (10kDa protein, 5mg/mL) (Beckman Coulter, catalog number: 390953) and 5μl 250mmol/L NEM solution (weigh Take 62mg of N-ethylmaleimide and dissolve it in 2ml of ultrapure water), mix well, heat at 70±2℃ for 10±2 minutes, cool to room temperature and transfer to sample bottle as test product Solution. Take the same volume of preparation buffer as the test product, and perform the same operation as described above to prepare a blank solution. Sample injection conditions: -5kV for 20 seconds; separation voltage: -15kV for 35 minutes. The capillary column temperature is controlled at 25°C, and the detection wavelength is 220nm.

電荷變異體(iCIEF) Charge variant (iCIEF)

採用成像毛細管等電聚焦電泳(iCIEF法)檢測。毛細管內徑100μm，總長5cm。樣品電泳前需分別使用0.5%甲基纖維素溶液(下文中也縮寫為MC溶液)、超純水沖洗毛細管管柱。採用真空進樣方式，預聚焦電壓及時間為1.5kV 1分鐘，聚焦電壓及時間為3kV 8分鐘，進樣時間55秒，樣品盤溫度為10℃，檢測波長為280nm。陰極穩定劑(Cathodic Stabilizer)為500mmol/L精胺酸溶液，0.5% MC溶液降低蛋白與毛細管之間的黏附。將供試品用水稀釋至1.0mg/ml，取稀釋後的供試品溶液20μl，向其中加入78μl預混液(預混液配比如下：70μl pI 0.5% MC溶液，4μl兩性電解質(pH 3-10)，2μl陰極穩定劑，1μl pI 5.85標誌物，1μl pI 9.99標誌物)，充分混勻製得待測樣品溶液。進樣分析，根據面積歸一化法，計算主成分、酸性組分及鹼性組分含量。 Using imaging capillary isoelectric focusing electrophoresis (iCIEF method) detection. The capillary has an inner diameter of 100μm and a total length of 5cm. Before sample electrophoresis, 0.5% methyl cellulose solution (hereinafter also abbreviated as MC solution) and ultrapure water should be used to rinse the capillary column. The vacuum sampling method is adopted, the pre-focusing voltage and time are 1.5kV for 1 minute, the focusing voltage and time are 3kV for 8 minutes, the sampling time is 55 seconds, the sample tray temperature is 10°C, and the detection wavelength is 280nm. Cathodic Stabilizer is 500mmol/L arginine solution, 0.5% MC solution reduces the adhesion between protein and capillary. Dilute the test product to 1.0 mg/ml with water, take 20 μl of the diluted test product solution, and add 78 μl of the pre-mixed solution to it (the pre-mixed solution is as follows: 70 μl pI 0.5% MC solution, 4 μl amphoteric electrolyte (pH 3-10) ), 2μl cathode stabilizer, 1μl pI 5.85 marker, 1μl pI 9.99 marker), mix well to prepare the sample solution to be tested. Sample injection analysis, according to the area normalization method, calculate the main component, acidic component and alkaline component content.

電荷變異體(CEX-HPLC法) Charge variant (CEX-HPLC method)

採用離子交換色譜法(CEX-HPLC法)檢測。使用離子交換色譜管柱分離，流動相為10mmol/L磷酸鹽緩衝液(稱取1.15g二水合磷酸二氫鈉，0.95g十二水合磷酸氫二鈉，用800ml超純水溶解後定容至1000ml)及10mmol/L磷酸鹽+200mmol/L氯化鈉緩衝液(稱取1.15g二水合磷酸二氫鈉，0.95g十二水合磷酸氫二鈉，11.69g氯化鈉用800ml超純水溶解後定容至1000ml)，流速0.5ml/分鐘，檢測波長280nm，管柱溫25℃。用流動相將樣品稀釋至2mg/ml，作為供試品溶液；將工作參比品用流動相稀釋至2mg/ml，作為系統適用性溶液。取製劑緩衝液用流動相稀釋相同倍數做為空白溶液。取空白溶液、系統適用性溶液、供試品溶液各50μl注入液相色譜儀，流動相流速1.0ml/min，採集時間35分鐘，管柱溫35℃，檢測波長280nm。 It is detected by ion exchange chromatography (CEX-HPLC method). Use ion exchange chromatography column to separate, the mobile phase is 10mmol/L phosphate buffer solution (weigh 1.15g sodium dihydrogen phosphate dihydrate, 0.95g disodium hydrogen phosphate dodecahydrate, dissolve in 800ml ultrapure water and dilute to 1000ml) and 10mmol/L phosphate+200mmol/L sodium chloride buffer (weigh 1.15g sodium dihydrogen phosphate dihydrate, 0.95g disodium hydrogen phosphate dodecahydrate, 11.69g sodium chloride dissolved in 800ml ultrapure water The volume is then adjusted to 1000ml), the flow rate is 0.5ml/min, the detection wavelength is 280nm, and the column temperature is 25°C. Dilute the sample to 2mg/ml with mobile phase as the test solution; dilute the working reference product to 2mg/ml with mobile phase as the system suitability solution. Take the preparation buffer and dilute the same multiple with the mobile phase as a blank solution. Take 50μl each of the blank solution, system suitability solution, and test solution into the liquid chromatograph, the mobile phase flow rate is 1.0ml/min, the collection time is 35 minutes, the column temperature is 35°C, and the detection wavelength is 280nm.

1.4 判斷標準 1.4 Judgment criteria

根據對產品的認識以及儀器和方法的精密度，設定了樣品檢測指標數值與初始值相比質量未發生變化的判斷標準，用以判斷樣品是否發生了變化，具體見下表。 According to the knowledge of the product and the precision of the instrument and method, the judgment standard of the quality of the sample detection index value and the initial value has not changed to determine whether the sample has changed. See the table below for details.

質量未發生變化的判斷標準 Judgment criteria for unchanged quality

Figure 110101453-A0101-12-0061-29
Figure 110101453-A0101-12-0061-29

Figure 110101453-A0101-12-0062-30
Figure 110101453-A0101-12-0062-30

實施例1. 製備和純化抗PD-1/PD-L1抗體 Example 1. Preparation and purification of anti-PD-1/PD-L1 antibodies

根據PCT申請號PCT/US2018/041205該，獲得特異性結合PD-1和PD-L1的新型抗體抗體v3.2、v3.0及v13844，抗體v3.2、v3.0及v13844的輕鏈、重鏈、輕鏈可變區及重鏈可變區的胺基酸序列的SEQ ID NO示於上表1(a)中。此外，抗體v3.2、v3.0及v13844的PD-L1及PD-1結合可變區的CDR的胺基酸序列的SEQ ID NO分別示於表1(b)及表1(c)中。 According to PCT application number PCT/US2018/041205, new antibody antibodies v3.2, v3.0 and v13844 that specifically bind PD-1 and PD-L1, light chains of antibodies v3.2, v3.0 and v13844, The SEQ ID NOs of the amino acid sequences of the heavy chain, light chain variable region, and heavy chain variable region are shown in Table 1(a) above. In addition, the SEQ ID NOs of the amino acid sequences of the CDRs of the PD-L1 and PD-1 binding variable regions of antibodies v3.2, v3.0 and v13844 are shown in Table 1(b) and Table 1(c), respectively .

本發明抗體包括但不限於抗體v3.2、v3.0及v13844，且基本上如下製備及純化。可使用四倍載體(即，編碼兩條輕鏈及兩條重鏈的表達的單一載體)、雙重載體(即，一起編碼兩條不同輕鏈及兩條不同重鏈的兩個載體)或四個單一載體(其中的兩個編碼不同輕鏈且其中的兩個編碼不同重鏈)用分泌抗體的表達系統瞬時或穩定轉染適當宿主細胞(例如CHO)。可使用許多常用技術中的任一純化抗體分泌於其中的培養基。舉例而言，可將培養基施加至MabSelect管柱(GE Healthcare)或KappaSelect管柱(GE Healthcare)用於Fab片段，該管柱已經用相容緩衝液(例如磷酸鹽緩衝鹽水 (pH 7.4))平衡。可洗滌管柱以移除非特異性結合組分。可例如借由pH梯度(例如20mM Tris緩衝液(pH 7)至10mM檸檬酸緩衝液(pH 3.0)，或生理鹽水(pH 7.4)至100mM甘胺酸緩衝液(pH 3.0))沖提結合的抗體。可例如借由SDS-PAGE檢測抗體抗體片段，且隨後可彙集。可溶性聚集體及多聚體可借由常見技術有效移除，該技術包括尺寸排除色譜、疏水相互作用色譜、離子交換色譜、多峰(multimodal)色譜或羥磷灰石色譜等。可使用常見技術濃縮及/或無菌過濾抗體。可將產物立即於-70℃下冷凍或可經凍乾。 The antibodies of the present invention include but are not limited to antibodies v3.2, v3.0 and v13844, and are basically prepared and purified as follows. Quadruple vectors (ie, a single vector encoding the expression of two light chains and two heavy chains), dual vectors (ie, two vectors encoding two different light chains and two different heavy chains together), or quadruple vectors can be used. A single vector (two of which code for different light chains and two of which code for different heavy chains) is transiently or stably transfected into an appropriate host cell (e.g. CHO) with an antibody-secreting expression system. The medium in which the purified antibody is secreted can be used by any of many commonly used techniques. For example, the medium can be applied to a MabSelect column (GE Healthcare) or a KappaSelect column (GE Healthcare) for Fab fragments, which has been used with a compatible buffer (such as phosphate buffered saline). (pH 7.4)) Equilibrium. The column can be washed to remove non-specific binding components. For example, by pH gradient (for example, 20mM Tris buffer (pH 7) to 10mM citrate buffer (pH 3.0), or physiological saline (pH 7.4) to 100mM glycine buffer (pH 3.0)) to extract the bound Antibody. The antibody antibody fragments can be detected, for example, by SDS-PAGE, and can then be pooled. Soluble aggregates and multimers can be effectively removed by common techniques, including size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, multimodal chromatography, or hydroxyapatite chromatography. Common techniques can be used to concentrate and/or sterile filter the antibody. The product can be frozen immediately at -70°C or can be lyophilized.

以下描述了v3.2的一種示例性製備方法。 An exemplary preparation method of v3.2 is described below.

