TW202246333A - An antigen-binding molecule that specifically binds bcma and cd3 and the pharmaceutical use thereof - Google Patents
An antigen-binding molecule that specifically binds bcma and cd3 and the pharmaceutical use thereof Download PDFInfo
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Abstract
Description
本申請要求2021年01月20日提交中國專利申請(申請號CN202110076465.5)和2021年07月14日提交的中國專利申請(申請號CN202110795647.8)的優先權。 This application claims the priority of the Chinese patent application (application number CN202110076465.5) submitted on January 20, 2021 and the Chinese patent application (application number CN202110795647.8) submitted on July 14, 2021.
本揭露屬於生物技術領域，更具體地，本揭露關於抗原結合分子及其應用。 The present disclosure belongs to the field of biotechnology, more specifically, the present disclosure relates to antigen-binding molecules and applications thereof.
這裡的陳述僅提供與本揭露有關的背景信息，而不必然地構成現有技術。 The statements herein merely provide background information related to the present disclosure and do not necessarily constitute prior art.
多發性骨髓瘤(Multiple myeloma，MM)是全球第二大常見血液癌症，其以骨髓中漿細胞的失控增殖為主要特徵，癌變的漿細胞迅速增殖擴散，進而導致單株免疫球蛋白的大量產生進而引起免疫抑制、骨質溶解和末端器官損傷。 Multiple myeloma (Multiple myeloma, MM) is the second most common blood cancer in the world. It is characterized by the uncontrolled proliferation of plasma cells in the bone marrow. Cancerous plasma cells proliferate and spread rapidly, which in turn leads to a large number of single immunoglobulins produced. This in turn causes immunosuppression, osteolysis, and end-organ damage.
在全球範圍內，有超過13.85萬名新確診MM患者。過去的數十年，由於蛋白酶體抑制劑、免疫調節劑和CD38抗體等新的治療手段出現，使MM患者的臨床治療效果有很大改善，將患者的預期壽命從3至4年提高到7至8年。但大多數患者仍會因耐藥而復發。即便MRD(minimal residual disease)陰性的患者，仍會復發。因此，臨床迫切需要更加有效的新治療手段。在靶向MM細胞的同時，重塑免疫細胞的抗腫瘤活性的免疫療法將是非常優異的治療MM的療法。 Worldwide, there are more than 138,500 newly diagnosed patients with MM. In the past few decades, due to the emergence of new treatments such as proteasome inhibitors, immunomodulators and CD38 antibodies, the clinical treatment effect of MM patients has been greatly improved, and the life expectancy of patients has increased from 3 to 4 years to 7 years. to 8 years. However, most patients still relapse due to drug resistance. Even patients with negative MRD (minimal residual disease) will still relapse. Therefore, there is an urgent need for new and more effective treatments. Immunotherapy that remodels the antitumor activity of immune cells while targeting MM cells would be an excellent therapy for MM.
BCMA(tumor necrosis factor receptor superfamily member 17，TNFRS17)是一個沒有信號肽的3型跨膜蛋白，其胞外區富含半胱胺酸，與TNFR超家族因子B細胞活化因子受體(BAFF-R)和TACI共同作用，調節B細胞的增殖、以及使B細胞分化成漿細胞。這些功能性受體藉由與配體BAFF或APRIL結合以支持B細胞的長期存活。僅在記憶性B細胞後期分化成漿細胞時，BCMA才開始被誘導表達，並僅表達在漿系母細胞和已分化的漿細胞表面；而在記憶性B細胞、naïve B細胞或者CD34+造血幹細胞、以及其他正常組織等無表達。藉由對BCMA基因剔除小鼠的研究證明，BCMA僅對長期存活的漿細胞有重要作用，並不調節B細胞的自穩。因此，和在正常組織(尤其免疫細胞和免疫器官)廣泛表達的CD38比，BCMA是一個非常特異的MM抗原。 BCMA (tumor necrosis factor receptor superfamily member 17, TNFRS17) is a type 3 transmembrane protein without signal peptide, its extracellular region is rich in cysteine, and TNFR superfamily factor B cell activating factor receptor (BAFF-R ) and TACI work together to regulate the proliferation of B cells and the differentiation of B cells into plasma cells. These functional receptors support long-term survival of B cells by binding to the ligands BAFF or APRIL. Only when memory B cells differentiate into plasma cells in the late stage, BCMA begins to be induced to express, and is only expressed on the surface of plasma blasts and differentiated plasma cells; while memory B cells, naïve B cells or CD34+ hematopoietic stem cells , and other normal tissues have no expression. Studies on BCMA knockout mice have demonstrated that BCMA only plays an important role in the long-term survival of plasma cells, and does not regulate the homeostasis of B cells. Therefore, compared with CD38, which is widely expressed in normal tissues (especially immune cells and immune organs), BCMA is a very specific MM antigen.
靶向CD3/TAA的雙特異性抗體是一種新型的免疫療法，可以同時結合T細胞和腫瘤細胞，模擬MHC和TCR的相互作用，使T細胞形成裂解性突觸(synapse)後釋放穿孔素和顆粒酶，從而特異性殺傷腫瘤細胞。活化後的T細胞可以釋放細胞因子，啟動其他免疫細胞並擴大針對腫 瘤的免疫反應，最終導致T細胞的增殖和殺傷腫瘤細胞的級聯反應。因此，CD3/BCMA雙特異性抗體可作為非常優異的針對MM的免疫治療手段。 The bispecific antibody targeting CD3/TAA is a new type of immunotherapy, which can simultaneously bind T cells and tumor cells, mimic the interaction between MHC and TCR, and make T cells release perforin and granzymes, thereby specifically killing tumor cells. Activated T cells can release cytokines, activate other immune cells and expand Tumor immune response, eventually leading to the proliferation of T cells and the cascade reaction of killing tumor cells. Therefore, CD3/BCMA bispecific antibody can be used as an excellent immunotherapy for MM.
本揭露提供了一種抗原結合分子。其包含至少一個特異性結合BCMA的第一抗原結合域和至少一個特異性結合CD3的第二抗原結合域。這些抗原結合分子能夠提供相比BCMA抗體和CD3抗體更好的治療活性。 The present disclosure provides an antigen binding molecule. It comprises at least one first antigen binding domain that specifically binds BCMA and at least one second antigen binding domain that specifically binds CD3. These antigen binding molecules can provide better therapeutic activity than BCMA antibody and CD3 antibody.
在一個方面，本揭露提供了一種抗原結合分子，其包含至少一個特異性結合BCMA的第一抗原結合域和至少一個特異性結合CD3的第二抗原結合域，該特異性結合BCMA的第一抗原結合域包含重鏈可變區BCMA-VH和輕鏈可變區BCMA-VL，其中， In one aspect, the present disclosure provides an antigen binding molecule comprising at least one first antigen binding domain that specifically binds BCMA and at least one second antigen binding domain that specifically binds CD3, the first antigen that specifically binds BCMA The binding domain comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, wherein,
(i)該BCMA-VH中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3分別包含SEQ ID NO：29中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的胺基酸序列；和該BCMA-VL中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3分別包含SEQ ID NO：30中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的胺基酸序列；或 (i) BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in the BCMA-VH respectively comprise the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 29; and the BCMA- BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in VL comprise the amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in SEQ ID NO: 30, respectively; or
(ii)該BCMA-VH中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3分別包含SEQ ID NO：31中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的胺基酸序列；和該BCMA-VL中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3分別包含SEQ ID NO：32中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的胺基酸序列；或 (ii) BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in the BCMA-VH respectively comprise the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 31; and the BCMA- BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in VL comprise the amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in SEQ ID NO: 32, respectively; or
(iii)該BCMA-VH中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3分別包含SEQ ID NO：33中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的胺基酸序列；和該BCMA-VL中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3分別包含SEQ ID NO：34中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的胺基酸序列；或 (iii) BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in the BCMA-VH respectively comprise the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 33; and the BCMA- BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in VL comprise the amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in SEQ ID NO: 34, respectively; or
(iv)該BCMA-VH中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3分別包含SEQ ID NO：35中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的胺基酸序列；和該BCMA-VL中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3分別包含SEQ ID NO：36中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的胺基酸序列。 (iv) BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in the BCMA-VH respectively comprise the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 35; and the BCMA- BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in VL comprise the amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in SEQ ID NO:36, respectively.
在一些實施方案中，該BCMA-HCDR1、BCMA-HCDR2、BCMA-HCDR3、BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3根據Kabat、IMGT、Chothia、AbM或Contact編號規則定義。 In some embodiments, the BCMA-HCDR1, BCMA-HCDR2, BCMA-HCDR3, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering convention.
在一個方面，本揭露提供了一種抗原結合分子，其包含至少一個特異性結合BCMA的第一抗原結合域和至少一個特異性結合CD3的第二抗原結合域，該特異性結合BCMA的第一抗原結合域包含重鏈可變區BCMA-VH和輕鏈可變區BCMA-VL，其中， In one aspect, the present disclosure provides an antigen binding molecule comprising at least one first antigen binding domain that specifically binds BCMA and at least one second antigen binding domain that specifically binds CD3, the first antigen that specifically binds BCMA The binding domain comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, wherein,
該重鏈可變區BCMA-VH具有BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3，並且該輕鏈可變區BCMA-VL具有BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3，其中， The heavy chain variable region BCMA-VH has BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3, and the light chain variable region BCMA-VL has BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3, wherein,
(i)該BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3分別包含SEQ ID NO：29中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的胺基酸序列；和該BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3 分別包含SEQ ID NO：30中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的胺基酸序列；或 (i) the BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 respectively comprise the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 29; and the BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 respectively comprising the amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in SEQ ID NO: 30; or
(ii)該BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3分別包含SEQ ID NO：31中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的胺基酸序列；和該BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3分別包含SEQ ID NO：32中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的胺基酸序列；或 (ii) the BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 respectively comprise the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 31; and the BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 respectively comprise the amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in SEQ ID NO: 32; or
(iii)該BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3分別包含SEQ ID NO：33中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的胺基酸序列；和該BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3分別包含SEQ ID NO：34中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的胺基酸序列；或 (iii) the BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 respectively comprise the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 33; and the BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 respectively comprise the amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in SEQ ID NO: 34; or
(iv)該BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3分別包含SEQ ID NO：35中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的胺基酸序列；和該BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3分別包含SEQ ID NO：36中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的胺基酸序列； (iv) the BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 respectively comprise the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 35; and the BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 respectively comprise the amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in SEQ ID NO: 36;
該BCMA-HCDR1、BCMA-HCDR2、BCMA-HCDR3、BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3根據Kabat、IMGT、Chothia、AbM或Contact編號規則定義。 The BCMA-HCDR1, BCMA-HCDR2, BCMA-HCDR3, BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering convention.
在一個方面，本揭露提供了一種抗原結合分子，其包含至少一個特異性結合BCMA的第一抗原結合域和至少一個特異性結合CD3的第二抗原結合域，該特異性結合BCMA的第一抗原結合域包含重鏈可變區BCMA-VH和輕鏈可變區BCMA-VL，其中， In one aspect, the present disclosure provides an antigen binding molecule comprising at least one first antigen binding domain that specifically binds BCMA and at least one second antigen binding domain that specifically binds CD3, the first antigen that specifically binds BCMA The binding domain comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, wherein,
(i)該重鏈可變區BCMA-VH包含SEQ ID NO：29中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的胺基酸序列，並且該輕鏈可變區BCMA-VL包含SEQ ID NO：30中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的胺基酸序列；或 (i) the heavy chain variable region BCMA-VH comprises the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 29, and the light chain variable region BCMA-VL comprises SEQ ID Amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in NO:30; or
(ii)該重鏈可變區BCMA-VH包含SEQ ID NO：31中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的胺基酸序列，並且該輕鏈可變區BCMA-VL包含SEQ ID NO：32中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的胺基酸序列；或 (ii) the heavy chain variable region BCMA-VH comprises the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 31, and the light chain variable region BCMA-VL comprises SEQ ID Amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in NO:32; or
(iii)該重鏈可變區BCMA-VH包含SEQ ID NO：33中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的胺基酸序列，並且該輕鏈可變區BCMA-VL包含SEQ ID NO：34中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的胺基酸序列；或 (iii) the heavy chain variable region BCMA-VH comprises the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 33, and the light chain variable region BCMA-VL comprises SEQ ID Amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in NO:34; or
(iv)該重鏈可變區BCMA-VH包含SEQ ID NO：35中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的胺基酸序列，並且該輕鏈可變區BCMA-VL包含SEQ ID NO：36中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的胺基酸序列； (iv) The heavy chain variable region BCMA-VH comprises the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 35, and the light chain variable region BCMA-VL comprises SEQ ID Amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in NO:36;
該BCMA-HCDR1、BCMA-HCDR2、BCMA-HCDR3、BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3根據Kabat、IMGT、Chothia、AbM或Contact編號規則定義。 The BCMA-HCDR1, BCMA-HCDR2, BCMA-HCDR3, BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering convention.
在一個方面，本揭露提供了一種抗原結合分子，其包含至少一個特異性結合BCMA的第一抗原結合域和至少一個特異性結合CD3的第二抗原結合域，該特異性結合BCMA的第一抗原結合域包含重鏈可變區BCMA-VH和輕鏈可變區BCMA-VL，其中， In one aspect, the present disclosure provides an antigen binding molecule comprising at least one first antigen binding domain that specifically binds BCMA and at least one second antigen binding domain that specifically binds CD3, the first antigen that specifically binds BCMA The binding domain comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, wherein,
(i)該重鏈可變區BCMA-VH具有：包含SEQ ID NO：5的胺基酸序列的BCMA-HCDR1，包含SEQ ID NO：6的胺基酸序列的BCMA-HCDR2，和包含SEQ ID NO：7的胺基酸序列的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：包含SEQ ID NO：8的胺基酸序列的BCMA-LCDR1，包含SEQ ID NO：9的胺基酸序列的BCMA-LCDR2，和包含SEQ ID NO：10的胺基酸序列的BCMA-LCDR3；或 (i) The heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 5, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 6, and comprising SEQ ID BCMA-HCDR3 of the amino acid sequence of NO: 7; and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 8, comprising the amino group of SEQ ID NO: 9 BCMA-LCDR2 of the acid sequence, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 10; or
(ii)該重鏈可變區BCMA-VH具有：包含SEQ ID NO：11的胺基酸序列的BCMA-HCDR1，包含SEQ ID NO：12的胺基酸序列的BCMA-HCDR2，和包含SEQ ID NO：13的胺基酸序列的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：包含SEQ ID NO：14的胺基酸序列的BCMA-LCDR1，包含SEQ ID NO：15的胺基酸序列的BCMA-LCDR2，和包含SEQ ID NO：16的胺基酸序列的BCMA-LCDR3；或 (ii) the heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 11, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and comprising SEQ ID BCMA-HCDR3 of the amino acid sequence of NO: 13; and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 14, comprising the amino group of SEQ ID NO: 15 BCMA-LCDR2 of the acid sequence, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 16; or
(iii)該重鏈可變區BCMA-VH具有：包含SEQ ID NO：17的胺基酸序列的BCMA-HCDR1，包含SEQ ID NO：18的胺基酸序列的BCMA-HCDR2，和包含SEQ ID NO：19的胺基酸序列的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：包含SEQ ID NO：20的胺基酸序列的BCMA-LCDR1，包含SEQ ID NO：21的胺基酸序列的BCMA-LCDR2，和包含SEQ ID NO：22的胺基酸序列的BCMA-LCDR3；或 (iii) the heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 17, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, and comprising SEQ ID BCMA-HCDR3 of the amino acid sequence of NO: 19; and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, comprising the amino group of SEQ ID NO: 21 BCMA-LCDR2 of the acid sequence, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 22; or
(iv)該重鏈可變區BCMA-VH具有：包含SEQ ID NO：23的胺基酸序列的BCMA-HCDR1，包含SEQ ID NO：24的胺基酸序列的BCMA-HCDR2，和包含SEQ ID NO：25的胺基酸序列的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：包含SEQ ID NO：26的胺基酸序列的BCMA-LCDR1，包含SEQ ID NO：27的胺基酸序列的BCMA-LCDR2，和包含SEQ ID NO：28的胺基酸序列的BCMA-LCDR3； (iv) The heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 23, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 24, and comprising SEQ ID NO: BCMA-HCDR3 of the amino acid sequence of 25; and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 26, comprising the amino group of SEQ ID NO: 27 The BCMA-LCDR2 of the acid sequence, and the BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 28;
該BCMA-HCDR1、BCMA-HCDR2、BCMA-HCDR3、BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3是根據Kabat編號規則定義的。 The BCMA-HCDR1, BCMA-HCDR2, BCMA-HCDR3, BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 are defined according to the Kabat numbering convention.
在一些實施方案中，如前所述的抗原結合分子，其中， In some embodiments, the antigen-binding molecule as described above, wherein,
(i)該重鏈可變區BCMA-VH具有：包含SEQ ID NO：5的胺基酸序列的BCMA-HCDR1，包含SEQ ID NO：6的胺基酸序列的BCMA-HCDR2，和包含SEQ ID NO：7的胺基酸序列的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：包含SEQ ID NO：8的胺基酸序列的BCMA-LCDR1，包含SEQ ID NO：9的胺基酸序列的BCMA-LCDR2，和包含SEQ ID NO：10的胺基酸序列的BCMA-LCDR3。在一些實施方案中，該重鏈可變區BCMA-VH和/或輕鏈可變區BCMA-VL是鼠源的或人源化的。在一些實施方案中，該重鏈可變區BCMA-VH和/或輕鏈可變區BCMA-VL是人源化的。 (i) The heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 5, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 6, and comprising SEQ ID BCMA-HCDR3 of the amino acid sequence of NO: 7; and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 8, comprising the amino group of SEQ ID NO: 9 BCMA-LCDR2 with an amino acid sequence, and BCMA-LCDR3 with an amino acid sequence of SEQ ID NO:10. In some embodiments, the heavy chain variable region BCMA-VH and/or the light chain variable region BCMA-VL is murine or humanized. In some embodiments, the heavy chain variable region BCMA-VH and/or the light chain variable region BCMA-VL is humanized.
在一些實施方案中，該重鏈可變區BCMA-VH具有來源於IGHV1-46*01的重鏈框架區，並且其是未被取代的或具有選自48I、67A、71A、73K、76T和93V組成的組中的一個或多個胺基酸取代；和/或該輕鏈可變區BCMA-VL具有來源於IGKV1-39*01的輕鏈框架區，並且其是未被取代的或具有選自43S、45Q、48V和71Y組成的組中的一個或多個胺基酸取代。在一些實施方案中，該BCMA-VH具有來源於IGHV1-46*01的FR1-3和來源於IGHJ6*01的FR4，並且其是未被取代的或具有選自1E、48I、67A、71A、73K、76T和93V組成的組中的一個或多個胺基酸取代；和/或該BCMA-VL具有來源於IGKV1-39*01的FR1-3和來源於IGKJ2*01的FR4，並且其是未被取代的或具有選自43S、45Q、48V和 71Y組成的組中的一個或多個胺基酸取代。在一些實施方案中，上述可變區和CDR是根據Kabat編號規則定義的。 In some embodiments, the heavy chain variable region BCMA-VH has a heavy chain framework region derived from IGHV1-46*01 and is unsubstituted or has a heavy chain framework region selected from 48I, 67A, 71A, 73K, 76T and One or more amino acid substitutions in the group consisting of 93V; and/or the light chain variable region BCMA-VL has a light chain framework region derived from IGKV1-39*01, and it is unsubstituted or has One or more amino acid substitutions selected from the group consisting of 43S, 45Q, 48V and 71Y. In some embodiments, the BCMA-VH has FR1-3 derived from IGHV1-46*01 and FR4 derived from IGHJ6*01, and is unsubstituted or has a compound selected from 1E, 48I, 67A, 71A, One or more amino acid substitutions in the group consisting of 73K, 76T and 93V; and/or the BCMA-VL has FR1-3 derived from IGKV1-39*01 and FR4 derived from IGKJ2*01, and it is Unsubstituted or with selected from 43S, 45Q, 48V and One or more amino acid substitutions in the group consisting of 71Y. In some embodiments, the variable regions and CDRs described above are defined according to the Kabat numbering convention.
在一些實施方案中，如前所述的抗原結合分子，其中， In some embodiments, the antigen-binding molecule as described above, wherein,
(i)該重鏈可變區BCMA-VH包含與SEQ ID NO：29具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列，和該輕鏈可變區BCMA-VL包含與SEQ ID NO：30具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列。在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：29的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：30的胺基酸序列。 (i) the heavy chain variable region BCMA-VH comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 29, and the The light chain variable region BCMA-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:30. In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO:29, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:30.
在一些實施方案中，如前所述的抗原結合分子，其中， In some embodiments, the antigen-binding molecule as described above, wherein,
(i)該重鏈可變區BCMA-VH包含與選自由SEQ ID NO：37、SEQ ID NO：38和SEQ ID NO：39組成的組的胺基酸序列分別具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列，和該輕鏈可變區BCMA-VL包含與選自由SEQ ID NO：40、SEQ ID NO：41、SEQ ID NO：42和SEQ ID NO：43組成的組的胺基酸序列分別具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列。在一些實施方案中，該重鏈可變區BCMA-VH包含選自由SEQ ID NO：37、SEQ ID NO：38和SEQ ID NO：39組成的組的胺基酸序列，和該輕鏈可變區BCMA-VL包含選自由SEQ ID NO：40、SEQ ID NO：41、SEQ ID NO：42和SEQ ID NO：43組成的組的胺基酸序列。在一些實施方案中： (i) the heavy chain variable region BCMA-VH comprises amino acid sequences selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 have at least 90%, 95%, Amino acid sequences with 96%, 97%, 98% or 99% sequence identity, and the light chain variable region BCMA-VL comprising and selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO Amino acid sequences of the group consisting of SEQ ID NO: 42 and SEQ ID NO: 43 having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity, respectively. In some embodiments, the heavy chain variable region BCMA-VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39, and the light chain variable Region BCMA-VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43. In some embodiments:
該重鏈可變區BCMA-VH包含SEQ ID NO：37的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：40的胺基酸序列，或 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 37, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 40, or
該重鏈可變區BCMA-VH包含SEQ ID NO：38的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：40的胺基酸序列，或 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 38, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 40, or
該重鏈可變區BCMA-VH包含SEQ ID NO：39的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：40的胺基酸序列，或 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 39, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 40, or
該重鏈可變區BCMA-VH包含SEQ ID NO：37的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：41的胺基酸序列，或 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 37, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 41, or
該重鏈可變區BCMA-VH包含SEQ ID NO：37的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：42的胺基酸序列，或 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 37, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 42, or
該重鏈可變區BCMA-VH包含SEQ ID NO：37的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：43的胺基酸序列。 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO:37, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:43.
在一些實施方案中，如前所述的抗原結合分子，其中， In some embodiments, the antigen-binding molecule as described above, wherein,
(ii)該重鏈可變區BCMA-VH具有：包含SEQ ID NO：11的胺基酸序列的BCMA-HCDR1，包含SEQ ID NO：12的胺基酸序列的BCMA-HCDR2，和包含SEQ ID NO：13的胺基酸序列的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：包含SEQ ID NO：14的胺基酸序列的BCMA-LCDR1，包含SEQ ID NO：15的胺基酸序列的BCMA-LCDR2，和包含SEQ ID NO：16的胺基酸序列的BCMA-LCDR3。在一些實施方案中，該重鏈可變區BCMA-VH和/或輕鏈可變區BCMA-VL是鼠源的或人源化的。在一些實施方案中，該重鏈可變區BCMA-VH和/或輕鏈可變區BCMA-VL是人源化的。在一些實施方案中，該重鏈可變區BCMA-VH具有來源於IGHV7-4-1*02的重鏈框架區，並且其是未被取代的或具有選自2I、44V、45F、46K、75A、76N和93L組成的組中的一個或多個胺基酸取代；和/或該輕鏈可變區BCMA-VL具有來源於IGKV1-8*01或IGKV1-27*01的輕鏈框架區，並且其是未被取代的或具有選自43S和66D組成的組中的一個或多個胺基酸取代。在一些實施方案中，該BCMA-VH具有來源於IGHV7-4-1*02的FR1-3和來源於IGHJ1*01的FR4，並且其是未被 取代的或具有選自1E、21I、44V、45F、46K、75A、76N和93L組成的組中的一個或多個胺基酸取代；和/或該BCMA-VL具有來源於IGKV1-8*01或IGKV1-27*01的FR1-3和來源於IGKJ4*01的FR4，並且其是未被取代的或具有選自43S和66D組成的組中的一個或多個胺基酸取代。在一些實施方案中，上述可變區和CDR是根據Kabat編號規則定義的。 (ii) the heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 11, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and comprising SEQ ID BCMA-HCDR3 of the amino acid sequence of NO: 13; and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 14, comprising the amino group of SEQ ID NO: 15 BCMA-LCDR2 with an amino acid sequence, and BCMA-LCDR3 with an amino acid sequence of SEQ ID NO:16. In some embodiments, the heavy chain variable region BCMA-VH and/or the light chain variable region BCMA-VL is murine or humanized. In some embodiments, the heavy chain variable region BCMA-VH and/or the light chain variable region BCMA-VL is humanized. In some embodiments, the heavy chain variable region BCMA-VH has a heavy chain framework region derived from IGHV7-4-1*02, and it is unsubstituted or has a structure selected from 2I, 44V, 45F, 46K, One or more amino acid substitutions in the group consisting of 75A, 76N and 93L; and/or the light chain variable region BCMA-VL has a light chain framework region derived from IGKV1-8*01 or IGKV1-27*01 , and it is unsubstituted or substituted with one or more amino acids selected from the group consisting of 43S and 66D. In some embodiments, the BCMA-VH has FR1-3 derived from IGHV7-4-1*02 and FR4 derived from IGHJ1*01, and is unmodified Substituted or with one or more amino acid substitutions selected from the group consisting of 1E, 21I, 44V, 45F, 46K, 75A, 76N, and 93L; and/or the BCMA-VL has a protein derived from IGKV1-8*01 Or FR1-3 of IGKV1-27*01 and FR4 derived from IGKJ4*01, which are unsubstituted or substituted with one or more amino acids selected from the group consisting of 43S and 66D. In some embodiments, the variable regions and CDRs described above are defined according to the Kabat numbering convention.
在一些實施方案中，如前所述的抗原結合分子，其中， In some embodiments, the antigen-binding molecule as described above, wherein,
(ii)該重鏈可變區BCMA-VH包含與SEQ ID NO：31具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列，和該輕鏈可變區BCMA-VL包含與SEQ ID NO：32具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列。在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：31的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：32的胺基酸序列。 (ii) the heavy chain variable region BCMA-VH comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 31, and the The light chain variable region BCMA-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:32. In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO:31, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:32.
在一些實施方案中，如前所述的抗原結合分子，其中， In some embodiments, the antigen-binding molecule as described above, wherein,
(ii)該重鏈可變區BCMA-VH包含與選自由SEQ ID NO：44、SEQ ID NO：45和SEQ ID NO：46組成的組的胺基酸序列分別具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列，和該輕鏈可變區BCMA-VL包含與選自由SEQ ID NO：47和SEQ ID NO：48組成的組的胺基酸序列分別具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列。在一些實施方案中，該重鏈可變區BCMA-VH包含選自由SEQ ID NO：44、SEQ ID NO：45和SEQ ID NO：46組成的組的胺基酸序列，和該輕鏈可變區BCMA-VL包含選自由SEQ ID NO：47和SEQ ID NO：48組成的組的胺基酸序列。在一些實施方案中： (ii) the heavy chain variable region BCMA-VH comprises at least 90%, 95%, respectively, An amino acid sequence with 96%, 97%, 98% or 99% sequence identity, and the light chain variable region BCMA-VL comprising and selected from the group consisting of SEQ ID NO: 47 and SEQ ID NO: 48 Amino acid sequences Amino acid sequences having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity, respectively. In some embodiments, the heavy chain variable region BCMA-VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46, and the light chain variable Region BCMA-VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO:47 and SEQ ID NO:48. In some embodiments:
該重鏈可變區BCMA-VH包含SEQ ID NO：44的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：47的胺基酸序列，或 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 44, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 47, or
該重鏈可變區BCMA-VH包含SEQ ID NO：45的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：47的胺基酸序列，或 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 45, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 47, or
該重鏈可變區BCMA-VH包含SEQ ID NO：46的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：47的胺基酸序列，或 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 46, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 47, or
該重鏈可變區BCMA-VH包含SEQ ID NO：44的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：48的胺基酸序列，或 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 44, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 48, or
該重鏈可變區BCMA-VH包含SEQ ID NO：45的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：48的胺基酸序列，或 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 45, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 48, or
該重鏈可變區BCMA-VH包含SEQ ID NO：46的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：48的胺基酸序列。 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO:46, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:48.
在一些實施方案中，如前所述的抗原結合分子，其中， In some embodiments, the antigen-binding molecule as described above, wherein,
(iii)該重鏈可變區BCMA-VH具有：包含SEQ ID NO：17的胺基酸序列的BCMA-HCDR1，包含SEQ ID NO：18的胺基酸序列的BCMA-HCDR2，和包含SEQ ID NO：19的胺基酸序列的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：包含SEQ ID NO：20的胺基酸序列的BCMA-LCDR1，包含SEQ ID NO：21的胺基酸序列的BCMA-LCDR2，和包含SEQ ID NO：22的胺基酸序列的BCMA-LCDR3；在一些實施方案中，該重鏈可變區BCMA-VH和/或輕鏈可變區BCMA-VL是鼠源的或人源化的。在一些實施方案中，該重鏈可變區BCMA-VH和/或輕鏈可變區BCMA-VL是人源化的。在一些實施方案中，該重鏈可變區BCMA-VH具有來源於IGHV3-11*01的重鏈框架區，並且其是未被取代的或具有選自30R、47R、49A和93T組成的組中的一個或多個胺基酸取代；和/或該輕鏈可變區BCMA-VL具有來源於IGKV1-39*01的輕鏈框架區，並且其是未被取代的或具有44V的胺基酸取代。在一些實施方案中，該BCMA-VH 具有來源於IGHV3-11*01的FR1-3和來源於IGHJ1*01的FR4，並且其是未被取代的或具有選自1E、30R、47R、49A和93T組成的組中的一個或多個胺基酸取代；和/或該BCMA-VL具有來源於IGKV1-39*01的FR1-3和來源於IGKJ2*01的FR4，並且其是未被取代的或具有44V的胺基酸取代。在一些實施方案中，上述可變區和CDR是根據Kabat編號規則定義的。 (iii) the heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 17, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, and comprising SEQ ID BCMA-HCDR3 of the amino acid sequence of NO: 19; and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, comprising the amino group of SEQ ID NO: 21 The BCMA-LCDR2 of the acid sequence, and the BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 22; In some embodiments, the heavy chain variable region BCMA-VH and/or the light chain variable region BCMA-VL Be murine or humanized. In some embodiments, the heavy chain variable region BCMA-VH and/or the light chain variable region BCMA-VL is humanized. In some embodiments, the heavy chain variable region BCMA-VH has a heavy chain framework region derived from IGHV3-11*01 and is unsubstituted or has a group selected from the group consisting of 30R, 47R, 49A and 93T One or more amino acid substitutions in; and/or the light chain variable region BCMA-VL has a light chain framework region derived from IGKV1-39*01, and it is unsubstituted or has an amino group of 44V Acid substitution. In some embodiments, the BCMA-VH have FR1-3 derived from IGHV3-11*01 and FR4 derived from IGHJ1*01, and which are unsubstituted or have one or more selected from the group consisting of 1E, 30R, 47R, 49A and 93T Amino acid substitution; and/or the BCMA-VL has FR1-3 derived from IGKV1-39*01 and FR4 derived from IGKJ2*01, and it is unsubstituted or has an amino acid substitution of 44V. In some embodiments, the variable regions and CDRs described above are defined according to the Kabat numbering convention.
在一些的實施方案中，如前所述的抗原結合分子，其中， In some embodiments, the antigen-binding molecule as described above, wherein,
(iii)該重鏈可變區BCMA-VH包含與SEQ ID NO：33具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列，和該輕鏈可變區BCMA-VL包含與SEQ ID NO：34具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列。在一些的實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：33的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：34的胺基酸序列。 (iii) the heavy chain variable region BCMA-VH comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 33, and the The light chain variable region BCMA-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:34. In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO:33, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:34.
在一些實施方案中，如前所述的抗原結合分子，其中， In some embodiments, the antigen-binding molecule as described above, wherein,
(iii)該重鏈可變區BCMA-VH包含與選自由SEQ ID NO：49和SEQ ID NO：50組成的組的胺基酸序列分別具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列，和該輕鏈可變區BCMA-VL包含與SEQ ID NO：51具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列。在一些實施方案中，該重鏈可變區BCMA-VH包含選自由SEQ ID NO：49和SEQ ID NO：50組成的組的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：51的胺基酸序列。在一些實施方案中， (iii) the heavy chain variable region BCMA-VH comprises at least 90%, 95%, 96%, 97%, An amino acid sequence of 98% or 99% sequence identity, and the light chain variable region BCMA-VL comprises at least 90%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO:51 Amino acid sequences with % sequence identity. In some embodiments, the heavy chain variable region BCMA-VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 49 and SEQ ID NO: 50, and the light chain variable region BCMA-VL comprises SEQ ID NO: 49 and SEQ ID NO: 50 Amino acid sequence of ID NO:51. In some embodiments,
該重鏈可變區BCMA-VH包含SEQ ID NO：49的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：51的胺基酸序列，或 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 49, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 51, or
該重鏈可變區BCMA-VH包含SEQ ID NO：50的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：51的胺基酸序列。 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO:50, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:51.