具體而言，合成編碼人源化抗體PD-1的重鏈的核苷酸序列和輕鏈核苷酸序列；合成編碼人源化抗體PD-L1的重鏈的核苷酸序列和輕鏈核苷酸序列。PD-1的重鏈、PD-L1的重鏈分別連接入真核質粒表達載體PEE6.4，PD-1的輕鏈、PD-L1的輕鏈分別連接入真核質粒表達載體PEE12.4。含有PD-1重鏈的中間表達載體和含有PD-1輕鏈的中間表達載體合併，得到含有PD-1重鏈和輕鏈的核苷酸序列的重組表達載體。含有PD-L1重鏈的中間表達載體和含有PD-L1輕鏈的中間表達載體合併，得到含有PD-L1重鏈和輕鏈的核苷酸序列的重組表達載體。培養，最後得到兩個重組表達載體用於細胞轉染至269M細胞(Lonza Biologics公司)，篩選壓力為無穀胺醯胺的LM7300培養基(禮來自主培養基)。採用生物反應器培養，接種密度不小於1.0×106個/ml；培養至第12~16天或細胞活率<60%時，進行收穫，細胞收穫液用於下游純化。 Specifically, synthesize the nucleotide sequence encoding the heavy chain and light chain nucleotide sequence of the humanized antibody PD-1; synthesize the nucleotide sequence encoding the heavy chain and light chain core of the humanized antibody PD-L1 Nucleotide sequence. The heavy chain of PD-1 and the heavy chain of PD-L1 are respectively connected to the eukaryotic plasmid expression vector PEE6.4, and the light chain of PD-1 and the light chain of PD-L1 are respectively connected to the eukaryotic plasmid expression vector PEE12.4. The intermediate expression vector containing the heavy chain of PD-1 and the intermediate expression vector containing the light chain of PD-1 are combined to obtain a recombinant expression vector containing the nucleotide sequences of the heavy chain and light chain of PD-1. The intermediate expression vector containing the heavy chain of PD-L1 and the intermediate expression vector containing the light chain of PD-L1 are combined to obtain a recombinant expression vector containing the nucleotide sequences of the heavy chain and light chain of PD-L1. After culturing, two recombinant expression vectors were finally obtained for cell transfection to 269M cells (Lonza Biologics), and LM7300 medium without glutamine-free pressure was selected (from the main medium). Use bioreactor culture, the inoculation density is not less than 1.0×10 6 cells/ml; when it is cultivated to the 12th to 16th day or the cell viability is less than 60%, harvest, and the cell harvest liquid is used for downstream purification.

細胞收穫液用吸附深層過濾膜包過濾，過濾收穫液。過濾過程中，保持生物反應器的通氣及攪拌參數設置不變，再用親和平衡液沖洗膜包，將沖洗液與濾過液合併，命名為澄清收集液。填裝Mabselect Sure LX層析管柱，用親和平衡液平衡，平衡後將澄清收集液加入層析管柱，再用平衡液沖洗，隨後再用親和沖洗液沖洗，再親和平衡液沖洗。然後開始沖提，收集沖提液，命名為親和收集液。親和收集液調節pH至約6.0，命名為病毒滅活收集液。病毒滅活收集液使用深層過濾膜包對病毒滅活收集液進行吸附深層過濾，收集濾出液及頂洗液，命名為吸附深層過濾收集液。填裝Poros XQ層析管柱，層析管柱用陰離子平衡液平衡，然後開始上樣，收集流穿液，命名為陰離子收集液。填裝Poros HS管柱，用陽離子平衡液平衡，上樣，完成上樣後用平衡液沖洗，用沖提液沖提，收集沖提液，命名為陽離子收集液。樣品具有約5.0-25.0mg/ml的抗PD-1/PD-L1抗體v3.2蛋白含量。 The cell harvest liquid is filtered with an adsorption depth filter membrane bag to filter the harvest liquid. During the filtration process, keep the aeration and stirring parameter settings of the bioreactor unchanged, and then rinse the membrane package with the affinity balance solution, combine the rinse solution and the filtrate, and name the clarified collection solution. Pack Mabselect Sure LX chromatography column, balance with affinity balance solution, after balance, add the clear collection solution to the chromatography column, then rinse with balance solution, then rinse with affinity washing solution, and then affinity balance solution. Then start the extraction, collect the extraction solution, named as the affinity collection solution. The pH of the affinity collection solution was adjusted to about 6.0, and it was named the virus inactivation collection solution. The virus inactivation collection solution uses a depth filtration membrane package to perform adsorption and deep filtration of the virus inactivation collection solution, and collects the filtrate and the top washing solution, which is named as the absorption depth filtration collection solution. Pack the Poros XQ chromatography column, equilibrate the chromatography column with an anion balance solution, then start to load the sample, collect the flow-through solution, and name it anion collection solution. Pack the Poros HS column, equilibrate with the cation balance solution, load the sample, rinse with the balance solution after loading the sample, extract with the eluent, collect the eluent, and name it as the cation collection solution. The sample has an anti-PD-1/PD-L1 antibody v3.2 protein content of about 5.0-25.0 mg/ml.

以下實施例以抗體v3.2為例，其產品代號為IBI318。 The following examples take antibody v3.2 as an example, and its product code is IBI318.

實施例2. pH對製劑的穩定性影響試驗 Example 2. Test of the influence of pH on the stability of the formulation

本實施例考察了包含抗PD-1/PD-L1抗體的製劑在pH 5.5至6.5的穩定性。共設計了3個pH值，分別為5.5、6.0和6.5。 This example examines the stability of formulations containing anti-PD-1/PD-L1 antibodies at pH 5.5 to 6.5. A total of 3 pH values were designed, namely 5.5, 6.0 and 6.5.

2.1 實驗步驟 2.1 Experimental steps

按照表5配製各個處方的緩衝液，用稀鹽酸將pH分別調節為5.5、6.0和6.5，將實施例1的經純化的抗PD-1/PD-L1抗體超濾置換到該不同pH值的溶液中。置換完成後，調節樣品中的抗PD-1/PD-L1抗體蛋白含量至約20mg/ml；然後加入聚山梨酯80，使聚山梨酯80的終濃度 為0.20mg/ml；過濾分裝至西林瓶中，加塞壓蓋後根據實驗方案見表6進行研究考察。檢測指標為外觀、蛋白含量、純度(SEC-HPLC法、非還原型CE-SDS法)和電荷變異體(iCIEF法)。 Prepare the buffer solution of each prescription according to Table 5, adjust the pH to 5.5, 6.0 and 6.5 respectively with dilute hydrochloric acid, and replace the purified anti-PD-1/PD-L1 antibody of Example 1 by ultrafiltration to the different pH value. In solution. After the replacement, adjust the anti-PD-1/PD-L1 antibody protein content in the sample to about 20mg/ml; then add polysorbate 80 to make the final concentration of polysorbate 80 It is 0.20mg/ml; it is filtered and aliquoted into vials, stoppered and capped, and studied according to the experimental scheme shown in Table 6. The detection indicators are appearance, protein content, purity (SEC-HPLC method, non-reduced CE-SDS method) and charge variants (iCIEF method).

Figure 110101453-A0101-12-0065-10
Figure 110101453-A0101-12-0065-10

Figure 110101453-A0101-12-0065-11
Figure 110101453-A0101-12-0065-11

2.2 實驗結果 2.2 Experimental results

(1)外觀 (1) Appearance

在40℃±2℃的條件下放置2周，各組樣品外觀均合格。 Placed for 2 weeks under the condition of 40℃±2℃, the appearance of each group of samples is qualified.

(2)蛋白含量 (2) Protein content

在40℃±2℃條件下，各組樣品的蛋白含量均未發生變化(見表7)。 Under the condition of 40℃±2℃, the protein content of each group of samples did not change (see Table 7).

Figure 110101453-A0101-12-0066-12
Figure 110101453-A0101-12-0066-12

(3)純度 (3) Purity

純度(SEC-HPLC法)：40℃±2℃條件下加速2週，各組樣品的純度均發生明顯變化，隨著pH升高，樣品純度下降速率越快，表現為聚體增多；加入甲硫胺酸能有減緩純度下降的速率。(見表8、圖1)。 Purity (SEC-HPLC method): Accelerated for 2 weeks at 40℃±2℃, the purity of samples in each group changed significantly. As the pH increases, the purity of the samples decreases faster, which is manifested as an increase in aggregates; Thiamine can slow down the rate of purity decline. (See Table 8, Figure 1).

純度(非還原型CE-SDS法)：40℃±2℃條件下加速2週，各pH條件下樣品的純度均未發生明顯變化。(見表9)。 Purity (non-reduced CE-SDS method): Accelerated for 2 weeks at 40°C±2°C, and the purity of the sample under each pH condition did not change significantly. (See Table 9).

Figure 110101453-A0101-12-0066-13
Figure 110101453-A0101-12-0066-13

Figure 110101453-A0101-12-0067-14
Figure 110101453-A0101-12-0067-14

(4)電荷變異體 (4) Charge variant

40℃±2℃條件下，各處方電荷變異體-酸性組分和主成分均發生明顯變化，各處方間趨勢一致，無明顯差異。(見表10和圖2) Under the condition of 40℃±2℃, the charge variant-acidic component and main component of each square have obvious changes, and the trend is the same among all squares, and there is no obvious difference. (See Table 10 and Figure 2)

Figure 110101453-A0101-12-0067-15
Figure 110101453-A0101-12-0067-15

Figure 110101453-A0101-12-0068-16
Figure 110101453-A0101-12-0068-16

處方前試驗結果表明蛋白在pH5.5-6.5間穩定，尤其是pH 6.0時較穩定。綜合以上結果，選定pH為6.0，進行下一輪處方篩選試驗。 The pre-prescription test results show that the protein is stable at pH 5.5-6.5, especially at pH 6.0. Based on the above results, the pH was selected as 6.0 for the next round of prescription screening test.

實施例3. 處方確定實驗(一) Example 3. Prescription determination experiment (1)

3.1 實驗步驟 3.1 Experimental steps

本實施例考察了不同緩衝體系和穩定劑對包含抗PD-1/PD-L1抗體製劑穩定性的影響。 This example examines the effects of different buffer systems and stabilizers on the stability of anti-PD-1/PD-L1 antibody formulations.

共設計了4個處方，詳細處方信息見表11。 A total of 4 prescriptions were designed, and the detailed prescription information is shown in Table 11.

按照表11配製各個處方的緩衝液，將IBI318蛋白超濾置換到各自的處方溶液中。置換完成後，將各處方蛋白濃度稀釋至約20mg/ml，並加入聚山梨酯80，使終濃度為0.2mg/ml。過濾分裝至西林瓶，加塞軋蓋後在40℃±2℃、25℃±2℃條件下進行加速穩定性考察，並考察其振盪和凍融穩定性。檢測指標為外觀、可見異物、蛋白含量、純度(SEC-HPLC法、非還原型CE-SDS法)和電荷變異體(CEX-HPLC法)。 Prepare the buffer solution of each prescription according to Table 11, and replace the IBI318 protein into the respective prescription solution by ultrafiltration. After the replacement, the protein concentration of each prescription was diluted to about 20mg/ml, and polysorbate 80 was added to make the final concentration 0.2mg/ml. After being filtered and dispensed into vials, stoppered and capped, the accelerated stability was investigated under the conditions of 40℃±2℃ and 25℃±2℃, and its shaking and freeze-thaw stability were also investigated. The detection indicators are appearance, visible foreign matter, protein content, purity (SEC-HPLC method, non-reduced CE-SDS method) and charge variant (CEX-HPLC method).

Figure 110101453-A0101-12-0069-17
Figure 110101453-A0101-12-0069-17

詳細試驗條件及取樣計劃見表12。 The detailed test conditions and sampling plan are shown in Table 12.

Figure 110101453-A0101-12-0069-18
Figure 110101453-A0101-12-0069-18

3.2 試驗結果 3.2 Test results

(1)外觀、可見異物 (1) Appearance, visible foreign matter

在40℃±2℃的條件下觀察至1個月，25℃±2℃條件下觀察至3個月，振盪5天，凍融6次，四組處方外觀、可見異物均合格。 Observed at 40℃±2℃ for 1 month, 25℃±2℃ for 3 months, shaking for 5 days, freezing and thawing 6 times, the appearance of the four prescriptions and visible foreign bodies were all qualified.

(2)蛋白含量 (2) Protein content

在40℃±2℃和25℃±2℃以及振盪和凍融條件下，4組處方的蛋白含量均未發生變化(見表13)。 Under the conditions of 40℃±2℃ and 25℃±2℃, as well as shaking and freeze-thaw conditions, the protein content of the four groups of prescriptions did not change (see Table 13).

Figure 110101453-A0101-12-0070-19
Figure 110101453-A0101-12-0070-19

(3)純度 (3) Purity

純度(SEC-HPL C法)：40℃±2℃條件下1個月、25℃±2℃條件下3個月，處方1(枸櫞酸緩衝體系)純度發生下降，主要表現為聚體增多，其餘處方純度均未發生明顯變化。振盪和凍融條件下各處方純度均未發生變化。詳見表14、圖3和圖4。 Purity (SEC-HPL C method): 1 month at 40℃±2℃, 3 months at 25℃±2℃, the purity of prescription 1 (citrate buffer system) decreases, mainly manifested by increased aggregates , The purity of other prescriptions has not changed significantly. The purity of each cube remained unchanged under shaking and freezing and thawing conditions. See Table 14, Figure 3 and Figure 4 for details.

純度(非還原型CE-SDS法)：40℃±2℃條件下，各處方純度均有下降趨勢，處方間無差異；25℃±2℃條件下，各處方純度沒有發生明顯變化。詳見表15、圖5。 Purity (non-reduced CE-SDS method): Under the condition of 40℃±2℃, the purity of each prescription has a downward trend, and there is no difference between the prescriptions; under the condition of 25℃±2℃, the purity of each prescription does not change significantly. See Table 15 and Figure 5 for details.

Figure 110101453-A0101-12-0071-20
Figure 110101453-A0101-12-0071-20

Figure 110101453-A0101-12-0071-21
Figure 110101453-A0101-12-0071-21

(4)電荷變異體 (4) Charge variant

40℃±2℃和25℃±2℃條件下，各處方電荷變異體-酸性組分和主成分均發生明顯變化，變化速度處方1>處方2>處方3>處方4，表明組胺酸體系優於枸櫞酸和枸櫞酸鈉體系，同時IBI318蛋白在山梨醇、甲硫胺酸、精胺酸的共同作用下穩定性最優(見表16、圖6~7)。振盪、凍融條件下，各處方間電荷變異體無明顯差異。 Under the conditions of 40°C±2°C and 25°C±2°C, the charge variant-acidic components and principal components of each prescription changed significantly. The rate of change is prescription 1> prescription 2> prescription 3> prescription 4, indicating the histidine system It is better than citric acid and sodium citrate system, and IBI318 protein has the best stability under the combined action of sorbitol, methionine and arginine (see Table 16, Figure 6-7). Under the conditions of oscillation and freezing and thawing, there is no obvious difference in charge variants between each square.

Figure 110101453-A0101-12-0072-22
Figure 110101453-A0101-12-0072-22

綜合上述各個試驗結果，選定處方4為IBI318製劑處方。為生產時避免使用濃鹽酸調節pH，將緩衝體系調整為10mmol/L的組胺酸和鹽酸組胺酸，即IBI318處方為：20mg/ml重組全人源抗程序性死亡受體1(PD-1)和抗程序性死亡配體1(PD-L1)雙特異性抗體、0.85mg/ml組胺酸、1.00mg/ml鹽酸組胺酸、30.00mg/ml山梨醇、7.49mg/ml甲硫胺酸、21.07mg/ml鹽酸精胺酸、0.20mg/ml聚山梨酯80，pH6.0。 Based on the above test results, prescription 4 was selected as the IBI318 formulation prescription. In order to avoid the use of concentrated hydrochloric acid to adjust the pH during production, the buffer system was adjusted to 10mmol/L histidine and histidine hydrochloride. 1) And anti-programmed death ligand 1 (PD-L1) bispecific antibody, 0.85mg/ml histidine, 1.00mg/ml histidine hydrochloride, 30.00mg/ml sorbitol, 7.49mg/ml methyl sulfide Amino acid, 21.07mg/ml arginine hydrochloride, 0.20mg/ml polysorbate 80, pH 6.0.

實施例4：IBI318高濃度液體製劑及其凍乾製劑穩定性實驗 Example 4: Stability test of IBI318 high-concentration liquid preparation and its freeze-dried preparation

本實驗用於考察該處方下IBI318高濃度液體製劑及其凍乾製劑的穩定性。發明人意外地發現，藉由該處方，不僅可以製備穩定的低濃度例如20mg/ml的液體抗體製劑，還可以用於製備穩定的高濃度例如100mg/ml的液體製劑及其凍乾製劑。 This experiment is used to investigate the stability of IBI318 high-concentration liquid preparation and its freeze-dried preparation under this prescription. The inventors unexpectedly discovered that with this prescription, not only a stable low concentration, for example, 20 mg/ml liquid antibody preparation, but also a stable high concentration, such as 100 mg/ml, liquid preparation and its lyophilized preparation can be prepared.

4.1. 試驗步驟 4.1. Test procedure

將抗體超濾換液至實施例3中的緩衝液(20mg/ml重組全人源抗程序性死亡受體1(PD-1)和抗程序性死亡配體1(PD-L1)雙特異性抗體、0.85mg/ml組胺酸、1.00mg/ml鹽酸組胺酸、30.00mg/ml山梨醇、7.49mg/ml甲硫胺酸、21.07mg/ml鹽酸精胺酸、pH6.0)中，濃縮至約100mg/ml後，加入聚山梨酯80，使其終濃度為0.2mg/ml。過濾分裝至西林瓶，每瓶0.5ml，共分裝22瓶。取11瓶，加塞壓蓋標記為PD20191109-1。另取11瓶，進行凍乾後，加塞壓蓋標記為PD20191109-2。根據實驗方案見表17進行穩定性考察。檢測指標為外觀、蛋白含量、純度(SEC-HPLC法、非還原型CE-SDS法)和電荷變異體(CEX-HPLC法)。 Change the antibody by ultrafiltration to the buffer in Example 3 (20mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific Antibody, 0.85mg/ml histidine, 1.00mg/ml histidine hydrochloride, 30.00mg/ml sorbitol, 7.49mg/ml methionine, 21.07mg/ml arginine hydrochloride, pH 6.0), After being concentrated to about 100 mg/ml, polysorbate 80 was added to make the final concentration 0.2 mg/ml. Filter and dispense into vials, 0.5ml each, 22 bottles in total. Take 11 bottles, stopper and cap and label them as PD20191109-1. Take another 11 bottles, freeze-dry them, stopper and cap and mark them as PD20191109-2. See Table 17 for stability investigation according to the experimental scheme. The detection indicators are appearance, protein content, purity (SEC-HPLC method, non-reduced CE-SDS method) and charge variants (CEX-HPLC method).

Figure 110101453-A0101-12-0073-23
Figure 110101453-A0101-12-0073-23

4.2 試驗結果 4.2 Test results

(1)外觀、可見異物 (1) Appearance, visible foreign matter

在40℃±2℃的條件下考察1個月，25℃±2℃條件下考察至1個月，高濃度製劑處方外觀、可見異物均合格；凍乾製劑外觀合格，複溶後外觀和可見異物均合格。 When inspected at 40℃±2℃ for 1 month, and at 25℃±2℃ for 1 month, the appearance of high-concentration preparations and visible foreign bodies are all qualified; the appearance of freeze-dried preparations is qualified, and the appearance after reconstitution is visible. All foreign objects are qualified.

(2)蛋白含量 (2) Protein content

在40℃±2℃和25℃±2℃條件下1個月，高濃度液體製劑蛋白含量均未發生變化；凍乾製劑複溶後進行蛋白含量檢測，均未發生變化。 Under the conditions of 40℃±2℃ and 25℃±2℃ for 1 month, the protein content of high-concentration liquid preparations did not change; the protein content of the lyophilized preparations was reconstituted, and there was no change.

Figure 110101453-A0101-12-0074-24
Figure 110101453-A0101-12-0074-24

K20180504為20mg/ml液體製劑；PD20191109-1為100mg/ml高濃度液體製劑；PD20191109-2為100mg/ml高濃度凍乾製劑。 K20180504 is a 20mg/ml liquid preparation; PD20191109-1 is a 100mg/ml high-concentration liquid preparation; PD20191109-2 is a 100mg/ml high-concentration lyophilized preparation.