在一些實施方案中，如前所述的抗原結合分子，其中， In some embodiments, the antigen-binding molecule as described above, wherein,
(iv)該重鏈可變區BCMA-VH具有：包含SEQ ID NO：23的胺基酸序列的BCMA-HCDR1，包含SEQ ID NO：24的胺基酸序列的BCMA-HCDR2，和包含SEQ ID NO：25的胺基酸序列的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：包含SEQ ID NO：26的胺基酸序列的BCMA-LCDR1，包含SEQ ID NO：27的胺基酸序列的BCMA-LCDR2，和包含SEQ ID NO：28的胺基酸序列的BCMA-LCDR3；在一些實施方案中，該重鏈可變區BCMA-VH和/或輕鏈可變區BCMA-VL是鼠源的或人源化的。在一些實施方案中，該重鏈可變區BCMA-VH和/或輕鏈可變區BCMA-VL是人源化的。在一些實施方案中，該重鏈可變區BCMA-VH具有來源於IGHV2-70*01的重鏈框架區，並且其是未被取代的或具有選自24V、30T、37V、49G、73N、80F和89R組成的組中的一個或多個胺基酸取代；和/或該輕鏈可變區BCMA-VL具有來源於IGKV1-33*01的輕鏈框架區。在一些實施方案中，該BCMA-VH具有來源於IGHV2-70*01的FR1-3和來源於IGHJ6*01的FR4，並且其是未被取代的或具有選自1E、24V、30T、37V、49G、73N、80F和89R組成的組中的一個或多個胺基酸取代；和/或該BCMA-VL具有來源於IGKV1-33*01的FR1-3和來源於IGKJ2*01的FR4。在一些實施方案中，上述可變區和CDR是根據Kabat編號規則定義的。 (iv) The heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 23, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 24, and comprising SEQ ID NO: BCMA-HCDR3 of the amino acid sequence of 25; and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 26, comprising the amino group of SEQ ID NO: 27 The BCMA-LCDR2 of the acid sequence, and the BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 28; In some embodiments, the heavy chain variable region BCMA-VH and/or the light chain variable region BCMA-VL Be murine or humanized. In some embodiments, the heavy chain variable region BCMA-VH and/or the light chain variable region BCMA-VL is humanized. In some embodiments, the heavy chain variable region BCMA-VH has a heavy chain framework region derived from IGHV2-70*01, and it is unsubstituted or has a structure selected from 24V, 30T, 37V, 49G, 73N, One or more amino acid substitutions in the group consisting of 80F and 89R; and/or the light chain variable region BCMA-VL has a light chain framework region derived from IGKV1-33*01. In some embodiments, the BCMA-VH has FR1-3 derived from IGHV2-70*01 and FR4 derived from IGHJ6*01, and is unsubstituted or has a compound selected from 1E, 24V, 30T, 37V, One or more amino acid substitutions in the group consisting of 49G, 73N, 80F and 89R; and/or the BCMA-VL has FR1-3 derived from IGKV1-33*01 and FR4 derived from IGKJ2*01. In some embodiments, the variable regions and CDRs described above are defined according to the Kabat numbering convention.
在一些的實施方案中，如前所述的抗原結合分子，其中， In some embodiments, the antigen-binding molecule as described above, wherein,
(iv)該重鏈可變區BCMA-VH包含與SEQ ID NO：35具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列，和該輕鏈可變 區BCMA-VL包含與SEQ ID NO：36具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列。在一些的實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：35的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：36的胺基酸序列。 (iv) the heavy chain variable region BCMA-VH comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 35, and the variable light chain Region BCMA-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:36. In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO:35, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:36.
在一些實施方案中，如前所述的抗原結合分子，其中， In some embodiments, the antigen-binding molecule as described above, wherein,
(iv)該重鏈可變區BCMA-VH包含與選自由SEQ ID NO：52和SEQ ID NO：53組成的組的胺基酸序列分別具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列，和該輕鏈可變區BCMA-VL包含與SEQ ID NO：54分別具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列。在一些實施方案中，該重鏈可變區BCMA-VH包含選自由SEQ ID NO：52和SEQ ID NO：53組成的組的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：54的胺基酸序列。在一些實施方案中， (iv) the heavy chain variable region BCMA-VH comprises at least 90%, 95%, 96%, 97%, An amino acid sequence with 98% or 99% sequence identity, and the light chain variable region BCMA-VL comprises at least 90%, 95%, 96%, 97%, 98%, or Amino acid sequences with 99% sequence identity. In some embodiments, the heavy chain variable region BCMA-VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 52 and SEQ ID NO: 53, and the light chain variable region BCMA-VL comprises SEQ ID NO: 52 and SEQ ID NO: 53 Amino acid sequence of ID NO:54. In some embodiments,
該重鏈可變區BCMA-VH包含SEQ ID NO：52的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：54的胺基酸序列，或 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 52, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 54, or
該重鏈可變區BCMA-VH包含SEQ ID NO：53的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：54的胺基酸序列。 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO:53, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:54.
在一些實施方案中，如前任一項所述的抗原結合分子，其中該特異性結合CD3的第二抗原結合域包含重鏈可變區CD3-VH和輕鏈可變區CD3-VL，其中， In some embodiments, the antigen-binding molecule of any one of the preceding, wherein the second antigen-binding domain that specifically binds CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, wherein,
(i)該CD3-VH中的CD3-HCDR1、CD3-HCDR2和CD3-HCDR3分別包含SEQ ID NO：63中的CD3-HCDR1、CD3-HCDR2和CD3-HCDR3的胺基酸序列；和該CD3-VL中的CD3-LCDR1、CD3-LCDR2和CD3- LCDR3分別包含SEQ ID NO：64中的CD3-LCDR1、CD3-LCDR2和CD3-LCDR3的胺基酸序列；或 (i) CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in the CD3-VH respectively comprise the amino acid sequences of CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in SEQ ID NO: 63; and the CD3- CD3-LCDR1, CD3-LCDR2 and CD3-LCDR1 in VL LCDR3 respectively comprises the amino acid sequences of CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 in SEQ ID NO: 64; or
(ii)該CD3-VH中的CD3-HCDR1、CD3-HCDR2和CD3-HCDR3分別包含SEQ ID NO：65中的CD3-HCDR1、CD3-HCDR2和CD3-HCDR3的胺基酸序列；和該CD3-VL中的CD3-LCDR1、CD3-LCDR2和CD3-LCDR3分別包含SEQ ID NO：66中的CD3-LCDR1、CD3-LCDR2和CD3-LCDR3的胺基酸序列。 (ii) CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in the CD3-VH respectively comprise the amino acid sequences of CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in SEQ ID NO: 65; and the CD3- CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 in VL comprise the amino acid sequences of CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 in SEQ ID NO:66, respectively.
在一些實施方案中，該CD3-HCDR1、CD3-HCDR2、CD3-HCDR3、CD3-LCDR1、CD3-LCDR2和CD3-LCDR3根據Kabat、IMGT、Chothia、AbM或Contact編號規則定義。 In some embodiments, the CD3-HCDR1, CD3-HCDR2, CD3-HCDR3, CD3-LCDR1, CD3-LCDR2, and CD3-LCDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering convention.
在一些實施方案中，如前任一項所述的抗原結合分子，其中該特異性結合CD3的第二抗原結合域包含重鏈可變區CD3-VH和輕鏈可變區CD3-VL，其中， In some embodiments, the antigen-binding molecule of any one of the preceding, wherein the second antigen-binding domain that specifically binds CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, wherein,
該重鏈可變區CD3-VH具有CD3-HCDR1、CD3-HCDR2和CD3-HCDR3，並且該輕鏈可變區CD3-VL具有CD3-LCDR1、CD3-LCDR2和CD3-LCDR3，其中， The heavy chain variable region CD3-VH has CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3, and the light chain variable region CD3-VL has CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3, wherein,
(i)該CD3-HCDR1、CD3-HCDR2和CD3-HCDR3分別包含SEQ ID NO：63中的CD3-HCDR1、CD3-HCDR2和CD3-HCDR3的胺基酸序列；和該CD3-LCDR1、CD3-LCDR2和CD3-LCDR3分別包含SEQ ID NO：64中的CD3-LCDR1、CD3-LCDR2和CD3-LCDR3的胺基酸序列；或 (i) the CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 respectively comprise the amino acid sequences of CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in SEQ ID NO: 63; and the CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 respectively comprising the amino acid sequences of CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 in SEQ ID NO: 64; or
(ii)該CD3-HCDR1、CD3-HCDR2和CD3-HCDR3分別包含SEQ ID NO：65中的CD3-HCDR1、CD3-HCDR2和CD3-HCDR3的胺基酸序列；和該CD3-LCDR1、CD3-LCDR2和CD3-LCDR3分別包含SEQ ID NO：66中的CD3-LCDR1、CD3-LCDR2和CD3-LCDR3的胺基酸序列； (ii) the CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 respectively comprise the amino acid sequences of CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in SEQ ID NO: 65; and the CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 respectively comprise the amino acid sequences of CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 in SEQ ID NO:66;
該CD3-HCDR1、CD3-HCDR2、CD3-HCDR3、CD3-LCDR1、CD3-LCDR2和CD3-LCDR3根據Kabat、IMGT、Chothia、AbM或Contact編號規則定義。 The CD3-HCDR1, CD3-HCDR2, CD3-HCDR3, CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering convention.
在一些實施方案中，如前任一項所述的抗原結合分子，其中該特異性結合CD3的第二抗原結合域包含重鏈可變區CD3-VH和輕鏈可變區CD3-VL，其中， In some embodiments, the antigen-binding molecule of any one of the preceding, wherein the second antigen-binding domain that specifically binds CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, wherein,
(i)該重鏈可變區CD3-VH包含SEQ ID NO：63中的CD3-HCDR1、CD3-HCDR2和CD3-HCDR3的胺基酸序列，並且該輕鏈可變區CD3-VL包含SEQ ID NO：64中的CD3-LCDR1、CD3-LCDR2和CD3-LCDR3的胺基酸序列；或 (i) the heavy chain variable region CD3-VH comprises the amino acid sequences of CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in SEQ ID NO: 63, and the light chain variable region CD3-VL comprises SEQ ID Amino acid sequences of CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 in NO:64; or
(ii)該重鏈可變區CD3-VH包含SEQ ID NO：65中的CD3-HCDR1、CD3-HCDR2和CD3-HCDR3的胺基酸序列，並且該輕鏈可變區CD3-VL包含SEQ ID NO：66中的CD3-LCDR1、CD3-LCDR2和CD3-LCDR3的胺基酸序列； (ii) the heavy chain variable region CD3-VH comprises the amino acid sequences of CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in SEQ ID NO: 65, and the light chain variable region CD3-VL comprises SEQ ID Amino acid sequences of CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 in NO:66;
該CD3-HCDR1、CD3-HCDR2、CD3-HCDR3、CD3-LCDR1、CD3-LCDR2和CD3-LCDR3根據Kabat、IMGT、Chothia、AbM或Contact編號規則定義。 The CD3-HCDR1, CD3-HCDR2, CD3-HCDR3, CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering convention.
在一些實施方案中，如前任一項所述的抗原結合分子，其中該特異性結合CD3的第二抗原結合域包含重鏈可變區CD3-VH和輕鏈可變區CD3-VL，其中， In some embodiments, the antigen-binding molecule of any one of the preceding, wherein the second antigen-binding domain that specifically binds CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, wherein,
(i)該重鏈可變區CD3-VH具有：包含SEQ ID NO：55的胺基酸序列的CD3-HCDR1，包含SEQ ID NO：56的胺基酸序列的CD3-HCDR2，和包含SEQ ID NO：57的胺基酸序列的CD3-HCDR3；並且該輕鏈可變區CD3-VL具有：包含SEQ ID NO：58的胺基酸序列的CD3-LCDR1，包含 SEQ ID NO：59的胺基酸序列的CD3-LCDR2，和包含SEQ ID NO：60的胺基酸序列的CD3-LCDR3；或 (i) the heavy chain variable region CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 55, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 56, and comprising SEQ ID CD3-HCDR3 of the amino acid sequence of NO: 57; and the light chain variable region CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 58, comprising CD3-LCDR2 of the amino acid sequence of SEQ ID NO: 59, and CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 60; or
(ii)該重鏈可變區CD3-VH具有：包含SEQ ID NO：55的胺基酸序列的CD3-HCDR1，包含SEQ ID NO：61的胺基酸序列的CD3-HCDR2，和包含SEQ ID NO：62的胺基酸序列的CD3-HCDR3；並且該輕鏈可變區CD3-VL具有：包含SEQ ID NO：58的胺基酸序列的CD3-LCDR1，包含SEQ ID NO：59的胺基酸序列的CD3-LCDR2，和包含SEQ ID NO：60的胺基酸序列的CD3-LCDR3； (ii) the heavy chain variable region CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 55, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 61, and comprising SEQ ID NO: CD3-HCDR3 of the amino acid sequence of 62; and the light chain variable region CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 58, comprising the amino group of SEQ ID NO: 59 The CD3-LCDR2 of the acid sequence, and the CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO:60;
該CD3-HCDR1、CD3-HCDR2、CD3-HCDR3、CD3-LCDR1、CD3-LCDR2和CD3-LCDR3是根據Kabat編號規則定義的。 The CD3-HCDR1, CD3-HCDR2, CD3-HCDR3, CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 are defined according to the Kabat numbering convention.
在一些實施方案中，如前任一項所述的抗原結合分子，其中，該重鏈可變區CD3-VH包含與SEQ ID NO：63具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列，和該輕鏈可變區CD3-VL包含與SEQ ID NO：64具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列；在一些實施方案中，該重鏈可變區CD3-VH包含SEQ ID NO：63的胺基酸序列，和該輕鏈可變區CD3-VL包含SEQ ID NO：64的胺基酸序列。 In some embodiments, the antigen binding molecule according to any one of the preceding, wherein the heavy chain variable region CD3-VH comprises at least 90%, 95%, 96%, 97%, 98% of SEQ ID NO: 63 An amino acid sequence of % or 99% sequence identity, and the light chain variable region CD3-VL comprises at least 90%, 95%, 96%, 97%, 98% or 99% of SEQ ID NO:64 the amino acid sequence of sequence identity; in some embodiments, the heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO: 63, and the light chain variable region CD3-VL comprises SEQ ID NO: Amino acid sequence of 64.
在一些實施方案中，如前任一項所述的抗原結合分子，其中，該重鏈可變區CD3-VH包含與SEQ ID NO：65具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列，和該輕鏈可變區CD3-VL包含與SEQ ID NO：66具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列；在一些實施方案中，該重鏈可變區CD3-VH包含SEQ ID NO：65的胺基酸序列，和該輕鏈可變區CD3-VL包含SEQ ID NO：66的胺基酸序列。 In some embodiments, the antigen binding molecule according to any one of the preceding, wherein the heavy chain variable region CD3-VH comprises at least 90%, 95%, 96%, 97%, 98% of SEQ ID NO: 65 The amino acid sequence of % or 99% sequence identity, and the light chain variable region CD3-VL comprises at least 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO:66 amino acid sequence of sequence identity; in some embodiments, the heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO: 65, and the light chain variable region CD3-VL comprises SEQ ID NO: Amino acid sequence of 66.
在一些實施方案中，如前任一項所述的抗原結合分子，其中該重鏈可變區BCMA-VH具有：包含SEQ ID NO：5的胺基酸序列的BCMA-HCDR1，包含SEQ ID NO：6的胺基酸序列的BCMA-HCDR2，和包含SEQ ID NO：7的胺基酸序列的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：包含SEQ ID NO：8的胺基酸序列的BCMA-LCDR1，包含SEQ ID NO：9的胺基酸序列的BCMA-LCDR2，和包含SEQ ID NO：10的胺基酸序列的BCMA-LCDR3，和 In some embodiments, the antigen-binding molecule of any one of the preceding, wherein the heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 5, comprising SEQ ID NO: BCMA-HCDR2 having an amino acid sequence of 6, and BCMA-HCDR3 comprising an amino acid sequence of SEQ ID NO: 7; and the light chain variable region BCMA-VL has: comprising an amino acid of SEQ ID NO: 8 BCMA-LCDR1 of the sequence, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 9, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 10, and
該重鏈可變區CD3-VH具有：包含SEQ ID NO：55的胺基酸序列的CD3-HCDR1，包含SEQ ID NO：56的胺基酸序列的CD3-HCDR2，和包含SEQ ID NO：57的胺基酸序列的CD3-HCDR3；並且該輕鏈可變區CD3-VL具有：包含SEQ ID NO：58的胺基酸序列的CD3-LCDR1，包含SEQ ID NO：59的胺基酸序列的CD3-LCDR2，和包含SEQ ID NO：60的胺基酸序列的CD3-LCDR3。在一些實施方案中，上述CDR是根據Kabat編號規則定義的。 The heavy chain variable region CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO:55, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:56, and comprising SEQ ID NO:57 and the light chain variable region CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 58, comprising the amino acid sequence of SEQ ID NO: 59 CD3-LCDR2, and CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO:60. In some embodiments, the aforementioned CDRs are defined according to the Kabat numbering convention.
在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：37的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：40的胺基酸序列，和 In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 37, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 40, and
該重鏈可變區CD3-VH包含SEQ ID NO：63的胺基酸序列，和該輕鏈可變區CD3-VL包含SEQ ID NO：64的胺基酸序列。 The heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO:63, and the light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:64.
在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：38的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：40的胺基酸序列，和 In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 38, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 40, and
該重鏈可變區CD3-VH包含SEQ ID NO：63的胺基酸序列，和該輕鏈可變區CD3-VL包含SEQ ID NO：64的胺基酸序列。 The heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO:63, and the light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:64.
在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：39的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：40的胺基酸序列，和 In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 39, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 40, and
該重鏈可變區CD3-VH包含SEQ ID NO：63的胺基酸序列，和該輕鏈可變區CD3-VL包含SEQ ID NO：64的胺基酸序列。 The heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO:63, and the light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:64.
在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：37的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：41的胺基酸序列，和 In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 37, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 41, and
該重鏈可變區CD3-VH包含SEQ ID NO：63的胺基酸序列，和該輕鏈可變區CD3-VL包含SEQ ID NO：64的胺基酸序列。 The heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO:63, and the light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:64.
在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：37的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：42的胺基酸序列，和 In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 37, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 42, and
該重鏈可變區CD3-VH包含SEQ ID NO：63的胺基酸序列，和該輕鏈可變區CD3-VL包含SEQ ID NO：64的胺基酸序列。 The heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO:63, and the light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:64.
在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：37的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：43的胺基酸序列，和 In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 37, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 43, and
該重鏈可變區CD3-VH包含SEQ ID NO：63的胺基酸序列，和該輕鏈可變區CD3-VL包含SEQ ID NO：64的胺基酸序列。 The heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO:63, and the light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:64.
在一些實施方案中，如前任一項所述的抗原結合分子，其中該重鏈可變區BCMA-VH具有：包含SEQ ID NO：5的胺基酸序列的BCMA-HCDR1，包含SEQ ID NO：6的胺基酸序列的BCMA-HCDR2，和包含SEQ ID NO：7的胺基酸序列的BCMA-HCDR3；並且該輕鏈可變 區BCMA-VL具有：包含SEQ ID NO：8的胺基酸序列的BCMA-LCDR1，包含SEQ ID NO：9的胺基酸序列的BCMA-LCDR2，和包含SEQ ID NO：10的胺基酸序列的BCMA-LCDR3，和 In some embodiments, the antigen-binding molecule of any one of the preceding, wherein the heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 5, comprising SEQ ID NO: BCMA-HCDR2 having an amino acid sequence of 6, and BCMA-HCDR3 comprising an amino acid sequence of SEQ ID NO: 7; and the light chain is variable Region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:8, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:9, and comprising the amino acid sequence of SEQ ID NO:10 BCMA-LCDR3, and
該重鏈可變區CD3-VH具有：包含SEQ ID NO：55的胺基酸序列的CD3-HCDR1，包含SEQ ID NO：61的胺基酸序列的CD3-HCDR2，和包含SEQ ID NO：62的胺基酸序列的CD3-HCDR3；並且該輕鏈可變區CD3-VL具有：包含SEQ ID NO：58的胺基酸序列的CD3-LCDR1，包含SEQ ID NO：59的胺基酸序列的CD3-LCDR2，和包含SEQ ID NO：60的胺基酸序列的CD3-LCDR3。在一些實施方案中，上述CDR是根據Kabat編號規則定義的。 The heavy chain variable region CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 55, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 61, and comprising SEQ ID NO: 62 and the light chain variable region CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 58, comprising the amino acid sequence of SEQ ID NO: 59 CD3-LCDR2, and CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO:60. In some embodiments, the aforementioned CDRs are defined according to the Kabat numbering convention.
在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：37的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：43的胺基酸序列，和 In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 37, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 43, and
該重鏈可變區CD3-VH包含SEQ ID NO：65的胺基酸序列，和該輕鏈可變區CD3-VL包含SEQ ID NO：66的胺基酸序列。 The heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO:65, and the light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:66.
在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：38的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：40的胺基酸序列，和 In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 38, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 40, and
該重鏈可變區CD3-VH包含SEQ ID NO：65的胺基酸序列，和該輕鏈可變區CD3-VL包含SEQ ID NO：66的胺基酸序列。 The heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO:65, and the light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:66.
在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：39的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：40的胺基酸序列，和 In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 39, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 40, and
該重鏈可變區CD3-VH包含SEQ ID NO：65的胺基酸序列，和該輕鏈可變區CD3-VL包含SEQ ID NO：66的胺基酸序列。 The heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO:65, and the light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:66.
在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：37的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：41的胺基酸序列，和 In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 37, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 41, and
該重鏈可變區CD3-VH包含SEQ ID NO：65的胺基酸序列，和該輕鏈可變區CD3-VL包含SEQ ID NO：66的胺基酸序列。 The heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO:65, and the light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:66.
在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：37的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：42的胺基酸序列，和 In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 37, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 42, and
該重鏈可變區CD3-VH包含SEQ ID NO：65的胺基酸序列，和該輕鏈可變區CD3-VL包含SEQ ID NO：66的胺基酸序列。 The heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO:65, and the light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:66.
在一些實施方案中，如前任一項所述的抗原結合分子，其中該重鏈可變區BCMA-VH具有：包含SEQ ID NO：11的胺基酸序列的BCMA-HCDR1，包含SEQ ID NO：12的胺基酸序列的BCMA-HCDR2，和包含SEQ ID NO：13的胺基酸序列的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：包含SEQ ID NO：14的胺基酸序列的BCMA-LCDR1，包含SEQ ID NO：15的胺基酸序列的BCMA-LCDR2，和包含SEQ ID NO：16的胺基酸序列的BCMA-LCDR3，和 In some embodiments, the antigen-binding molecule of any one of the preceding, wherein the heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 11, comprising SEQ ID NO: BCMA-HCDR2 having an amino acid sequence of 12, and BCMA-HCDR3 comprising an amino acid sequence of SEQ ID NO: 13; and the light chain variable region BCMA-VL has: comprising an amino acid of SEQ ID NO: 14 BCMA-LCDR1 of the sequence, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 16, and
該重鏈可變區CD3-VH具有：包含SEQ ID NO：55的胺基酸序列的CD3-HCDR1，包含SEQ ID NO：56的胺基酸序列的CD3-HCDR2，和包含SEQ ID NO：57的胺基酸序列的CD3-HCDR3；並且該輕鏈可變區CD3-VL具有：包含SEQ ID NO：58的胺基酸序列的CD3-LCDR1，包含SEQ ID NO：59的胺基酸序列的CD3-LCDR2，和包含SEQ ID NO：60的胺基酸序列的CD3-LCDR3。 The heavy chain variable region CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO:55, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:56, and comprising SEQ ID NO:57 CD3-HCDR3 of the amino acid sequence; and the light chain variable region CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 58, comprising SEQ CD3-LCDR2 having the amino acid sequence of ID NO:59, and CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO:60.
在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：44的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：47的胺基酸序列，和 In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 44, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 47, and
該重鏈可變區CD3-VH包含SEQ ID NO：63的胺基酸序列，和該輕鏈可變區CD3-VL包含SEQ ID NO：64的胺基酸序列。 The heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO:63, and the light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:64.
在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：45的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：47的胺基酸序列，和 In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 45, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 47, and
該重鏈可變區CD3-VH包含SEQ ID NO：63的胺基酸序列，和該輕鏈可變區CD3-VL包含SEQ ID NO：64的胺基酸序列。 The heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO:63, and the light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:64.
在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：46的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：47的胺基酸序列，和 In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 46, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 47, and
該重鏈可變區CD3-VH包含SEQ ID NO：63的胺基酸序列，和該輕鏈可變區CD3-VL包含SEQ ID NO：64的胺基酸序列。 The heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO:63, and the light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:64.
在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：44的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：48的胺基酸序列，和 In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 44, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 48, and
該重鏈可變區CD3-VH包含SEQ ID NO：63的胺基酸序列，和該輕鏈可變區CD3-VL包含SEQ ID NO：64的胺基酸序列。 The heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO:63, and the light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:64.
在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：45的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：48的胺基酸序列，和 In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 45, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 48, and
該重鏈可變區CD3-VH包含SEQ ID NO：63的胺基酸序列，和該輕鏈可變區CD3-VL包含SEQ ID NO：64的胺基酸序列。 The heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO:63, and the light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:64.
在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：46的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：48的胺基酸序列，和 In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 46, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 48, and
該重鏈可變區CD3-VH包含SEQ ID NO：63的胺基酸序列，和該輕鏈可變區CD3-VL包含SEQ ID NO：64的胺基酸序列。 The heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO:63, and the light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:64.
在一些實施方案中，如前任一項所述的抗原結合分子，其中該重鏈可變區BCMA-VH具有：包含SEQ ID NO：17的胺基酸序列的BCMA-HCDR1，包含SEQ ID NO：18的胺基酸序列的BCMA-HCDR2，和包含SEQ ID NO：19的胺基酸序列的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：包含SEQ ID NO：20的胺基酸序列的BCMA-LCDR1，包含SEQ ID NO：21的胺基酸序列的BCMA-LCDR2，和包含SEQ ID NO：22的胺基酸序列的BCMA-LCDR3，和 In some embodiments, the antigen-binding molecule of any one of the preceding, wherein the heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 17, comprising SEQ ID NO: BCMA-HCDR2 having an amino acid sequence of 18, and BCMA-HCDR3 comprising an amino acid sequence of SEQ ID NO: 19; and the light chain variable region BCMA-VL has: comprising an amino acid of SEQ ID NO: 20 BCMA-LCDR1 of the sequence, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 21, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 22, and
該重鏈可變區CD3-VH具有：包含SEQ ID NO：55的胺基酸序列的CD3-HCDR1，包含SEQ ID NO：56的胺基酸序列的CD3-HCDR2，和包含SEQ ID NO：57的胺基酸序列的CD3-HCDR3；並且該輕鏈可變區CD3-VL具有：包含SEQ ID NO：58的胺基酸序列的CD3-LCDR1，包含SEQ ID NO：59的胺基酸序列的CD3-LCDR2，和包含SEQ ID NO：60的胺基酸序列的CD3-LCDR3。 The heavy chain variable region CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO:55, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:56, and comprising SEQ ID NO:57 and the light chain variable region CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 58, comprising the amino acid sequence of SEQ ID NO: 59 CD3-LCDR2, and CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO:60.
在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：49的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：51的胺基酸序列，和 In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 49, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 51, and
該重鏈可變區CD3-VH包含SEQ ID NO：63的胺基酸序列，和該輕鏈可變區CD3-VL包含SEQ ID NO：64的胺基酸序列。 The heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO:63, and the light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:64.
在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：50的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：51的胺基酸序列，和 In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 50, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 51, and
該重鏈可變區CD3-VH包含SEQ ID NO：63的胺基酸序列，和該輕鏈可變區CD3-VL包含SEQ ID NO：64的胺基酸序列。 The heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO:63, and the light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:64.
在一些實施方案中，如前任一項所述的抗原結合分子，其中該重鏈可變區BCMA-VH具有：該重鏈可變區BCMA-VH具有：包含SEQ ID NO：17的胺基酸序列的BCMA-HCDR1，包含SEQ ID NO：18的胺基酸序列的BCMA-HCDR2，和包含SEQ ID NO：19的胺基酸序列的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：包含SEQ ID NO：20的胺基酸序列的BCMA-LCDR1，包含SEQ ID NO：21的胺基酸序列的BCMA-LCDR2，和包含SEQ ID NO：22的胺基酸序列的BCMA-LCDR3，和 In some embodiments, the antigen-binding molecule of any one of the preceding, wherein the heavy chain variable region BCMA-VH has: the heavy chain variable region BCMA-VH has: an amino acid comprising SEQ ID NO: 17 sequence BCMA-HCDR1, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, and BCMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 19; and the light chain variable region BCMA-VL has : BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 21, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 22, with
該重鏈可變區CD3-VH具有：包含SEQ ID NO：55的胺基酸序列的CD3-HCDR1，包含SEQ ID NO：61的胺基酸序列的CD3-HCDR2，和包含SEQ ID NO：62的胺基酸序列的CD3-HCDR3；並且該輕鏈可變區CD3-VL具有：包含SEQ ID NO：58的胺基酸序列的CD3-LCDR1，包含SEQ ID NO：59的胺基酸序列的CD3-LCDR2，和包含SEQ ID NO：60的胺基酸序列的CD3-LCDR3。 The heavy chain variable region CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 55, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 61, and comprising SEQ ID NO: 62 and the light chain variable region CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 58, comprising the amino acid sequence of SEQ ID NO: 59 CD3-LCDR2, and CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO:60.
在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：49的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：51的胺基酸序列，和 In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 49, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 51, and
該重鏈可變區CD3-VH包含SEQ ID NO：65的胺基酸序列，和該輕鏈可變區CD3-VL包含SEQ ID NO：66的胺基酸序列。 The heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO:65, and the light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:66.
在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：50的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：51的胺基酸序列，和 In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 50, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 51, and
該重鏈可變區CD3-VH包含SEQ ID NO：65的胺基酸序列，和該輕鏈可變區CD3-VL包含SEQ ID NO：66的胺基酸序列。 The heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO:65, and the light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:66.
在一些實施方案中，如前任一項所述的抗原結合分子，其中該重鏈可變區BCMA-VH具有：該重鏈可變區BCMA-VH具有：包含SEQ ID NO：23的胺基酸序列的BCMA-HCDR1，包含SEQ ID NO：24的胺基酸序列的BCMA-HCDR2，和包含SEQ ID NO：25的胺基酸序列的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：包含SEQ ID NO：26的胺基酸序列的BCMA-LCDR1，包含SEQ ID NO：27的胺基酸序列的BCMA-LCDR2，和包含SEQ ID NO：28的胺基酸序列的BCMA-LCDR3，和 In some embodiments, the antigen-binding molecule of any one of the preceding, wherein the heavy chain variable region BCMA-VH has: the heavy chain variable region BCMA-VH has: an amino acid comprising SEQ ID NO: 23 sequence BCMA-HCDR1, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 24, and BCMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 25; and the light chain variable region BCMA-VL has : BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 26, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 27, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 28, with
該重鏈可變區CD3-VH具有：包含SEQ ID NO：55的胺基酸序列的CD3-HCDR1，包含SEQ ID NO：56的胺基酸序列的CD3-HCDR2，和包含SEQ ID NO：57的胺基酸序列的CD3-HCDR3；並且該輕鏈可變區CD3-VL具有：包含SEQ ID NO：58的胺基酸序列的CD3-LCDR1，包含SEQ ID NO：59的胺基酸序列的CD3-LCDR2，和包含SEQ ID NO：60的胺基酸序列的CD3-LCDR3。 The heavy chain variable region CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO:55, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:56, and comprising SEQ ID NO:57 and the light chain variable region CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 58, comprising the amino acid sequence of SEQ ID NO: 59 CD3-LCDR2, and CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO:60.
在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：52的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：54的胺基酸序列，和 In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO:52, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:54, and
該重鏈可變區CD3-VH包含SEQ ID NO：63的胺基酸序列，和該輕鏈可變區CD3-VL包含SEQ ID NO：64的胺基酸序列。 The heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO:63, and the light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:64.
在一些實施方案中，如前任一項所述的抗原結合分子，其中該重鏈可變區BCMA-VH具有：該重鏈可變區BCMA-VH具有：包含SEQ ID NO：17的胺基酸序列的BCMA-HCDR1，包含SEQ ID NO：18的胺基酸序列的BCMA-HCDR2，和包含SEQ ID NO：19的胺基酸序列的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：包含SEQ ID NO：20的胺基酸序列的BCMA-LCDR1，包含SEQ ID NO：21的胺基酸序列的BCMA-LCDR2，和包含SEQ ID NO：22的胺基酸序列的BCMA-LCDR3，和 In some embodiments, the antigen-binding molecule of any one of the preceding, wherein the heavy chain variable region BCMA-VH has: the heavy chain variable region BCMA-VH has: an amino acid comprising SEQ ID NO: 17 sequence BCMA-HCDR1, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, and BCMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 19; and the light chain variable region BCMA-VL has : BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 21, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 22, with
該重鏈可變區CD3-VH具有：包含SEQ ID NO：55的胺基酸序列的CD3-HCDR1，包含SEQ ID NO：61的胺基酸序列的CD3-HCDR2，和包含SEQ ID NO：62的胺基酸序列的CD3-HCDR3；並且該輕鏈可變區CD3-VL具有：包含SEQ ID NO：58的胺基酸序列的CD3-LCDR1，包含SEQ ID NO：59的胺基酸序列的CD3-LCDR2，和包含SEQ ID NO：60的胺基酸序列的CD3-LCDR3。 The heavy chain variable region CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 55, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 61, and comprising SEQ ID NO: 62 and the light chain variable region CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 58, comprising the amino acid sequence of SEQ ID NO: 59 CD3-LCDR2, and CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO:60.
在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：52的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：54的胺基酸序列，和 In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO:52, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:54, and
該重鏈可變區CD3-VH包含SEQ ID NO：65的胺基酸序列，和該輕鏈可變區CD3-VL包含SEQ ID NO：66的胺基酸序列。 The heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO:65, and the light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:66.
在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：53的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：54的胺基酸序列，和 In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 53, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 54, and
該重鏈可變區CD3-VH包含SEQ ID NO：65的胺基酸序列，和該輕鏈可變區CD3-VL包含SEQ ID NO：66的胺基酸序列。 The heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO:65, and the light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:66.
在一些實施方案中，如前任一項所述的抗原結合分子，其中該抗原結合分子還包含Fc區，該Fc區包含能夠締合的兩個亞基。在一些實施方案中，該兩個亞基是相同或不同的第一亞基和第二亞基。在一些實施方案中，該Fc區是IgG Fc區，特別是IgG1 Fc區。在一些實施方案中，該Fc區包含一個或多個胺基酸取代，該胺基酸取代能夠減少其與Fc受體的結合。特別地，該胺基酸取代能夠減少其與Fcγ受體的結合。在一些實施方案中，該Fc區相比野生型Fc區，包含一個或多個胺基酸取代，該胺基酸取代能夠減少與Fc受體的結合，特別是與Fcγ受體的結合。在一些實施方案中，該Fc區是人IgG1 Fc區，並且在234和235位置的胺基酸殘基為A，編號依據為EU索引。在一些實施方案中，該Fc區包含SEQ ID NO：69的胺基酸序列。 In some embodiments, the antigen binding molecule of any one of the preceding, wherein the antigen binding molecule further comprises an Fc region comprising two subunits capable of associating. In some embodiments, the two subunits are the same or different first and second subunits. In some embodiments, the Fc region is an IgG Fc region, particularly an IgG 1 Fc region. In some embodiments, the Fc region comprises one or more amino acid substitutions that reduce its binding to an Fc receptor. In particular, this amino acid substitution can reduce its binding to Fcγ receptors. In some embodiments, the Fc region comprises one or more amino acid substitutions that reduce binding to Fc receptors, particularly Fcγ receptors, compared to wild-type Fc regions. In some embodiments, the Fc region is a human IgG 1 Fc region, and the amino acid residues at positions 234 and 235 are A, numbered according to the EU index. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO:69.