(3)純度 (3) Purity

純度(SEC-HPL C法)：40℃和25℃條件下高濃度液體製劑純度均發生下降，主要表現為聚體增多，其下降速度略快於20mg/ml液體製劑，但40℃條件下1個月純度仍大於95%，穩定性可以接受。相較於 高濃度液體製劑，凍乾製劑表現出更佳的穩定性。詳見表19。 Purity (SEC-HPL C method): The purity of high-concentration liquid preparations decreases at 40°C and 25°C, which is mainly manifested by the increase in aggregates, and the decline rate is slightly faster than that of 20mg/ml liquid preparations, but at 40°C 1 The monthly purity is still greater than 95%, and the stability is acceptable. Compared to High-concentration liquid formulations and freeze-dried formulations show better stability. See Table 19 for details.

純度(非還原型CE-SDS法)：40℃條件下高濃度液體製劑純度發生下降，其下降速度與20mg/ml液體製劑相當，40℃條件下1個月純度仍大於90%，穩定性可以接受。相較於高濃度液體製劑，凍乾製劑表現出更佳的穩定性，在40℃條件下放置1個月，純度(非還原型CE-SDS法)無明顯變化。詳見表20。 Purity (non-reduced CE-SDS method): The purity of high-concentration liquid preparations decreases at 40°C, and the rate of decrease is equivalent to that of 20mg/ml liquid preparations. The purity is still greater than 90% at 40°C for 1 month, and the stability is good. accept. Compared with high-concentration liquid preparations, freeze-dried preparations show better stability, and the purity (non-reduced CE-SDS method) does not change significantly after being placed at 40°C for 1 month. See Table 20 for details.

Figure 110101453-A0101-12-0075-25
Figure 110101453-A0101-12-0075-25

Figure 110101453-A0101-12-0075-26
Figure 110101453-A0101-12-0075-26

(4)電荷變異體 (4) Charge variant

40℃條件下高濃度液體製劑酸性組分增加，主成分下降，其變化速度與20mg/ml液體製劑相當，凍乾製劑電荷變異體未發生明顯變化。25℃條件下高濃度液體製劑和凍乾製劑電荷變異體均未發生明顯變化。詳見表21。 Under the condition of 40℃, the acidic components of high-concentration liquid preparations increased, and the main components decreased. The rate of change was equivalent to that of 20mg/ml liquid preparations. The charge variants of freeze-dried preparations did not change significantly. The charge variants of high-concentration liquid preparations and freeze-dried preparations did not change significantly at 25°C. See Table 21 for details.

Figure 110101453-A0101-12-0076-27
Figure 110101453-A0101-12-0076-27

試驗結論Test Conclusions

綜合上述各個試驗現有結果可知，高濃度製劑處方(100mg/ml重組全人源抗程序性死亡受體1(PD-1)和抗程序性死亡配體1(PD-L1)雙特異性抗體、0.85mg/ml組胺酸、1.00mg/ml鹽酸組胺酸、30.00 mg/ml山梨醇、7.49mg/ml甲硫胺酸、21.07mg/ml鹽酸精胺酸、0.20mg/ml聚山梨酯80，pH6.0)製備的液體製劑和凍乾製劑穩定性均符合要求。 Based on the existing results of the above-mentioned various experiments, it can be known that the high-concentration preparation prescription (100mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibodies, 0.85mg/ml histidine, 1.00mg/ml histidine hydrochloride, 30.00 mg/ml sorbitol, 7.49mg/ml methionine, 21.07mg/ml arginine hydrochloride, 0.20mg/ml polysorbate 80, pH 6.0) The stability of the prepared liquid preparations and freeze-dried preparations meet the requirements .

實施例5：IBI318凍乾製劑複溶實驗以及黏度測試 Example 5: Reconstitution test and viscosity test of IBI318 freeze-dried preparation

凍乾製劑藉由加入更少的水可以複溶製備更高濃度的液體製劑。IBI318凍乾製劑PD20191109-2可以複溶形成蛋白濃度高至170mg/ml的液體製劑，黏度低於10cP，在液體製劑可接受的注射黏度範圍內。且在常溫下(25℃±2℃，60%RH±5%RH,500Lux±50Lux)放置一天，各項檢測指標均未發生變化。以上結果表明，以凍乾製劑複溶製備高濃度液體製劑進行皮下注射是可行的。 The lyophilized preparation can be reconstituted to prepare a higher concentration liquid preparation by adding less water. The IBI318 freeze-dried preparation PD20191109-2 can be reconstituted to form a liquid preparation with a protein concentration as high as 170mg/ml, with a viscosity of less than 10cP, and within the acceptable injection viscosity range of the liquid preparation. And when placed at room temperature (25℃±2℃, 60%RH±5%RH, 500Lux±50Lux) for one day, there is no change in all test indexes. The above results indicate that it is feasible to reconstitute a lyophilized preparation to prepare a high-concentration liquid preparation for subcutaneous injection.

Figure 110101453-A0101-12-0077-28
Figure 110101453-A0101-12-0077-28

以上描述了本發明的示例性實施方案，本領域技術人員應當理解的是，這些公開內容僅是示例性的，在本發明的範圍內可以進行各種其它替換、適應和修改。因此，本發明不限於文中列舉的具體實施方案。 The exemplary embodiments of the present invention have been described above, and those skilled in the art should understand that these disclosures are only exemplary, and various other substitutions, adaptations and modifications can be made within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments listed in the text.

Claims (27)

一種抗PD-1/PD-L1抗體或其抗原結合片段的凍乾製劑，其包含： A freeze-dried preparation of anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof, which comprises: (i)抗PD-1/PD-L1抗體或其抗原結合片段； (i) Anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof; (ii)緩衝體系； (ii) Buffer system; (iii)穩定劑，該穩定劑包括多元醇和/或胺基酸；和 (iii) a stabilizer, which includes a polyol and/or an amino acid; and (iv)表面活性劑， (iv) Surfactants, 其中該製劑當重構時具有5.5-6.5之間的pH，例如，pH約為5.5、6.0或6.5。 Wherein the formulation has a pH between 5.5-6.5 when reconstituted, for example, the pH is about 5.5, 6.0 or 6.5. 如請求項1所述的凍乾製劑，其中該製劑當重構時具有約6.0的pH。 The lyophilized formulation of claim 1, wherein the formulation has a pH of about 6.0 when reconstituted. 如請求項1或2所述的凍乾製劑，其中該製劑能夠在約1mg/mL至約150mg/mL之間的濃度重構該抗體或其抗原結合片段，例如該製劑能夠在約1mg/mL至約200mg/mL的濃度，例如約1、10、15、20、25、30、35、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190或200mg/ml的濃度重構該抗體或其抗原結合片段；較佳地，該製劑能夠在約20mg/mL至約170mg/mL之間的濃度重構該抗體或其抗原結合片段。 The freeze-dried preparation according to claim 1 or 2, wherein the preparation can reconstitute the antibody or antigen-binding fragment thereof at a concentration between about 1 mg/mL and about 150 mg/mL, for example, the preparation can be at a concentration of about 1 mg/mL To a concentration of about 200 mg/mL, such as about 1, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, The antibody or antigen-binding fragment thereof is reconstituted at a concentration of 170, 180, 190, or 200 mg/ml; preferably, the preparation can reconstitute the antibody or its antigen binding at a concentration between about 20 mg/mL and about 170 mg/mL Fragment. 如請求項1至3中任一項所述的凍乾製劑，其中該表面活性劑選自聚山梨酯80、聚山梨酯20、泊洛沙姆、聚乙二醇聚山梨酯80或其組合，且以約0.01%(w/v)-1%(w/v)，例如0.01、0.05、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1%(w/v)的重量比存在；較佳地，該表面活 性劑為聚山梨酯80，更佳地，該聚山梨酯80以約0.02%(w/v)的重量比存在。 The lyophilized formulation according to any one of claims 1 to 3, wherein the surfactant is selected from polysorbate 80, polysorbate 20, poloxamer, polyethylene glycol polysorbate 80, or a combination thereof , And at about 0.01%(w/v)-1%(w/v), such as 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1%(w/v) The weight ratio exists; preferably, the surface activity The sex agent is polysorbate 80, and more preferably, the polysorbate 80 is present in a weight ratio of about 0.02% (w/v). 如請求項1至4中任一項所述的凍乾製劑，其中該緩衝體系選自組胺酸緩衝體系、組胺酸和鹽酸組胺酸緩衝體系、枸櫞酸和枸櫞酸鈉緩衝體系、醋酸醋酸鈉緩衝體系、磷酸鹽緩衝體系，且以約0.01%(w/v)-1%(w/v)的重量比存在，例如約0.01、0.05、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1%(w/v)的重量比存在；較佳地，該緩衝體系以約0.01%(w/v)-0.2%(w/v)的重量比存在； The lyophilized preparation according to any one of claims 1 to 4, wherein the buffer system is selected from the group consisting of histidine buffer system, histidine and histidine hydrochloride buffer system, citric acid and sodium citrate buffer system , Sodium acetate acetate buffer system, phosphate buffer system, and exist in a weight ratio of about 0.01%(w/v)-1%(w/v), such as about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 , 0.6, 0.7, 0.8, 0.9, 1% (w/v) by weight ratio; preferably, the buffer system is present at a weight ratio of about 0.01% (w/v)-0.2% (w/v); 較佳地，該緩衝體系為組胺酸緩衝體系；更佳地，該組胺酸以約0.01-1%(w/v)，例如0.01、0.05、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1%(w/v)的重量比存在；更佳地，該組胺酸以約0.155%(w/v)的重量比存在； Preferably, the buffer system is a histidine buffer system; more preferably, the histidine is about 0.01-1% (w/v), such as 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 , 0.7, 0.8, 0.9, 1% (w/v) by weight ratio; more preferably, the histidine acid is present at a weight ratio of about 0.155% (w/v); 較佳地，該緩衝體系為組胺酸和鹽酸組胺酸緩衝體系；更佳地，該組胺酸和該鹽酸組胺酸分別以約0.01-1%(w/v)，例如0.01、0.05、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1%(w/v)的重量比存在；更佳地，該組胺酸和鹽酸組胺酸分別以約0.085%(w/v)和約0.1%(w/v)的重量比存在。 Preferably, the buffer system is a histidine and histidine hydrochloride buffer system; more preferably, the histidine and the histidine hydrochloride are respectively about 0.01-1% (w/v), such as 0.01, 0.05 , 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1% (w/v) by weight ratio; more preferably, the histidine and hydrochloric acid histidine are respectively about 0.085% ( w/v) and about 0.1% (w/v) by weight ratio. 如請求項1至5中任一項所述的凍乾製劑，其中該多元醇選自山梨醇、甘露醇或其組合，該胺基酸包括精胺酸、鹽酸精胺酸、甲硫胺酸、甘胺酸、脯胺酸或其組合； The freeze-dried preparation according to any one of claims 1 to 5, wherein the polyol is selected from sorbitol, mannitol or a combination thereof, and the amino acid includes arginine, arginine hydrochloride, and methionine , Glycine, Proline or a combination thereof; 較佳地，該穩定劑包括山梨醇和精胺酸；更佳地，該山梨醇以約1-10%(w/v)，例如約1、2、3、4、5、6、7、8、9、10%(w/v)的重量比存在， 該精胺酸以約1-10%(w/v)，例如約1、2、3、4、5、6、7、8、9、10%(w/v)的重量比存在；更佳地，該山梨醇和該精胺酸分別以約3%(w/v)和約1.742%(w/v)的重量比存在； Preferably, the stabilizer includes sorbitol and arginine; more preferably, the sorbitol contains about 1-10% (w/v), such as about 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10% (w/v) weight ratio exists, The arginine is present in a weight ratio of about 1-10% (w/v), such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10% (w/v); more preferably Ground, the sorbitol and the arginine are respectively present in a weight ratio of about 3% (w/v) and about 1.742% (w/v); 較佳地，該穩定劑包括山梨醇和鹽酸精胺酸；更佳地，該山梨醇以約1-10%(w/v)，例如約1、2、3、4、5、6、7、8、9、10%(w/v)的重量比存在，以約1-10%(w/v)，例如約1、2、3、4、5、6、7、8、9、10%(w/v)的重量比存在；更佳地，該山梨醇和該鹽酸精胺酸分別以約3%(w/v)和約2.107%(w/v)的重量比存在。 Preferably, the stabilizer includes sorbitol and arginine hydrochloride; more preferably, the sorbitol is about 1-10% (w/v), such as about 1, 2, 3, 4, 5, 6, 7, The weight ratio of 8, 9, 10% (w/v) is present, with about 1-10% (w/v), for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10% (w/v) is present in a weight ratio; more preferably, the sorbitol and the arginine hydrochloride are present in a weight ratio of about 3% (w/v) and about 2.107% (w/v), respectively. 如請求項1至6中任一項所述的凍乾製劑，其中該穩定劑還包括甲硫胺酸；較佳地，該甲硫胺酸以約0.1-10%(w/v)的重量比存在，例如以約0.1、0.5、1、2、3、4、5、6、7、8、9、10%(w/v)的重量比存在；更佳地，該甲硫胺酸以約0.749%(w/v)的重量比存在。 The lyophilized formulation according to any one of claims 1 to 6, wherein the stabilizer further comprises methionine; preferably, the weight of the methionine is about 0.1-10% (w/v) Ratio exists, for example in a weight ratio of about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10% (w/v); more preferably, the methionine is About 0.749% (w/v) weight ratio exists. 一種抗PD-1/PD-L1抗體或其抗原結合片段的凍乾製劑，其藉由凍乾水溶液製成，該水溶液包含： A freeze-dried preparation of an anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof, which is prepared by a freeze-dried aqueous solution, the aqueous solution comprising: (i)抗PD-1/PD-L1抗體或其抗原結合片段； (i) Anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof; (ii)緩衝體系； (ii) Buffer system; (iii)穩定劑，該穩定劑包括多元醇和/或胺基酸；和 (iii) a stabilizer, which includes a polyol and/or an amino acid; and (iv)表面活性劑， (iv) Surfactants, 該液體製劑具有約5.5-6.5的pH，例如，pH約為5.5、6.0或6.5；較佳地，該液體製劑具有約6.0的pH。 The liquid preparation has a pH of about 5.5-6.5, for example, the pH is about 5.5, 6.0 or 6.5; preferably, the liquid preparation has a pH of about 6.0. 一種抗PD-1/PD-L1抗體或其抗原結合片段的液體製劑，其包含水溶液，該水溶液包含： A liquid preparation of an anti-PD-1/PD-L1 antibody or an antigen-binding fragment thereof, which comprises an aqueous solution, the aqueous solution comprising: (i)抗PD-1/PD-L1抗體或其抗原結合片段； (i) Anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof; (ii)緩衝體系； (ii) Buffer system; (iii)穩定劑，該穩定劑包括多元醇和/或胺基酸；和 (iii) a stabilizer, which includes a polyol and/or an amino acid; and (iv)表面活性劑， (iv) Surfactants, 該液體製劑具有約5.5-6.5的pH，例如，pH約為5.5、6.0或6.5；較佳地，該液體製劑具有約6.0的pH。 The liquid preparation has a pH of about 5.5-6.5, for example, the pH is about 5.5, 6.0 or 6.5; preferably, the liquid preparation has a pH of about 6.0. 如請求項8所述的凍乾製劑或如請求項9所述的液體製劑，其中該抗PD-1/PD-L1抗體或其抗原結合片段以約1mg/mL至約200mg/mL的濃度存在於水溶液中，例如以約1、5、10、20、25、30、35、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190或200mg/ml的濃度存在於水溶液中；較佳地，該抗PD-1/PD-L1抗體或其抗原結合片段以約20mg/mL至約170mg/mL之間的濃度存在於水溶液中。 The lyophilized preparation according to claim 8 or the liquid preparation according to claim 9, wherein the anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof is present at a concentration of about 1 mg/mL to about 200 mg/mL In an aqueous solution, for example, about 1, 5, 10, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180 , 190 or 200 mg/ml in the aqueous solution; preferably, the anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof is present in the aqueous solution at a concentration between about 20 mg/mL to about 170 mg/mL . 