在一些實施方案中，如前任一項所述的抗原結合分子，其包含至少兩個特異性結合BCMA的第一抗原結合域、至少兩個特異性結合CD3的第二抗原結合域和Fc區。在一些實施方案中，其包含兩個特異性結合BCMA的第一抗原結合域、兩個特異性結合CD3的第二抗原結合域和一個Fc區。 In some embodiments, the antigen-binding molecule of any one of the preceding items, comprising at least two first antigen-binding domains that specifically bind BCMA, at least two second antigen-binding domains that specifically bind CD3, and an Fc region. In some embodiments, it comprises two first antigen binding domains that specifically bind BCMA, two second antigen binding domains that specifically bind CD3, and an Fc region.
在一些實施方案中，該特異性結合BCMA的第一抗原結合域是Fab，和/或該特異性結合CD3的第二抗原結合域是scFv。在一些實施方案中，該特異性結合BCMA的第一抗原結合域是Fab，和/或該特異性結 合CD3的第二抗原結合域是Fab。在一些實施方案中，該特異性結合BCMA的第一抗原結合域是scFv，和/或該特異性結合CD3的第二抗原結合域是scFv。在一些實施方案中，該特異性結合BCMA的第一抗原結合域是scFv，和/或該特異性結合CD3的第二抗原結合域是Fab。 In some embodiments, the first antigen binding domain that specifically binds BCMA is a Fab, and/or the second antigen binding domain that specifically binds CD3 is a scFv. In some embodiments, the first antigen binding domain that specifically binds BCMA is a Fab, and/or the specific binding The second antigen binding domain of CD3 is Fab. In some embodiments, the first antigen binding domain that specifically binds BCMA is a scFv, and/or the second antigen binding domain that specifically binds CD3 is a scFv. In some embodiments, the first antigen binding domain that specifically binds BCMA is a scFv, and/or the second antigen binding domain that specifically binds CD3 is a Fab.
在一些實施方案中，該特異性結合BCMA的第一抗原結合域、該特異性結合CD3的第二抗原結合域、和Fc區的一個亞基是以任意的順序偶聯的。在一些實施方案中，該特異性結合BCMA的第一抗原結合域、該特異性結合CD3的第二抗原結合域、和Fc區的一個亞基按N端至C端的順序直接或藉由連接子連接。在一些實施方案中，該特異性結合CD3的第二抗原結合域、該特異性結合BCMA的第一抗原結合域、和Fc區的一個亞基按N端至C端的順序直接或藉由連接子連接。在一些實施方案中，該特異性結合BCMA的第一抗原結合域、Fc區的一個亞基、該特異性結合CD3的第二抗原結合域按N端至C端的順序直接或藉由連接子連接。在一些實施方案中，該特異性結合CD3的第二抗原結合域、Fc區的一個亞基、該特異性結合BCMA的第一抗原結合域按N端至C端的順序直接或藉由連接子連接。 In some embodiments, the first antigen binding domain that specifically binds BCMA, the second antigen binding domain that specifically binds CD3, and a subunit of the Fc region are coupled in any order. In some embodiments, the first antigen-binding domain that specifically binds BCMA, the second antigen-binding domain that specifically binds CD3, and a subunit of the Fc region are in N-terminal to C-terminal order directly or via a linker connect. In some embodiments, the second antigen-binding domain that specifically binds CD3, the first antigen-binding domain that specifically binds BCMA, and a subunit of the Fc region are in N-terminal to C-terminal order directly or via a linker connect. In some embodiments, the first antigen-binding domain that specifically binds BCMA, a subunit of the Fc region, and the second antigen-binding domain that specifically binds CD3 are connected directly or via a linker in the order from N-terminal to C-terminal . In some embodiments, the second antigen-binding domain that specifically binds CD3, a subunit of the Fc region, and the first antigen-binding domain that specifically binds BCMA are connected directly or via a linker in the order from N-terminal to C-terminal .
在一些實施方案中，如前任一項所述的抗原結合分子，其包含兩個特異性結合BCMA的第一抗原結合域、兩個特異性結合CD3的第二抗原結合域和Fc區；其中該特異性結合BCMA的第一抗原結合域是Fab，該特異性結合CD3的第二抗原結合域是scFv。在一些實施方案中，該特異性結合BCMA的第一抗原結合域、該特異性結合CD3的第二抗原結合域和Fc區的一個亞基按N端至C端的順序直接或藉由連接子連接。在一些實施方案中，該Fab重鏈的C端直接或藉由連接子融合至該scFv 的N端。在一些實施方案中，該Fab的重鏈、所scFv和Fc區的一個亞基按N端至C端的順序直接或藉由連接子連接。 In some embodiments, the antigen-binding molecule of any one of the preceding, comprising two first antigen-binding domains that specifically bind BCMA, two second antigen-binding domains that specifically bind CD3, and an Fc region; wherein the The first antigen binding domain that specifically binds BCMA is a Fab and the second antigen binding domain that specifically binds CD3 is a scFv. In some embodiments, the first antigen-binding domain that specifically binds BCMA, the second antigen-binding domain that specifically binds CD3, and a subunit of the Fc region are connected directly or via a linker in the order from N-terminus to C-terminus . In some embodiments, the C-terminus of the Fab heavy chain is fused to the scFv directly or via a linker the N-terminus. In some embodiments, the heavy chain of the Fab, the scFv and a subunit of the Fc region are connected in N-terminal to C-terminal order directly or via a linker.
在一些實施方案中，如前任一項所述的抗原結合分子，其中該抗原結合分子包含兩條具有式(a)所示結構的第一鏈和兩條具有式(b)所示結構的第二鏈， In some embodiments, the antigen-binding molecule according to any one of the preceding, wherein the antigen-binding molecule comprises two first chains having a structure represented by formula (a) and two first chains having a structure represented by formula (b) second chain,
(a)[BCMA-VH]-[CH1]-[CD3-VH]-[連接子]-[CD3-VL]-[連接子]-[Fc區的一個亞基]；式(a)中的連接子較佳為相同或不同的肽連接子； (a) [BCMA-VH]-[CH1]-[CD3-VH]-[linker]-[CD3-VL]-[linker]-[a subunit of the Fc region]; in formula (a) The linkers are preferably the same or different peptide linkers;
(b)[BCMA-VL]-[CL]。 (b) [BCMA-VL]-[CL].
在一些實施方案中，該式(a)和式(b)各自是從N端到C端排列的。 In some embodiments, the formula (a) and formula (b) are each arranged from N-terminus to C-terminus.
在一些實施方案中，該肽連接子是柔性肽連接子。在一些實施方案中，該肽連接子的長度為3-15個胺基酸殘基。在一些實施方案中，該肽連接子獨立的具有L1-(GGGGS)n-L2的結構，其中，L1是鍵、A、GS、GGS或GGGS(SEQ ID NO：177)，n是0、1、2、3、4、5、6、7、8、9或10，L2是鍵、G、GG、GGG或GGGG(SEQ ID NO：177)。在一些實施方案中，該肽連接子不是鍵。在一些實施方案中，該肽連接子是GGGGSGGGGSGGGGS(SEQ ID NO：155)或GGG(SEQ ID NO：156)。在一些實施方案中，該第一鏈具有如下的結構： In some embodiments, the peptide linker is a flexible peptide linker. In some embodiments, the peptide linker is 3-15 amino acid residues in length. In some embodiments, the peptide linker independently has the structure L 1 -(GGGGS)nL 2 , wherein L 1 is a bond, A, GS, GGS or GGGS (SEQ ID NO: 177), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, L2 is a bond, G, GG, GGG or GGGG (SEQ ID NO: 177). In some embodiments, the peptide linker is not a bond. In some embodiments, the peptide linker is GGGGSGGGGSGGGGS (SEQ ID NO: 155) or GGG (SEQ ID NO: 156). In some embodiments, the first strand has the structure:
[BCMA-VH]-[CH1]-[CD3-VH]-[GGGGSGGGGSGGGGS]-[CD3-VL]-[GGG]-[Fc區的一個亞基]。 [BCMA-VH]-[CH1]-[CD3-VH]-[GGGGSGGGGSGGGGS]-[CD3-VL]-[GGG]-[a subunit of the Fc region].
在一些實施方案中，如前任一項所述的抗原結合分子，其具有： In some embodiments, the antigen-binding molecule of any one of the preceding items, which has:
包含SEQ ID NO：76的胺基酸序列的第一鏈，和包含SEQ ID NO：72的胺基酸序列的第二鏈；或 A first strand comprising the amino acid sequence of SEQ ID NO: 76, and a second strand comprising the amino acid sequence of SEQ ID NO: 72; or
包含SEQ ID NO：77的胺基酸序列的第一鏈，和包含SEQ ID NO：72的胺基酸序列的第二鏈；或 A first strand comprising the amino acid sequence of SEQ ID NO: 77, and a second strand comprising the amino acid sequence of SEQ ID NO: 72; or
包含SEQ ID NO：78的胺基酸序列的第一鏈，和包含SEQ ID NO：72的胺基酸序列的第二鏈；或 A first strand comprising the amino acid sequence of SEQ ID NO: 78, and a second strand comprising the amino acid sequence of SEQ ID NO: 72; or
包含SEQ ID NO：76的胺基酸序列的第一鏈，和包含SEQ ID NO：73的胺基酸序列的第二鏈；或 A first strand comprising the amino acid sequence of SEQ ID NO: 76, and a second strand comprising the amino acid sequence of SEQ ID NO: 73; or
包含SEQ ID NO：76的胺基酸序列的第一鏈，和包含SEQ ID NO：74的胺基酸序列的第二鏈；或 A first strand comprising the amino acid sequence of SEQ ID NO: 76, and a second strand comprising the amino acid sequence of SEQ ID NO: 74; or
包含SEQ ID NO：76的胺基酸序列的第一鏈，和包含SEQ ID NO：75的胺基酸序列的第二鏈；或 A first strand comprising the amino acid sequence of SEQ ID NO: 76, and a second strand comprising the amino acid sequence of SEQ ID NO: 75; or
包含SEQ ID NO：80的胺基酸序列的第一鏈，和包含SEQ ID NO：72的胺基酸序列的第二鏈；或 A first strand comprising the amino acid sequence of SEQ ID NO: 80, and a second strand comprising the amino acid sequence of SEQ ID NO: 72; or
包含SEQ ID NO：81的胺基酸序列的第一鏈，和包含SEQ ID NO：72的胺基酸序列的第二鏈；或 A first strand comprising the amino acid sequence of SEQ ID NO: 81, and a second strand comprising the amino acid sequence of SEQ ID NO: 72; or
包含SEQ ID NO：79的胺基酸序列的第一鏈，和包含SEQ ID NO：73的胺基酸序列的第二鏈；或 A first strand comprising the amino acid sequence of SEQ ID NO: 79, and a second strand comprising the amino acid sequence of SEQ ID NO: 73; or
包含SEQ ID NO：79的胺基酸序列的第一鏈，和包含SEQ ID NO：74的胺基酸序列的第二鏈；或 A first strand comprising the amino acid sequence of SEQ ID NO: 79, and a second strand comprising the amino acid sequence of SEQ ID NO: 74; or
包含SEQ ID NO：79的胺基酸序列的第一鏈，和包含SEQ ID NO：75的胺基酸序列的第二鏈；或 A first strand comprising the amino acid sequence of SEQ ID NO: 79, and a second strand comprising the amino acid sequence of SEQ ID NO: 75; or
包含SEQ ID NO：84的胺基酸序列的第一鏈，和包含SEQ ID NO：82的胺基酸序列的第二鏈；或 A first strand comprising the amino acid sequence of SEQ ID NO: 84, and a second strand comprising the amino acid sequence of SEQ ID NO: 82; or
包含SEQ ID NO：85的胺基酸序列的第一鏈，和包含SEQ ID NO：82的胺基酸序列的第二鏈；或 A first strand comprising the amino acid sequence of SEQ ID NO: 85, and a second strand comprising the amino acid sequence of SEQ ID NO: 82; or
包含SEQ ID NO：86的胺基酸序列的第一鏈，和包含SEQ ID NO：82的胺基酸序列的第二鏈；或 A first strand comprising the amino acid sequence of SEQ ID NO: 86, and a second strand comprising the amino acid sequence of SEQ ID NO: 82; or
包含SEQ ID NO：84的胺基酸序列的第一鏈，和包含SEQ ID NO：83的胺基酸序列的第二鏈；或 A first strand comprising the amino acid sequence of SEQ ID NO: 84, and a second strand comprising the amino acid sequence of SEQ ID NO: 83; or
包含SEQ ID NO：85的胺基酸序列的第一鏈，和包含SEQ ID NO：83的胺基酸序列的第二鏈；或 A first strand comprising the amino acid sequence of SEQ ID NO: 85, and a second strand comprising the amino acid sequence of SEQ ID NO: 83; or
包含SEQ ID NO：86的胺基酸序列的第一鏈，和包含SEQ ID NO：83的胺基酸序列的第二鏈；或 A first strand comprising the amino acid sequence of SEQ ID NO: 86, and a second strand comprising the amino acid sequence of SEQ ID NO: 83; or
包含SEQ ID NO：88的胺基酸序列的第一鏈，和包含SEQ ID NO：87的胺基酸序列的第二鏈；或 A first strand comprising the amino acid sequence of SEQ ID NO: 88, and a second strand comprising the amino acid sequence of SEQ ID NO: 87; or
包含SEQ ID NO：89的胺基酸序列的第一鏈，和包含SEQ ID NO：87的胺基酸序列的第二鏈；或 A first strand comprising the amino acid sequence of SEQ ID NO: 89, and a second strand comprising the amino acid sequence of SEQ ID NO: 87; or
包含SEQ ID NO：90的胺基酸序列的第一鏈，和包含SEQ ID NO：87的胺基酸序列的第二鏈；或 A first strand comprising the amino acid sequence of SEQ ID NO: 90, and a second strand comprising the amino acid sequence of SEQ ID NO: 87; or
包含SEQ ID NO：91的胺基酸序列的第一鏈，和包含SEQ ID NO：87的胺基酸序列的第二鏈；或 A first strand comprising the amino acid sequence of SEQ ID NO: 91, and a second strand comprising the amino acid sequence of SEQ ID NO: 87; or
包含SEQ ID NO：95的胺基酸序列的第一鏈，和包含SEQ ID NO：94的胺基酸序列的第二鏈；或 A first strand comprising the amino acid sequence of SEQ ID NO: 95, and a second strand comprising the amino acid sequence of SEQ ID NO: 94; or
包含SEQ ID NO：96的胺基酸序列的第一鏈，和包含SEQ ID NO：94的胺基酸序列的第二鏈；或 A first strand comprising the amino acid sequence of SEQ ID NO: 96, and a second strand comprising the amino acid sequence of SEQ ID NO: 94; or
包含SEQ ID NO：97的胺基酸序列的第一鏈，和包含SEQ ID NO：94的胺基酸序列的第二鏈；或 A first strand comprising the amino acid sequence of SEQ ID NO: 97, and a second strand comprising the amino acid sequence of SEQ ID NO: 94; or
在一些實施方案中，如前任一項所述的抗原結合分子，其具有： In some embodiments, the antigen-binding molecule of any one of the preceding items, which has:
包含SEQ ID NO：76的胺基酸序列的第一鏈，和包含SEQ ID NO：72的胺基酸序列的第二鏈；或 A first strand comprising the amino acid sequence of SEQ ID NO: 76, and a second strand comprising the amino acid sequence of SEQ ID NO: 72; or
包含SEQ ID NO：79的胺基酸序列的第一鏈，和包含SEQ ID NO：75的胺基酸序列的第二鏈。 A first strand comprising the amino acid sequence of SEQ ID NO:79, and a second strand comprising the amino acid sequence of SEQ ID NO:75.
在一些實施方案中，如前所述的抗原結合分子，其中該特異性結合BCMA的第一抗原結合域是Fab，該特異性結合CD3的第二抗原結合域是經替換的Fab，該經替換的Fab包含分別與可變區的兩條多肽鏈直接或藉由連接子連接的Titin鏈和Obscurin鏈，不包含輕鏈恆定區與重鏈恆定區CH1；或該特異性結合CD3的第二抗原結合域是Fab，該特異性結合BCMA的第一抗原結合域是經替換的Fab，該經替換的Fab包含分別與可變區的兩條多肽鏈直接或藉由連接子連接的Titin鏈和Obscurin鏈，不包含輕鏈恆定區與重鏈恆定區CH1。 In some embodiments, the antigen-binding molecule as described above, wherein the first antigen-binding domain that specifically binds BCMA is a Fab, the second antigen-binding domain that specifically binds CD3 is a substituted Fab, and the substituted The Fab comprises a Titin chain and an Obscurin chain connected directly or via a linker to the two polypeptide chains of the variable region, and does not contain the light chain constant region and the heavy chain constant region CH1; or the second antigen that specifically binds CD3 The binding domain is Fab, and the first antigen-binding domain that specifically binds BCMA is a substituted Fab, which comprises a Titin chain and an Obscurin that are respectively connected to the two polypeptide chains of the variable region directly or through a linker chain, excluding the light chain constant region and the heavy chain constant region CH1.
在一些實施方案中，如前所述的抗原結合分子，其中該特異性結合BCMA的第一抗原結合域是Fab，該特異性結合CD3的第二抗原結合域是經替換的Fab，該經替換的Fab中，重鏈恆定區CH1和輕鏈恆定區被Titin鏈和Obscurin鏈替換；或該特異性結合CD3的第二抗原結合域是Fab，該特異性結合BCMA的第一抗原結合域是經替換的Fab，該經替換的Fab中，重鏈恆定區CH1和輕鏈恆定區被Titin鏈和Obscurin鏈替換。 In some embodiments, the antigen-binding molecule as described above, wherein the first antigen-binding domain that specifically binds BCMA is a Fab, the second antigen-binding domain that specifically binds CD3 is a substituted Fab, and the substituted In the Fab, the heavy chain constant region CH1 and the light chain constant region are replaced by Titin chain and Obscurin chain; or the second antigen-binding domain that specifically binds CD3 is Fab, and the first antigen-binding domain that specifically binds BCMA is obtained by Substituted Fab in which the heavy chain constant region CH1 and the light chain constant region are replaced by Titin chain and Obscurin chain.
在一些實施方案中，該特異性結合BCMA的第一抗原結合域是Fab；該特異性結合CD3的第二抗原結合域包含重鏈可變區CD3-VH和輕鏈可變區CD3-VL，並且該重鏈可變區CD3-VH的C端直接或藉由連接子融合至Titin鏈的N端，以及該輕鏈可變區CD3-VL的C端直接或藉由連接子融合至Obscurin鏈的N端。在一些實施方案中，該特異性結合 BCMA的第一抗原結合域是Fab；該特異性結合CD3的第二抗原結合域包含重鏈可變區CD3-VH和輕鏈可變區CD3-VL，並且該重鏈可變區CD3-VL的C端直接或藉由連接子融合至Titin鏈的N端，以及該輕鏈可變區CD3-VH的C端直接或藉由連接子融合至Obscurin鏈的N端。在一些實施方案中，該特異性結合CD3的第二抗原結合域是Fab；該特異性結合BCMA的第一抗原結合域包含重鏈可變區BCMA-VH和輕鏈可變區BCMA-VL，並且該重鏈可變區BCMA-VH的C端直接或藉由連接子融合至Titin鏈的N端，以及該輕鏈可變區BCMA-VL的C端直接或藉由連接子融合至Obscurin鏈的N端。在一些實施方案中，該特異性結合CD3的第二抗原結合域是Fab；該特異性結合BCMA的第一抗原結合域包含重鏈可變區BCMA-VH和輕鏈可變區BCMA-VL，並且該重鏈可變區BCMA-VL的C端直接或藉由連接子融合至Titin鏈的N端，以及該輕鏈可變區BCMA-VH的C端直接或藉由連接子融合至Obscurin鏈的N端。 In some embodiments, the first antigen-binding domain that specifically binds BCMA is Fab; the second antigen-binding domain that specifically binds CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, And the C-terminal of the heavy chain variable region CD3-VH is fused directly or via a linker to the N-terminal of the Titin chain, and the C-terminal of the light chain variable region CD3-VL is directly or via a linker fused to the Obscurin chain the N-terminus. In some embodiments, the specific binding The first antigen-binding domain of BCMA is Fab; The second antigen-binding domain that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and the heavy chain variable region CD3-VL The C-terminal of the CD3-VH of the light chain variable region is fused directly or via a linker to the N-terminal of the Titin chain, and the C-terminal of the light chain variable region CD3-VH is directly or via a linker fused to the N-terminal of the Obscurin chain. In some embodiments, the second antigen-binding domain that specifically binds CD3 is Fab; the first antigen-binding domain that specifically binds BCMA comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, And the C-terminal of the heavy chain variable region BCMA-VH is fused directly or via a linker to the N-terminal of the Titin chain, and the C-terminal of the light chain variable region BCMA-VL is directly or via a linker fused to the Obscurin chain the N-terminus. In some embodiments, the second antigen-binding domain that specifically binds CD3 is Fab; the first antigen-binding domain that specifically binds BCMA comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, And the C-terminal of the heavy chain variable region BCMA-VL is fused to the N-terminal of the Titin chain directly or through a linker, and the C-terminal of the light chain variable region BCMA-VH is fused to the Obscurin chain directly or through a linker the N-terminus.
在一些實施方案中，該抗原結合分子還包含Fc區，該Fc區包含能夠締合的第一亞基與第二亞基，該第一亞基和第二亞基具有一個或多個減少Fc區同源二聚化的胺基酸取代。在一些實施方案中，該抗原結合分子還包含Fc區，該Fc區相比野生型Fc區，包含能夠締合的第一亞基與第二亞基，該第一亞基和第二亞基具有一個或多個減少Fc區同源二聚化的胺基酸取代。在一些實施方案中，該第一亞基具有根據杵臼技術的凸起結構，該第二亞基具有根據杵臼技術的孔結構。在一些實施方案中，該Titin鏈的C端直接或藉由連接子融合至第一亞基的N端，以及該Fab的CH1的C端直接或藉由連接子融合至第二亞基的N端。在一些實施方案中，該Titin鏈的C端直接或藉由連接子融合至第二亞基的N端，以及該Fab的CH1的C端直接或藉由連接子融合至第一亞基的N端。在一些實施方案 中，該Obscurin鏈的C端直接或藉由連接子融合至第一亞基的N端，以及該Fab的CH1的C端直接或藉由連接子融合至第二亞基的N端。在一些實施方案中，該Obscurin鏈的C端直接或藉由連接子融合至第二亞基的N端，以及該Fab的CH1的C端直接或藉由連接子融合至第一亞基的N端。 In some embodiments, the antigen binding molecule further comprises an Fc region comprising a first subunit and a second subunit capable of associating, the first subunit and the second subunit having one or more reduced Fc Amino acid substitutions in the homodimerization region. In some embodiments, the antigen-binding molecule further comprises an Fc region that, compared to a wild-type Fc region, comprises a first subunit and a second subunit capable of associating, the first subunit and the second subunit Having one or more amino acid substitutions that reduce homodimerization of the Fc region. In some embodiments, the first subunit has a convex structure according to the knob-and-hole technique and the second subunit has a hole structure according to the knob-and-hole technique. In some embodiments, the C-terminus of the Titin chain is fused directly or via a linker to the N-terminus of the first subunit, and the C-terminus of CH1 of the Fab is fused directly or via a linker to the N-terminus of the second subunit. end. In some embodiments, the C-terminus of the Titin chain is fused directly or via a linker to the N-terminus of the second subunit, and the C-terminus of the CH1 of the Fab is fused directly or via a linker to the N-terminus of the first subunit. end. in some embodiments In this method, the C-terminal of the Obscurin chain is fused directly or via a linker to the N-terminal of the first subunit, and the C-terminal of the CH1 of the Fab is fused directly or via a linker to the N-terminal of the second subunit. In some embodiments, the C-terminus of the Obscurin chain is fused directly or via a linker to the N-terminus of the second subunit, and the C-terminus of the CH1 of the Fab is fused directly or via a linker to the N-terminus of the first subunit. end.
在一些實施方案中，如前所述的抗原結合分子，該第一亞基的胺基酸殘基取代包括366位點的一個或多個胺基酸取代，該第二亞基的胺基酸殘基包括選自366、368和407的位點的一個或多個胺基酸取代。在一些實施方案中，該第一亞基包括366W的一個或多個胺基酸取代，該第二亞基包括選自366S、368A和407V的一個或多個胺基酸取代。在一些實施方案中，該第一亞基包括366W的胺基酸取代，該第二亞基包括366S、368A和407V的胺基酸取代。 In some embodiments, the antigen-binding molecule as described above, the amino acid residue substitution of the first subunit includes one or more amino acid substitutions at position 366, and the amino acid residue substitution of the second subunit Residues include one or more amino acid substitutions at positions selected from 366, 368 and 407. In some embodiments, the first subunit includes one or more amino acid substitutions of 366W and the second subunit includes one or more amino acid substitutions selected from 366S, 368A, and 407V. In some embodiments, the first subunit includes amino acid substitutions of 366W and the second subunit includes amino acid substitutions of 366S, 368A, and 407V.
在一些實施方案中，如前所述的抗原結合分子，其包含具有式(c)所示結構的第一鏈、具有式(b)所示結構的第二鏈、具有式(d)所示結構的第三鏈和具有式(e)所示結構的第四鏈， In some embodiments, the aforementioned antigen-binding molecule comprises a first chain having a structure represented by formula (c), a second chain having a structure represented by formula (b), and a chain having a structure represented by formula (d). The 3rd chain of structure and the 4th chain with structure shown in formula (e),
(c)[BCMA-VH]-[CH1]-[Fc區的第二亞基]； (c) [BCMA-VH]-[CH1]-[the second subunit of the Fc region];
(b)[BCMA-VL]-[CL]； (b) [BCMA-VL]-[CL];
(d)[CD3-VH]-[連接子]-[Titin鏈]-[Fc區的第一亞基]； (d) [CD3-VH]-[linker]-[Titin chain]-[the first subunit of the Fc region];
(e)[CD3-VL]-[連接子]-[Obscurin鏈]。 (e) [CD3-VL]-[Linker]-[Obscurin chain].
在一些實施方案中，該Fc區的第一亞基具有根據杵臼技術的凸起結構，和該Fc區的第二亞基具有根據杵臼技術的孔結構。在一些實施方案中，式(d)和(e)中的連接子較佳為相同或不同的肽連接子。 In some embodiments, the first subunit of the Fc region has a bulge structure according to the knob-and-hole technique, and the second subunit of the Fc region has a pore structure according to the knob-and-hole technique. In some embodiments, the linkers in formulas (d) and (e) are preferably the same or different peptide linkers.
在一些實施方案中，該式(c)、式(b)、式(d)和式(e)各自是從N端到C端排列的。 In some embodiments, each of formula (c), formula (b), formula (d) and formula (e) is arranged from N-terminus to C-terminus.
在一些實施方案中，如前所述的抗原結合分子，其包含具有式(f)所示結構的第一鏈、具有式(b)所示結構的第二鏈、具有式(g)所示結構的第三鏈和具有式(h)所示結構的第四鏈， In some embodiments, the aforementioned antigen-binding molecule comprises a first chain having a structure represented by formula (f), a second chain having a structure represented by formula (b), and a chain having a structure represented by formula (g). The 3rd chain of structure and the 4th chain with structure shown in formula (h),
(f)[BCMA-VH]-[CH1]-[Fc區的第一亞基]； (f) [BCMA-VH]-[CH1]-[the first subunit of the Fc region];
(b)[BCMA-VL]-[CL]； (b) [BCMA-VL]-[CL];
(g)[CD3-VH]-[連接子]-[Obscurin鏈]-[Fc區的第二亞基]； (g) [CD3-VH]-[Linker]-[Obscurin chain]-[Second subunit of Fc region];
(h)[CD3-VL]-[連接子]-[Titin鏈]； (h) [CD3-VL]-[Linker]-[Titin chain];
其中，該Fc區的第一亞基具有根據杵臼技術的凸起結構，和該Fc區的第二亞基具有根據杵臼技術的孔結構；式(g)和(h)中的連接子較佳為相同或不同的肽連接子。 Wherein, the first subunit of the Fc region has a protrusion structure according to the knob-and-hole technique, and the second subunit of the Fc region has a hole structure according to the knob-and-hole technique; the linkers in formulas (g) and (h) are preferred be the same or different peptide linkers.
在一些實施方案中，該式(f)、式(b)、式(g)和式(h)各自是從N端到C端排列的。 In some embodiments, each of formula (f), formula (b), formula (g) and formula (h) is arranged from N-terminus to C-terminus.
在一些實施方案中，該肽連接子是柔性肽連接子。在一些實施方案中，該肽連接子的長度為3-15個胺基酸殘基。在一些實施方案中，該肽連接子獨立的具有L1-(GGGGS)n-L2的結構，其中，L1是鍵、A、GS、GGS或GGGS，n是0、1、2、3、4、5、6、7、8、9或10，L2是鍵、G、GG、GGG或GGGG。在一些實施方案中，該肽連接子不是鍵。在一些實施方案中，該肽連接子是GGGGS(SEQ ID NO：157)。 In some embodiments, the peptide linker is a flexible peptide linker. In some embodiments, the peptide linker is 3-15 amino acid residues in length. In some embodiments, the peptide linker independently has the structure L1-(GGGGS) nL2 , wherein L1 is a bond, A, GS, GGS or GGGS, and n is 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10, L2 is a bond, G, GG, GGG or GGGG. In some embodiments, the peptide linker is not a bond. In some embodiments, the peptide linker is GGGGS (SEQ ID NO: 157).
在一些實施方案中，該Titin鏈包含選自由SEQ ID NO：98至SEQ ID NO：116組成的組的胺基酸序列，該Obscurin鏈包含選自由SEQ ID NO：117至SEQ ID NO：152和SEQ ID NO：162至SEQ ID NO：166組成的組的胺基酸序列。在一些實施方案中，該Titin鏈包含SEQ ID NO：114的胺基酸序列，該Obscurin鏈包含SEQ ID NO：152的胺基酸序列。 In some embodiments, the Titin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 98 to SEQ ID NO: 116, the Obscurin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 117 to SEQ ID NO: 152 and Amino acid sequences of the group consisting of SEQ ID NO: 162 to SEQ ID NO: 166. In some embodiments, the Titin chain comprises the amino acid sequence of SEQ ID NO:114, and the Obscurin chain comprises the amino acid sequence of SEQ ID NO:152.
在一些實施方案中，該抗原結合分子具有： In some embodiments, the antigen binding molecule has:
包含SEQ ID NO：92的胺基酸序列的第一鏈、包含SEQ ID NO：87的胺基酸序列的第二鏈、包含SEQ ID NO：70的胺基酸序列的第三鏈、和包含SEQ ID NO：71的胺基酸序列的第四鏈；或 A first strand comprising the amino acid sequence of SEQ ID NO: 92, a second strand comprising the amino acid sequence of SEQ ID NO: 87, a third strand comprising the amino acid sequence of SEQ ID NO: 70, and comprising the fourth strand of the amino acid sequence of SEQ ID NO: 71; or
包含SEQ ID NO：93的胺基酸序列的第一鏈、包含SEQ ID NO：87的胺基酸序列的第二鏈、包含SEQ ID NO：70的胺基酸序列的第三鏈、和包含SEQ ID NO：71的胺基酸序列的第四鏈。 A first strand comprising the amino acid sequence of SEQ ID NO: 93, a second strand comprising the amino acid sequence of SEQ ID NO: 87, a third strand comprising the amino acid sequence of SEQ ID NO: 70, and comprising The fourth strand of the amino acid sequence of SEQ ID NO:71.
在一些實施方案中，該抗原結合分子具有： In some embodiments, the antigen binding molecule has:
包含SEQ ID NO：171的胺基酸序列的第一鏈、包含SEQ ID NO：83的胺基酸序列的第二鏈、包含SEQ ID NO：167的胺基酸序列的第三鏈、和包含SEQ ID NO：168的胺基酸序列的第四鏈；或 The first strand comprising the amino acid sequence of SEQ ID NO: 171, the second strand comprising the amino acid sequence of SEQ ID NO: 83, the third strand comprising the amino acid sequence of SEQ ID NO: 167, and comprising the fourth strand of the amino acid sequence of SEQ ID NO: 168; or
包含SEQ ID NO：172的胺基酸序列的第一鏈、包含SEQ ID NO：87的胺基酸序列的第二鏈、包含SEQ ID NO：167的胺基酸序列的第三鏈、和包含SEQ ID NO：168的胺基酸序列的第四鏈。 The first strand comprising the amino acid sequence of SEQ ID NO: 172, the second strand comprising the amino acid sequence of SEQ ID NO: 87, the third strand comprising the amino acid sequence of SEQ ID NO: 167, and comprising The fourth strand of the amino acid sequence of SEQ ID NO:168.
在另一個方面，本揭露提供了一種抗原結合分子，其與前述任一項所述的抗原結合分子競爭性結合人BCMA和人CD3。 In another aspect, the present disclosure provides an antigen-binding molecule that competes with the antigen-binding molecule described in any one of the foregoing for binding to human BCMA and human CD3.
在一些實施方案中，如前任一項所述的抗原結合分子，其較佳為雙特異性抗體。 In some embodiments, the antigen-binding molecule according to any one of the preceding items is preferably a bispecific antibody.
在另一個方面，本揭露提供了抗BCMA抗體，其包含重鏈可變區BCMA-VH和輕鏈可變區BCMA-VL，其中， In another aspect, the disclosure provides an anti-BCMA antibody comprising a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, wherein,
(i)該BCMA-VH中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3分別包含SEQ ID NO：29中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的胺基酸序列；和該BCMA-VL中的BCMA-LCDR1、 BCMA-LCDR2和BCMA-LCDR3分別包含SEQ ID NO：30中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的胺基酸序列；或 (i) BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in the BCMA-VH respectively comprise the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 29; and the BCMA- BCMA-LCDR1 in VL, BCMA-LCDR2 and BCMA-LCDR3 respectively comprise the amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in SEQ ID NO: 30; or
(ii)該BCMA-VH中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3分別包含SEQ ID NO：31中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的胺基酸序列；和該BCMA-VL中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3分別包含SEQ ID NO：32中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的胺基酸序列；或 (ii) BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in the BCMA-VH respectively comprise the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 31; and the BCMA- BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in VL comprise the amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in SEQ ID NO: 32, respectively; or
(iii)該BCMA-VH中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3分別包含SEQ ID NO：33中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的胺基酸序列；和該BCMA-VL中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3分別包含SEQ ID NO：34中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的胺基酸序列；或 (iii) BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in the BCMA-VH respectively comprise the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 33; and the BCMA- BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in VL comprise the amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in SEQ ID NO: 34, respectively; or
(iv)該BCMA-VH中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3分別包含SEQ ID NO：35中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的胺基酸序列；和該BCMA-VL中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3分別包含SEQ ID NO：36中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的胺基酸序列。在一些實施方案中，該BCMA-HCDR1、BCMA-HCDR2、BCMA-HCDR3、BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3是根據Kabat、IMGT、Chothia、AbM或Contact編號規則定義的。 (iv) BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in the BCMA-VH respectively comprise the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 35; and the BCMA- BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in VL comprise the amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in SEQ ID NO:36, respectively. In some embodiments, the BCMA-HCDR1, BCMA-HCDR2, BCMA-HCDR3, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering convention.
在另一個方面，本揭露提供了抗BCMA抗體，其包含重鏈可變區BCMA-VH和輕鏈可變區BCMA-VL，該重鏈可變區BCMA-VH具有BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3，並且該輕鏈可變 區BCMA-VL具有BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3，其中， In another aspect, the disclosure provides an anti-BCMA antibody comprising a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, the heavy chain variable region BCMA-VH having BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3, and the light chain variable Region BCMA-VL has BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3, where,
(i)該BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3分別包含SEQ ID NO：29中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的胺基酸序列；和該BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3分別包含SEQ ID NO：30中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的胺基酸序列；或 (i) the BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 respectively comprise the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 29; and the BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 respectively comprise the amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in SEQ ID NO: 30; or
(ii)該BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3分別包含SEQ ID NO：31中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的胺基酸序列；和該BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3分別包含SEQ ID NO：32中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的胺基酸序列；或 (ii) the BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 respectively comprise the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 31; and the BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 respectively comprise the amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in SEQ ID NO: 32; or
(iii)該BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3分別包含SEQ ID NO：33中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的胺基酸序列；和該BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3分別包含SEQ ID NO：34中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的胺基酸序列；或 (iii) the BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 respectively comprise the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 33; and the BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 respectively comprise the amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in SEQ ID NO: 34; or
(iv)該BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3分別包含SEQ ID NO：35中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的胺基酸序列；和該BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3分別包含SEQ ID NO：36中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的胺基酸序列； (iv) the BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 respectively comprise the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 35; and the BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 respectively comprise the amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in SEQ ID NO: 36;
該BCMA-HCDR1、BCMA-HCDR2、BCMA-HCDR3、BCMA- LCDR1、BCMA-LCDR2和BCMA-LCDR3是根據Kabat、IMGT、Chothia、AbM或Contact編號規則定義的。 The BCMA-HCDR1, BCMA-HCDR2, BCMA-HCDR3, BCMA- LCDR1, BCMA-LCDR2, and BCMA-LCDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering conventions.
在另一個方面，本揭露提供了抗BCMA抗體，其包含重鏈可變區BCMA-VH和輕鏈可變區BCMA-VL，其中， In another aspect, the disclosure provides an anti-BCMA antibody comprising a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, wherein,
(i)該重鏈可變區BCMA-VH包含SEQ ID NO：29中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的胺基酸序列，並且該輕鏈可變區BCMA-VL包含SEQ ID NO：30中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的胺基酸序列；或 (i) the heavy chain variable region BCMA-VH comprises the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 29, and the light chain variable region BCMA-VL comprises SEQ ID Amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in NO:30; or
(ii)該重鏈可變區BCMA-VH包含SEQ ID NO：31中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的胺基酸序列，並且該輕鏈可變區BCMA-VL包含SEQ ID NO：32中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的胺基酸序列；或 (ii) the heavy chain variable region BCMA-VH comprises the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 31, and the light chain variable region BCMA-VL comprises SEQ ID Amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in NO:32; or
(iii)該重鏈可變區BCMA-VH包含SEQ ID NO：33中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的胺基酸序列，並且該輕鏈可變區BCMA-VL包含SEQ ID NO：34中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的胺基酸序列；或 (iii) the heavy chain variable region BCMA-VH comprises the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 33, and the light chain variable region BCMA-VL comprises SEQ ID Amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in NO:34; or
(iv)該重鏈可變區BCMA-VH包含SEQ ID NO：35中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的胺基酸序列，並且該輕鏈可變區BCMA-VL包含SEQ ID NO：36中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的胺基酸序列； (iv) The heavy chain variable region BCMA-VH comprises the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 35, and the light chain variable region BCMA-VL comprises SEQ ID Amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in NO:36;
該BCMA-HCDR1、BCMA-HCDR2、BCMA-HCDR3、BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3根據Kabat、IMGT、Chothia、AbM或Contact編號規則定義。 The BCMA-HCDR1, BCMA-HCDR2, BCMA-HCDR3, BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering convention.
在另一個方面，本揭露提供了抗BCMA抗體，其包含重鏈可變區BCMA-VH和輕鏈可變區BCMA-VL，其中， In another aspect, the disclosure provides an anti-BCMA antibody comprising a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, wherein,
(i)該重鏈可變區BCMA-VH具有：包含SEQ ID NO：5的胺基酸序列的BCMA-HCDR1，包含SEQ ID NO：6的胺基酸序列的BCMA-HCDR2，和包含SEQ ID NO：7的胺基酸序列的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：包含SEQ ID NO：8的胺基酸序列的BCMA-LCDR1，包含SEQ ID NO：9的胺基酸序列的BCMA-LCDR2，和包含SEQ ID NO：10的胺基酸序列的BCMA-LCDR3；或 (i) The heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 5, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 6, and comprising SEQ ID BCMA-HCDR3 of the amino acid sequence of NO: 7; and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 8, comprising the amino group of SEQ ID NO: 9 BCMA-LCDR2 of the acid sequence, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 10; or
(ii)該重鏈可變區BCMA-VH具有：包含SEQ ID NO：11的胺基酸序列的BCMA-HCDR1，包含SEQ ID NO：12的胺基酸序列的BCMA-HCDR2，和包含SEQ ID NO：13的胺基酸序列的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：包含SEQ ID NO：14的胺基酸序列的BCMA-LCDR1，包含SEQ ID NO：15的胺基酸序列的BCMA-LCDR2，和包含SEQ ID NO：16的胺基酸序列的BCMA-LCDR3；或 (ii) the heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 11, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and comprising SEQ ID BCMA-HCDR3 of the amino acid sequence of NO: 13; and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 14, comprising the amino group of SEQ ID NO: 15 BCMA-LCDR2 of the acid sequence, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 16; or
(iii)該重鏈可變區BCMA-VH具有：包含SEQ ID NO：17的胺基酸序列的BCMA-HCDR1，包含SEQ ID NO：18的胺基酸序列的BCMA-HCDR2，和包含SEQ ID NO：19的胺基酸序列的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：包含SEQ ID NO：20的胺基酸序列的BCMA-LCDR1，包含SEQ ID NO：21的胺基酸序列的BCMA-LCDR2，和包含SEQ ID NO：22的胺基酸序列的BCMA-LCDR3；或 (iii) the heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 17, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, and comprising SEQ ID BCMA-HCDR3 of the amino acid sequence of NO: 19; and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, comprising the amino group of SEQ ID NO: 21 BCMA-LCDR2 of the acid sequence, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 22; or
(iv)該重鏈可變區BCMA-VH具有：包含SEQ ID NO：23的胺基酸序列的BCMA-HCDR1，包含SEQ ID NO：24的胺基酸序列的BCMA-HCDR2，和包含SEQ ID NO：25的胺基酸序列的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：包含SEQ ID NO：26的胺基酸序列的 BCMA-LCDR1，包含SEQ ID NO：27的胺基酸序列的BCMA-LCDR2，和包含SEQ ID NO：28的胺基酸序列的BCMA-LCDR3； (iv) The heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 23, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 24, and comprising SEQ ID NO: BCMA-HCDR3 of the amino acid sequence of 25; and the light chain variable region BCMA-VL has: comprising the amino acid sequence of SEQ ID NO: 26 BCMA-LCDR1, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 27, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 28;
該BCMA-HCDR1、BCMA-HCDR2、BCMA-HCDR3、BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3是根據Kabat編號規則定義的。 The BCMA-HCDR1, BCMA-HCDR2, BCMA-HCDR3, BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 are defined according to the Kabat numbering convention.
在一些實施方案中，如前所述的抗BCMA抗體，其中， In some embodiments, the anti-BCMA antibody as described above, wherein,
該重鏈可變區BCMA-VH具有：包含SEQ ID NO：5的胺基酸序列的BCMA-HCDR1，包含SEQ ID NO：6的胺基酸序列的BCMA-HCDR2，和包含SEQ ID NO：7的胺基酸序列的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：包含SEQ ID NO：8的胺基酸序列的BCMA-LCDR1，包含SEQ ID NO：9的胺基酸序列的BCMA-LCDR2，和包含SEQ ID NO：10的胺基酸序列的BCMA-LCDR3；在一些實施方案中，該重鏈可變區BCMA-VH和/或輕鏈可變區BCMA-VL是鼠源的或人源化的。在一些實施方案中，該重鏈可變區BCMA-VH和/或輕鏈可變區BCMA-VL是人源化的。在一些實施方案中，該重鏈可變區BCMA-VH具有來源於IGHV1-46*01的重鏈框架區，並且其是未被取代的或具有選自48I、67A、71A、73K、76T和93V組成的組中的一個或多個胺基酸取代；和/或該輕鏈可變區BCMA-VL具有來源於IGKV1-39*01的輕鏈框架區，並且其是未被取代的或具有選自43S、45Q、48V和71Y組成的組中的一個或多個胺基酸取代。在一些實施方案中，該BCMA-VH具有來源於IGHV1-46*01的FR1-3和來源於IGHJ6*01的FR4，並且其是未被取代的或具有選自1E、48I、67A、71A、73K、76T和93V組成的組中的一個或多個胺基酸取代；和/或該BCMA-VL具有來源於IGKV1-39*01的FR1-3和來源於IGKJ2*01的FR4，並且其是未被取代的或具有選自43S、45Q、48V和71Y組成的 組中的一個或多個胺基酸取代。在一些實施方案中，上述可變區和CDR是根據Kabat編號規則定義的。 The heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 5, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 6, and comprising SEQ ID NO: 7 and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 8, comprising the amino acid sequence of SEQ ID NO: 9 BCMA-LCDR2, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 10; in some embodiments, the heavy chain variable region BCMA-VH and/or light chain variable region BCMA-VL are of murine origin or humanized. In some embodiments, the heavy chain variable region BCMA-VH and/or the light chain variable region BCMA-VL is humanized. In some embodiments, the heavy chain variable region BCMA-VH has a heavy chain framework region derived from IGHV1-46*01 and is unsubstituted or has a heavy chain framework region selected from 48I, 67A, 71A, 73K, 76T and One or more amino acid substitutions in the group consisting of 93V; and/or the light chain variable region BCMA-VL has a light chain framework region derived from IGKV1-39*01, and it is unsubstituted or has One or more amino acid substitutions selected from the group consisting of 43S, 45Q, 48V and 71Y. In some embodiments, the BCMA-VH has FR1-3 derived from IGHV1-46*01 and FR4 derived from IGHJ6*01, and is unsubstituted or has a compound selected from 1E, 48I, 67A, 71A, One or more amino acid substitutions in the group consisting of 73K, 76T and 93V; and/or the BCMA-VL has FR1-3 derived from IGKV1-39*01 and FR4 derived from IGKJ2*01, and it is Unsubstituted or having a composition selected from 43S, 45Q, 48V and 71Y One or more amino acid substitutions in the group. In some embodiments, the variable regions and CDRs described above are defined according to the Kabat numbering convention.
在一些實施方案中，如前所述的抗BCMA抗體，其中， In some embodiments, the anti-BCMA antibody as described above, wherein,
(i)該重鏈可變區BCMA-VH包含與SEQ ID NO：29具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列，和該輕鏈可變區BCMA-VL包含與SEQ ID NO：30具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列。在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：29的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：30的胺基酸序列。 (i) the heavy chain variable region BCMA-VH comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 29, and the The light chain variable region BCMA-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:30. In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO:29, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:30.
在一些實施方案中，如前所述的抗BCMA抗體，其中， In some embodiments, the anti-BCMA antibody as described above, wherein,
(i)該重鏈可變區BCMA-VH包含與選自由SEQ ID NO：37、SEQ ID NO：38和SEQ ID NO：39組成的組的胺基酸序列具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列，和該輕鏈可變區BCMA-VL包含與選自由SEQ ID NO：40、SEQ ID NO：41、SEQ ID NO：42和SEQ ID NO：43組成的組的胺基酸序列具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列。在一些實施方案中，該重鏈可變區BCMA-VH包含選自由SEQ ID NO：37、SEQ ID NO：38和SEQ ID NO：39組成的組的胺基酸序列，和該輕鏈可變區BCMA-VL包含選自由SEQ ID NO：40、SEQ ID NO：41、SEQ ID NO：42和SEQ ID NO：43組成的組的胺基酸序列。在一些實施方案中， (i) the heavy chain variable region BCMA-VH comprises at least 90%, 95%, 96% of the amino acid sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 %, 97%, 98% or 99% of the amino acid sequence of sequence identity, and the light chain variable region BCMA-VL comprising and selected from SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: Amino acid sequences that have at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences of the group consisting of 42 and SEQ ID NO:43. In some embodiments, the heavy chain variable region BCMA-VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39, and the light chain variable Region BCMA-VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43. In some embodiments,
該重鏈可變區BCMA-VH包含SEQ ID NO：37的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：40的胺基酸序列，或 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 37, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 40, or
該重鏈可變區BCMA-VH包含SEQ ID NO：38的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：40的胺基酸序列，或 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 38, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 40, or
該重鏈可變區BCMA-VH包含SEQ ID NO：39的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：40的胺基酸序列，或 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 39, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 40, or
該重鏈可變區BCMA-VH包含SEQ ID NO：37的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：41的胺基酸序列，或 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 37, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 41, or
該重鏈可變區BCMA-VH包含SEQ ID NO：37的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：42的胺基酸序列，或 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 37, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 42, or
該重鏈可變區BCMA-VH包含SEQ ID NO：37的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：43的胺基酸序列。 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO:37, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:43.
在一些實施方案中，如前所述的抗BCMA抗體，其中， In some embodiments, the anti-BCMA antibody as described above, wherein,
(ii)該重鏈可變區BCMA-VH具有：包含SEQ ID NO：11的胺基酸序列的BCMA-HCDR1，包含SEQ ID NO：12的胺基酸序列的BCMA-HCDR2，和包含SEQ ID NO：13的胺基酸序列的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：包含SEQ ID NO：14的胺基酸序列的BCMA-LCDR1，包含SEQ ID NO：15的胺基酸序列的BCMA-LCDR2，和包含SEQ ID NO：16的胺基酸序列的BCMA-LCDR3；在一些實施方案中，該重鏈可變區BCMA-VH和/或輕鏈可變區BCMA-VL是鼠源的或人源化的。在一些實施方案中，該重鏈可變區BCMA-VH和/或輕鏈可變區BCMA-VL是人源化的。在一些實施方案中，該重鏈可變區BCMA-VH具有來源於IGHV7-4-1*02的重鏈框架區，並且其是未被取代的或具有選自21I、44V、45F、46K、75A、76N和93L組成的組中的一個或多個胺基酸取代；和/或該輕鏈可變區BCMA-VL具有來源於IGKV1-8*01或IGKV1-27*01的輕鏈框架區，並且其是未被取代的或具有選自43S和66D組成的組中的一個或多個胺基酸取代。在一些實施方案中，該BCMA-VH具有來源於IGHV7-4-1*02的FR1-3和來源於IGHJ1*01的FR4，並且其是未被 取代的或具有選自1E、2I、44V、45F、46K、75A、76N和93L組成的組中的一個或多個胺基酸取代；和/或該BCMA-VL具有來源於IGKV1-8*01或IGKV1-27*01的FR1-3和來源於IGKJ4*01的FR4，並且其是未被取代的或具有選自43S和66D組成的組中的一個或多個胺基酸取代。在一些實施方案中，上述可變區和CDR是根據Kabat編號規則定義的。 (ii) the heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 11, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and comprising SEQ ID BCMA-HCDR3 of the amino acid sequence of NO: 13; and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 14, comprising the amino group of SEQ ID NO: 15 The BCMA-LCDR2 of the acid sequence, and the BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 16; In some embodiments, the heavy chain variable region BCMA-VH and/or the light chain variable region BCMA-VL Be murine or humanized. In some embodiments, the heavy chain variable region BCMA-VH and/or the light chain variable region BCMA-VL is humanized. In some embodiments, the heavy chain variable region BCMA-VH has a heavy chain framework region derived from IGHV7-4-1*02, and it is unsubstituted or has a structure selected from 21I, 44V, 45F, 46K, One or more amino acid substitutions in the group consisting of 75A, 76N and 93L; and/or the light chain variable region BCMA-VL has a light chain framework region derived from IGKV1-8*01 or IGKV1-27*01 , and it is unsubstituted or substituted with one or more amino acids selected from the group consisting of 43S and 66D. In some embodiments, the BCMA-VH has FR1-3 derived from IGHV7-4-1*02 and FR4 derived from IGHJ1*01, and is unmodified Substituted or having one or more amino acid substitutions selected from the group consisting of 1E, 2I, 44V, 45F, 46K, 75A, 76N, and 93L; and/or the BCMA-VL has a protein derived from IGKV1-8*01 Or FR1-3 of IGKV1-27*01 and FR4 derived from IGKJ4*01, which are unsubstituted or substituted with one or more amino acids selected from the group consisting of 43S and 66D. In some embodiments, the variable regions and CDRs described above are defined according to the Kabat numbering convention.
在一些實施方案中，如前所述的抗BCMA抗體，其中， In some embodiments, the anti-BCMA antibody as described above, wherein,
(ii)該重鏈可變區BCMA-VH包含與SEQ ID NO：31具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列，和該輕鏈可變區BCMA-VL包含與SEQ ID NO：32具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列。在一些實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：31的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：32的胺基酸序列。 (ii) the heavy chain variable region BCMA-VH comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 31, and the The light chain variable region BCMA-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:32. In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO:31, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:32.
在一些實施方案中，如前所述的抗BCMA抗體，其中， In some embodiments, the anti-BCMA antibody as described above, wherein,
(ii)該重鏈可變區BCMA-VH包含與選自由SEQ ID NO：44、SEQ ID NO：45和SEQ ID NO：46組成的組的胺基酸序列具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列，和該輕鏈可變區BCMA-VL包含與選自由SEQ ID NO：47和SEQ ID NO：48組成的組的胺基酸序列具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列。在一些實施方案中，該重鏈可變區BCMA-VH包含選自由SEQ ID NO：44、SEQ ID NO：45和SEQ ID NO：46組成的組的胺基酸序列，和該輕鏈可變區BCMA-VL包含選自由SEQ ID NO：47和SEQ ID NO：48組成的組的胺基酸序列。在一些實施方案中， (ii) the heavy chain variable region BCMA-VH comprises at least 90%, 95%, 96% of the amino acid sequence selected from the group consisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 %, 97%, 98% or 99% sequence identity of amino acid sequences, and the light chain variable region BCMA-VL comprises an amine selected from the group consisting of SEQ ID NO: 47 and SEQ ID NO: 48 An amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the heavy chain variable region BCMA-VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46, and the light chain variable Region BCMA-VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO:47 and SEQ ID NO:48. In some embodiments,
該重鏈可變區BCMA-VH包含SEQ ID NO：44的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：47的胺基酸序列，或 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 44, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 47, or
該重鏈可變區BCMA-VH包含SEQ ID NO：45的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：47的胺基酸序列，或 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 45, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 47, or
該重鏈可變區BCMA-VH包含SEQ ID NO：46的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：47的胺基酸序列，或 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 46, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 47, or
該重鏈可變區BCMA-VH包含SEQ ID NO：44的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：48的胺基酸序列，或 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 44, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 48, or
該重鏈可變區BCMA-VH包含SEQ ID NO：45的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：48的胺基酸序列，或 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 45, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 48, or
該重鏈可變區BCMA-VH包含SEQ ID NO：46的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：48的胺基酸序列。 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO:46, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:48.
在一些實施方案中，如前所述的抗BCMA抗體，其中， In some embodiments, the anti-BCMA antibody as described above, wherein,
(iii)該重鏈可變區BCMA-VH具有：包含SEQ ID NO：17的胺基酸序列的BCMA-HCDR1，包含SEQ ID NO：18的胺基酸序列的BCMA-HCDR2，和包含SEQ ID NO：19的胺基酸序列的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：包含SEQ ID NO：20的胺基酸序列的BCMA-LCDR1，包含SEQ ID NO：21的胺基酸序列的BCMA-LCDR2，和包含SEQ ID NO：22的胺基酸序列的BCMA-LCDR3；在一些實施方案中，該重鏈可變區BCMA-VH和/或輕鏈可變區BCMA-VL是鼠源的或人源化的。在一些實施方案中，該重鏈可變區BCMA-VH和/或輕鏈可變區BCMA-VL是人源化的。在一些實施方案中，該重鏈可變區BCMA-VH具有來源於IGHV3-11*01的重鏈框架區，並且其是未被取代的或具有選自30R、47R、49A和93T組成的組中的一個或多個胺基酸取代；和/或該輕鏈可變區BCMA-VL具有來源於IGKV1-39*01的輕鏈框架區，並且其是未被取代的或具有44V的胺基酸取代。在一些實施方案中，該BCMA-VH 具有來源於IGHV3-11*01的FR1-3和來源於IGHJ1*01的FR4，並且其是未被取代的或具有選自1E、30R、47R、49A和93T組成的組中的一個或多個胺基酸取代；和/或該BCMA-VL具有來源於IGKV1-39*01的FR1-3和來源於IGKJ2*01的FR4，並且其是未被取代的或具有44V的胺基酸取代。在一些實施方案中，上述可變區和CDR是根據Kabat編號規則定義的。 (iii) the heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 17, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, and comprising SEQ ID BCMA-HCDR3 of the amino acid sequence of NO: 19; and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, comprising the amino group of SEQ ID NO: 21 The BCMA-LCDR2 of the acid sequence, and the BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 22; In some embodiments, the heavy chain variable region BCMA-VH and/or the light chain variable region BCMA-VL Be murine or humanized. In some embodiments, the heavy chain variable region BCMA-VH and/or the light chain variable region BCMA-VL is humanized. In some embodiments, the heavy chain variable region BCMA-VH has a heavy chain framework region derived from IGHV3-11*01 and is unsubstituted or has a group selected from the group consisting of 30R, 47R, 49A and 93T One or more amino acid substitutions in; and/or the light chain variable region BCMA-VL has a light chain framework region derived from IGKV1-39*01, and it is unsubstituted or has an amino group of 44V acid substitution. In some embodiments, the BCMA-VH have FR1-3 derived from IGHV3-11*01 and FR4 derived from IGHJ1*01, and which are unsubstituted or have one or more selected from the group consisting of 1E, 30R, 47R, 49A and 93T Amino acid substitution; and/or the BCMA-VL has FR1-3 derived from IGKV1-39*01 and FR4 derived from IGKJ2*01, and it is unsubstituted or has an amino acid substitution of 44V. In some embodiments, the variable regions and CDRs described above are defined according to the Kabat numbering convention.
在一些的實施方案中，如前所述的抗BCMA抗體，其中， In some embodiments, the anti-BCMA antibody as described above, wherein,
(iii)該重鏈可變區BCMA-VH包含與SEQ ID NO：33具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列，和該輕鏈可變區BCMA-VL包含與SEQ ID NO：34具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列。在一些的實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：33的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：34的胺基酸序列。 (iii) the heavy chain variable region BCMA-VH comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 33, and the The light chain variable region BCMA-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:34. In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO:33, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:34.
在一些實施方案中，如前所述的抗BCMA抗體，其中， In some embodiments, the anti-BCMA antibody as described above, wherein,
(iii)該重鏈可變區BCMA-VH包含與選自由SEQ ID NO：49和SEQ ID NO：50組成的組的胺基酸序列具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列，和該輕鏈可變區BCMA-VL包含與SEQ ID NO：51具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列。在一些實施方案中，該重鏈可變區BCMA-VH包含選自由SEQ ID NO：49和SEQ ID NO：50組成的組的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：51的胺基酸序列。在一些實施方案中， (iii) the heavy chain variable region BCMA-VH comprises at least 90%, 95%, 96%, 97%, 98% of the amino acid sequence selected from the group consisting of SEQ ID NO: 49 and SEQ ID NO: 50 An amino acid sequence of % or 99% sequence identity, and the light chain variable region BCMA-VL comprises at least 90%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO:51 The sequence identity of the amino acid sequence. In some embodiments, the heavy chain variable region BCMA-VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 49 and SEQ ID NO: 50, and the light chain variable region BCMA-VL comprises SEQ ID NO: 49 and SEQ ID NO: 50 Amino acid sequence of ID NO:51. In some embodiments,
該重鏈可變區BCMA-VH包含SEQ ID NO：49的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：51的胺基酸序列，或 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 49, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 51, or
該重鏈可變區BCMA-VH包含SEQ ID NO：50的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：51的胺基酸序列。 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO:50, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:51.
在一些實施方案中，如前所述的抗BCMA抗體，其中， In some embodiments, the anti-BCMA antibody as described above, wherein,
(iv)該重鏈可變區BCMA-VH具有：包含SEQ ID NO：23的胺基酸序列的BCMA-HCDR1，包含SEQ ID NO：24的胺基酸序列的BCMA-HCDR2，和包含SEQ ID NO：25的胺基酸序列的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：包含SEQ ID NO：26的胺基酸序列的BCMA-LCDR1，包含SEQ ID NO：27的胺基酸序列的BCMA-LCDR2，和包含SEQ ID NO：28的胺基酸序列的BCMA-LCDR3；在一些實施方案中，該重鏈可變區BCMA-VH和/或輕鏈可變區BCMA-VL是鼠源的或人源化的。在一些實施方案中，該重鏈可變區BCMA-VH和/或輕鏈可變區BCMA-VL是人源化的。在一些實施方案中，該重鏈可變區BCMA-VH具有來源於IGHV2-70*01的重鏈框架區，並且其是未被取代的或具有選自24V、30T、37V、49G、73N、80F和89R組成的組中的一個或多個胺基酸取代；和/或該輕鏈可變區BCMA-VL具有來源於IGKV1-33*01的輕鏈框架區。在一些實施方案中，該BCMA-VH具有來源於IGHV2-70*01的FR1-3和來源於IGHJ6*01的FR4，並且其是未被取代的或具有選自1E、24V、30T、37V、49G、73N、80F和89R組成的組中的一個或多個胺基酸取代；和/或該BCMA-VL具有來源於IGKV1-33*01的FR1-3和來源於IGKJ2*01的FR4。在一些實施方案中，上述可變區和CDR是根據Kabat編號規則定義的。 (iv) The heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 23, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 24, and comprising SEQ ID BCMA-HCDR3 of the amino acid sequence of NO: 25; and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 26, comprising the amino group of SEQ ID NO: 27 The BCMA-LCDR2 of the acid sequence, and the BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 28; In some embodiments, the heavy chain variable region BCMA-VH and/or the light chain variable region BCMA-VL Be murine or humanized. In some embodiments, the heavy chain variable region BCMA-VH and/or the light chain variable region BCMA-VL is humanized. In some embodiments, the heavy chain variable region BCMA-VH has a heavy chain framework region derived from IGHV2-70*01, and it is unsubstituted or has a structure selected from 24V, 30T, 37V, 49G, 73N, One or more amino acid substitutions in the group consisting of 80F and 89R; and/or the light chain variable region BCMA-VL has a light chain framework region derived from IGKV1-33*01. In some embodiments, the BCMA-VH has FR1-3 derived from IGHV2-70*01 and FR4 derived from IGHJ6*01, and is unsubstituted or has a compound selected from 1E, 24V, 30T, 37V, One or more amino acid substitutions in the group consisting of 49G, 73N, 80F and 89R; and/or the BCMA-VL has FR1-3 derived from IGKV1-33*01 and FR4 derived from IGKJ2*01. In some embodiments, the variable regions and CDRs described above are defined according to the Kabat numbering convention.
在一些的實施方案中，如前所述的抗BCMA抗體，其中， In some embodiments, the anti-BCMA antibody as described above, wherein,
(iv)該重鏈可變區BCMA-VH包含與SEQ ID NO：35具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列，和該輕鏈可變 區BCMA-VL包含與SEQ ID NO：36具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列。在一些的實施方案中，該重鏈可變區BCMA-VH包含SEQ ID NO：35的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：36的胺基酸序列。 (iv) the heavy chain variable region BCMA-VH comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 35, and the variable light chain Region BCMA-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:36. In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO:35, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:36.
在一些實施方案中，如前所述的抗BCMA抗體，其中， In some embodiments, the anti-BCMA antibody as described above, wherein,
(iv)該重鏈可變區BCMA-VH包含與選自由SEQ ID NO：52和SEQ ID NO：53組成的組的胺基酸序列具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列，和該輕鏈可變區BCMA-VL包含與SEQ ID NO：54具有至少90%、95%、96%、97%、98%或99%的序列同一性的胺基酸序列。在一些實施方案中，該重鏈可變區BCMA-VH包含選自由SEQ ID NO：52和SEQ ID NO：53組成的組的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：54的胺基酸序列。在一些實施方案中， (iv) the heavy chain variable region BCMA-VH comprises at least 90%, 95%, 96%, 97%, 98% of the amino acid sequence selected from the group consisting of SEQ ID NO: 52 and SEQ ID NO: 53 An amino acid sequence of % or 99% sequence identity, and the light chain variable region BCMA-VL comprises at least 90%, 95%, 96%, 97%, 98% or 99% of SEQ ID NO:54 The sequence identity of the amino acid sequence. In some embodiments, the heavy chain variable region BCMA-VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 52 and SEQ ID NO: 53, and the light chain variable region BCMA-VL comprises SEQ ID NO: 52 and SEQ ID NO: 53 Amino acid sequence of ID NO:54. In some embodiments,
該重鏈可變區BCMA-VH包含SEQ ID NO：52的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：54的胺基酸序列，或 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO: 52, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 54, or
該重鏈可變區BCMA-VH包含SEQ ID NO：53的胺基酸序列，和該輕鏈可變區BCMA-VL包含SEQ ID NO：54的胺基酸序列。 The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO:53, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:54.
在另一個方面，本揭露提供了一種醫藥組成物，其含有：治療有效量的前述任一項所述的抗原結合分子或抗BCMA抗體，以及一種或更多種藥學上可接受的載體、稀釋劑、緩衝劑或賦形劑。 In another aspect, the present disclosure provides a pharmaceutical composition comprising: a therapeutically effective amount of the antigen-binding molecule or anti-BCMA antibody described in any one of the foregoing, and one or more pharmaceutically acceptable carriers, diluted agents, buffers or excipients.
在一些實施方案中，該醫藥組成物中還包含至少一種第二治療劑。 In some embodiments, the pharmaceutical composition also includes at least one second therapeutic agent.
在另一個方面，本揭露還提供分離的核酸，其編碼前述任一項所述的抗原結合分子或抗BCMA抗體。 In another aspect, the present disclosure also provides an isolated nucleic acid encoding the antigen-binding molecule or anti-BCMA antibody of any one of the foregoing.
在另一個方面，本揭露還提供一種宿主細胞，其包含前述的核酸。 In another aspect, the present disclosure also provides a host cell comprising the aforementioned nucleic acid.
在另一個方面，本揭露還提供一種治療疾病的方法，該方法包括向受試者施用治療有效量的前述任一項所述的抗原結合分子、抗BCMA抗體、核酸或組成物。 In another aspect, the present disclosure also provides a method for treating a disease, the method comprising administering to a subject a therapeutically effective amount of the antigen-binding molecule, anti-BCMA antibody, nucleic acid or composition described in any one of the foregoing.
在一些實施方案中，該疾病為B細胞障礙和自身免疫性疾病。在一些實施方案中，該疾病為與BCMA表達有關的B細胞障礙和自身免疫性疾病。在另一些實施方案中，該與BCMA表達有關的B細胞障礙是漿細胞障礙；該自身免疫性疾病是全身性紅斑狼瘡。在其中一些實施方案中，該漿細胞障礙選自：多發性骨髓瘤、漿細胞瘤、漿細胞白血病、巨球蛋白血症、澱粉樣變性、華氏巨球蛋白血症、孤立性骨漿細胞瘤、髓外漿細胞瘤、骨硬化性骨髓瘤、重鏈病、意義不明確的單株γ球蛋白病以及鬱積型多發性骨髓瘤。 In some embodiments, the disease is a B cell disorder and an autoimmune disease. In some embodiments, the disease is a B cell disorder and an autoimmune disease associated with BCMA expression. In other embodiments, the B cell disorder associated with BCMA expression is a plasma cell disorder; the autoimmune disease is systemic lupus erythematosus. In some of these embodiments, the plasma cell disorder is selected from the group consisting of multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, Waldenström macroglobulinemia, solitary plasmacytoma , extramedullary plasmacytoma, osteosclerotic myeloma, heavy chain disease, monoclonal gammopathy of undetermined significance, and smoldering multiple myeloma.
在另一個方面，本揭露還提供前述任一項所述的抗原結合分子、抗BCMA抗體、核酸或組成物在製備治療或預防疾病的藥物中的用途。在一些實施方案中，該疾病為B細胞障礙和自身免疫性疾病。在一些實施方案中，該疾病為與BCMA表達有關的B細胞障礙和自身免疫性疾病。在另一些實施方案中，該與BCMA表達有關的B細胞障礙是漿細胞障礙；該自身免疫性疾病是全身性紅斑狼瘡。在其中一些實施方案中，該漿細胞障礙選自：多發性骨髓瘤、漿細胞瘤、漿細胞白血病、巨球蛋白血症、澱粉樣變性、華氏巨球蛋白血症、孤立性骨漿細胞瘤、髓外漿細胞瘤、骨硬化性骨髓瘤、重鏈病、意義不明確的單株γ球蛋白病以及鬱積型多發性骨髓瘤。 In another aspect, the present disclosure also provides the use of the antigen-binding molecule, anti-BCMA antibody, nucleic acid or composition described in any one of the foregoing in the preparation of a medicament for treating or preventing a disease. In some embodiments, the disease is a B cell disorder and an autoimmune disease. In some embodiments, the disease is a B cell disorder and an autoimmune disease associated with BCMA expression. In other embodiments, the B cell disorder associated with BCMA expression is a plasma cell disorder; the autoimmune disease is systemic lupus erythematosus. In some of these embodiments, the plasma cell disorder is selected from the group consisting of multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, Waldenström macroglobulinemia, solitary plasmacytoma , extramedullary plasmacytoma, osteosclerotic myeloma, heavy chain disease, monoclonal gammopathy of undetermined significance, and smoldering multiple myeloma.