如請求項8所述的凍乾製劑或如請求項9至10中任一項所述的液體製劑，其中該表面活性劑選自聚山梨酯80、聚山梨酯20、泊洛沙姆、聚乙二醇聚山梨酯80或其組合，且以約0.1-10mg/ml，例如約0.1、0.5、1、2、3、4、5、6、7、8、9、10mg/ml的濃度存在；較佳地，該表面活性劑為聚山梨酯80，更佳地，該聚山梨酯80以約0.2mg/ml的濃度存在。 The lyophilized formulation according to claim 8 or the liquid formulation according to any one of claims 9 to 10, wherein the surfactant is selected from polysorbate 80, polysorbate 20, poloxamer, polysorbate Ethylene glycol polysorbate 80 or a combination thereof, and is present at a concentration of about 0.1-10 mg/ml, such as about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/ml ; Preferably, the surfactant is polysorbate 80, more preferably, the polysorbate 80 is present at a concentration of about 0.2 mg/ml. 如請求項8所述的凍乾製劑或如請求項9至11中任一項所述的液體製劑，其中該緩衝體系選自組胺酸緩衝體系、組胺酸和鹽酸組胺酸緩衝體系、枸櫞酸和枸櫞酸鈉緩衝體系、醋酸醋酸鈉緩衝體系、磷酸 鹽緩衝體系，且以約1-100mM，例如，約1、5、10、15、20、30、40、50、60、70、80、90、100mM的濃度存在；較佳地，該緩衝體系以約1-20mM，例如，1、5、10、15、20mM的濃度存在； The lyophilized preparation according to claim 8 or the liquid preparation according to any one of claims 9 to 11, wherein the buffer system is selected from the group consisting of histidine buffer system, histidine and histidine hydrochloride buffer system, Citrate and sodium citrate buffer system, sodium acetate acetate buffer system, phosphoric acid The salt buffer system is present at a concentration of about 1-100 mM, for example, about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 mM; preferably, the buffer system Exist at a concentration of about 1-20 mM, for example, 1, 5, 10, 15, 20 mM; 較佳地，該緩衝體系為組胺酸緩衝體系；更佳地，該組胺酸以約0.1-10mg/mL，例如約0.1、0.5、1、2、3、4、5、6、7、8、9、10mg/ml的濃度存在；更佳地，該組胺酸以約1.55mg/mL的濃度存在； Preferably, the buffer system is a histidine buffer system; more preferably, the histidine is about 0.1-10 mg/mL, such as about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/ml is present; more preferably, the histidine is present at a concentration of about 1.55 mg/ml; 較佳地，該緩衝體系為組胺酸和鹽酸組胺酸緩衝體系；更佳地，該組胺酸和該鹽酸組胺酸分別以約0.1-10mg/mL，例如約0.1、0.5、1、2、3、4、5、6、7、8、9、10mg/ml的濃度存在；更佳地，該組胺酸和該鹽酸組胺酸分別以約0.85mg/mL和約1.00mg/mL的濃度存在。 Preferably, the buffer system is a histidine and histidine hydrochloride buffer system; more preferably, the histidine and the histidine hydrochloride are respectively about 0.1-10 mg/mL, such as about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/ml are present; more preferably, the histidine and the hydrochloric acid histidine are respectively about 0.85 mg/mL and about 1.00 mg/mL The concentration exists. 如請求項8所述的凍乾製劑或如請求項9至12中任一項所述的液體製劑，其中該多元醇選自山梨醇、甘露醇或其組合，該胺基酸包括精胺酸、鹽酸精胺酸、甲硫胺酸、甘胺酸、脯胺酸或其組合； The freeze-dried formulation according to claim 8 or the liquid formulation according to any one of claims 9 to 12, wherein the polyol is selected from sorbitol, mannitol or a combination thereof, and the amino acid includes arginine , Arginine hydrochloride, methionine, glycine, proline or a combination thereof; 較佳地，該穩定劑包括山梨醇和精胺酸；更佳地，該山梨醇以約10-100mg/mL，例如約10、20、30、40、50、60、70、80、90、100mg/mL的濃度存在，該精胺酸以例如約10、20、30、40、50、60、70、80、90、100mg/mL存在；更佳地，該山梨醇和該精胺酸分別以約30mg/mL和約17.42mg/mL的濃度存在； Preferably, the stabilizer includes sorbitol and arginine; more preferably, the sorbitol is about 10-100 mg/mL, such as about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg. /mL, the arginine is present at a concentration of, for example, about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg/mL; more preferably, the sorbitol and the arginine are present at about 10, 20, 30, 40, 50, 60, 100 mg/mL; Exist at a concentration of 30mg/mL and about 17.42mg/mL; 較佳地，該穩定劑包括山梨醇和鹽酸精胺酸；更佳地，該山梨醇和該鹽酸精胺酸分別以約10-100mg/mL，例如約10、20、30、40、50、60、70、80、90、100mg/mL的濃度存在；更佳地，該山梨醇和該鹽酸精胺酸分別以約30mg/mL和約21.07mg/mL的濃度存在。 Preferably, the stabilizer includes sorbitol and arginine hydrochloride; more preferably, the sorbitol and the arginine hydrochloride are respectively about 10-100 mg/mL, such as about 10, 20, 30, 40, 50, 60, It is present at a concentration of 70, 80, 90, 100 mg/mL; more preferably, the sorbitol and the arginine hydrochloride are present at a concentration of about 30 mg/mL and about 21.07 mg/mL, respectively. 如請求項8所述的凍乾製劑或如請求項9至13中任一項所述的液體製劑，其中該穩定劑還包括甲硫胺酸；較佳地，該甲硫胺酸以約1-100mg/mL，例如約1、5、10、20、30、40、50、60、70、80、90、100mg/mL的濃度存在；更佳地，該甲硫胺酸以約1-50mg/mL；更佳地，該甲硫胺酸以約7.49mg/mL的濃度存在。 The freeze-dried formulation according to claim 8 or the liquid formulation according to any one of claims 9 to 13, wherein the stabilizer further comprises methionine; preferably, the methionine is about 1 -100mg/mL, such as about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100mg/mL; more preferably, the methionine is present at a concentration of about 1-50mg /mL; More preferably, the methionine is present at a concentration of about 7.49 mg/mL. 如請求項8所述的凍乾製劑或如請求項9至14中任一項所述的液體製劑，其中： The lyophilized formulation according to claim 8 or the liquid formulation according to any one of claims 9 to 14, wherein: 該水溶液包含：約20-170mg/ml重組全人源抗程序性死亡受體1(PD-1)和抗程序性死亡配體1(PD-L1)雙特異性抗體、約2.94mg/ml枸櫞酸鈉、約50mg/ml山梨醇、約0.20mg/ml聚山梨酯80，該水溶液具有約5.5的pH； The aqueous solution contains: about 20-170mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibodies, about 2.94mg/ml citrate Sodium nitrate, about 50mg/ml sorbitol, about 0.20mg/ml polysorbate 80, the aqueous solution has a pH of about 5.5; 或該水溶液包含：約20-170mg/ml重組全人源抗程序性死亡受體1(PD-1)和抗程序性死亡配體1(PD-L1)雙特異性抗體、約2.94mg/ml枸櫞酸鈉、約50mg/ml山梨醇、約0.20mg/ml聚山梨酯80，該水溶液具有約6.0的pH； Or the aqueous solution contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibodies, about 2.94 mg/ml Sodium citrate, about 50mg/ml sorbitol, about 0.20mg/ml polysorbate 80, the aqueous solution has a pH of about 6.0; 或該水溶液包含：約20-170mg/ml重組全人源抗程序性死亡受體1(PD-1)和抗程序性死亡配體1(PD-L1)雙特異性抗體、約2.94mg/ml枸櫞酸鈉、約50mg/ml山梨醇、約2.94mg/ml甲硫胺酸、約0.20mg/ml聚山梨酯80，該水溶液具有約6.0的pH； Or the aqueous solution contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibodies, about 2.94 mg/ml Sodium citrate, about 50mg/ml sorbitol, about 2.94mg/ml methionine, about 0.20mg/ml polysorbate 80, the aqueous solution has a pH of about 6.0; 或該水溶液包含：約20-170mg/ml重組全人源抗程序性死亡受體1(PD-1)和抗程序性死亡配體1(PD-L1)雙特異性抗體、約2.94mg/ml 枸櫞酸鈉、約50mg/ml山梨醇、約0.20mg/ml聚山梨酯80，該水溶液具有約6.5的pH； Or the aqueous solution contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibodies, about 2.94 mg/ml Sodium citrate, about 50mg/ml sorbitol, about 0.20mg/ml polysorbate 80, the aqueous solution has a pH of about 6.5; 或該水溶液包含：約20-170mg/ml重組全人源抗程序性死亡受體1(PD-1)和抗程序性死亡配體1(PD-L1)雙特異性抗體、約2.94mg/ml枸櫞酸鈉、約50mg/ml山梨醇、約0.20mg/ml聚山梨酯80，該水溶液具有約6.0的pH； Or the aqueous solution contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibodies, about 2.94 mg/ml Sodium citrate, about 50mg/ml sorbitol, about 0.20mg/ml polysorbate 80, the aqueous solution has a pH of about 6.0; 或該水溶液包含：約20-170mg/ml重組全人源抗程序性死亡受體1(PD-1)和抗程序性死亡配體1(PD-L1)雙特異性抗體、約1.55mg/ml組胺酸、約50mg/ml山梨醇、約0.20mg/ml聚山梨酯80，該水溶液具有約6.0的pH； Or the aqueous solution contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibodies, about 1.55 mg/ml Histidine, about 50mg/ml sorbitol, about 0.20mg/ml polysorbate 80, the aqueous solution has a pH of about 6.0; 或該水溶液包含：約20-170mg/ml重組全人源抗程序性死亡受體1(PD-1)和抗程序性死亡配體1(PD-L1)雙特異性抗體、約1.55mg/ml組胺酸、約50mg/ml山梨醇、約7.49mg/ml甲硫胺酸、約0.20mg/ml聚山梨酯80，該水溶液具有約6.0的pH； Or the aqueous solution contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibodies, about 1.55 mg/ml Histidine, about 50mg/ml sorbitol, about 7.49mg/ml methionine, about 0.20mg/ml polysorbate 80, the aqueous solution has a pH of about 6.0; 更佳地，該水溶液包含：20-170mg/ml重組全人源抗程序性死亡受體1(PD-1)和抗程序性死亡配體1(PD-L1)雙特異性抗體、約1.55mg/ml組胺酸、約30mg/ml山梨醇、約7.49mg/ml甲硫胺酸、約17.42mg/ml精胺酸、約0.20mg/ml聚山梨酯80，該水溶液具有約6.0的pH； More preferably, the aqueous solution contains: 20-170mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibodies, about 1.55 mg /ml histidine, about 30mg/ml sorbitol, about 7.49mg/ml methionine, about 17.42mg/ml arginine, about 0.20mg/ml polysorbate 80, the aqueous solution has a pH of about 6.0; 更佳地，該水溶液包含：約20-170mg/ml重組全人源抗程序性死亡受體1(PD-1)和抗程序性死亡配體1(PD-L1)雙特異性抗體、約0.85mg/ml組胺酸、約1.00mg/ml鹽酸組胺酸、約30.00mg/ml山梨醇、約7.49mg/ml 甲硫胺酸、約21.07mg/ml鹽酸精胺酸、約0.20mg/ml聚山梨酯80，該水溶液具有約6.0的pH。 More preferably, the aqueous solution contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibodies, about 0.85 mg/ml histidine, about 1.00mg/ml histidine hydrochloride, about 30.00mg/ml sorbitol, about 7.49mg/ml Methionine, about 21.07 mg/ml arginine hydrochloride, about 0.20 mg/ml polysorbate 80, the aqueous solution has a pH of about 6.0. 