在另一個方面，本揭露還提供用作藥物的前述任一項所述的抗原結合分子、抗BCMA抗體、核酸或組成物。在一些實施方案中，該疾病為B細胞障礙和自身免疫性疾病。在一些實施方案中，該藥物用於治療或預防與BCMA表達有關的B細胞障礙和自身免疫性疾病。在另一些實施方案中，該與BCMA表達有關的B細胞障礙是漿細胞障礙；該自身免疫性疾病是全身性紅斑狼瘡。在其中一些實施方案中，該漿細胞障礙選自：多發性骨髓瘤、漿細胞瘤、漿細胞白血病、巨球蛋白血症、澱粉樣變性、華氏巨球蛋白血症、孤立性骨漿細胞瘤、髓外漿細胞瘤、骨硬化性骨髓瘤、重鏈病、意義不明確的單株γ球蛋白病以及鬱積型多發性骨髓瘤。 In another aspect, the present disclosure also provides the antigen-binding molecule, anti-BCMA antibody, nucleic acid or composition described in any one of the foregoing for use as a medicament. In some embodiments, the disease is a B cell disorder and an autoimmune disease. In some embodiments, the medicament is used to treat or prevent B cell disorders and autoimmune diseases associated with BCMA expression. In other embodiments, the B cell disorder associated with BCMA expression is a plasma cell disorder; the autoimmune disease is systemic lupus erythematosus. In some of these embodiments, the plasma cell disorder is selected from the group consisting of multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, Waldenström macroglobulinemia, solitary plasmacytoma , extramedullary plasmacytoma, osteosclerotic myeloma, heavy chain disease, monoclonal gammopathy of undetermined significance, and smoldering multiple myeloma.
本揭露提供的抗原結合分子，具有治療活性、安全性、藥物代謝動力學特性和成藥性(如穩定性)好的特點。 The antigen-binding molecule provided by the present disclosure has the characteristics of good therapeutic activity, safety, pharmacokinetic properties and druggability (such as stability).
圖1A為Format-11和Format-11-6164的結構示意圖； Figure 1A is a schematic structural diagram of Format-11 and Format-11-6164;
圖1B為Format-14的結構示意圖； Fig. 1B is a structural schematic diagram of Format-14;
圖1C為Format-15的結構示意圖。 Figure 1C is a schematic diagram of the structure of Format-15.
術語the term
本文所用的術語只是為了描述實施方案的目的，並非旨在進行限制。除非另外定義，本文所用的全部技術術語和科學術語具有與本揭露所屬技術領域具有通常知識者通常所理解的相同意義。 The terminology used herein is for the purpose of describing the embodiments only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
說明書和申請專利範圍中所用的單數形式“一個”、“一種”和“該”包括複數指代，除非上下文清楚表明並非如此。 As used in the specification and claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
除非上下文另外清楚要求，否則在整個說明書和申請專利範圍中，應將詞語“包含”、“具有”、“包括”等理解為具有包含意義，而不是排他性或窮舉性意義；也即，“包括但不僅限於”的意義。除非另有說明，“包含”包括了“由……組成”。例如，對於包含SEQ ID NO：5的胺基酸序列的BCMA-HCDR1，其明確的包含胺基酸序列如SEQ ID NO：5所示的BCMA-HCDR1。 Unless the context clearly requires otherwise, throughout the specification and scope of the patent application, the words "comprises", "has", "comprising", etc. should be understood as having an inclusive meaning rather than an exclusive or exhaustive meaning; that is, " including but not limited to ". Unless otherwise stated, "comprising" includes "consisting of". For example, for the BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:5, it definitely comprises the BCMA-HCDR1 whose amino acid sequence is shown in SEQ ID NO:5.
本揭露所用胺基酸三字母代碼和單字母代碼如J.biol.chem，243，p3558(1968)中所述。 The three-letter and one-letter codes for amino acids used in this disclosure are as described in J.biol.chem , 243, p3558 (1968).
術語“和/或”，例如“X和/或Y”應當理解為意指“X和Y”或“X或Y”並且應當被用來提供對兩種含義或任一含義的明確支持。 The term "and/or", eg "X and/or Y" should be understood to mean "X and Y" or "X or Y" and should be used to provide explicit support for both or either meaning.
術語“胺基酸”是指天然存在的和合成的胺基酸，以及以與天然存在的胺基酸類似的方式起作用的胺基酸類似物和胺基酸模擬物。天然存在的胺基酸是由遺傳密碼編碼的那些胺基酸，以及後來修飾的那些胺基酸，例如羥脯胺酸、γ-羧基谷胺酸和O-磷酸絲胺酸。胺基酸類似物是指與天然存在的胺基酸具有相同基本化學結構(即與氫、羧基、胺基和R基團結合的α碳)的化合物，例如高絲胺酸、正亮胺酸、甲硫胺酸亞碸、甲硫胺酸甲基鋶。此類類似物具有修飾的R基團(例如，正亮胺酸)或修飾的肽骨架，但保留與天然存在的胺基酸相同的基本化學結構。胺基酸模擬物是指具有與胺基酸的一般化學結構不同的結構，但是以與天然存在的胺基酸類似的方式起作用的化學化合物。 The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, eg, hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure (i.e., alpha carbon bonded to hydrogen, carboxyl, amine and R groups) as naturally occurring amino acids, such as homoserine, norleucine, Methionine imine, methionine methyl peroxide. Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. An amino acid mimetic refers to a chemical compound that has a structure that differs from the general chemical structure of an amino acid, but functions in a manner similar to a naturally occurring amino acid.
術語“胺基酸突變”包括胺基酸取代，缺失，插入和修飾。可以進行取代、缺失、插入和修飾的任意組合來實現最終構建體，只要最終構 建體擁有期望的特性，例如降低或對Fc受體的結合。胺基酸序列缺失和插入包括在多肽鏈的胺基端和/或羧基端的缺失和插入。具體的胺基酸突變可以是胺基酸取代。在一個實施方式中，胺基酸突變是非保守性的胺基酸取代，即將一個胺基酸用具有不同結構和/或化學特性的另一種胺基酸替換。胺基酸取代包括由非天然存在的胺基酸或由20種天然胺基酸的衍生物(例如4-羥脯胺酸、3-甲基組胺酸、鳥胺酸、高絲胺酸、5-羥賴胺酸)替換。可以使用本領域中公知的遺傳或化學方法生成胺基酸突變。遺傳方法可以包括定點誘變、PCR，基因合成等。預計基因工程以外的改變胺基酸側鏈基團的方法，如化學修飾也可能是可用的。本文中可使用各種名稱來指示同一胺基酸突變。本文中，可採用位置+胺基酸殘基的方式表示特定位點的胺基酸殘基，例如366W，則表示在366位點上的胺基酸殘基為W。T366W則表示第366位點上的胺基酸殘基由原來的T突變為了W。 The term "amino acid mutation" includes amino acid substitutions, deletions, insertions and modifications. Any combination of substitutions, deletions, insertions and modifications can be made to achieve the final construct, as long as the final construct Constructs possess desirable properties, such as reduced or binding to Fc receptors. Amino acid sequence deletions and insertions include deletions and insertions at the amino terminus and/or carboxyl terminus of the polypeptide chain. Specific amino acid mutations may be amino acid substitutions. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, ie, replacing one amino acid with another amino acid having different structural and/or chemical properties. Amino acid substitutions include non-naturally occurring amino acids or derivatives of 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5 -Hydroxylysine) replacement. Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like. It is anticipated that methods other than genetic engineering to alter amino acid side chain groups, such as chemical modification, may also be available. Various names may be used herein to refer to mutations of the same amino acid. Herein, the amino acid residue at a specific position can be expressed in the form of position + amino acid residue, for example, 366W means that the amino acid residue at position 366 is W. T366W means that the amino acid residue at position 366 is mutated from the original T to W.
術語“抗原結合分子”以最廣義使用，涵蓋各種特異性結合抗原的分子，包括但不限於抗體、其他具有抗原結合活性的多肽以及兩者融合而成的抗體融合蛋白。示例性的，本文中的抗原結合分子是雙特異性抗原結合分子(例如：雙特異性抗體)，其可包含兩條相同的第一鏈和兩條相同的第二鏈；或互不相同的第一鏈、第二鏈、第三鏈和第四鏈。示例性的，該鏈是多肽鏈。示例性的，該第一多肽鏈或第三多肽鏈可以是抗體的重鏈或包含Fc區的多肽，該第二多肽鏈或第四多肽鏈可以是抗體的輕鏈或經改造的抗體輕鏈。 The term "antigen-binding molecule" is used in the broadest sense and covers various molecules that specifically bind to an antigen, including but not limited to antibodies, other polypeptides with antigen-binding activity, and antibody fusion proteins fused thereto. Exemplary, the antigen-binding molecule herein is a bispecific antigen-binding molecule (for example: a bispecific antibody), which may comprise two identical first chains and two identical second chains; or mutually different First strand, second strand, third strand and fourth strand. Exemplarily, the chain is a polypeptide chain. Exemplarily, the first polypeptide chain or the third polypeptide chain may be a heavy chain of an antibody or a polypeptide comprising an Fc region, and the second or fourth polypeptide chain may be a light chain of an antibody or an engineered antibody light chains.
術語“雙特異性抗原結合分子”指能夠對兩個不同抗原或同一抗原的至少兩個不同抗原表位特異性結合的抗原結合分子。 The term "bispecific antigen binding molecule" refers to an antigen binding molecule capable of specifically binding to two different antigens or at least two different epitopes of the same antigen.
術語“抗體”以最廣義使用，並且涵蓋各種抗體結構，包括但不限於單株抗體，多株抗體；單特異性抗體，多特異性抗體(例如雙特異 性抗體)，全長抗體和抗體片段(或抗原結台片段，或抗原結合部分)，只要它們展現出期望的抗原結合活性。“天然抗體”指天然存在的免疫球蛋白分子。例如，天然IgG抗體是約150,000道爾頓的異四聚蛋白，由二硫鍵結合的兩條輕鏈和兩條重鏈構成。從N至C端，每條重鏈具有一個可變區(VH，又稱作可變重域、重鏈可變區)，接著是三個恆定域(CH1、CH2和CH3)。類似地，從N至C端，每條輕鏈具有一個可變區(VL，又稱作可變輕域，或輕鏈可變域)，接著是一個恆定輕域(輕鏈恆定區、CL)。術語“全長抗體”、“完整抗體”和“全抗體”在本文可互換使用，指具有與天然抗體結構基本類似的結構或具有含有Fc區的重鏈的抗體。 The term "antibody" is used in the broadest sense and encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific Antibodies), full-length antibodies and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. "Native antibody" refers to a naturally occurring immunoglobulin molecule. For example, native IgG antibodies are heterotetrameric proteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains joined by disulfide bonds. From N to C-terminus, each heavy chain has a variable region (VH, also called variable heavy domain, heavy chain variable region) followed by three constant domains (CH1, CH2 and CH3). Similarly, from N to C-terminus, each light chain has a variable region (VL, also known as variable light domain, or light chain variable domain), followed by a constant light domain (light chain constant region, CL ). The terms "full-length antibody", "intact antibody" and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody or having a heavy chain containing an Fc region.
術語“雙特異性抗體”指能夠對兩個不同抗原或同一抗原的兩個不同抗原表位特異性結合的抗體(包括抗體或其抗原結合片段，如單鏈抗體)。現有技術已公開了各種結構的雙特異性抗體，根據IgG分子的完整性可分為IgG樣雙特異性抗體和抗體片段型雙特異性抗體；根據抗原結合區域的數量可分為二價、三價、四價或更多價的雙特異性抗體；根據結構是否對稱可分為對稱結構雙特異性抗體和不對稱結構雙特異性抗體。其中，片段型雙特異性抗體，例如缺乏Fc片段的Fab片段，其藉由將2個或多個Fab片段結合在一個分子中形成雙特異性抗體，其具有較低的免疫原性，且分子量小，具有較高的腫瘤組織滲透性；該類型的典型的抗體結構如F(ab)2、scFv-Fab、(scFv)2-Fab等。IgG樣雙特異性抗體(例如具有Fc片段)，這類抗體相對分子量較大，Fc片段有助於抗體的純化，並提高其溶解性、穩定性，Fc部分還可能會與受體FcRn結合，增加抗體血清半衰期，典型的雙特異性抗體結構模型如KiH、CrossMAb、Triomab quadroma、Fc△Adp、ART-Ig、BiMAb、Biclonics、BEAT、DuoBody、Azymetric、XmAb、2：1 TCBs、1Fab-IgG TDB、FynomAb、two-in-one/DAF、scFv-
Fab-IgG、DART-Fc、LP-DART、CODV-Fab-TL、HLE-BiTE、F(ab)2-CrossMAb、IgG-(scFv)2、Bs4Ab、DVD-Ig、Tetravalent-DART-Fc、(scFv)4-Fc、CODV-Ig、mAb2、F(ab)4-CrossMAb等(參見Aran F.Labrijn等，Nature Reviews Drug Discovery volume 18，pages585-608(2019)；Chen S1等，J Immunol Res.2019 Feb 11；2019：4516041)。
The term "bispecific antibody" refers to an antibody (including an antibody or an antigen-binding fragment thereof, such as a single-chain antibody) capable of specifically binding to two different antigens or two different epitopes of the same antigen. The prior art has disclosed bispecific antibodies of various structures, which can be divided into IgG-like bispecific antibodies and antibody fragment bispecific antibodies according to the integrity of the IgG molecule; according to the number of antigen-binding regions, they can be divided into bivalent and trivalent bispecific antibodies. Bispecific antibodies with valence, tetravalent or more; according to whether the structure is symmetrical, they can be divided into bispecific antibodies with symmetrical structure and bispecific antibodies with asymmetric structure. Among them, fragment-type bispecific antibodies, such as Fab fragments lacking Fc fragments, which form bispecific antibodies by combining two or more Fab fragments in one molecule, have lower immunogenicity, and have a lower molecular weight. Small, with high tumor tissue permeability; typical antibody structures of this type are F(ab)2, scFv-Fab, (scFv)2-Fab, etc. IgG-like bispecific antibody (for example, with Fc fragment), this type of antibody has a relatively large molecular weight, and the Fc fragment helps to purify the antibody and improve its solubility and stability. The Fc part may also bind to the receptor FcRn, Increase antibody serum half-life, typical bispecific antibody structure models such as KiH, CrossMAb, Triomab quadroma, Fc△Adp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCBs, 1Fab-IgG TDB , FynomAb, two-in-one/DAF, scFv-
Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb, IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent-DART-Fc, ( scFv)4-Fc, CODV-Ig, mAb2, F(ab)4-CrossMAb, etc. (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages585-608 (2019); Chen S1 et al., J Immunol Res. 2019
術語“可變區”或“可變域”指抗體重鏈或輕鏈中參與抗體結合抗原的域。本文中，特異性結合BCMA的第一抗原結合域中的重鏈可變區標示為BCMA-VH，輕鏈可變區標示為BCMA-VL；特異性結合CD3的第二抗原結合域中的重鏈可變區標示為CD3-VH，輕鏈可變區標示為CD3-VL。VH和VL各包含四個保守的框架區(FR)和三個互補決定區(CDR)。其中，術語“互補決定區”或“CDR”指可變結構域內主要促成與抗原結合的區域；“框架”或“FR”是指除CDR殘基之外的可變結構域殘基。VH包含3個CDR區：HCDR1、HCDR2和HCDR3；VL包含3個CDR區：LCDR1、LCDR2和LCDR3。本文中，BCMA-VH中的3個CDR區分別標示為BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3；BCMA-VL中的3個CDR區分別標示為BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3；CD3-VH中的3個CDR區分別標示為CD3-HCDR1、CD3-HCDR2和CD3-HCDR3；CD3-VL中的3個CDR區分別標示為CD3-LCDR1、CD3-LCDR2和CD3-LCDR3。每個VH和VL由從胺基末端排到羧基末端按以下順序排列的三個CDR和四個FR構成：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。單個VH或VL可能足以賦予抗原結合特異性。 The term "variable region" or "variable domain" refers to the domains of an antibody heavy or light chain that are involved in binding the antibody to antigen. Herein, the heavy chain variable region in the first antigen-binding domain that specifically binds BCMA is marked as BCMA-VH, and the light chain variable region is marked as BCMA-VL; the heavy chain variable region in the second antigen-binding domain that specifically binds CD3 The chain variable region is denoted as CD3-VH and the light chain variable region as CD3-VL. VH and VL each contain four conserved framework regions (FRs) and three complementarity determining regions (CDRs). Among them, the term "complementarity determining region" or "CDR" refers to the region in the variable domain that mainly contributes to binding to the antigen; "framework" or "FR" refers to the variable domain residues other than the CDR residues. VH contains 3 CDR regions: HCDR1, HCDR2 and HCDR3; VL contains 3 CDR regions: LCDR1, LCDR2 and LCDR3. In this paper, the three CDR regions in BCMA-VH are marked as BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3; the three CDR regions in BCMA-VL are marked as BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 ; The three CDR regions in CD3-VH are respectively marked as CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3; the three CDR regions in CD3-VL are respectively marked as CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3. Each VH and VL consists of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL may be sufficient to confer antigen binding specificity.
可以藉由各種公知方案來確定CDR的胺基酸序列邊界，例如：“Kabat”編號規則(參見Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”編號規則、“ABM”編號規則、“contact”編號規則(參見Martin,ACR.Protein Sequence and Structure Analysis of Antibody Variable Domains[J].2001)和ImMunoGenTics(IMGT)編號規則(Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003)；Front Immunol.2018 Oct 16；9：2278)等；各種編號系統之間的對應關係是所屬技術領域具有通常知識者熟知的，示例性的，如下表1中所示。 The amino acid sequence boundaries of CDRs can be determined by various known schemes, for example: "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD), "Chothia" numbering sequence, "ABM" numbering sequence, "contact" numbering sequence (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001) and ImMunoGenTics ( IMGT) numbering rules (Lefranc, M.P., etc., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol.2018 Oct 16; 9: 2278), etc.; the corresponding relationship between various numbering systems is the technical Domains are well known to those of ordinary skill, exemplary, as shown in Table 1 below.
表1. CDR編號系統之間的關係
除非另有說明，本揭露實施例中的可變區和CDR序列均適用“Kabat”編號規則。 Unless otherwise stated, the "Kabat" numbering convention applies to the variable regions and CDR sequences in the examples of the present disclosure.
術語“抗體片段”指不同於完整抗體的分子，其包含完整抗體的部分，該部分保留了完整抗體的抗原結合能力。抗體片段的實例包括但不限於Fv、Fab、Fab’、Fab’-SH、F(ab')2、單域抗體、單鏈Fab(scFab)、雙抗體、線性抗體、單鏈抗體分子(例如scFv)；以及由抗體片段形成的多特異性抗體。 The term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that retains the antigen-binding ability of the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab ' ) 2 , single domain antibodies, single chain Fab (scFab), diabodies, linear antibodies, single chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
術語“Fc區”或“片段可結晶區”用於定義抗體重鏈的C末端區域，包括天然Fc區和改造的Fc區。在一些實施方式中，Fc區包含了相同或不同的兩個亞基。在一些實施方式中，人IgG重鏈的Fc區定義為從Cys226位置處的胺基酸殘基或從Pro230延伸至其羧基末端。用於本文所述抗體的合適天然序列Fc區包括人IgG1、IgG2(IgG2A、IgG2B)、IgG3和IgG4。除非另有說明，Fc區的編號規則為EU編號規則。 The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, including native and engineered Fc regions. In some embodiments, the Fc region comprises the same or different two subunits. In some embodiments, the Fc region of a human IgG heavy chain is defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. Suitable native sequence Fc regions for the antibodies described herein include human IgGl, IgG2 (IgG2A, IgG2B), IgG3 and IgG4. Unless otherwise stated, the numbering convention for the Fc region is the EU numbering convention.
術語“嵌合”抗體指這樣的一種抗體，其中重和/或輕鏈的一部分源自特定的來源或物種，而重和/或輕鏈的剩餘部分源自不同來源或物種。 The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chains is derived from a particular source or species and the remaining portion of the heavy and/or light chains is derived from a different source or species.
術語“人源化”抗體是保留非人抗體的反應性同時在人中具有較低免疫原性的抗體。例如，可以藉由保留非人CDR區、並用人對應物(即，恆定區以及可變區的框架區部分)替換抗體的其餘部分來實現人源化。 The term "humanized" antibody is an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. Humanization can be achieved, for example, by retaining the non-human CDR regions and replacing the remainder of the antibody with their human counterparts (ie, the constant regions and the framework portion of the variable regions).
術語“親和力(affinity)”是指分子(例如，抗體)的單個結合部位與其結合配體(例如，抗原)之間非共價相互作用的總體的強度。除非另外指明，如本文所用，“結合親和力”是指內部結合親和力，其反映出結合對(例如，抗體與抗原)的成員之間1：1相互作用。分子X對其配體Y的親和力通常可以由解離常數(KD)表示。親和力可以藉由本領域已知的常規方法(包括本文所述的那些)測量。術語“kassoc”或“ka”指特定抗體-抗原相互作用的締合速率，而如本文所使用的術語“kdis”或“kd”意在是指特定抗體-抗原相互作用的解離速率。如本文所使用的，術語“KD”指解離常數，其獲得自kd與ka的比率(即kd/ka)並且表示為莫耳濃度(M)。可以使用本領域已知的方法測定抗體的KD值，例如：用於測定抗體KD的 方法包括使用生物傳感系統例如系統測量表面電漿共振，或藉由溶液平衡滴定法(SET)測量溶液中的親和力。 The term "affinity" refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its ligand Y can generally be expressed by a dissociation constant (KD). Affinity can be measured by routine methods known in the art, including those described herein. The term "kassoc" or "ka" refers to the on-rate of a particular antibody-antigen interaction, while the term "kdis" or "kd" as used herein is intended to refer to the off-rate of a particular antibody-antigen interaction. As used herein, the term "KD" refers to the dissociation constant, which is obtained from the ratio of kd to ka (ie, kd/ka) and is expressed as a molar concentration (M). The KD value of the antibody can be determined using methods known in the art, for example: for determining the KD of the antibody Methods include measuring surface plasmon resonance using biosensing systems such as the system, or measuring affinity in solution by solution equilibrium titration (SET).
術語“效應子功能”指那些可歸於抗體Fc區(天然序列Fc區或胺基酸序列變體Fc區)且隨抗體同種型而變化的生物學活性。抗體效應子功能的例子包括：C1q結合和補體依賴性細胞毒性；Fc受體結合；抗體依賴性細胞介導的細胞毒性(ADCC)；吞噬作用；細胞表面受體(例如B細胞受體)下調；和B細胞活化。 The term "effector functions" refers to those biological activities attributable to an antibody Fc region (either native sequence Fc region or amino acid sequence variant Fc region) and which vary with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; ; and B cell activation.
術語“單株抗體”指基本上均質的抗體的群或其成員，即在該群中包含的抗體分子的胺基酸序列是相同的，除了可能少量存在的天然突變以外。相比之下，“多株抗體”製劑通常包含在其可變結構域具有不同胺基酸序列的多種不同抗體，其通常特異性針對不同表位。“單株”表示從基本上均質的抗體群體獲得的抗體的特徵，並且不應解釋為要求藉由特定方法來生產抗體。在一些實施方式中，本揭露提供的抗體是單株抗體。 The term "monoclonal antibody" refers to a population of antibodies or members thereof that are substantially homogeneous, ie, the antibody molecules comprised in the population are identical in amino acid sequence, except for natural mutations that may be present in minor amounts. In contrast, a "polyclonal antibody" preparation typically comprises a number of different antibodies with different amino acid sequences in their variable domains, often specific for different epitopes. "Monoclonal" denotes the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by a particular method. In some embodiments, the antibodies provided by the present disclosure are monoclonal antibodies.
術語“抗原”是指能夠由抗原結合蛋白(包括例如抗體)所選擇性識別或結合的分子或分子部分。抗原可具有一個或多個能夠與不同的抗原結合蛋白(例如抗體)相互作用的表位。 The term "antigen" refers to a molecule or portion of a molecule capable of being selectively recognized or bound by an antigen binding protein, including, for example, an antibody. An antigen may have one or more epitopes capable of interacting with different antigen binding proteins (eg antibodies).
術語“表位”指能夠與抗體或其抗原結合片段特異性結合的抗原上的區域(area或region)。表位可以由連續胺基酸(線性表位)形成；或包含非連續胺基酸(構象表位)，例如因抗原的折疊(即藉由蛋白質性質的抗原的三級折疊)而使得非連續的胺基酸在空間上得以接近。構象表位和線性表位的差別在於：在變性溶劑的存在下，抗體對構象表位的結合喪失。表位包含處於獨特空間構象的至少3、至少4、至少5、至少6、至少7、或8-10個胺基酸。可以使用本領域例行方法來篩選結合特定表位的抗體(即那些結合相同表位的)，例如但不限於丙胺酸掃描，肽印跡(見 Meth.Mol.Biol.248(2004)443-463)，肽切割分析，表位切除，表位提取，抗原的化學修飾(見Prot.Sci.9(2000)487-496)，和交叉阻斷(見“Antibodies”，Harlow and Lane(Cold Spring Harbor Press,Cold Spring Harb.,NY))。 The term "epitope" refers to an area (area or region) on an antigen capable of specifically binding to an antibody or antigen-binding fragment thereof. An epitope may be formed from contiguous amino acids (linear epitope); or contain discontinuous amino acids (conformational epitope), for example due to the folding of the antigen (i.e., tertiary folding of the antigen by proteinaceous nature) The amino acids are in close proximity in space. The difference between a conformational epitope and a linear epitope is that antibody binding to a conformational epitope is lost in the presence of denaturing solvents. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Antibodies that bind to a particular epitope (i.e., those that bind to the same epitope) can be screened using methods routine in the art, such as, but not limited to, alanine scanning, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443-463 ), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigen (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)).
與參照抗體“競爭結合的抗體”指在競爭測定法中，將參照抗體對抗原的結合阻斷50%或更多的抗體，或其對抗原的結合被參照抗體阻斷50%或更多的抗體。 An antibody that "competes for binding" with a reference antibody is an antibody that, in a competition assay, blocks the binding of the antigen by 50% or more of the reference antibody, or whose binding to the antigen is blocked by 50% or more by the reference antibody Antibody.
術語“能夠特異性結合”、“特異性結合”或“結合”是指相比其他抗原或表位，抗體能夠以更高的親和力結合至某個抗原或該抗原內的表位。通常，抗體以約1×10-7M或更小(例如約1×10-8M或更小)的平衡解離常數(KD)結合抗原或抗原內的表位。在一些實施方式中，抗體與抗原結合的KD為該抗體結合至非特異性抗原(例如BSA、酪蛋白)的KD的10%或更低(例如1%)。可使用已知的方法來測量KD，例如藉由BIACORE®表面電漿共振測定法所測量的。然而，特異性結合至抗原或抗原內的表位的抗體可能對其它相關的抗原具有交叉反應性，例如，對來自其它物種(同源)(諸如人或猴，例如食蟹獼猴(Macaca fascicularis)(cynomolgus，cyno)、黑猩猩(Pan troglodytes)(chimpanzee，chimp))或狨猴(Callithrix jacchus)(commonmarmoset，marmoset)的相應抗原具有交叉反應性。 The term "capable of specifically binding", "specifically binds" or "binds" means that an antibody is capable of binding to a certain antigen or an epitope within the antigen with a higher affinity than to other antigens or epitopes. Typically, an antibody binds the antigen or an epitope within the antigen with an equilibrium dissociation constant (KD) of about 1 x 10 -7 M or less (eg, about 1 x 10 -8 M or less). In some embodiments, the antibody binds an antigen with a KD that is 10% or less (eg, 1%) of the antibody's KD for binding to a non-specific antigen (eg, BSA, casein). KD can be measured using known methods, such as by BIACORE® surface plasmon resonance assay. However, antibodies that specifically bind to an antigen or an epitope within an antigen may have cross-reactivity to other related antigens , e.g. (cynomolgus, cyno), chimpanzee ( Pan troglodytes ) (chimpanzee, chimp)) or marmoset ( Callithrix jacchus ) (commonmarmoset, marmoset) are cross-reactive.
術語“抗BCMA抗體”和“特異性結合BCMA的抗原結合域”是指能夠以足夠的親和力結合BCMA的抗體或結構域，使得抗BCMA抗體或含有該結構域的分子可用作靶向BCMA的診斷劑和/或治療劑。在某些實施例中，與BCMA結合的抗體或結構域具有以下解離常數(KD)<約1μM、<約100nM、<約10nM，其是藉由FACS方法測量的。在某些 實施例中，抗BCMA抗體或特異性結合BCMA的抗原結合域結合來自不同物種的BCMA中保守的BCMA表位。 The terms "anti-BCMA antibody" and "antigen binding domain that specifically binds BCMA" refer to an antibody or domain that is capable of binding BCMA with sufficient affinity such that an anti-BCMA antibody or a molecule containing the domain can be used as a BCMA-targeting Diagnostic and/or therapeutic agents. In certain embodiments, an antibody or domain that binds BCMA has a dissociation constant (KD) of <about 1 μM, <about 100 nM, <about 10 nM, as measured by a FACS method. in some In embodiments, the anti-BCMA antibody or the antigen binding domain that specifically binds BCMA binds a BCMA epitope that is conserved among BCMA from different species.
術語“特異性結合CD3的抗原結合域”是指能夠以足夠的親和力結合CD3的結構域，使得含有該結構域的分子可用作靶向CD3的診斷劑和/或治療劑。在某些實施例中，特異性結合CD3的抗原結合域結合來自不同物種的CD3中保守的CD3表位。本文中，抗原結合域可以指抗原結合模塊，例如Fab、經替換的Fab或scFv。 The term "antigen binding domain that specifically binds CD3" refers to a domain that is capable of binding CD3 with sufficient affinity such that molecules containing this domain are useful as diagnostic and/or therapeutic agents targeting CD3. In certain embodiments, the antigen binding domain that specifically binds CD3 binds a CD3 epitope that is conserved among CD3 from different species. Herein, an antigen binding domain may refer to an antigen binding moiety, such as a Fab, substituted Fab or scFv.
術語“連接子linker”指連接兩個多肽片段的連接單元。在本文中，同一結構中出現的連接子可以是相同或不同的。連接子可以是肽連接子，其包含一個或多個胺基酸，典型的約1-30個、2-24個或3-15個胺基酸。應用於本文的連接子可以是相同或不同的。當“-”出現在結構式中，其表示兩側的單元直接藉由共價鍵連接。當術語“鍵”出現在結構單元，其表示該單元沒有胺基酸，單元兩側的單元直接連接。 The term "linker" refers to a linking unit that joins two polypeptide fragments. Herein, linkers appearing in the same structure may be the same or different. The linker may be a peptide linker comprising one or more amino acids, typically about 1-30, 2-24 or 3-15 amino acids. The linkers used herein may be the same or different. When "-" appears in the structural formula, it means that the units on both sides are directly linked by covalent bonds. When the term "bond" appears in a structural unit, it means that the unit has no amino acids, and the units on either side of the unit are directly connected.
術語“抗體依賴性細胞的細胞毒性”、“抗體依賴性細胞介導的細胞毒性”或“ADCC”是誘導細胞死亡的機制，該機制依賴於抗體包被的靶細胞與具有裂解活性的效應細胞(諸如自然殺傷細胞(NK)、單核細胞、巨噬細胞和中性粒細胞)經由效應細胞上表達的Fcγ受體(FcγR)發生的相互作用。例如，NK細胞表達FcγRIIIa，而單核細胞表達FcγRI、FcγRII和FcγRIIIa。本文提供的抗體的ADCC活性可使用體外測定，使用表達抗原的細胞作為靶細胞和NK細胞作為效應細胞進行評定。根據從裂解的細胞中釋放的標記物(例如放射性受質、螢光染料或天然胞內蛋白)來檢測細胞裂解。 The terms "antibody-dependent cellular cytotoxicity", "antibody-dependent cell-mediated cytotoxicity" or "ADCC" are mechanisms for inducing cell death that rely on the interaction of antibody-coated target cells with lytically active effector cells (such as natural killer cells (NK), monocytes, macrophages, and neutrophils) via Fcγ receptors (FcγRs) expressed on effector cells. For example, NK cells express FcyRIIIa, while monocytes express FcyRI, FcyRII, and FcyRIIIa. The ADCC activity of the antibodies provided herein can be assessed using an in vitro assay using antigen-expressing cells as target cells and NK cells as effector cells. Cell lysis is detected based on markers released from lysed cells, such as radioacceptors, fluorescent dyes, or native intracellular proteins.
術語“抗體依賴性細胞吞噬作用”(“ADCP”)是指藉由吞噬細胞(諸如巨噬細胞或樹突狀細胞)的內化作用消除抗體包被的靶細胞的機制。 The term "antibody-dependent cellular phagocytosis" ("ADCP") refers to the mechanism by which antibody-coated target cells are eliminated by internalization by phagocytes, such as macrophages or dendritic cells.
術語“補體依賴性細胞毒性”或“CDC”是指誘導細胞死亡的機制，其中靶結合抗體的Fc效應域結合並激活補體成分C1q，C1q繼而激活補體級聯，從而導致靶細胞死亡。補體的激活也可導致補體成分沉積在靶細胞表面上，這些補體成分藉由結合白細胞上的補體受體(例如，CR3)來促進CDC。 The term "complement-dependent cytotoxicity" or "CDC" refers to a cell death-inducing mechanism in which the Fc effector domain of a target-binding antibody binds and activates the complement component C1q, which in turn activates the complement cascade, resulting in target cell death. Activation of complement can also result in the deposition of complement components on the surface of target cells that promote CDC by binding to complement receptors (eg, CR3) on leukocytes.
術語“核酸”在本文中可與術語“多核苷酸”互換使用，並且是指呈單鏈或雙鏈形式的脫氧核糖核苷酸或核糖核苷酸及其聚合物。該術語涵蓋含有已知核苷酸類似物或修飾的骨架殘基或連接的核酸，該核酸是合成的、天然存在的和非天然存在的，具有與參考核酸相似的結合特性，並且以類似於參考核苷酸的方式代謝。此類類似物的實例包括但不限於硫代磷酸酯、胺基磷酸酯、甲基膦酸酯、手性-甲基膦酸酯、2-O-甲基核糖核苷酸、肽-核酸(PNA)。“分離的”核酸指已經與其天然環境的組分分開的核酸分子。分離的核酸包括在下述細胞中含有的核酸分子，該細胞通常含有該核酸分子，但該核酸分子存在於染色體外或存在於不同於其天然染色體位置的染色體位置處。編碼抗BCMA抗體或該抗原結合分子的分離的核酸指編碼抗體重鏈和輕鏈(或其片段)的一個或更多個核酸分子，包括在單一載體或分開的載體中的這樣的一個或更多個核酸分子，和存在於宿主細胞中一個或更多個位置的這樣的一個或更多個核酸分子。除非另有說明，否則特定的核酸序列還隱含地涵蓋其保守修飾的變體(例如，簡並密碼子取代)和互補序列以及明確指明的序列。具體地，如下詳述，簡並密碼子 取代可以藉由產生如下序列而獲得，在這些序列中，一個或多個所選的(或全部)密碼子的第三位被簡並鹼基和/或脫氧肌苷殘基取代。 The term "nucleic acid" is used herein interchangeably with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, synthetic, naturally occurring and non-naturally occurring, having similar binding properties to a reference nucleic acid, and operating in a manner similar to Metabolized in the manner of reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioate, phosphoroamidate, methylphosphonate, chiral-methylphosphonate, 2-O-methylribonucleotide, peptide-nucleic acid ( PNA). An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location other than its natural chromosomal location. An isolated nucleic acid encoding an anti-BCMA antibody or the antigen-binding molecule refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including such one or more in a single vector or in separate vectors. A plurality of nucleic acid molecules, and such one or more nucleic acid molecules present at one or more locations in a host cell. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (eg, degenerate codon substitutions) and complementary sequences as well as the explicitly indicated sequence. Specifically, as detailed below, degenerate codons Substitutions may be made by generating sequences in which one or more selected (or all) codons are substituted at the third position by a degenerate base and/or a deoxyinosine residue.
術語“多肽”和“蛋白質”在本文中可互換使用，指胺基酸殘基的聚合物。該術語適用於胺基酸聚合物，其中一個或多個胺基酸殘基是天然存在的胺基酸相應的人工化學模擬物，以及適用於天然存在的胺基酸聚合物和非天然存在的胺基酸聚合物。除非另外說明，否則特定的多肽序列還隱含地涵蓋其保守修飾的變體。 The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are the corresponding artificial chemical mimics of a naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. Unless otherwise stated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
術語序列“同一性”指，當對兩條序列進行最佳比對時，兩條序列的胺基酸/核酸在等價位置相同的程度(百分比)；其中在比對過程中，必要時允許引入間隙以獲取最大序列同一性百分比，但任何保守性取代不視為構成序列同一性的一部分。為測定序列同一性百分比，比對可以藉由本領域技術已知的技術來實現，例如使用公開可得到的計算機軟體，諸如BLAST、BLAST-2、ALIGN、ALIGN-2或Megalign(DNASTAR)軟體。所屬技術領域具有通常知識者可確定適用於測量比對的參數，包括在所比較的序列全長上達成最大比對所需的任何算法。 The term sequence "identity" refers to the degree (percentage) to which the amino acids/nucleic acids of the two sequences are identical at equivalent positions when the two sequences are optimally aligned; Gaps are introduced to achieve the maximum percent sequence identity, but any conservative substitutions are not considered to form part of the sequence identity. To determine percent sequence identity, alignment can be achieved by techniques known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those of ordinary skill in the art can determine suitable parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
術語“融合”或“連接”是指部件(例如抗原結合結構域和Fc結構域)直接地或經由一個或多個連接子而藉由共價鍵連接。當連接子是肽連接子時，該共價鍵是肽鍵。 The terms "fused" or "linked" refer to the linking of components such as an antigen binding domain and an Fc domain by a covalent bond, either directly or via one or more linkers. When the linker is a peptide linker, the covalent bond is a peptide bond.
術語“載體vector”意指能夠遞送與其連接的另一多核苷酸的多核苷酸分子。一種類型的載體是“質粒”，其是指環狀雙鏈DNA環，其中可以連接附加的DNA區段。另一種類型的載體是病毒載體，例如腺相關病毒載體(AAV或AAV2)，其中另外的DNA區段可以連接到病毒基因組中。某些載體能夠在引入它們的宿主細胞中自主複製(例如，具有細菌複製起點的細菌載體和附加型哺乳動物載體)。其他載體(例如，非附加型 哺乳動物載體)可以在引入宿主細胞中後整合到宿主細胞的基因組中，從而與宿主基因組一起複製。術語“表達載體”或“表達構建體”是指適用於對宿主細胞進行轉化且含有指導和/或控制(連同宿主細胞一起)與其可操作地連接的一個或多個異源編碼區的表達的核酸序列的載體。表達構建體可以包括但不限於影響或控制轉錄、翻譯且在存在內含子時影響與其可操作地連接的編碼區的RNA剪接的序列。 The term "vector vector" means a polynucleotide molecule capable of delivering another polynucleotide to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, such as an adeno-associated viral vector (AAV or AAV2), in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (eg, bacterial vectors and episomal mammalian vectors with a bacterial origin of replication). Other vectors (e.g., non-epsomal mammalian vector) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome. The term "expression vector" or "expression construct" refers to a vector suitable for transformation of a host cell and containing the expression of one or more heterologous coding regions operably linked thereto to direct and/or control (along with the host cell). A vector of nucleic acid sequences. Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and, when an intron is present, RNA splicing of the coding region to which it is operably linked.
術語“宿主細胞”，“宿主細胞系”，和“宿主細胞培養物”可互換使用，並且指已經導入外源核酸的細胞，包括此類細胞的後代。宿主細胞包括“轉化體”和“經轉化的細胞”，其包括原代primary經轉化的細胞及自其衍生的後代，而不考慮傳代(passage)的次數。後代在核酸內容物上可以與親本(parent)細胞不完全相同，而是可以含有突變。本文中，該術語包括突變體後代，其與在原代轉化細胞中篩選或選擇的細胞具有相同的功能或生物學活性。宿主細胞包括原核和真核宿主細胞，其中真核宿主細胞包括但不限於哺乳動物細胞、昆蟲細胞系植物細胞和真菌細胞。哺乳動物宿主細胞包括人、小鼠、大鼠、犬、猴、豬、山羊、牛、馬和倉鼠細胞，包括但不限於中國倉鼠卵巢(CHO)細胞、NSO、SP2細胞、HeLa細胞、幼倉鼠腎(BHK)細胞、猴腎細胞(COS)、人肝細胞癌細胞(例如，HepG2)、A549細胞、3T3細胞和HEK-293細胞。真菌細胞包括酵母和絲狀真菌細胞，包括例如巴氏畢赤酵母(Pichiapastoris)、芬蘭畢赤酵母(Pichia finlandica)、海藻畢赤酵母(Pichia trehalophila)、科克拉馬畢赤酵母(Pichia koclamae)、膜狀畢赤酵母(Pichia membranaefaciens)、小畢赤酵母(Pichia minuta)(Ogataea minuta、Pichia lindneri)、仙人掌畢赤酵母(Pichiaopuntiae)、耐熱畢赤酵母(Pichia thermotolerans)、柳畢赤酵母(Pichia salictaria)、Pichia guercuum、皮傑普畢赤酵母(Pichia pijperi)、具柄畢赤酵母(Pichia stiptis)、甲醇畢赤酵母(Pichia methanolica)、畢赤酵母屬、釀酒酵母(Saccharomycescerevisiae)、釀酒酵母屬、多形漢遜酵母(Hansenula polymorpha)、克魯維酵母屬、乳酸克魯維酵母(Kluyveromyces lactis)、白色念珠菌(Candida albicans)、構巢麯黴(Aspergillus nidulans)、黑麯黴(Aspergillus niger)、米麯黴(Aspergillus oryzae)、裡氏木黴(Trichoderma reesei)、勒克氏菌(Chrysosporium lucknowense)、鐮刀菌屬(Fusarium sp.)、禾穀鐮刀菌(Fusarium gramineum)、菜鐮刀菌(Fusarium venenatum)、小立碗蘚(Physcomitrella patens)和粗糙脈孢菌(Neurospora crassa)。畢赤酵母屬、任何釀酒酵母屬、多形漢遜酵母(Hansenula polymorpha)、任何克魯維酵母屬、白色念珠菌(Candida albicans)、任何麯黴屬、裡氏木黴(Trichoderma reesei)、勒克黴菌(Chrysosporium lucknowense)、任何鐮刀菌屬、解脂耶氏酵母(Yarrowia lipolytica)和粗糙脈孢菌(Neurospora crassa)。 The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Herein, the term includes mutant progeny that have the same function or biological activity as the cells screened or selected for among the primary transformed cells. Host cells include prokaryotic and eukaryotic host cells, where eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster cells Kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg, HepG2), A549 cells, 3T3 cells, and HEK-293 cells. Fungal cells include yeast and filamentous fungal cells including, for example, Pichia pastoris, Pichia finlandica , Pichia trehalophila , Pichia koclamae , Pichia membranaefaciens , Pichia minuta ( Ogataea minuta , Pichia lindneri ), Pichia puntiae , Pichia thermotolerans , Pichia salictaria ), Pichia guercuum , Pichia pijperi , Pichia stiptis , Pichia methanolica , Pichia, Saccharomycescerevisiae , Saccharomyces, Hansenula polymorpha , Kluyveromyces, Kluyveromyces lactis , Candida albicans , Aspergillus nidulans , Aspergillus niger , rice Aspergillus oryzae , Trichoderma reesei , Chrysosporium lucknowense , Fusarium sp., Fusarium gramineum , Fusarium venenatum , Physcomitrella patens and Neurospora crassa . Pichia, any Saccharomyces, Hansenula polymorpha , any Kluyveromyces, Candida albicans , any Aspergillus, Trichoderma reesei , Luke Mold ( Chrysosporium lucknowense ), any Fusarium species, Yarrowia lipolytica ( Yarrowia lipolytica ) and Neurospora crassa ( Neurospora crassa ).
如在本申請中所使用的，表述“細胞”、“細胞系”和“細胞培養物”可以互換使用，並且所有這樣的名稱均包括子代。因而，詞語“轉化體”和“轉化的細胞”包括原代受試者細胞和來源於其的培養物，而與傳代的次數無關。還應理解的是，由於有意或無意的突變，使得並非所有子代均具有完全相同的DNA內容物。包括與篩選出其的原始轉化細胞具有相同功能或生物活性的突變子代。 As used in this application, the expressions "cell", "cell line" and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include primary subject cells and cultures derived therefrom, regardless of the number of passages. It should also be understood that not all progeny will have the exact same DNA content due to deliberate or unintentional mutations. Mutant progeny having the same function or biological activity as the original transformed cell from which they were screened are included.
“視需要”或“視需要地”意味著隨後所描述地特徵可以但不必發生，將指明包括該特徵發生或不發生的場合。 "Optional" or "optionally" means that the subsequently described feature may but need not occur, and the indication includes instances where the feature occurs or does not occur.
術語“醫藥組成物”表示含有一種或多種本文所述的抗體或抗原結合分子與其他化學組分的混合物，該其他組分例如生理學/可藥用的載體和賦形劑。 The term "pharmaceutical composition" means a mixture comprising one or more of the antibodies or antigen-binding molecules described herein and other chemical components such as physiological/pharmaceutically acceptable carriers and excipients.
術語“藥學上可接受的載體(vehicle)”指藥學製劑(formulation)中與活性成分不同的，且對受試者無毒的成分。藥學上可接受的載體包括但不限於緩衝劑、賦形劑、穩定劑或防腐劑。 The term "pharmaceutically acceptable carrier (vehicle)" refers to a pharmaceutical formulation (formulation) that is different from the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
術語“受試者”或“個體”包括人類和非人類動物。非人動物包括所有脊椎動物(例如哺乳動物和非哺乳動物)例如非人靈長類(例如，食蟹猴)、綿羊、狗、牛、雞、兩棲動物和爬行動物。除非明確指出，否則該術語“患者”或“受試者”在本文中可互換地使用。如本文所使用的，術語“食蟹猴(cyno)”或“食蟹猴(cynomolgus)”是指食蟹猴(Macaca fascicularis)。在某些實施方案中，個體或受試者是人。 The term "subject" or "individual" includes humans and non-human animals. Non-human animals include all vertebrates (eg, mammals and non-mammals) such as non-human primates (eg, cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles. The terms "patient" or "subject" are used interchangeably herein unless expressly stated otherwise. As used herein, the term "cyno" or "cynomolgus" refers to Macaca fascicularis . In certain embodiments, the individual or subject is a human.
“施用”或“給予”，當其應用於動物、人、實驗受試者、細胞、組織、器官或生物流體時，是指外源性藥物、治療劑、診斷劑或組成物與動物、人、受試者、細胞、組織、器官或生物流體的接觸。 "Administration" or "administration", when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids, refers to the interaction of exogenous drugs, therapeutic agents, diagnostic agents or compositions with animals, humans , subjects, cells, tissues, organs or biological fluids.
術語“樣本”是指從受試者分離的採集物(如流體、細胞、或組織)，以及存在於受試者體內的流體、細胞或組織。示例性樣本為生物流體，諸如血液、血清和漿膜液、血漿、淋巴液、尿液、唾液、囊液、淚液、排泄物、痰、分泌組織的或分泌器官的黏膜分泌物、陰道分泌物、腹水、胸膜、心包、腹膜、腹腔和其它體腔的流體、由支氣管灌洗液收集的流體、滑液、與受試者或生物來源接觸的液體溶液，例如培養基(包括條件培養基)、灌洗液等，組織活檢樣本、細針穿刺、手術切除的組織、器官培養物或細胞培養物。 The term "sample" refers to a collection (such as a fluid, cell, or tissue) isolated from a subject, as well as a fluid, cell, or tissue present in a subject. Exemplary samples are biological fluids such as blood, serum and serosal fluids, plasma, lymph, urine, saliva, cyst fluid, tears, faeces, sputum, mucous secretions of secretory tissues or organs, vaginal secretions, Ascites, pleura, pericardium, peritoneum, fluids of the peritoneal cavity and other body cavities, fluid collected from bronchial lavage, synovial fluid, liquid solutions in contact with the subject or biological source, such as culture medium (including conditioned medium), lavage fluid etc., tissue biopsy samples, fine needle aspirations, surgically removed tissue, organ cultures, or cell cultures.
“治療(treatment或treat)”和“處理”(及其語法變型)指試圖施加至所治療個體的臨床干預，並且可以為了預防目的、或者在臨床病理學的過程期間進行實施。治療的期望效果包括但不限於預防疾病的發生或復發，減輕症狀，減輕/減少疾病的任何直接或間接病理後果，預防轉移，降低疾病進展速率，改善或減輕疾病狀態，和消退或改善的預後。在一些實施方案中，使用本揭露的分子來延遲疾病的形成或減緩疾病的進展。 "Treatment" and "treatment" (and grammatical variants thereof) refer to clinical intervention intended to be applied to the individual being treated, and may be performed for prophylactic purposes, or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, prevention of occurrence or recurrence of disease, alleviation of symptoms, alleviation/reduction of any direct or indirect pathological consequences of disease, prevention of metastasis, reduction of rate of disease progression, amelioration or palliation of disease state, and regression or improved prognosis . In some embodiments, the molecules of the present disclosure are used to delay the development of a disease or slow the progression of a disease.
“有效量”一般是足以降低症狀的嚴重程度和/或頻率、消除這些症狀和/或潛在病因、預防症狀和/或其潛在病因出現、和/或改良或改善由疾病狀態引起或與其相關的損傷的量。在一些實施例中，有效量是治療有效量或預防有效量。“治療有效量”是這樣的量，其足以治療疾病狀態或症狀、尤其與該疾病狀態相關的狀態或症狀，或者以其他方式阻礙、延遲或逆轉該疾病狀態、或與該疾病相關的任何不理想症狀的進展。“預防有效量”是這樣的量，當被給予受試者時，將具有預定的預防效應，例如預防或延遲該疾病狀態的發作(或復發)，或者降低該疾病狀態或相關症狀的發作(或復發)可能性。完全治療或預防效未必在給予一個劑量之後立即發生，可能在給予一系列劑量之後發生。因而，治療或預防有效量可以一次或多次給予的方式給予。“治療有效量”和“預防有效量”可取決於多種因素變化：諸如個體的疾病狀態、年齡、性別和體重，以及治療劑或治療劑組合在個體中引發期望的應答的能力。有效治療劑或治療劑組合的示例性指標包括例如患者改善的健康狀況。 An "effective amount" is generally sufficient to reduce the severity and/or frequency of symptoms, eliminate these symptoms and/or underlying causes, prevent the occurrence of symptoms and/or their underlying causes, and/or ameliorate or improve the amount of damage. In some embodiments, the effective amount is a therapeutically or prophylactically effective amount. A "therapeutically effective amount" is an amount sufficient to treat a disease state or symptom, especially a state or symptom associated with the disease state, or otherwise hinder, delay or reverse the disease state, or any adverse effect associated with the disease state. Progression of desired symptoms. A "prophylactically effective amount" is an amount that, when administered to a subject, will have a predetermined prophylactic effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the onset (or recurrence) of the disease state or associated symptoms ( or recurrence) possibility. Complete therapeutic or prophylactic effect does not necessarily occur immediately after administration of one dose, but may occur after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount may be administered in one or more administrations. "Therapeutically effective amount" and "prophylactically effective amount" can vary depending on factors such as the disease state, age, sex and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status of a patient.
本揭露的抗原結合分子Antigen binding molecules of the present disclosure
本揭露提供了抗原結合分子，其具有諸多有利的特性，例如親和力、對細胞表面BCMA的特異性、在BCMA存在下特異性激活T細 胞的活性、治療活性、安全性(如更低的細胞因子釋放)、藥物代謝動力學特性和成藥性(如產率、純度和穩定性等)。 The present disclosure provides antigen-binding molecules with favorable properties such as affinity, specificity for cell surface BCMA, specific activation of T cells in the presence of BCMA, Cell activity, therapeutic activity, safety (such as lower cytokine release), pharmacokinetic properties and druggability (such as yield, purity and stability, etc.).
示例性的抗原結合分子Exemplary Antigen Binding Molecules
在一個方面，本揭露提供了一種抗原結合分子，其包含至少一個特異性結合BCMA的第一抗原結合域和至少一個特異性結合CD3的第二抗原結合域。特別的，本揭露的抗原結合分子具有： In one aspect, the present disclosure provides an antigen binding molecule comprising at least one first antigen binding domain that specifically binds BCMA and at least one second antigen binding domain that specifically binds CD3. In particular, the antigen-binding molecules of the present disclosure have:
a．針對膜表面BCMA的高親和力。在一些實施方式中，該抗原結合分子以小於20nM的親和力EC50與過表達人BCMA的穩轉細胞株K562表達的BCMA蛋白結合，該親和力EC50是藉由FACS方法測定的。在一些實施方式中，該抗原結合分子以小於100nM的親和力EC50與過表達食蟹猴BCMA的穩轉細胞株CHOK1表達的BCMA蛋白結合，該親和力EC50是藉由FACS方法測定的。具體測試方法見測試例1。 a. High affinity for BCMA on the membrane surface. In some embodiments, the antigen-binding molecule binds to the BCMA protein expressed by the stable cell line K562 overexpressing human BCMA with an affinity EC50 of less than 20 nM, and the affinity EC50 is determined by FACS method. In some embodiments, the antigen-binding molecule binds to the BCMA protein expressed by the stable transgenic cell line CHOK1 overexpressing cynomolgus BCMA with an affinity EC50 of less than 100 nM, the affinity EC50 being determined by FACS method. See Test Example 1 for specific test methods.
b．不受高濃度可溶性BCMA影響而優先結合膜表面BCMA的能力，在一些實施方式中，200ng/ml可溶性BCMA存在條件下與無可溶性BCMA條件下相比，該抗原結合分子與內源性表達BCMA的骨髓瘤細胞系NCIH929表達的BCMA蛋白結合的親和力EC50變化不超過30%，該親和力EC50是藉由FACS方法測定的。具體測試方法見測試例2。 b. The ability to preferentially bind BCMA to a membrane surface independent of high concentrations of soluble BCMA, in some embodiments, the antigen-binding molecule is associated with endogenously expressed BCMA in the presence of 200 ng/ml soluble BCMA compared to the absence of soluble BCMA The EC50 of binding affinity of BCMA protein expressed by the myeloma cell line NCIH929 did not vary by more than 30%, and the affinity EC50 was determined by FACS method. See Test Example 2 for specific test methods.
c．體外特異性激活T細胞的活性。在一些實施方式中，該抗原結合分子在表達BCMA的細胞時可激活T細胞，但在不表達BCMA的細胞存在時不激活T細胞。具體測試方法見測試例3。 c. In vitro specific activation of T cell activity. In some embodiments, the antigen binding molecule activates T cells in the presence of cells expressing BCMA but does not activate T cells in the presence of cells not expressing BCMA. See Test Example 3 for specific test methods.
d．針對表達BCMA細胞的體外特異性殺傷活性。在一些實施方式中，該抗原結合分子可特異性殺傷表達BCMA的細胞，但不能殺傷不表達BCMA的細胞。具體測試方法見測試例4。 d. In vitro specific killing activity against BCMA-expressing cells. In some embodiments, the antigen binding molecule can specifically kill cells expressing BCMA but not cells not expressing BCMA. See Test Example 4 for specific test methods.
e．誘導低水平的細胞因子(IL6和IFNγ)釋放。在一些實施方式中，該抗原結合分子在100nM濃度條件下，殺傷表達BCMA的細胞系U266B時產生不高於2ng/mL的IL-6，該細胞因子釋放是藉由ELISA方法檢測的。在一些實施方式中，該抗原結合分子在濃度20nM或更低條件下，殺傷表達BCMA的細胞系U266B時產生不高於10ng/mL的IFNγ，該細胞因子釋放是藉由HTRF方法檢測的。具體測試方法見測試例6。 e. Induces low levels of cytokine (IL6 and IFNγ) release. In some embodiments, the antigen-binding molecule produces no more than 2 ng/mL of IL-6 when killing the BCMA-expressing cell line U266B at a concentration of 100 nM, and the cytokine release is detected by an ELISA method. In some embodiments, the antigen-binding molecule produces no more than 10 ng/mL of IFNγ when killing the BCMA-expressing cell line U266B at a concentration of 20 nM or lower, and the cytokine release is detected by the HTRF method. See Test Example 6 for specific test methods.
f．更強的體內治療活性。(測試例7、8)。 f. Stronger in vivo therapeutic activity. (Test examples 7, 8).
在一個方面，本揭露提供了一種抗原結合分子，其包含至少一個特異性結合BCMA的第一抗原結合域和至少一個特異性結合CD3的第二抗原結合域，該特異性結合BCMA的第一抗原結合域包含重鏈可變區BCMA-VH和輕鏈可變區BCMA-VL，其中， In one aspect, the present disclosure provides an antigen binding molecule comprising at least one first antigen binding domain that specifically binds BCMA and at least one second antigen binding domain that specifically binds CD3, the first antigen that specifically binds BCMA The binding domain comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, wherein,
(i)該重鏈可變區BCMA-VH具有：胺基酸序列如SEQ ID NO：5所示的BCMA-HCDR1，胺基酸序列如SEQ ID NO：6所示的BCMA-HCDR2，和胺基酸序列如SEQ ID NO：7所示的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：胺基酸序列如SEQ ID NO：8所示的BCMA-LCDR1，胺基酸序列如SEQ ID NO：9所示的BCMA-LCDR2，和胺基酸序列如SEQ ID NO：10所示的BCMA-LCDR3；或 (i) The heavy chain variable region BCMA-VH has: BCMA-HCDR1 having an amino acid sequence as shown in SEQ ID NO: 5, BCMA-HCDR2 having an amino acid sequence as shown in SEQ ID NO: 6, and amine The amino acid sequence is BCMA-HCDR3 as shown in SEQ ID NO: 7; and the light chain variable region BCMA-VL has: the amino acid sequence is BCMA-LCDR1 as shown in SEQ ID NO: 8, and the amino acid sequence is as BCMA-LCDR2 as shown in SEQ ID NO: 9, and BCMA-LCDR3 with the amino acid sequence as shown in SEQ ID NO: 10; or
(ii)該重鏈可變區BCMA-VH具有：胺基酸序列如SEQ ID NO：11所示的BCMA-HCDR1，胺基酸序列如SEQ ID NO：12所示的BCMA-HCDR2，和胺基酸序列如SEQ ID NO：13所示的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：胺基酸序列如SEQ ID NO：14所示的BCMA-LCDR1，胺基酸序列如SEQ ID NO：15所示的BCMA-LCDR2，和胺基酸序列如SEQ ID NO：16所示的BCMA-LCDR3；或 (ii) The heavy chain variable region BCMA-VH has: BCMA-HCDR1 having an amino acid sequence as shown in SEQ ID NO: 11, BCMA-HCDR2 having an amino acid sequence as shown in SEQ ID NO: 12, and amine The amino acid sequence is BCMA-HCDR3 as shown in SEQ ID NO: 13; and the light chain variable region BCMA-VL has: the amino acid sequence is BCMA-LCDR1 as shown in SEQ ID NO: 14, and the amino acid sequence is as BCMA-LCDR2 shown in SEQ ID NO: 15, and BCMA-LCDR3 whose amino acid sequence is shown in SEQ ID NO: 16; or
(iii)該重鏈可變區BCMA-VH具有：胺基酸序列如SEQ ID NO：17所示的BCMA-HCDR1，胺基酸序列如SEQ ID NO：18所示的BCMA-HCDR2，和胺基酸序列如SEQ ID NO：19所示的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：胺基酸序列如SEQ ID NO：20所示的BCMA-LCDR1，胺基酸序列如SEQ ID NO：21所示的BCMA-LCDR2，和胺基酸序列如SEQ ID NO：22所示的BCMA-LCDR3；或 (iii) the heavy chain variable region BCMA-VH has: BCMA-HCDR1 having an amino acid sequence as shown in SEQ ID NO: 17, BCMA-HCDR2 having an amino acid sequence as shown in SEQ ID NO: 18, and amine The amino acid sequence is BCMA-HCDR3 as shown in SEQ ID NO: 19; and the light chain variable region BCMA-VL has: the amino acid sequence is BCMA-LCDR1 as shown in SEQ ID NO: 20, and the amino acid sequence is as BCMA-LCDR2 shown in SEQ ID NO: 21, and BCMA-LCDR3 whose amino acid sequence is shown in SEQ ID NO: 22; or
(iv)該重鏈可變區BCMA-VH具有：胺基酸序列如SEQ ID NO：23所示的BCMA-HCDR1，胺基酸序列如SEQ ID NO：24所示的BCMA-HCDR2，和胺基酸序列如SEQ ID NO：25所示的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：胺基酸序列如SEQ ID NO：26所示的BCMA-LCDR1，胺基酸序列如SEQ ID NO：27所示的BCMA-LCDR2，和胺基酸序列如SEQ ID NO：28所示的BCMA-LCDR3。 (iv) The heavy chain variable region BCMA-VH has: BCMA-HCDR1 having an amino acid sequence as shown in SEQ ID NO: 23, BCMA-HCDR2 having an amino acid sequence as shown in SEQ ID NO: 24, and amine The amino acid sequence is BCMA-HCDR3 as shown in SEQ ID NO: 25; and the light chain variable region BCMA-VL has: the amino acid sequence is BCMA-LCDR1 as shown in SEQ ID NO: 26, and the amino acid sequence is as BCMA-LCDR2 shown in SEQ ID NO: 27, and BCMA-LCDR3 with amino acid sequence shown in SEQ ID NO: 28.
該BCMA-HCDR1、BCMA-HCDR2、BCMA-HCDR3、BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3是根據Kabat編號規則定義的。 The BCMA-HCDR1, BCMA-HCDR2, BCMA-HCDR3, BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 are defined according to the Kabat numbering convention.
在一些實施方案中，如前所述的抗原結合分子，其中 In some embodiments, the antigen binding molecule as described above, wherein
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：29所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：30所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 29, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 30, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：37所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：40所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 40, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：38所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：40所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 38, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 40, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：39所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：40所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 39, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 40, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：37所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：41所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 41, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：37所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：42所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 42, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：37所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：43所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 43, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：31所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：32所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 32, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：44所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：47所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 47, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：45所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：47所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 45, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 47, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：46所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：47所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 47, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：44所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：48所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 48, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：45所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：48所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 45, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 48, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：46所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：48所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 48, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：33所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：34所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 34, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：49所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：51所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 49, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 51, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：50所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：51所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 50, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 51, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：35所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：36所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 36, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：52所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：54所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 52, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 54, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：53所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：54所示。 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO:53, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO:54.
在一些實施方案中，如前任一項所述的抗原結合分子，其中該特異性結合CD3的第二抗原結合域包含重鏈可變區CD3-VH和輕鏈可變區CD3-VL，其中， In some embodiments, the antigen-binding molecule of any one of the preceding, wherein the second antigen-binding domain that specifically binds CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, wherein,
(i)該重鏈可變區CD3-VH具有：胺基酸序列如SEQ ID NO：55所示的CD3-HCDR1，胺基酸序列如SEQ ID NO：56所示的CD3-HCDR2，和胺基酸序列如SEQ ID NO：57所示的CD3-HCDR3；並且該輕鏈可變區CD3-VL具有：胺基酸序列如SEQ ID NO：58所示的CD3-LCDR1，胺基酸序列如SEQ ID NO：59所示的CD3-LCDR2，和胺基酸序列如SEQ ID NO：60所示的CD3-LCDR3；或 (i) the heavy chain variable region CD3-VH has: the amino acid sequence of CD3-HCDR1 shown in SEQ ID NO: 55, the amino acid sequence of CD3-HCDR2 shown in SEQ ID NO: 56, and amine The amino acid sequence is CD3-HCDR3 as shown in SEQ ID NO: 57; and the light chain variable region CD3-VL has: the amino acid sequence is CD3-LCDR1 as shown in SEQ ID NO: 58, and the amino acid sequence is as CD3-LCDR2 shown in SEQ ID NO: 59, and CD3-LCDR3 whose amino acid sequence is shown in SEQ ID NO: 60; or
(ii)該重鏈可變區CD3-VH具有：胺基酸序列如SEQ ID NO：55所示的CD3-HCDR1，胺基酸序列如SEQ ID NO：61所示的CD3-HCDR2，和胺基酸序列如SEQ ID NO：62所示的CD3-HCDR3；並且該輕鏈可變區CD3-VL具有：胺基酸序列如SEQ ID NO：58所示的CD3-LCDR1，胺基酸序列如SEQ ID NO：59所示的CD3-LCDR2，和胺基酸序列如SEQ ID NO：60所示的CD3-LCDR3。 (ii) the heavy chain variable region CD3-VH has: the amino acid sequence of CD3-HCDR1 shown in SEQ ID NO: 55, the amino acid sequence of CD3-HCDR2 shown in SEQ ID NO: 61, and amine The amino acid sequence is CD3-HCDR3 as shown in SEQ ID NO: 62; and the light chain variable region CD3-VL has: the amino acid sequence is CD3-LCDR1 as shown in SEQ ID NO: 58, and the amino acid sequence is as CD3-LCDR2 shown in SEQ ID NO: 59, and CD3-LCDR3 with amino acid sequence shown in SEQ ID NO: 60.
在一些實施方案中，該CD3-HCDR1、CD3-HCDR2、CD3-HCDR3、CD3-LCDR1、CD3-LCDR2和CD3-LCDR3是根據Kabat編號規則定義的。 In some embodiments, the CD3-HCDR1, CD3-HCDR2, CD3-HCDR3, CD3-LCDR1, CD3-LCDR2, and CD3-LCDR3 are defined according to the Kabat numbering convention.
在一些實施方案中，如前任一項所述的抗原結合分子，該重鏈可變區CD3-VH的胺基酸序列如SEQ ID NO：63所示，和該輕鏈可變區CD3-VL的胺基酸序列如SEQ ID NO：64所示；或該重鏈可變區CD3-VH的胺基酸序列如SEQ ID NO：65所示，和該輕鏈可變區CD3-VL的胺基酸序列如SEQ ID NO：66所示。 In some embodiments, the antigen-binding molecule according to any one of the preceding items, the amino acid sequence of the heavy chain variable region CD3-VH is as shown in SEQ ID NO: 63, and the light chain variable region CD3-VL The amino acid sequence of the heavy chain variable region CD3-VH is shown in SEQ ID NO: 64; or the amino acid sequence of the heavy chain variable region CD3-VH is shown in SEQ ID NO: 65, and the amine of the light chain variable region CD3-VL The amino acid sequence is shown in SEQ ID NO:66.
在一些實施方案中，如前任一項所述的抗原結合分子，其中該重鏈可變區BCMA-VH具有：胺基酸序列如SEQ ID NO：5所示的BCMA-HCDR1，胺基酸序列如SEQ ID NO：6所示的BCMA-HCDR2，和胺基酸序列如SEQ ID NO：7所示的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：胺基酸序列如SEQ ID NO：8所示的BCMA-LCDR1，胺基酸序列如SEQ ID NO：9所示的BCMA-LCDR2，和胺基酸序列如SEQ ID NO：10所示的BCMA-LCDR3，和 In some embodiments, the antigen-binding molecule according to any one of the preceding items, wherein the heavy chain variable region BCMA-VH has: the amino acid sequence of BCMA-HCDR1 as shown in SEQ ID NO: 5, the amino acid sequence BCMA-HCDR2 as shown in SEQ ID NO: 6, and BCMA-HCDR3 with an amino acid sequence as shown in SEQ ID NO: 7; and the light chain variable region BCMA-VL has: an amino acid sequence such as SEQ ID BCMA-LCDR1 shown in NO: 8, BCMA-LCDR2 with amino acid sequence shown in SEQ ID NO: 9, and BCMA-LCDR3 with amino acid sequence shown in SEQ ID NO: 10, and
該重鏈可變區CD3-VH具有：胺基酸序列如SEQ ID NO：55所示的CD3-HCDR1，胺基酸序列如SEQ ID NO：56所示的CD3-HCDR2，和包含SEQ ID NO：胺基酸序列如SEQ ID NO：57所示的CD3-HCDR3；並且該輕鏈可變區CD3-VL具有：胺基酸序列如SEQ ID NO：58所示的CD3-LCDR1，胺基酸序列如SEQ ID NO：59所示的CD3-LCDR2，和胺基酸序列如SEQ ID NO：60所示的CD3-LCDR3。 The heavy chain variable region CD3-VH has: the amino acid sequence of CD3-HCDR1 shown in SEQ ID NO: 55, the amino acid sequence of CD3-HCDR2 shown in SEQ ID NO: 56, and comprising SEQ ID NO : the amino acid sequence of CD3-HCDR3 shown in SEQ ID NO: 57; and the light chain variable region CD3-VL has: the amino acid sequence of CD3-LCDR1 shown in SEQ ID NO: 58, amino acid CD3-LCDR2 whose sequence is shown in SEQ ID NO:59, and CD3-LCDR3 whose amino acid sequence is shown in SEQ ID NO:60.
在一些實施方案中，該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：37所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：40所示，和 In some embodiments, the amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 40 shown, and
該重鏈可變區CD3-VH的胺基酸序列如SEQ ID NO：63所示，和該輕鏈可變區CD3-VL的胺基酸序列如SEQ ID NO：64所示。 The amino acid sequence of the heavy chain variable region CD3-VH is shown in SEQ ID NO:63, and the amino acid sequence of the light chain variable region CD3-VL is shown in SEQ ID NO:64.