如請求項1至8中任一項所述的凍乾製劑或如請求項9至15中任一項所述的液體製劑，其中該抗體或其抗原結合片段結合人類PD-L1(SEQ ID NO：1)及人類PD-1(SEQ ID NO：2)； The lyophilized preparation according to any one of claims 1 to 8 or the liquid preparation according to any one of claims 9 to 15, wherein the antibody or antigen-binding fragment thereof binds to human PD-L1 (SEQ ID NO :1) and human PD-1 (SEQ ID NO: 2); 較佳地，該抗體或其抗原結合片段包含第一重鏈(HC1)，其包含第一重鏈可變區(HCVR1)和恆定區；第一輕鏈(LC1)，其包含第一輕鏈可變區(LCVR1)和恆定區；第二重鏈(HC2)，其包含第二重鏈可變區(HCVR2)和恆定區；以及第二輕鏈(LC2)，其包含第二輕鏈可變區(LCVR2)和恆定區，其中： Preferably, the antibody or antigen-binding fragment thereof includes a first heavy chain (HC1), which includes a first heavy chain variable region (HCVR1) and a constant region; and a first light chain (LC1), which includes a first light chain The variable region (LCVR1) and the constant region; the second heavy chain (HC2), which includes the second heavy chain variable region (HCVR2) and the constant region; and the second light chain (LC2), which includes the second light chain Variable region (LCVR2) and constant region, where: 1)該HCVR1包含SEQ ID NO：3所示的重鏈可變區所含的三個互補決定區域HCDR1、HCDR2和HCDR3，並且該LCVR1包含SEQ ID NO：4所示的輕鏈可變區所含的LCDR1、LCDR2和LCDR3；以及 1) The HCVR1 includes the three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 3, and the LCVR1 includes the light chain variable region shown in SEQ ID NO: 4 LCDR1, LCDR2 and LCDR3 included; and 2)該HCVR2包含SEQ ID NO：5所示的重鏈可變區中所含的三個互補決定區域(CDR)HCDR1、HCDR2和HCDR3，並且該LCVR2包含SEQ ID NO：8所示的輕鏈可變區所含的三個互補決定區域LCDR1、LCDR2和LCDR3。 2) The HCVR2 includes the three complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 5, and the LCVR2 includes the light chain shown in SEQ ID NO: 8 The variable region contains three complementary determining regions LCDR1, LCDR2 and LCDR3. 如請求項1至8中任一項所述的凍乾製劑或如請求項9至15中任一項所述的液體製劑，其中該抗體或其抗原結合片段包含第一重鏈(HC1)，其包含第一重鏈可變區(HCVR1)和恆定區；第一輕鏈(LC1)，其包含第一輕鏈可變區(LCVR1)和恆定區；第二重鏈(HC2)，其包含第 二重鏈可變區(HCVR2)和恆定區；以及第二輕鏈(LC2)，其包含第二輕鏈可變區(LCVR2)和恆定區，其中： The lyophilized preparation according to any one of claims 1 to 8 or the liquid preparation according to any one of claims 9 to 15, wherein the antibody or antigen-binding fragment thereof comprises a first heavy chain (HC1), It includes a first heavy chain variable region (HCVR1) and a constant region; a first light chain (LC1), which includes a first light chain variable region (LCVR1) and a constant region; a second heavy chain (HC2), which includes NS The variable region of the double heavy chain (HCVR2) and the constant region; and the second light chain (LC2), which includes the variable region of the second light chain (LCVR2) and the constant region, wherein: a)該HCVR1包含具有SEQ ID NO：16所示的胺基酸序列的互補決定區域CDR1、具有SEQ ID NO：17所示的胺基酸序列的互補決定區域CDR2及具有SEQ ID NO：18所示的胺基酸序列的互補決定區域CDR3； a) The HCVR1 includes the complementarity determining region CDR1 having the amino acid sequence shown in SEQ ID NO: 16, the complementarity determining region CDR2 having the amino acid sequence shown in SEQ ID NO: 17, and the complementarity determining region CDR2 having the amino acid sequence shown in SEQ ID NO: 18; The complementarity determining region CDR3 of the amino acid sequence shown; b)該LCVR1包含具有SEQ ID NO：19所示的胺基酸序列的互補決定區域CDR1、具有SEQ ID NO：20所示的胺基酸序列的互補決定區域CDR2及具有SEQ ID NO：21所示的胺基酸序列的互補決定區域CDR3； b) The LCVR1 includes the complementarity determining region CDR1 having the amino acid sequence shown in SEQ ID NO: 19, the complementarity determining region CDR2 having the amino acid sequence shown in SEQ ID NO: 20, and the complementarity determining region CDR2 having the amino acid sequence shown in SEQ ID NO: 21. The complementarity determining region CDR3 of the amino acid sequence shown; c)該HCVR2包含具有SEQ ID NO：22所示的胺基酸序列的互補決定區域CDR1、具有SEQ ID NO：23或SEQ ID NO：24所示的胺基酸序列的互補決定區域CDR2及具有SEQ ID NO：25所示的胺基酸序列的互補決定區域CDR3；以及 c) The HCVR2 includes the complementarity determining region CDR1 having the amino acid sequence shown in SEQ ID NO: 22, the complementarity determining region CDR2 having the amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 24, and The complementarity determining region CDR3 of the amino acid sequence shown in SEQ ID NO: 25; and d)該LCVR2包含具有SEQ ID NO：26所示的胺基酸序列的互補決定區域CDR1、具有SEQ ID NO：27所示的胺基酸序列的互補決定區域CDR2及具有SEQ ID NO：28所示的胺基酸序列的互補決定區域CDR3。 d) The LCVR2 includes the complementarity determining region CDR1 having the amino acid sequence shown in SEQ ID NO: 26, the complementarity determining region CDR2 having the amino acid sequence shown in SEQ ID NO: 27, and the complementarity determining region CDR2 having the amino acid sequence shown in SEQ ID NO: 28. The complementarity determining region of the amino acid sequence shown is CDR3. 如請求項1至8中任一項所述的凍乾製劑或如請求項9至15中任一項所述的液體製劑，其中該抗體或其抗原結合片段包含第一重鏈(HC1)，其包含第一重鏈可變區(HCVR1)和恆定區；第一輕鏈(LC1)，其包含第一輕鏈可變區(LCVR1)和恆定區；第二重鏈(HC2)，其包含第二重鏈可變區(HCVR2)和恆定區；以及第二輕鏈(LC2)，其包含第二輕鏈可變區(LCVR2)和恆定區，其中： The lyophilized preparation according to any one of claims 1 to 8 or the liquid preparation according to any one of claims 9 to 15, wherein the antibody or antigen-binding fragment thereof comprises a first heavy chain (HC1), It includes a first heavy chain variable region (HCVR1) and a constant region; a first light chain (LC1), which includes a first light chain variable region (LCVR1) and a constant region; a second heavy chain (HC2), which includes The second heavy chain variable region (HCVR2) and constant region; and the second light chain (LC2), which includes the second light chain variable region (LCVR2) and the constant region, wherein: 該抗體或其抗原結合片段包含與SEQ ID NO：3具有至少90%,95%,98%或99%或更高同一性的HCVR1；和/或與SEQ ID NO：4具有至少90%,95%,98%或99%或更高同一性的LCVR1；和/或與SEQ ID NO：5具有至少90%,95%,98%或99%或更高同一性的HCVR2；和/或與SEQ ID NO：8具有至少90%,95%,98%或99%或更高同一性的LCVR2； The antibody or antigen-binding fragment thereof comprises HCVR1 having at least 90%, 95%, 98%, or 99% or more identity with SEQ ID NO: 3; and/or having at least 90%, 95% identity with SEQ ID NO: 4, 95 %, 98% or 99% or higher identity LCVR1; and/or HCVR2 with at least 90%, 95%, 98% or 99% identity or higher identity with SEQ ID NO: 5; and/or with SEQ ID NO: 5 ID NO: 8 LCVR2 with at least 90%, 95%, 98% or 99% or higher identity; 較佳地，a)該HCVR1包含具有SEQ ID NO：3所示的胺基酸序列；b)該LCVR1包含具有SEQ ID NO：4所示的胺基酸序列；c)該HCVR2包含具有SEQ ID NO：5所示的胺基酸序列；及d)該LCVR2包含具有SEQ ID NO：8所示的胺基酸序列。 Preferably, a) the HCVR1 includes the amino acid sequence shown in SEQ ID NO: 3; b) the LCVR1 includes the amino acid sequence shown in SEQ ID NO: 4; c) the HCVR2 includes the amino acid sequence shown in SEQ ID NO: 4; NO: the amino acid sequence shown in 5; and d) the LCVR2 includes the amino acid sequence shown in SEQ ID NO: 8. 如請求項1至8中任一項所述的凍乾製劑或如請求項9至15中任一項所述的液體製劑，其中該抗體或其抗原結合片段包含第一重鏈(HC1)，其包含第一重鏈可變區(HCVR1)和恆定區；第一輕鏈(LC1)，其包含第一輕鏈可變區(LCVR1)和恆定區；第二重鏈(HC2)，其包含第二重鏈可變區(HCVR2)和恆定區；以及第二輕鏈(LC2)，其包含第二輕鏈可變區(LCVR2)和恆定區，其中： The lyophilized preparation according to any one of claims 1 to 8 or the liquid preparation according to any one of claims 9 to 15, wherein the antibody or antigen-binding fragment thereof comprises a first heavy chain (HC1), It includes a first heavy chain variable region (HCVR1) and a constant region; a first light chain (LC1), which includes a first light chain variable region (LCVR1) and a constant region; a second heavy chain (HC2), which includes The second heavy chain variable region (HCVR2) and constant region; and the second light chain (LC2), which includes the second light chain variable region (LCVR2) and the constant region, wherein: 該抗體或其抗原結合片段包含與SEQ ID NO：49或SEQ ID NO：29具有至少90%,95%,98%或99%或更高同一性的HC1；和/或與SEQ ID NO：30具有至少90%,95%,98%或99%或更高同一性的LC1；和/或與SEQ ID NO：33或SEQ ID NO：31或SEQ ID NO：32具有至少90%,95%,98%或99%或更高同一性的HC2；和/或與SEQ ID NO：34具有至少90%,95%,98%或99%或更高同一性的LC2； The antibody or antigen-binding fragment thereof comprises HC1 having at least 90%, 95%, 98% or 99% or more identity with SEQ ID NO: 49 or SEQ ID NO: 29; and/or with SEQ ID NO: 30 LC1 having at least 90%, 95%, 98% or 99% or higher identity; and/or at least 90%, 95% with SEQ ID NO: 33 or SEQ ID NO: 31 or SEQ ID NO: 32, HC2 with 98% or 99% or higher identity; and/or LC2 with at least 90%, 95%, 98% or 99% or higher identity with SEQ ID NO: 34; 較佳地，該HC1、該LC1、該HC2及該LC2分別包含SEQ ID NO：49、SEQ ID NO：30、SEQ ID NO：31及SEQ ID NO：34所示的胺基酸序列；或者分別包含SEQ ID NO：49、SEQ ID NO：30、SEQ ID NO：32及SEQ ID NO：34所示的胺基酸序列；或者分別包含SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：33及SEQ ID NO：34所示的胺基酸序列； Preferably, the HC1, the LC1, the HC2 and the LC2 respectively comprise the amino acid sequences shown in SEQ ID NO: 49, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 34; or respectively Contains the amino acid sequences shown in SEQ ID NO: 49, SEQ ID NO: 30, SEQ ID NO: 32, and SEQ ID NO: 34; or SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO, respectively : 33 and the amino acid sequence shown in SEQ ID NO: 34; 更佳地，該抗體或其抗原結合片段含有衍生自人IgG1的Fc部分變體。 