在一些實施方案中，如前任一項所述的抗原結合分子，其中該重鏈可變區BCMA-VH具有：胺基酸序列如SEQ ID NO：5所示的BCMA-HCDR1，胺基酸序列如SEQ ID NO：6所示的BCMA-HCDR2，和胺基酸序列如SEQ ID NO：7所示的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：胺基酸序列如SEQ ID NO：8所示的BCMA-LCDR1，胺基酸序列如SEQ ID NO：9所示的BCMA-LCDR2，和胺基酸序列如SEQ ID NO：10所示的BCMA-LCDR3，和 In some embodiments, the antigen-binding molecule according to any one of the preceding items, wherein the heavy chain variable region BCMA-VH has: the amino acid sequence of BCMA-HCDR1 as shown in SEQ ID NO: 5, the amino acid sequence BCMA-HCDR2 as shown in SEQ ID NO: 6, and BCMA-HCDR3 with an amino acid sequence as shown in SEQ ID NO: 7; and the light chain variable region BCMA-VL has: an amino acid sequence such as SEQ ID BCMA-LCDR1 shown in NO: 8, BCMA-LCDR2 with amino acid sequence shown in SEQ ID NO: 9, and BCMA-LCDR3 with amino acid sequence shown in SEQ ID NO: 10, and
該重鏈可變區CD3-VH具有：胺基酸序列如SEQ ID NO：55所示的CD3-HCDR1，胺基酸序列如SEQ ID NO：61所示的CD3-HCDR2，和胺基酸序列如SEQ ID NO：62所示的CD3-HCDR3；並且該輕鏈可變區CD3-VL具有：胺基酸序列如SEQ ID NO：58所示的CD3-LCDR1，胺基酸序列如SEQ ID NO：59所示的CD3-LCDR2，和胺基酸序列如SEQ ID NO：60所示的CD3-LCDR3。 The heavy chain variable region CD3-VH has: the amino acid sequence of CD3-HCDR1 shown in SEQ ID NO: 55, the amino acid sequence of CD3-HCDR2 shown in SEQ ID NO: 61, and the amino acid sequence CD3-HCDR3 as shown in SEQ ID NO: 62; and the light chain variable region CD3-VL has: CD3-LCDR1 with an amino acid sequence such as SEQ ID NO: 58, and an amino acid sequence such as SEQ ID NO :59 for CD3-LCDR2, and the amino acid sequence for CD3-LCDR3 as shown in SEQ ID NO:60.
在一些實施方案中，該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：37所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：43所示，和 In some embodiments, the amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 43 shown, and
該重鏈可變區CD3-VH的胺基酸序列如SEQ ID NO：65所示，和該輕鏈可變區CD3-VL的胺基酸序列如SEQ ID NO：66所示。 The amino acid sequence of the heavy chain variable region CD3-VH is shown in SEQ ID NO:65, and the amino acid sequence of the light chain variable region CD3-VL is shown in SEQ ID NO:66.
在一些實施方案中，如前任一項所述的抗原結合分子，其中該抗原結合分子還包含Fc區，該Fc區包含能夠締合的兩個亞基。在一些實施方案中，該兩個亞基是相同或不同的第一亞基和第二亞基。在一些實施方案中，該Fc區是IgG Fc區，特別是IgG1 Fc區。 In some embodiments, the antigen binding molecule of any one of the preceding, wherein the antigen binding molecule further comprises an Fc region comprising two subunits capable of associating. In some embodiments, the two subunits are the same or different first and second subunits. In some embodiments, the Fc region is an IgG Fc region, particularly an IgG 1 Fc region.
抗原結合分子的結構Structure of Antigen Binding Molecule
本揭露提供一種雙價的特異性結合BCMA和雙價的特異性結合CD3的抗原結合分子(2+2 format)。 The present disclosure provides a bivalent antigen-binding molecule (2+2 format) that specifically binds to BCMA and bivalently specifically binds to CD3.
示例性的，抗原結合分子包含兩條具有式(a)所示結構的第一鏈和兩條具有式(b)所示結構的第二鏈， Exemplarily, the antigen-binding molecule comprises two first chains having a structure represented by formula (a) and two second chains having a structure represented by formula (b),
(a)[BCMA-VH]-[CH1]-[CD3-VH]-[連接子]-[CD3-VL]-[連接子]-[Fc區的一個亞基]；式(a)中的連接子較佳為相同或不同的肽連接子； (a) [BCMA-VH]-[CH1]-[CD3-VH]-[linker]-[CD3-VL]-[linker]-[a subunit of the Fc region]; in formula (a) The linkers are preferably the same or different peptide linkers;
(b)[BCMA-VL]-[CL]。 (b) [BCMA-VL]-[CL].
示例性的分子包括，一種抗原結合分子，其具有： Exemplary molecules include, an antigen binding molecule having:
包含SEQ ID NO：76的胺基酸序列的第一鏈，和包含SEQ ID NO：72的胺基酸序列的第二鏈；或 A first strand comprising the amino acid sequence of SEQ ID NO: 76, and a second strand comprising the amino acid sequence of SEQ ID NO: 72; or
包含SEQ ID NO：79的胺基酸序列的第一鏈，和包含SEQ ID NO：75的胺基酸序列的第二鏈。 A first strand comprising the amino acid sequence of SEQ ID NO:79, and a second strand comprising the amino acid sequence of SEQ ID NO:75.
示例性的，該抗原結合分子包含兩條具有式(a-1)所示結構的第一鏈和兩條具有式(b-1)所示結構的第二鏈， Exemplarily, the antigen-binding molecule comprises two first chains having a structure represented by formula (a-1) and two second chains having a structure represented by formula (b-1),
(a-1)[CD3-VH]-[CH1]-[BCMA-VH]-[連接子]-[BCMA-VL]-[連接子]-[Fc區的一個亞基]；式(a)中的連接子較佳為相同或不同的肽連接子； (a-1) [CD3-VH]-[CH1]-[BCMA-VH]-[linker]-[BCMA-VL]-[linker]-[a subunit of the Fc region]; formula (a) The linkers in are preferably the same or different peptide linkers;
(b-1)[CD3-VL]-[CL]。 (b-1) [CD3-VL]-[CL].
本揭露還提供一種單價的特異性結合BCMA和單價的特異性結合CD3的抗原結合分子(1+1 format)。 The present disclosure also provides a monovalent antigen-binding molecule (1+1 format) that specifically binds BCMA and that specifically binds CD3.
示例性的，其包含具有式(c)所示結構的第一鏈、具有式(b)所示結構的第二鏈、具有式(d)所示結構的第三鏈和具有式(e)所示結構的第四鏈， Exemplarily, it comprises a first chain having a structure shown in formula (c), a second chain having a structure shown in formula (b), a third chain having a structure shown in formula (d) and a chain having a structure shown in formula (e) The fourth strand of the structure shown,
(c)[BCMA-VH]-[CH1]-[Fc區的第二亞基]； (c) [BCMA-VH]-[CH1]-[the second subunit of the Fc region];
(b)[BCMA-VL]-[CL]； (b) [BCMA-VL]-[CL];
(d)[CD3-VH]-[連接子]-[Titin鏈]-[Fc區的第一亞基]； (d) [CD3-VH]-[linker]-[Titin chain]-[the first subunit of the Fc region];
(e)[CD3-VL]-[連接子]-[Obscurin鏈]； (e) [CD3-VL]-[Linker]-[Obscurin Chain];
式(d)和(e)中的連接子較佳為相同或不同的肽連接子。 The linkers in formulas (d) and (e) are preferably the same or different peptide linkers.
示例性的，該抗原結合分子包含具有式(c)所示結構的第一鏈、具有式(b)所示結構的第二鏈、具有式(d-1)所示結構的第三鏈和具有式(e-1)所示結構的第四鏈， Exemplarily, the antigen-binding molecule comprises a first chain having a structure represented by formula (c), a second chain having a structure represented by formula (b), a third chain having a structure represented by formula (d-1) and A fourth chain having a structure shown in formula (e-1),
(c)[BCMA-VH]-[CH1]-[Fc區的第二亞基]； (c) [BCMA-VH]-[CH1]-[the second subunit of the Fc region];
(b)[BCMA-VL]-[CL]； (b) [BCMA-VL]-[CL];
(d-1)[CD3-VH]-[連接子]-[Obscurin鏈]-[Fc區的第一亞基]； (d-1) [CD3-VH]-[Linker]-[Obscurin chain]-[First subunit of Fc region];
(e-1)[CD3-VL]-[連接子]-[Titin鏈]； (e-1)[CD3-VL]-[Linker]-[Titin chain];
式(d-1)和(e-1)中的連接子較佳為相同或不同的肽連接子。 The linkers in formulas (d-1) and (e-1) are preferably the same or different peptide linkers.
示例性的，該抗原結合分子包含具有式(c-1)所示結構的第一鏈、具有式(b-1)所示結構的第二鏈、具有式(d-2)所示結構的第三鏈和具有式(e-2)所示結構的第四鏈， Exemplarily, the antigen-binding molecule comprises a first chain having a structure represented by formula (c-1), a second chain having a structure represented by formula (b-1), and a chain having a structure represented by formula (d-2). A third chain and a fourth chain having a structure shown in formula (e-2),
(c-1)[CD3-VH]-[CH1]-[Fc區的第二亞基]； (c-1) [CD3-VH]-[CH1]-[the second subunit of the Fc region];
(b-1)[CD3-VL]-[CL]； (b-1)[CD3-VL]-[CL];
(d-2)[BCMA-VH]-[連接子]-[Titin鏈]-[Fc區的第一亞基]； (d-2) [BCMA-VH]-[linker]-[Titin chain]-[the first subunit of the Fc region];
(e-2)[BCMA-VL]-[連接子]-[Obscurin鏈]； (e-2) [BCMA-VL]-[Linker]-[Obscurin Chain];
式(d-2)和(e-2)中的連接子較佳為相同或不同的肽連接子。 The linkers in formulas (d-2) and (e-2) are preferably the same or different peptide linkers.
示例性的，該抗原結合分子包含具有式(c-1)所示結構的第一鏈、具有式(b-1)所示結構的第二鏈、具有式(d-3)所示結構的第三鏈和具有式(e-3)所示結構的第四鏈， Exemplarily, the antigen-binding molecule comprises a first chain having a structure represented by formula (c-1), a second chain having a structure represented by formula (b-1), and a chain having a structure represented by formula (d-3). A third chain and a fourth chain having a structure shown in formula (e-3),
(c-1)[CD3-VH]-[CH1]-[Fc區的第二亞基]； (c-1) [CD3-VH]-[CH1]-[the second subunit of the Fc region];
(b-1)[CD3-VL]-[CL]； (b-1)[CD3-VL]-[CL];
(d-3)[BCMA-VH]-[連接子]-[Obscurin鏈]-[Fc區的第一亞基]； (d-3) [BCMA-VH]-[Linker]-[Obscurin chain]-[First subunit of Fc region];
(e-3)[BCMA-VL]-[連接子]-[Titin鏈]； (e-3) [BCMA-VL]-[Linker]-[Titin chain];
式(d-3)和(e-3)中的連接子較佳為相同或不同的肽連接子。 The linkers in formulas (d-3) and (e-3) are preferably the same or different peptide linkers.
示例性的，該抗原結合分子包含具有式(f)所示結構的第一鏈、具有式(b)所示結構的第二鏈、具有式(g)所示結構的第三鏈和具有式(h)所示結構的第四鏈， Exemplarily, the antigen-binding molecule comprises a first chain having a structure represented by formula (f), a second chain having a structure represented by formula (b), a third chain having a structure represented by formula (g) and having a structure represented by formula (h) the fourth strand of the structure shown,
(f)[BCMA-VH]-[CH1]-[Fc區的第一亞基]； (f) [BCMA-VH]-[CH1]-[the first subunit of the Fc region];
(b)[BCMA-VL]-[CL]； (b) [BCMA-VL]-[CL];
(g)[CD3-VH]-[連接子]-[Obscurin鏈]-[Fc區的第二亞基]； (g) [CD3-VH]-[Linker]-[Obscurin chain]-[Second subunit of Fc region];
(h)[CD3-VL]-[連接子]-[Titin鏈]； (h) [CD3-VL]-[Linker]-[Titin chain];
式(g)和(h)中的連接子較佳為相同或不同的肽連接子。 The linkers in formulas (g) and (h) are preferably the same or different peptide linkers.
示例性的，該抗原結合分子包含具有式(f)所示結構的第一鏈、具有式(b)所示結構的第二鏈、具有式(g-1)所示結構的第三鏈和具有式(h-1)所示結構的第四鏈， Exemplarily, the antigen-binding molecule comprises a first chain having a structure represented by formula (f), a second chain having a structure represented by formula (b), a third chain having a structure represented by formula (g-1) and A fourth chain having a structure shown in formula (h-1),
(f)[BCMA-VH]-[CH1]-[Fc區的第一亞基]； (f) [BCMA-VH]-[CH1]-[the first subunit of the Fc region];
(b)[BCMA-VL]-[CL]； (b) [BCMA-VL]-[CL];
(g-1)[CD3-VH]-[連接子]-[Titin鏈]-[Fc區的第二亞基]； (g-1) [CD3-VH]-[Linker]-[Titin chain]-[Second subunit of Fc region];
(h-1)[CD3-VL]-[連接子]-[Obscurin鏈]； (h-1)[CD3-VL]-[Linker]-[Obscurin Chain];
式(g-1)和(h-1)中的連接子較佳為相同或不同的肽連接子。 The linkers in formulas (g-1) and (h-1) are preferably the same or different peptide linkers.
示例性的，該抗原結合分子包含具有式(c-2)所示結構的第一鏈、具有式(b-1)所示結構的第二鏈、具有式(d-4)所示結構的第三鏈和具有式(e-2)所示結構的第四鏈， Exemplarily, the antigen-binding molecule comprises a first chain having a structure represented by formula (c-2), a second chain having a structure represented by formula (b-1), and a chain having a structure represented by formula (d-4). A third chain and a fourth chain having a structure shown in formula (e-2),
(c-2)[CD3-VH]-[CH1]-[Fc區的第一亞基]； (c-2) [CD3-VH]-[CH1]-[the first subunit of the Fc region];
(b-1)[CD3-VL]-[CL]； (b-1)[CD3-VL]-[CL];
(d-4)[BCMA-VH]-[連接子]-[Titin鏈]-[Fc區的第二亞基]； (d-4) [BCMA-VH]-[linker]-[Titin chain]-[second subunit of Fc region];
(e-2)[BCMA-VL]-[連接子]-[Obscurin鏈]； (e-2) [BCMA-VL]-[Linker]-[Obscurin Chain];
式(d-4)和(e-2)中的連接子較佳為相同或不同的肽連接子。 The linkers in formulas (d-4) and (e-2) are preferably the same or different peptide linkers.
示例性的，該抗原結合分子包含具有式(c-2)所示結構的第一鏈、具有式(b-1)所示結構的第二鏈、具有式(d-5)所示結構的第三鏈和具有式(e-3)所示結構的第四鏈， Exemplarily, the antigen-binding molecule comprises a first chain having a structure represented by formula (c-2), a second chain having a structure represented by formula (b-1), and a chain having a structure represented by formula (d-5). A third chain and a fourth chain having a structure shown in formula (e-3),
(c-2)[CD3-VH]-[CH1]-[Fc區的第一亞基]； (c-2) [CD3-VH]-[CH1]-[the first subunit of the Fc region];
(b-1)[CD3-VL]-[CL]； (b-1)[CD3-VL]-[CL];
(d-5)[BCMA-VH]-[連接子]-[Obscurin鏈]-[Fc區的第二亞基]； (d-5) [BCMA-VH]-[Linker]-[Obscurin chain]-[Second subunit of Fc region];
(e-3)[BCMA-VL]-[連接子]-[Titin鏈]； (e-3) [BCMA-VL]-[Linker]-[Titin chain];
式(d-5)和(e-3)中的連接子較佳為相同或不同的肽連接子。 The linkers in formulas (d-5) and (e-3) are preferably the same or different peptide linkers.
示例性的抗BCMA抗體Exemplary anti-BCMA antibodies
在一個方面，本揭露提供了一種抗BCMA抗體，其包含重鏈可變區BCMA-VH和輕鏈可變區BCMA-VL，其中， In one aspect, the disclosure provides an anti-BCMA antibody comprising a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, wherein,
(i)該重鏈可變區BCMA-VH具有：胺基酸序列如SEQ ID NO：5所示的BCMA-HCDR1，胺基酸序列如SEQ ID NO：6所示的BCMA-HCDR2，和胺基酸序列如SEQ ID NO：7所示的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：胺基酸序列如SEQ ID NO：8所示的BCMA-LCDR1，胺基酸序列如SEQ ID NO：9所示的BCMA-LCDR2，和胺基酸序列如SEQ ID NO：10所示的BCMA-LCDR3；或 (i) The heavy chain variable region BCMA-VH has: BCMA-HCDR1 having an amino acid sequence as shown in SEQ ID NO: 5, BCMA-HCDR2 having an amino acid sequence as shown in SEQ ID NO: 6, and amine The amino acid sequence is BCMA-HCDR3 as shown in SEQ ID NO: 7; and the light chain variable region BCMA-VL has: the amino acid sequence is BCMA-LCDR1 as shown in SEQ ID NO: 8, and the amino acid sequence is as BCMA-LCDR2 as shown in SEQ ID NO: 9, and BCMA-LCDR3 with the amino acid sequence as shown in SEQ ID NO: 10; or
(ii)該重鏈可變區BCMA-VH具有：胺基酸序列如SEQ ID NO：11所示的BCMA-HCDR1，胺基酸序列如SEQ ID NO：12所示的BCMA-HCDR2，和胺基酸序列如SEQ ID NO：13所示的BCMA-HCDR3；並且 該輕鏈可變區BCMA-VL具有：胺基酸序列如SEQ ID NO：14所示的BCMA-LCDR1，胺基酸序列如SEQ ID NO：15所示的BCMA-LCDR2，和胺基酸序列如SEQ ID NO：16所示的BCMA-LCDR3；或 (ii) The heavy chain variable region BCMA-VH has: BCMA-HCDR1 having an amino acid sequence as shown in SEQ ID NO: 11, BCMA-HCDR2 having an amino acid sequence as shown in SEQ ID NO: 12, and amine The amino acid sequence is BCMA-HCDR3 as shown in SEQ ID NO: 13; and The light chain variable region BCMA-VL has: BCMA-LCDR1 whose amino acid sequence is shown in SEQ ID NO: 14, BCMA-LCDR2 whose amino acid sequence is shown in SEQ ID NO: 15, and the amino acid sequence BCMA-LCDR3 as shown in SEQ ID NO: 16; or
(iii)該重鏈可變區BCMA-VH具有：胺基酸序列如SEQ ID NO：17所示的BCMA-HCDR1，胺基酸序列如SEQ ID NO：18所示的BCMA-HCDR2，和胺基酸序列如SEQ ID NO：19所示的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：胺基酸序列如SEQ ID NO：20所示的BCMA-LCDR1，胺基酸序列如SEQ ID NO：21所示的BCMA-LCDR2，和胺基酸序列如SEQ ID NO：22所示的BCMA-LCDR3；或 (iii) the heavy chain variable region BCMA-VH has: BCMA-HCDR1 having an amino acid sequence as shown in SEQ ID NO: 17, BCMA-HCDR2 having an amino acid sequence as shown in SEQ ID NO: 18, and amine The amino acid sequence is BCMA-HCDR3 as shown in SEQ ID NO: 19; and the light chain variable region BCMA-VL has: the amino acid sequence is BCMA-LCDR1 as shown in SEQ ID NO: 20, and the amino acid sequence is as BCMA-LCDR2 shown in SEQ ID NO: 21, and BCMA-LCDR3 whose amino acid sequence is shown in SEQ ID NO: 22; or
(iv)該重鏈可變區BCMA-VH具有：胺基酸序列如SEQ ID NO：23所示的BCMA-HCDR1，胺基酸序列如SEQ ID NO：24所示的BCMA-HCDR2，和胺基酸序列如SEQ ID NO：25所示的BCMA-HCDR3；並且該輕鏈可變區BCMA-VL具有：胺基酸序列如SEQ ID NO：26所示的BCMA-LCDR1，胺基酸序列如SEQ ID NO：27所示的BCMA-LCDR2，和胺基酸序列如SEQ ID NO：28所示的BCMA-LCDR3。 (iv) The heavy chain variable region BCMA-VH has: BCMA-HCDR1 having an amino acid sequence as shown in SEQ ID NO: 23, BCMA-HCDR2 having an amino acid sequence as shown in SEQ ID NO: 24, and amine The amino acid sequence is BCMA-HCDR3 as shown in SEQ ID NO: 25; and the light chain variable region BCMA-VL has: the amino acid sequence is BCMA-LCDR1 as shown in SEQ ID NO: 26, and the amino acid sequence is as BCMA-LCDR2 shown in SEQ ID NO: 27, and BCMA-LCDR3 with amino acid sequence shown in SEQ ID NO: 28.
在一些實施方案中，如前所述的抗BCMA抗體，其包含： In some embodiments, the anti-BCMA antibody as previously described, comprising:
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：29所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：30所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 29, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 30, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：37所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：40所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 40, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：38所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：40所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 38, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 40, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：39所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：40所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 39, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 40, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：37所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：41所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 41, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：37所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：42所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 42, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：37所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：43所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 43, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：31所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：32所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 32, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：44所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：47所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 47, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：45所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：47所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 45, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 47, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：46所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：47所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 47, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：44所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：48所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 48, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：45所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：48所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 45, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 48, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：46所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：48所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 48, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：33所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：34所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 34, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：49所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：51所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 49, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 51, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：50所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：51所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 50, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 51, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：35所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：36所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 36, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：52所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：54所示，或 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 52, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 54, or
該重鏈可變區BCMA-VH的胺基酸序列如SEQ ID NO：53所示，和該輕鏈可變區BCMA-VL的胺基酸序列如SEQ ID NO：54所示。 The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO:53, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO:54.
抗原結合分子或抗BCMA抗體的變體Antigen-binding molecules or variants of anti-BCMA antibodies
在某些實施方案中，涵蓋本文中提供的抗原結合分子的胺基酸序列變體。例如，可以期望改善抗體的結合親和力和/或其它生物學特性。可以藉由將合適的修飾引入編碼抗體的核苷酸序列中，或者藉由肽合成來製備抗體的胺基酸序列變體。此類修飾包括例如對抗原結合分子的胺基酸序列內的殘基的刪除、和/或插入、和/或取代。可以進行刪除、插入、和取代的任何組合以得到最終的構建體，只要最終的構建體擁有期望的特徵，例如抗原結合特性。 In certain embodiments, amino acid sequence variants of the antigen binding molecules provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions, and/or insertions, and/or substitutions of residues within the amino acid sequence of the antigen-binding molecule. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, so long as the final construct possesses the desired characteristics, such as antigen-binding properties.
取代、插入、和刪除變體Substitution, insertion, and deletion variants
在某些實施方案中，提供了具有一處或多處胺基酸取代的抗原結合分子變體。在感興趣的位點進行取代，包括CDR和FR。保守取代在表2中在“較佳的取代”的標題下顯示。更實質的變化在表2中在“示例性取代”的標題下提供，並且如下文參照胺基酸側鏈類別進一步描述的。可以將胺基酸取代引入感興趣的抗體中，並且對產物篩選期望的活性，例如保留/改善的抗原結合，降低的免疫原性，或改善的ADCC或CDC。 In certain embodiments, antigen binding molecule variants having one or more amino acid substitutions are provided. Make substitutions at sites of interest, including CDRs and FRs. Conservative substitutions are shown in Table 2 under the heading "Preferred Substitutions". More substantial variations are provided in Table 2 under the heading "Exemplary Substitutions" and are described further below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into an antibody of interest, and the products screened for desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.
表2. 胺基酸的取代
依照常見的側鏈特性，胺基酸可以如下分組： According to common side chain properties, amino acids can be grouped as follows:
(1)疏水性的：Nle，Met，Ala，Val，Leu，Ile； (1) Hydrophobic: Nle, Met, Ala, Val, Leu, Ile;
(2)中性，親水性的：Cys，Ser，Thr，Asn，Gln； (2) Neutral, hydrophilic: Cys, Ser, Thr, Asn, Gln;
(3)酸性的：Asp，Glu； (3) acidic: Asp, Glu;
(4)鹼性的：His，Lys，Arg； (4) Basic: His, Lys, Arg;
(5)影響鏈取向(orientation)的殘基：Gly，Pro； (5) Residues affecting chain orientation: Gly, Pro;
(6)芳香族的：Trp，Tyr，Phe。 (6) Aromatic: Trp, Tyr, Phe.
非保守取代是指，用一個類別的成員替換另一個類別的成員。 Non-conservative substitutions refer to the substitution of a member of one class for a member of another class.
一類取代變體涉及取代親本抗體(例如人源化或人抗體)的一個或多個CDR殘基。一般地，經選擇用於進一步研究的所得變體，相對於親本抗體，會具有某些生物學特性(例如升高的親和力，降低的免疫原性)的改變(例如改善)，和/或會基本上保留親本抗體的某些生物學特性。一種例示性的取代變體是親和力成熟的抗體，可以例如使用基於噬菌體展示的親和力成熟技術(如本文所述的那些技術)，便利地產生該抗體。簡言之，將一個或多個CDR殘基突變，並將變體抗體在噬菌體上展示，並對其篩選特定的生物學活性(例如結合親和力)。可以對CDR做出改變(例如取代)，例如以改善抗體親和力。可以對CDR“熱點”(即在體細胞成熟過程期間，以高頻率經歷突變的密碼子所編碼的殘基，和/或接觸抗原的殘基)做出此類改變，同時對所得的變體VH或VL測試結合親和力。在親和力成熟的一些實施方案中，藉由多種方法(例如易錯PCR、鏈改組、或寡核苷酸指導的誘變)的任一種，將多樣性引入所選擇用於成熟的可變基因中。然後，創建次級文庫。然後，篩選文庫以鑑定具有期望的親和力的任何抗體變體。另一種引入多樣性的方法涉及CDR定向的方法，其中將幾個CDR殘基(例如一次4-6個殘基)隨機化。可以例如使用丙胺酸掃描誘變或建 模來特異性鑑定涉及抗原結合的CDR殘基。特別地，HCDR3和LCDR3經常被作為靶點。 One type of substitutional variant involves substituting one or more CDR residues of a parent antibody (eg, a humanized or human antibody). Typically, the resulting variants selected for further study will have altered (eg improved) certain biological properties (eg increased affinity, decreased immunogenicity) relative to the parental antibody, and/or Certain biological properties of the parental antibody are substantially retained. An exemplary substitution variant is an affinity matured antibody, which can be conveniently produced, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated, and the variant antibodies are displayed on phage and screened for specific biological activity (eg, binding affinity). Alterations (eg, substitutions) can be made to the CDRs, eg, to improve antibody affinity. Such changes can be made to CDR "hotspots" (i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process, and/or residues that contact antigen) while modifying the resulting variant Binding affinity was tested for VH or VL. In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods, such as error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis . Then, create secondary libraries. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves a CDR-directed approach, in which several CDR residues (eg, 4-6 residues at a time) are randomized. One can e.g. use alanine scanning mutagenesis or build Modeling specifically identifies CDR residues involved in antigen binding. In particular, HCDR3 and LCDR3 are frequently targeted.
在某些實施方案中，取代、插入或缺失可以在一個或多個CDR內發生，只要此類變化不實質性降低抗體結合抗原的能力。例如，可以對CDR做出保守變化(例如保守取代，如本文中提供的)，其不實質性降低結合親和力。此類變化不發生在抗原接觸殘基。在上文提供的變體VH和VL序列的某些實施方案中，每個CDR是未改變的，或者含有不超過1、2或3處胺基酸取代。 In certain embodiments, substitutions, insertions or deletions may occur within one or more CDRs, so long as such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes (eg, conservative substitutions, as provided herein) can be made to the CDRs that do not substantially reduce binding affinity. Such changes do not occur at antigen contact residues. In certain embodiments of the variant VH and VL sequences provided above, each CDR is unchanged, or contains no more than 1, 2 or 3 amino acid substitutions.
一種可用於鑑定抗體中可以作為誘變靶位的殘基或區域的方法稱作“丙胺酸掃描誘變”。在這種方法中，鑑定一個殘基或殘基組(例如帶電荷的殘基，諸如Arg、Asp、His、Lys和Glu)，並且替換為中性或帶負電荷的胺基酸(例如，Ala或聚丙胺酸)，以確定該抗體與抗原的相互作用是否受影響。可以在對初始取代顯示功能敏感性的胺基酸位置引入進一步的取代。此外，可藉由研究抗原-抗體複合物的晶體結構來鑑定抗體與抗原間的接觸點。這些接觸殘基及鄰近殘基可以作為取代候選物被打靶或消除。可以篩選變體以確定它們是否含有期望的特性。 One method that can be used to identify residues or regions of an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis". In this approach, a residue or group of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., Ala or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions showing functional sensitivity to the initial substitution. In addition, contact points between antibody and antigen can be identified by studying the crystal structure of the antigen-antibody complex. These contact residues and neighboring residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain desired properties.
胺基酸序列插入包括：在胺基和/或羧基端融合1個殘基或長度為100或更多個殘基的多肽；和單個或多個胺基酸殘基的序列內插入。在末端的插入的例子包括具有N端甲硫胺醯基殘基的抗體。抗體分子的其它插入變體包括，在抗體的N或C端融合有酶(或延長抗體的血清半衰期的多肽)的融合物。 Amino acid sequence insertions include: amino and/or carboxy terminus fusions of 1 residue or polypeptides of 100 or more residues in length; and intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusions with enzymes (or polypeptides that extend the serum half-life of antibodies) at the N- or C-terminus of the antibody.
Fab的改造Fab Transformation
在一個方面，本揭露的抗原結合分子中，該特異性結合BCMA的第一抗原結合域是Fab，該特異性結合CD3的第二抗原結合域是經替換 的Fab，該經替換的Fab包含與可變區連接的Titin鏈和Obscurin鏈，也即Fab原有的CH1和CL被Titin鏈和Obscurin鏈替換；或 In one aspect, in the antigen-binding molecules of the present disclosure, the first antigen-binding domain that specifically binds BCMA is Fab, and the second antigen-binding domain that specifically binds CD3 is substituted Fab, the substituted Fab comprises a Titin chain and an Obscurin chain connected to the variable region, that is, the original CH1 and CL of the Fab are replaced by a Titin chain and an Obscurin chain; or
該特異性結合CD3的第二抗原結合域是Fab，該特異性結合BCMA的第一抗原結合域是經替換的Fab，該經替換的Fab包含與可變區連接的Titin鏈和Obscurin鏈，也即Fab原有的CH1和CL被Titin鏈和Obscurin鏈替換。 The second antigen-binding domain that specifically binds CD3 is a Fab, the first antigen-binding domain that specifically binds BCMA is a substituted Fab, and the substituted Fab comprises a Titin chain and an Obscurin chain connected to the variable region, and also That is, the original CH1 and CL of Fab are replaced by Titin chain and Obscurin chain.
該Titin鏈和Obscurin鏈的替換技術詳細記載於PCT/CN2021/070832和CN202110527339.7及將其作為優先權文件的專利，在此全文援引加入本文。可選的Titin鏈和Obscurin鏈見下表，其中該Titin鏈包含選自由SEQ ID NO：98至SEQ ID NO：116組成的組的胺基酸序列，Obscurin鏈包含選自由SEQ ID NO：117至SEQ ID NO：152和SEQ ID NO：162至SEQ ID NO：166組成的組的胺基酸序列。 The replacement technology of the Titin chain and the Obscurin chain is described in detail in PCT/CN2021/070832 and CN202110527339.7 and the patents as priority documents, which are incorporated herein by reference in their entirety. The optional Titin chain and Obscurin chain are shown in the following table, wherein the Titin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 98 to SEQ ID NO: 116, and the Obscurin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 117 to Amino acid sequences of SEQ ID NO: 152 and the group consisting of SEQ ID NO: 162 to SEQ ID NO: 166.
表3-1. Titin鏈的胺基酸序列
表3-2. Obscurin鏈的胺基酸序列
Fc區的改造Modification of the Fc region
在一個方面，本揭露的抗原結合分子的Fc區包含一個或多個胺基酸取代，該一個或多個胺基酸取代減少其與Fc受體的結合，例如其與Fcγ受體的結合，並且降低或消除效應子功能。天然IgG Fc區，具體地是IgG1 Fc區或IgG4 Fc區，可能導致本揭露的抗原結合分子靶向表達Fc受體的細胞，而不是表達抗原的細胞。本揭露改造的Fc區表現出降低的對Fc受體的結合親和力和/或降低的效應子功能。在一些實施方案中，改造的Fc區與天然Fc區相比，對Fc受體的結合親和力下降50%、80%、90%或95%以上。在一些實施方案中，該Fc受體是Fcγ受體。在一些實施方案中，該Fc受體是人Fcγ受體，例如FcγRI、FcγRIIa、FcγRIIB、FcγRIIIa。在一些實施方案中，改造的Fc區與天然Fc區相比，對補體(如C1q)的結合親和力降低。在一些實施方案中，改造的Fc區與天然Fc區相比，對新生兒Fc受體(FcRn)的結合親和力不降低。在一些實施例中，改造的Fc區具有降低的效應子功能，該降低的效應子功能可以包括但不限於以下中的一個或多個：降低的補體依賴性細胞毒性(CDC)、降低的抗體依賴性細胞介導的細胞毒性(ADCC)、降低的抗體依賴性細胞吞噬(ADCP)、減少的細胞因子分泌、減少的免疫複合物介導的抗原呈遞細胞的抗原攝取、減少的與NK細胞的結合、減少的與巨噬細胞的結合、減少的與單核細胞 的結合、減少的與多形核細胞的結合、減少的直接信號傳導誘導性細胞凋亡、降低的樹突細胞成熟或減少的T細胞引發。 In one aspect, the Fc region of an antigen binding molecule of the disclosure comprises one or more amino acid substitutions that reduce its binding to an Fc receptor, such as its binding to an Fcγ receptor, And reduce or eliminate effector function. A native IgG Fc region, specifically an IgG 1 Fc region or an IgG 4 Fc region, may result in targeting of an antigen binding molecule of the present disclosure to cells expressing Fc receptors, rather than cells expressing antigen. The engineered Fc regions of the present disclosure exhibit reduced binding affinity to Fc receptors and/or reduced effector functions. In some embodiments, the engineered Fc region has a reduced binding affinity for Fc receptors by more than 50%, 80%, 90% or more than 95% compared to a native Fc region. In some embodiments, the Fc receptor is an Fc gamma receptor. In some embodiments, the Fc receptor is a human Fc gamma receptor, eg, FcγRI, FcγRIIa, FcγRIIB, FcγRIIIa. In some embodiments, the engineered Fc region has reduced binding affinity for complement (eg, C1q) compared to a native Fc region. In some embodiments, the engineered Fc region has no reduced binding affinity for neonatal Fc receptor (FcRn) compared to a native Fc region. In some embodiments, the engineered Fc region has reduced effector function, which may include, but is not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody Dependent cell-mediated cytotoxicity (ADCC), decreased antibody-dependent cellular phagocytosis (ADCP), decreased cytokine secretion, decreased immune complex-mediated antigen uptake by antigen-presenting cells, decreased interaction with NK cells Binding, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced apoptosis, reduced dendritic cell maturation, or reduced T cell priming.