More preferably, the antibody or antigen-binding fragment thereof contains a variant of the Fc portion derived from human IgG1. 如請求項1至8中任一項所述的凍乾製劑或如請求項9至15中任一項所述的液體製劑，其中該抗體選自：PD-1/PD-L1雙特異性Ab v13844、Ab v3.0或Ab v3.2；較佳地，該抗體維PD-1/PD-L1雙特異性Ab v3.2。 The lyophilized preparation according to any one of claims 1 to 8 or the liquid preparation according to any one of claims 9 to 15, wherein the antibody is selected from: PD-1/PD-L1 bispecific Ab v13844, Ab v3.0 or Ab v3.2; preferably, the antibody is PD-1/PD-L1 bispecific Ab v3.2. 如請求項1至8中任一項所述的凍乾製劑或如請求項9至15中任一項所述的液體製劑，其中該抗體或其抗原結合片段在至少約12個月內表現出穩定性，較佳地，該抗體或其抗原結合片段在至少約24個月內表現出穩定性，更佳地，該抗體或其抗原結合片段在至少約36個月內表現出穩定性。 The lyophilized preparation according to any one of claims 1 to 8 or the liquid preparation according to any one of claims 9 to 15, wherein the antibody or antigen-binding fragment thereof exhibits Stability. Preferably, the antibody or antigen-binding fragment thereof exhibits stability for at least about 24 months, and more preferably, the antibody or antigen-binding fragment thereof exhibits stability for at least about 36 months. 如請求項1至8中任一項所述的凍幹製劑或如請求項9至15中任一項所述的液體製劑，其中該製劑在儲存後，例如在2-8℃儲存至少24個月後，或在室溫儲存至少6個月後，或在40℃±2℃儲存1個月後，是穩定的，較佳地具有如下特徵之一或多項： The freeze-dried preparation according to any one of claims 1 to 8 or the liquid preparation according to any one of claims 9 to 15, wherein the preparation is stored, for example, at least 24 at 2-8°C It is stable after one month, or after storage at room temperature for at least 6 months, or after storage at 40°C±2°C for 1 month, and preferably has one or more of the following characteristics: (i)藉由SEC-HPLC法測量，製劑具有大於或等於90%的純度，較佳大於或等於92%、94%、95%、96%、98%或99%的純度； (i) Measured by the SEC-HPLC method, the preparation has a purity greater than or equal to 90%, preferably greater than or equal to 92%, 94%, 95%, 96%, 98% or 99% purity; (ii)藉由還原型或非還原型CE-SDS法測量，製劑具有大於或等於 90%的純度，較佳大於或等於92%、94%、96%、98%的純度； (ii) Measured by the reduced or non-reduced CE-SDS method, the preparation has greater than or equal to 90% purity, preferably greater than or equal to 92%, 94%, 96%, 98% purity; (iii)藉由iCIEF或CEX-HPLC法測量，主成分
Figure 110101453-A0101-13-0012-40
35%，較佳大於或等於35%、40%、45%或50%。 (iii) Measured by iCIEF or CEX-HPLC method, the main component
Figure 110101453-A0101-13-0012-40
35%, preferably greater than or equal to 35%, 40%, 45% or 50%. 一種遞送裝置，其包含如請求項1至8中任一項所述的凍乾製劑或如請求項9至15中任一項所述的液體製劑，用於靜脈內、皮下、皮內或者肌內注射，或靜脈內輸注。 A delivery device comprising the lyophilized preparation according to any one of claims 1 to 8 or the liquid preparation according to any one of claims 9 to 15 for intravenous, subcutaneous, intradermal or intramuscular Intravenous injection, or intravenous infusion. 一種預填裝注射器，其包含如請求項1至8中任一項所述的凍乾製劑或如請求項9至15中任一項所述的液體製劑，用於靜脈內、皮下、皮內或者肌內注射，或靜脈內輸注。 A pre-filled syringe comprising the freeze-dried preparation according to any one of claims 1 to 8 or the liquid preparation according to any one of claims 9 to 15, for intravenous, subcutaneous, and intradermal use Or intramuscular injection, or intravenous infusion. 一種如請求項1至8中任一項所述的凍乾製劑或如請求項9至15中任一項所述的液體製劑的用途，用於製備在受試者中預防和/或治療腫瘤的遞送裝置或預填裝注射器或藥物；例如，該腫瘤是癌症，較佳地，該癌症為霍奇金或非霍奇金淋巴瘤、黑色素瘤、腎細胞癌、腎癌、肺癌、膀胱癌、胃及食道癌、結腸直腸癌、肝癌、肝細胞癌、膽管癌、胰臟癌、乳癌、三陰性乳癌、卵巢癌、子宮內膜癌、***癌、小細胞肺癌(SCLC)、非小細胞肺癌(NSCLC)、間皮瘤、頭頸的鱗狀細胞癌(SCCHN)、軟組織肉瘤或多形性神經膠母細胞瘤；更佳地，該肺癌是NSCLC、小細胞肺癌或間皮瘤；較佳地，其中該遞送裝置或預填裝注射器或藥物可與一或多種選自由以下組成的群組的抗腫瘤劑同時、分開或依序給藥：順鉑(cisplatin)、卡鉑(carboplatin)、達卡巴嗪(dacarbazine)、脂質體多柔比星(liposomal doxorubicin)、多西他賽(docetaxel)、環磷醯胺(cyclophosphamide)及多柔比星、諾維本(navelbine)、艾瑞布林(eribulin)、太平洋紫杉醇(paclitaxel)、用於可注射懸浮液的太平洋紫杉醇蛋白質結合顆粒、伊沙匹隆 (ixabepilone)、卡培他濱(capecitabine)、FOLFOX(亞葉酸鈣(leucovorin)、氟尿嘧啶(fluorouracil)及奧沙利鉑(oxaliplatin))、FOLFIRI(亞葉酸鈣、氟尿嘧啶及伊立替康(irinotecan))、吉西他濱(gemcitabine)、托泊替康(topotecan)、脂質體伊立替康、培美曲塞(pemetrexed)、西妥昔單抗(cetuximab)、尼沃魯單抗(nivolumab)、伊匹單抗(ipilimumab)、匹利珠單抗(pidilizumab)、派姆單抗(pembrolizumab)、曲美木單抗(tremelimumab)、烏瑞魯單抗(urelumab)、利麗單抗(lirilumab)、阿替珠單抗(atezolizumab)、愛帕司他(epacadostat)及德瓦魯單抗(durvalumab)；更佳地，該遞送裝置或預填裝注射器或藥物可與電離輻射同時、分開或依次施用。 A use of the freeze-dried preparation according to any one of claims 1 to 8 or the liquid preparation according to any one of claims 9 to 15 for preparing the prevention and/or treatment of tumors in a subject Delivery device or pre-filled syringe or drug; for example, the tumor is cancer, preferably, the cancer is Hodgkin or non-Hodgkin’s lymphoma, melanoma, renal cell carcinoma, kidney cancer, lung cancer, bladder cancer , Stomach and esophageal cancer, colorectal cancer, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, pancreatic cancer, breast cancer, triple negative breast cancer, ovarian cancer, endometrial cancer, prostate cancer, small cell lung cancer (SCLC), non-small cell Lung cancer (NSCLC), mesothelioma, squamous cell carcinoma of the head and neck (SCCHN), soft tissue sarcoma or glioblastoma multiforme; more preferably, the lung cancer is NSCLC, small cell lung cancer or mesothelioma; preferably Wherein, the delivery device or pre-filled syringe or drug can be administered simultaneously, separately or sequentially with one or more anti-tumor agents selected from the group consisting of: cisplatin, carboplatin, Dacarbazine, liposomal doxorubicin, docetaxel, cyclophosphamide and doxorubicin, navelbine, eribulin (eribulin), paclitaxel (paclitaxel), paclitaxel protein-binding particles for injectable suspension, ixabepilone (ixabepilone), capecitabine, FOLFOX (leucovorin, fluorouracil and oxaliplatin), FOLFIRI (calcium leucovorin, fluorouracil and irinotecan) , Gemcitabine, topotecan, liposomal irinotecan, pemetrexed, cetuximab, nivolumab, ipilimumab (ipilimumab), pidilizumab, pembrolizumab, tremelimumab, urelumab, lirilumab, atezumab Monoclonal antibody (atezolizumab), epacadostat (epacadostat) and devalumumab (durvalumab); more preferably, the delivery device or pre-filled syringe or drug can be administered simultaneously, separately or sequentially with ionizing radiation. 一種預防和/或治療腫瘤的方法，包括向受試者施用有效量的如請求項1至8中任一項所述的凍乾製劑或如請求項9至15中任一項所述的液體製劑；例如，該腫瘤是癌症，較佳地，該癌症為霍奇金或非霍奇金淋巴瘤、黑色素瘤、腎細胞癌、腎癌、肺癌、膀胱癌、胃及食道癌、結腸直腸癌、肝癌、肝細胞癌、膽管癌、胰臟癌、乳癌、三陰性乳癌、卵巢癌、子宮內膜癌、***癌、小細胞肺癌(SCLC)、非小細胞肺癌(NSCLC)、間皮瘤、頭頸的鱗狀細胞癌(SCCHN)、軟組織肉瘤或多形性神經膠母細胞瘤；更較佳地，該肺癌是NSCLC、小細胞肺癌或間皮瘤。 A method for preventing and/or treating tumors, comprising administering to a subject an effective amount of the freeze-dried preparation according to any one of claims 1 to 8 or the liquid according to any one of claims 9 to 15 Preparation; for example, the tumor is cancer, preferably, the cancer is Hodgkin or non-Hodgkin's lymphoma, melanoma, renal cell carcinoma, kidney cancer, lung cancer, bladder cancer, stomach and esophageal cancer, colorectal cancer , Liver cancer, hepatocellular carcinoma, cholangiocarcinoma, pancreatic cancer, breast cancer, triple negative breast cancer, ovarian cancer, endometrial cancer, prostate cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), mesothelioma, Squamous cell carcinoma of the head and neck (SCCHN), soft tissue sarcoma or glioblastoma multiforme; more preferably, the lung cancer is NSCLC, small cell lung cancer or mesothelioma. 如請求項26所述的方法，還包括同時，分開或依次給予一種或多種抗腫瘤劑，該抗腫瘤劑選自順鉑(cisplatin)、卡鉑(carboplatin)、達卡巴嗪(dacarbazine)、脂質體多柔比星(liposomal doxorubicin)、多西他賽(docetaxel)、環磷醯胺(cyclophosphamide)及多柔比星、諾維本(navelbine)、艾瑞布林(eribulin)、太平洋紫杉醇(paclitaxel)、用於可注射懸浮液的太平洋紫杉醇蛋白質結合科粒、伊沙匹隆(ixabepilone)、卡培他濱 (capecitabine)、FOLFOX(亞葉酸鈣(leucovorin)、氟尿嘧啶(fluorouracil)及奥沙利鉑(oxaliplatin))、FOLFIRI(亞葉酸鈣、氟尿嘧啶及伊立替康(irinotecan))、吉西他濱(gemcitabine)、托泊替康(topotecan)、脂質體伊立替康、培美曲塞(pemetrexed)、西妥昔單抗(cetuximab)、尼沃鲁單抗(nivolumab)、伊匹單抗(ipilimumab)、匹利珠單抗(pidilizumab)、派姆單抗(pembrolizumab)、曲美木單抗(tremelimumab)、烏瑞鲁單抗(urelumab)、利兩單抗(lirilumab)、阿替珠單抗(atezolizumab)、愛帕司他(epacadostat)及德瓦鲁單抗(durvalumab)。 The method according to claim 26, further comprising simultaneously, separately or sequentially administering one or more anti-tumor agents selected from the group consisting of cisplatin, carboplatin, dacarbazine, lipid Liposomal doxorubicin, docetaxel, cyclophosphamide and doxorubicin, navelbine, eribulin, paclitaxel ), paclitaxel protein-binding pellets, ixabepilone, capecitabine for injectable suspension (capecitabine), FOLFOX (leucovorin, fluorouracil and oxaliplatin), FOLFIRI (calcium folinate, fluorouracil and irinotecan), gemcitabine, Topo Topotecan, liposomal irinotecan, pemetrexed, cetuximab, nivolumab, ipilimumab, pilizumab Anti-(pidilizumab), pembrolizumab, tremelimumab, urelumab, lirilumab, atezolizumab, Aipa Epacadostat and durvalumab.
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