對於IgG1 Fc區，在238、265、269、270、297、327和329等位置的胺基酸殘基取代可降低的效應子功能。在一些實施方案中，該Fc區是人IgG1 Fc區，並且在234和235位置的胺基酸殘基為A，編號依據為EU編號規則。對於IgG4 Fc區，在228等位置的胺基酸殘基取代可降低的效應子功能。 For the IgG 1 Fc region, amino acid residue substitutions at positions 238, 265, 269, 270, 297, 327, and 329 may reduce effector function. In some embodiments, the Fc region is a human IgG 1 Fc region, and the amino acid residues at positions 234 and 235 are A, and the numbering is according to the EU numbering convention. For the IgG 4 Fc region, substitution of amino acid residues at positions such as 228 may reduce effector function.
抗原結合分子還可包含二硫鍵改造，例如第一亞基的354C和第二亞基的349C。基於不同來源，該Fc區的第356位胺基酸殘基可以是E或D，第358位胺基酸殘基可以是M或L。在一些實施例方式中，該Fc區的第356位胺基酸殘基可以是E，第358位胺基酸殘基是M。在一些實施例方式中，該Fc區的第356位胺基酸殘基可以是D，第358位胺基酸殘基是L。 Antigen binding molecules may also comprise disulfide bond engineering, eg, 354C of the first subunit and 349C of the second subunit. Based on different sources, the 356th amino acid residue of the Fc region can be E or D, and the 358th amino acid residue can be M or L. In some embodiments, the 356th amino acid residue of the Fc region may be E, and the 358th amino acid residue may be M. In some embodiments, the 356th amino acid residue of the Fc region may be D, and the 358th amino acid residue may be L.
當抗原結合分子包含與Fc區的兩個亞基融合的結合域時，可能導致不期望的同源二聚化。為了提高產率和純度，因此在本公開的抗原結合分子的Fc區中引入促進異源二聚化的修飾將是有利的。在一些實施方式中，本揭露的Fc區包含根據杵臼(knob-into-hole，KIH)技術的改造，該方法涉及在第一亞基的界面處引入凸起結構(knob)以及在第二亞基的界面處引入孔結構(hole)。使得該凸起結構可以定位在孔結構中，促進異源二聚體的形成並抑制同源二聚體的產生。凸起結構是藉由用較大側鏈(例如酪胺酸或色胺酸)取代來自第一亞基的界面的小胺基酸側鏈而構建的。而孔結構是藉由用較小的胺基酸側鏈(例如丙胺酸或蘇胺酸)取代大胺基酸側鏈而在第二亞基的界面中創建的。凸起結構和孔結構藉由改變編碼多肽的核酸來製備，可選的胺基酸取代如下表所示： Undesirable homodimerization may result when the antigen binding molecule comprises a binding domain fused to two subunits of the Fc region. In order to increase yield and purity, it would therefore be advantageous to introduce modifications in the Fc region of the antigen binding molecules of the disclosure that promote heterodimerization. In some embodiments, the Fc region of the present disclosure comprises a modification according to the knob-into-hole (KIH) technique, which involves the introduction of a knob at the interface of the first subunit and the introduction of a knob at the interface of the second subunit. A hole structure (hole) is introduced at the interface of the base. This allows the protrusion to be positioned in the pore structure, promoting the formation of heterodimers and inhibiting the generation of homodimers. The bulge structure is constructed by replacing small amino acid side chains from the interface of the first subunit with larger side chains such as tyrosine or tryptophan. The pore structure is created in the interface of the second subunit by replacing large amino acid side chains with smaller amino acid side chains such as alanine or threonine. Protrusion and pore structures are prepared by altering the nucleic acid encoding the polypeptide, with optional amino acid substitutions as shown in the table below:
表4. KIH突變組合
除了杵臼技術外，用於修飾重鏈CH3結構域以實現異源二聚化的其他技術也是本領域中已知的，例如WO96/27011、WO98/050431、EP1870459、WO2007/110205、WO 007/147901、WO2009/089004、WO2010/129304、WO2011/90754、WO2011/143545、WO2012/058768、WO2013/157954和WO 013/096291。 In addition to the knob-and-hole technique, other techniques for modifying heavy chain CH3 domains to achieve heterodimerization are known in the art, for example WO96/27011, WO98/050431, EP1870459, WO2007/110205, WO 007/147901 , WO2009/089004, WO2010/129304, WO2011/90754, WO2011/143545, WO2012/058768, WO2013/157954 and WO 013/096291.
Fc區的C末端可以是以胺基酸殘基PGK結束的完整C末端；也可以是截短的(truncated)C末端，例如在該截短的C末端中去除了一個或兩個C末端胺基酸殘基。在一個較佳的方面中，Fc區的C末端是以PG結束的縮短的C末端。因此，在一些實施方式中，完整抗體的組成物可以包括去除了所有K447殘基和/或G446+K447殘基的抗體群體。在一些實施方式中，完整抗體的組成物可以包括沒有去除K447殘基和/或G446+K447殘基的抗體群體。在一些實施方式中，完整抗體的組成物具有帶有和不帶有K447殘基和/或G446+K447殘基的抗體混合物的抗體群體。 The C-terminus of the Fc region can be a complete C-terminus ending with the amino acid residue PGK; it can also be a truncated (truncated) C-terminus, for example, one or two C-terminal amines have been removed from the truncated C-terminus amino acid residues. In a preferred aspect, the C-terminus of the Fc region is a shortened C-terminus ending with PG. Thus, in some embodiments, a composition of intact antibodies can include a population of antibodies from which all K447 residues and/or G446+K447 residues have been removed. In some embodiments, the composition of intact antibodies can include a population of antibodies without removal of the K447 residue and/or the G446+K447 residues. In some embodiments, the composition of intact antibodies has a population of antibodies with and without the K447 residue and/or the antibody mixture of G446+K447 residues.
重組方法Recombination method
抗體可以使用重組方法來產生。對於這些方法，提供編碼抗體的一個或更多個分離的核酸。 Antibodies can be produced using recombinant methods. For these methods, one or more isolated nucleic acids encoding the antibodies are provided.
在天然抗體、天然抗體片段或具有同源二聚體重鏈的雙特異性抗體的情況下，需要兩個核酸，一個用於輕鏈或其片段，一個用於重鏈 或其片段。此類核酸編碼包含抗體VL的胺基酸序列和/或包含抗體VH的胺基酸序列(例如抗體的輕鏈和/或重鏈)。這些核酸可以在相同的表達載體上或在不同的表達載體上。 In the case of native antibodies, native antibody fragments or bispecific antibodies with homodimeric heavy chains, two nucleic acids are required, one for the light chain or fragment thereof and one for the heavy chain or a fragment thereof. Such nucleic acids encode an amino acid sequence comprising the VL of the antibody and/or an amino acid sequence comprising the VH of the antibody (eg, the light and/or heavy chains of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors.
在具有異二聚體重鏈的雙特異性抗體的情況下，需要四個核酸，一個用於第一輕鏈，一個用於包含第一異源單體Fc區多肽的第一重鏈，一個用於第二輕鏈，並且一個用於包含第二異源單體Fc區多肽的第二重鏈。這四個核酸可包含在一個或更多個核酸分子或表達載體中，通常這些核酸位於兩個或三個表達載體上，即一個載體可包含這些核酸中的多於一個。 In the case of a bispecific antibody with a heterodimeric heavy chain, four nucleic acids are required, one for the first light chain, one for the first heavy chain comprising the first heteromonomeric Fc region polypeptide, and one for the first heteromeric Fc region polypeptide. for the second light chain, and one for the second heavy chain comprising a second heterologous monomeric Fc region polypeptide. These four nucleic acids may be contained in one or more nucleic acid molecules or expression vectors, usually these nucleic acids are located on two or three expression vectors, ie one vector may contain more than one of these nucleic acids.
在一個實施方案中，本揭露提供了編碼如前所述的抗體的分離的核酸。此類核酸可以獨立地編碼前述的任一多肽鏈。在另一方面中，本揭露提供了包含此類核酸的一種或多種載體(例如表達載體)。 In one embodiment, the present disclosure provides an isolated nucleic acid encoding an antibody as previously described. Such nucleic acids may independently encode any of the aforementioned polypeptide chains. In another aspect, the present disclosure provides one or more vectors (eg, expression vectors) comprising such nucleic acids.
在另一方面中，本揭露提供了包含此類核酸的宿主細胞。在一個實施方案中，提供製備抗原結合分子、抗BCMA抗體的方法；其中該方法包括，在適合抗體表達的條件下，培養包含編碼該抗體的核酸的宿主細胞，如上文所提供的，和視需要地從宿主細胞(或宿主細胞培養基)回收該抗體。 In another aspect, the present disclosure provides host cells comprising such nucleic acids. In one embodiment, there is provided a method of making an antigen-binding molecule, an anti-BCMA antibody; wherein the method comprises, under conditions suitable for expression of the antibody, culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, and optionally The antibody is recovered from the host cell (or host cell culture medium) as desired.
為了重組產生抗原結合分子或抗BCMA抗體，將編碼蛋白的核酸分離並插入一個或更多個載體中，用於在宿主細胞中進一步選殖和/或表達。此類核酸可以使用常規程序容易地分離和測序(例如藉由使用能夠與編碼抗體重鏈和輕鏈的基因特異性結合的寡核苷酸探針)，或者藉由重組方法產生或藉由化學合成獲得。 For recombinant production of antigen-binding molecules or anti-BCMA antibodies, nucleic acid encoding the protein is isolated and inserted into one or more vectors for further cloning and/or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of the antibody), or produced recombinantly or by chemical Obtained by synthesis.
用於選殖或表達編碼抗體的載體的適當宿主細胞包括本文描述的原核或真核細胞。例如，抗體可在細菌中產生，特別是當抗體不需要 糖基化和Fc效應子功能時。在表達後，抗體可以在可溶級分中從細菌細胞糊狀物分離，並且可進一步純化。 Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, especially when antibodies do not require Glycosylation and Fc effector function. After expression, antibodies can be isolated from bacterial cell paste in a soluble fraction and can be further purified.
除了原核生物以外，真核微生物諸如絲狀真菌或酵母也是用於編碼抗體的載體的合適的選殖或表達宿主，包括真菌和酵母菌株，其糖基化途徑已經“人源化”，導致產生具有部分或完全人糖基化模式的抗體。適於表達(糖基化)抗體的合適的宿主細胞也可源自多細胞生物體(無脊椎動物和脊椎動物)；無脊椎動物細胞的例子包括植物和昆蟲細胞。已經鑑定了許多杆狀病毒株，其可與昆蟲細胞聯合使用，特別是用於草地貪夜蛾(Spodoptera frugiperda)細胞的轉染；還可利用植物細胞培養物作為宿主，例如US5959177、US 6040498、US6420548、US 7125978和US6417429；也可將脊椎動物細胞用作宿主，例如適應於在懸浮液中生長的哺乳動物細胞系。適宜的哺乳動物宿主細胞系的其它例子是經SV40轉化的猴腎CV1系(COS-7)；人胚腎系(293或293T細胞)；幼倉鼠腎細胞(BHK)；小鼠塞托利(sertoli)細胞(TM4細胞)；猴腎細胞(CV1)；非洲綠猴腎細胞(VERO-76)；人宮頸癌細胞(HELA)；犬腎細胞(MDCK)；水牛鼠(buffalo rat)肝細胞(BRL3A)；人肺細胞(W138)；人肝細胞(Hep G2)；小鼠乳房腫瘤(MMT 060562)；TRI細胞；MRC 5細胞；和FS4細胞。其它適宜的哺乳動物宿主細胞系包括中國倉鼠卵巢(CHO)細胞，包括DHFR-CHO細胞；以及骨髓瘤細胞系，如Y0、NS0和Sp2/0。關於適合產生抗體的某些哺乳動物宿主細胞系的綜述參見例如Yazaki，P.和Wu，A.M.，Methods in Molecular Biology，Vol.248，Lo，B.K.C.(編)，Humana Press，Totowa，NJ(2004)，第255-268頁。 In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable selection or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of Antibodies with partially or fully human glycosylation patterns. Suitable host cells for expressing (glycosylated) antibodies may also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified for use in combination with insect cells, especially for the transfection of Spodoptera frugiperda cells; plant cell cultures can also be used as hosts, e.g. US5959177, US 6040498, US6420548, US7125978 and US6417429; vertebrate cells can also be used as hosts, eg mammalian cell lines adapted for growth in suspension. Other examples of suitable mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7); the human embryonic kidney line (293 or 293T cells); baby hamster kidney cells (BHK); Sertoli) cells (TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat (buffalo rat) liver cells ( BRL3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Other suitable mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines, such as YO, NSO and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production see, e.g., Yazaki, P. and Wu, AM, Methods in Molecular Biology , Vol. 248, Lo, BKC (eds.), Humana Press, Totowa, NJ (2004) , pp. 255-268.
測定determination
本文提供的抗原結合分子或抗BCMA抗體可以藉由本領域已知的多種測定法對其物理/化學特徵和/或生物學活性進行鑑定、篩選或表徵。 Antigen binding molecules or anti-BCMA antibodies provided herein can be identified, screened or characterized for their physical/chemical characteristics and/or biological activity by a variety of assays known in the art.
在一個方面中，例如藉由已知方法如ELISA、蛋白印跡法等，測試本揭露的抗體的抗原結合活性。 In one aspect, the antibodies of the present disclosure are tested for antigen binding activity, eg, by known methods such as ELISA, Western blot, and the like.
在另一個方面，競爭測定法可用於鑑定與BCMA競爭結合的抗體。在某些實施方案中，該競爭性抗體與抗原結合分子或抗BCMA抗體結合相同的表位(例如線性或構象表位)在示例性的競爭測定中，在溶液中溫育固定化的BCMA，該溶液包含結合BCMA的第一標記抗體和第二未標記抗體，該第二未標記抗體被檢測其與第一標記抗體競爭結合BCMA的能力。第二未標記抗體可以存在於融合瘤上清中。作為對照，將固定的BCMA在包含第一標記抗體而不包含第二未標記的抗體的溶液中溫育，去除過量的未結合的抗體，並且測量與固定的BCMA締合的標記量。如果相對於對照樣品，在測試樣品中，與固定的BCMA締合的標記量實質性減少，則指示第二抗體與第一抗體競爭對BCMA的結合。 In another aspect, competition assays can be used to identify antibodies that compete for binding to BCMA. In certain embodiments, the competing antibody binds to the same epitope (e.g., a linear or conformational epitope) as the antigen-binding molecule or anti-BCMA antibody. In an exemplary competition assay, immobilized BCMA is incubated in solution, The solution contains a first labeled antibody that binds BCMA and a second unlabeled antibody that is tested for its ability to compete with the first labeled antibody for binding to BCMA. A second unlabeled antibody can be present in the supernatant of the fusion tumor. As a control, immobilized BCMA was incubated in a solution containing the first labeled antibody but not the second unlabeled antibody, excess unbound antibody was removed, and the amount of label associated with immobilized BCMA was measured. A substantial decrease in the amount of label associated with immobilized BCMA in the test sample relative to the control sample indicates that the second antibody is competing with the first antibody for binding to BCMA.
在一個方面中，提供了用於鑑定具有生物學活性的抗BCMA抗體的測定法。詳見本揭露的測試例。 In one aspect, assays for identifying biologically active anti-BCMA antibodies are provided. See the test examples in this disclosure for details.
免疫綴合物Immunoconjugate
本揭露還提供免疫綴合物，其包含與一種或多種細胞毒性劑綴合的抗原結合分子或抗BCMA抗體，該一種或多種細胞毒性劑為諸如化學治療劑或藥物、生長抑制劑、毒素(例如細菌、真菌、植物或動物來源的蛋白質毒素、酶活性毒素，或它們的片段)、或放射性同位素。 The present disclosure also provides immunoconjugates comprising an antigen binding molecule or an anti-BCMA antibody conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitors, toxins ( For example protein toxins of bacterial, fungal, vegetable or animal origin, enzymatically active toxins, or fragments thereof), or radioactive isotopes.
診斷與治療組成物Diagnostic and Therapeutic Composition
在某些實施方案中，本揭露提供的抗原結合分子可用於檢測生物學樣品中BCMA或CD3的存在，以及本揭露提供的抗BCMA抗體可用於檢測生物學樣品中BCMA的存在。在用於本文時，術語“檢測”涵蓋定量或定性檢測。在某些實施方案中，生物學樣品包含細胞或組織，諸如腫瘤組織。 In certain embodiments, the antigen binding molecules provided by the present disclosure can be used to detect the presence of BCMA or CD3 in a biological sample, and the anti-BCMA antibodies provided by the present disclosure can be used to detect the presence of BCMA in a biological sample. As used herein, the term "detection" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissue, such as tumor tissue.
在一個實施方案中，提供了在診斷或檢測方法中使用的抗原結合分子或抗BCMA抗體。在又一方面，提供了檢測生物學樣品中BCMA或CD3的存在的方法。在某些實施方案中，該方法包括在適宜條件下使生物學樣品與抗原結合分子或抗BCMA抗體接觸，並檢測是否在檢測試劑與抗原之間形成複合物。此類方法可以是體外或體內方法。在一個實施方案中，使用抗原結合分子或抗BCMA抗體來選擇適合治療的受試者，例如BCMA或CD3是用於選擇患者的生物標誌物。 In one embodiment, an antigen binding molecule or an anti-BCMA antibody for use in a method of diagnosis or detection is provided. In yet another aspect, methods of detecting the presence of BCMA or CD3 in a biological sample are provided. In certain embodiments, the method comprises contacting the biological sample with the antigen binding molecule or anti-BCMA antibody under suitable conditions and detecting whether a complex is formed between the detection reagent and the antigen. Such methods can be in vitro or in vivo methods. In one embodiment, an antigen binding molecule or an anti-BCMA antibody is used to select subjects suitable for treatment, eg BCMA or CD3 are biomarkers used to select patients.
可使用本揭露的抗體來診斷的示例性病症，例如與BCMA表達有關的B細胞障礙、漿細胞障礙、多發性骨髓瘤、漿細胞瘤、漿細胞白血病、巨球蛋白血症、澱粉樣變性、華氏巨球蛋白血症、孤立性骨漿細胞瘤、髓外漿細胞瘤、骨硬化性骨髓瘤、重鏈病、意義不明確的單株γ球蛋白病以及鬱積型多發性骨髓瘤；自身免疫性疾病、全身性紅斑狼瘡。 Exemplary disorders that can be diagnosed using the antibodies of the disclosure, such as B cell disorders associated with BCMA expression, plasma cell disorders, multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, Waldenstrom's macroglobulinemia, solitary plasmacytoma, extramedullary plasmacytoma, osteosclerotic myeloma, heavy chain disease, monoclonal gammopathy of undetermined significance, and smoldering multiple myeloma; autoimmune disease, systemic lupus erythematosus.
在某些實施方案中，提供了經標記的抗原結合分子或抗BCMA抗體。標記物包括但不限於直接檢測的標記物或模塊(諸如螢光、發色、電子緻密、化學發光、和放射性標記物)，和間接檢測的模塊(例如，經由酶反應或分子相互作用間接檢測的模塊，諸如酶或配體)。 In certain embodiments, a labeled antigen binding molecule or anti-BCMA antibody is provided. Labels include, but are not limited to, markers or moieties for direct detection (such as fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), and moieties for indirect detection (e.g., indirect detection via enzymatic reactions or molecular interactions). modules, such as enzymes or ligands).
在另外的方面，提供包含該抗原結合分子或抗BCMA抗體的醫藥組成物，例如，用於以下任何治療方法。在一個方面，醫藥組成物包 含本文提供的任何抗體和藥學上可接受的載體。在另一個方面，醫藥組成物包含本文提供的任何抗體和至少一種另外的治療劑。 In a further aspect, pharmaceutical compositions comprising the antigen-binding molecule or anti-BCMA antibody are provided, eg, for use in any of the following methods of treatment. In one aspect, the pharmaceutical composition comprises comprising any of the antibodies provided herein and a pharmaceutically acceptable carrier. In another aspect, a pharmaceutical composition comprises any of the antibodies provided herein and at least one additional therapeutic agent.
本揭露所述的抗原結合分子或抗BCMA抗體的醫藥組成物藉由以下製備：將具有所需純度的此類抗體與一種或更多種視需要的藥學上可接受的載體接觸，該醫藥組成物為凍乾組成物或水溶液的形式。用於體內施用的製劑一般是無菌的。無菌性可容易地實現，例如藉由穿過無菌濾膜過濾。 Pharmaceutical compositions of antigen-binding molecules or anti-BCMA antibodies described in the present disclosure are prepared by contacting such antibodies having the desired purity with one or more optional pharmaceutically acceptable carriers, the pharmaceutical composition The product is in the form of a lyophilized composition or an aqueous solution. Formulations for in vivo administration are generally sterile. Sterility is readily achieved, for example, by filtration through sterile filters.
治療方法與施用途徑Methods of treatment and routes of administration
本文提供的任何抗原結合分子或抗BCMA抗體可用於治療方法。 Any of the antigen binding molecules or anti-BCMA antibodies provided herein can be used in methods of treatment.
在又一個方面，本揭露提供抗原結合分子或抗BCMA抗體在藥物的製造或製備中的用途。在一個實施方案中，該藥物用於治療BCMA相關疾病，例如與BCMA表達有關的B細胞障礙、漿細胞障礙、多發性骨髓瘤、漿細胞瘤、漿細胞白血病、巨球蛋白血症、澱粉樣變性、華氏巨球蛋白血症、孤立性骨漿細胞瘤、髓外漿細胞瘤、骨硬化性骨髓瘤、重鏈病、意義不明確的單株γ球蛋白病以及鬱積型多發性骨髓瘤；自身免疫性疾病、全身性紅斑狼瘡。並且該藥物是以對上述疾病的有效量的形式存在的。在一些實施方式中，該有效量是單位劑量(如，日單位劑量或週單位劑量)。在一個此類實施方案中，該用途進一步包括向受試者施用有效量的至少一種另外的治療劑(例如一種、兩種、三種、四種、五種或六種另外的治療劑)。根據任意以上實施方案的“受試者”可以是人。 In yet another aspect, the present disclosure provides the use of an antigen-binding molecule or an anti-BCMA antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of BCMA-associated diseases, such as B-cell disorders associated with BCMA expression, plasma cell disorders, multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloid Degeneration, WM, solitary plasmacytoma, extramedullary plasmacytoma, osteosclerotic myeloma, heavy chain disease, monoclonal gammopathy of undetermined significance, and smoldering multiple myeloma; Autoimmune diseases, systemic lupus erythematosus. And the medicine is in the form of an effective amount for the above-mentioned diseases. In some embodiments, the effective amount is a unit dose (eg, a daily unit dose or a weekly unit dose). In one such embodiment, the use further comprises administering to the subject an effective amount of at least one additional therapeutic agent (eg, one, two, three, four, five, or six additional therapeutic agents). A "subject" according to any of the above embodiments may be a human.
在又一個的方面，提供包含該抗原結合分子或抗BCMA抗體的醫藥組成物，例如，其用於以上任何製藥用途或治療方法。在一個實施 方案中，醫藥組成物包含本文提供的任何抗BCMA抗體和藥學上可接受的載體。在另一個實施方案中，醫藥組成物還包含至少一種另外的治療劑。 In yet another aspect, there is provided a pharmaceutical composition comprising the antigen binding molecule or anti-BCMA antibody, eg, for any of the above pharmaceutical uses or methods of treatment. in an implementation In the aspect, the pharmaceutical composition comprises any anti-BCMA antibody provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent.
本揭露的抗原結合分子或抗BCMA抗體可單獨使用或與其他試劑聯合用於治療。例如，本揭露的抗體可與至少一種另外的治療劑聯合施用(同時、或先後施用)。 The antigen binding molecules or anti-BCMA antibodies of the present disclosure can be used alone or in combination with other agents for therapy. For example, an antibody of the present disclosure can be administered in combination (simultaneously, or sequentially) with at least one additional therapeutic agent.
本揭露的抗原結合分子或抗BCMA抗體(和任何另外的治療劑)可藉由任何合適的手段施用，包括腸胃外、肺內和鼻內，並且如果需要局部治療，則病灶內施用。腸胃外輸注包括肌肉內、靜脈內、動脈內、腹膜內或皮下施用。給藥可以藉由任何適當的途徑，例如，藉由注射，諸如靜脈內或皮下注射，這部分取決於施用是短期的還是長期的。本文考慮多種給藥時間方案，包括但不限於，單次或在多個時間點多次施用，推注施用和脈衝輸注。 Antigen binding molecules or anti-BCMA antibodies of the present disclosure (and any additional therapeutic agents) may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, eg, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or chronic. A variety of dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulse infusion.
本揭露的抗原結合分子或抗BCMA抗體將以符合GOOD MEDICAL PRACTICE(GMP)Guideline的方式配製、給藥和施用。在此背景下考慮的因素包括所治療的具體病症、所治療的具體哺乳動物、個體患者的臨床狀況、病症的起因、試劑的遞送部位、施用方法、施用時間安排以及醫學從業者已知的其他因素。抗原結合分子或抗BCMA抗體視需要地與目前用於預防或治療該病症的一種或更多種試劑一起配製。此類其它試劑的有效量取決於醫藥組成物中存在的抗原結合分子或抗BCMA抗體的量、病症或治療的類型以及上文討論的其它因素。這些通常以與本文所述相同的劑量和施用路徑使用，或以本文所述劑量的約1至99%使用，或以任何劑量使用，並藉由經驗/臨床確定為合適的任何途徑使用。 Antigen binding molecules or anti-BCMA antibodies of the present disclosure will be formulated, dosed and administered in a manner consistent with GOOD MEDICAL PRACTICE (GMP) Guidelines. Factors considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and others known to the medical practitioner. factor. Antigen binding molecules or anti-BCMA antibodies are optionally formulated with one or more agents currently used to prevent or treat the disorder. The effective amount of such other agents depends on the amount of antigen binding molecule or anti-BCMA antibody present in the pharmaceutical composition, the type of disorder or treatment, and other factors discussed above. These are generally used at the same dosages and routes of administration as described herein, or at about 1 to 99% of the dosages described herein, or at any dosage, and by any route empirically/clinically determined to be suitable.
為了預防或治療疾病，本揭露的抗原結合分子或抗BCMA抗體(當單獨使用或與一種或更多種其他另外的治療劑組合使用時)的適當 的劑量將取決於待治療的疾病的類型，治療分子的類型，疾病的嚴重性和病程，是為預防還是治療目的施用，之前的治療，患者的臨床病史和對治療分子的響應，和主治醫師的判斷。治療分子恰當地以一次或經過一系列治療施用於患者。取決於疾病的類型和嚴重性，約1μg/kg至15mg/kg的抗原結合分子或抗BCMA抗體可以是用於施用至患者的初始候選劑量，不管例如是藉由一次或更多次分開的施用還是藉由連續輸注。一種典型的每日劑量可能在約1μg/kg至100mg/kg或更多的範圍內，這取決於上文提及的因素。相應的，以50kg體重為例，示例性的單位日劑量為50μg-5g。 For the prophylaxis or treatment of diseases, the antigen-binding molecules or anti-BCMA antibodies of the present disclosure (when used alone or in combination with one or more other additional therapeutic agents) are suitable The dosage will depend on the type of disease being treated, the type of therapeutic molecule, the severity and course of the disease, whether it is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the therapeutic molecule, and the attending physician judgment. The therapeutic molecule is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg of the antigen binding molecule or anti-BCMA antibody may be an initial candidate dose for administration to the patient, whether for example by one or more separate administrations Or by continuous infusion. A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. Correspondingly, taking a body weight of 50kg as an example, the exemplary unit daily dose is 50 μg-5g.
製品products
在本揭露的另一方面中，提供一種製品，該製品包含可用於治療、預防和/或診斷上述病症的材料。該製品包含容器、和在容器上或與容器聯合的標簽或包裝插頁(package insert)。合適的容器包括，例如，瓶子、管形瓶、注射器、IV溶液袋等。容器可以自各種材料諸如玻璃或塑料形成。容器裝有單獨或與另一種組成物組合有效治療，預防和/或診斷疾患的組成物，並且可具有無菌的存取口(例如，容器可以是具有由皮下注射針可刺穿的塞子的靜脈內溶液袋或管形瓶)。組成物中的至少一種活性試劑是本揭露的抗原結合分子或抗BCMA抗體。標簽或包裝插頁指示使用該組成物是來治療選擇的病況。此外，製品可以包含：(a)其中裝有組成物的第一容器，其中該組成物包含本揭露的抗原結合分子或抗BCMA抗體；和(b)其中裝有組成物的第二容器，其中該組成物包含另外的細胞毒性劑或其他方面的治療劑。本揭露的該實施方案中的製品可進一步包含包裝插頁，該包裝插頁指示該組成物可以用於治療特定病況。備選地，或另外地，製品可進一步包含第二(或第三)容器，該第二(或第三)容器包含藥學上 可接受的緩衝液。從商業和用戶立場，它可進一步包括所需的其他材料，包括其他緩衝劑、稀釋劑、濾器、針頭和注射器。 In another aspect of the present disclosure, an article of manufacture comprising materials useful for the treatment, prevention and/or diagnosis of the disorders described above is provided. The article comprises a container, and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. Containers can be formed from various materials such as glass or plastic. The container contains a composition effective alone or in combination with another composition for the treatment, prophylaxis and/or diagnosis of a condition, and may have a sterile access port (e.g., the container may be a vein with a stopper pierceable by a hypodermic needle) inner solution bag or vial). At least one active agent in the composition is an antigen binding molecule of the present disclosure or an anti-BCMA antibody. The label or package insert indicates that the composition is used to treat the condition of choice. In addition, the article of manufacture may comprise: (a) a first container having a composition therein, wherein the composition comprises an antigen binding molecule or an anti-BCMA antibody of the present disclosure; and (b) a second container having a composition therein, wherein The composition comprises an additional cytotoxic or otherwise therapeutic agent. The articles of manufacture of this embodiment of the present disclosure may further comprise a package insert indicating that the composition may be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer. It may further comprise other materials as desired from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.
實施例與測試例Example and test case
以下結合實施例和測試例進一步描述本揭露，但這些實施例和測試例並非限制著本揭露的範圍。本揭露實施例和測試例中未註明具體條件的實驗方法，通常按照常規條件，如冷泉港的抗體技術實驗手冊，分子選殖手冊；或按照原料或商品製造廠商所建議的條件。未註明具體來源的試劑，為市場購買的常規試劑。 The present disclosure is further described below in conjunction with examples and test examples, but these examples and test examples do not limit the scope of the present disclosure. For the experimental methods not specified in the examples and test examples of this disclosure, conventional conditions are usually followed, such as Cold Spring Harbor Antibody Technology Experiment Manual, Molecular Breeding Manual; or the conditions suggested by raw material or commodity manufacturers. Reagents without specific sources indicated are conventional reagents purchased in the market.
實施例1. 人BCMA和食蟹猴BCMA的表達Example 1. Expression of human BCMA and cynomolgus monkey BCMA
編碼帶人IgG1-Fc標簽的人BCMA和食蟹猴BCMA胞外區序列插入phr載體中，構建成表達質粒，然後轉染HEK293。編碼帶His標簽的人BCMA胞外區序列插入phr載體中，構建成表達質粒，然後轉染HEK293。具體轉染步驟為：將HEK293E細胞以1×106/mL接種於freestyle表達培養基(含有1% FBS，Gibco，12338-026)，放置於37度恆溫搖床(120rpm)繼續培養24小時。24小時後，將轉染質粒和轉染試劑PEI用0.22μm的濾器除菌，然後將轉染質粒調整為100μg/100mL細胞，PEI(1mg/mL)和質粒的質量比為3：1，取10mL的Opti-MEM和200μg質粒混勻，靜置5min；另取10mL的Opti-MEM和400μg PEI混勻，靜置5min。將質粒和PEI進行混勻，靜置15min。將質粒和PEI混合物緩慢加入200mL HEK293E的細胞中，放入8% CO2、120rpm、37℃的搖床中培養。轉染第3天，補充10%體積的補料培養基(20mM葡萄糖+2mM L-谷胺酸)。待轉染第6天，取樣4500rpm離心10min收集細胞上清，參照測試例2的方法純化。純化後的蛋白可用於下述各實施例或測試例實驗中。 The sequences encoding human BCMA with human IgG1-Fc tag and cynomolgus BCMA extracellular region were inserted into the phr vector to construct expression plasmids, which were then transfected into HEK293. The sequence encoding the human BCMA extracellular region with His tag was inserted into the phr vector to construct an expression plasmid, which was then transfected into HEK293. The specific transfection steps are as follows: HEK293E cells were inoculated in freestyle expression medium (containing 1% FBS, Gibco, 12338-026) at 1×10 6 /mL, and placed in a 37-degree constant temperature shaker (120 rpm) for 24 hours. After 24 hours, the transfection plasmid and transfection reagent PEI were sterilized with a 0.22 μm filter, and then the transfection plasmid was adjusted to 100 μg/100 mL cells, and the mass ratio of PEI (1 mg/mL) and plasmid was 3:1, which was taken as Mix 10 mL of Opti-MEM with 200 μg of plasmid and let it stand for 5 minutes; take another 10 mL of Opti-MEM and 400 μg of PEI, mix it and let it stand for 5 minutes. The plasmid and PEI were mixed and allowed to stand for 15 minutes. The plasmid and PEI mixture was slowly added to 200mL HEK293E cells, and cultured in a shaker with 8% CO 2 , 120 rpm, and 37°C. On day 3 of transfection, 10% volume of feed medium (20 mM glucose + 2 mM L-glutamate) was supplemented. On the 6th day after transfection, samples were taken and centrifuged at 4500 rpm for 10 minutes to collect the cell supernatant, and purified according to the method in Test Example 2. The purified protein can be used in the experiments of the following examples or test examples.
其中humanBCMA-ECD-Fc、humanBCMA-ECD-his、cynoBCMA-ECD-his和cynoBCMA-ECD-Fc的胺基酸序列如下所示。cynoBCMA-ECD-his購自ACRO公司。 The amino acid sequences of humanBCMA-ECD-Fc, humanBCMA-ECD-his, cynoBCMA-ECD-his and cynoBCMA-ECD-Fc are shown below. cynoBCMA-ECD-his was purchased from ACRO.
(1)帶人Fc標簽的人BCMA：humanBCMA-ECD-Fc(SEQ ID NO：1) (1) Human BCMA with human Fc tag: humanBCMA-ECD-Fc (SEQ ID NO: 1)
註：下劃線為信號肽序列；斜體部分為human Fc-linker-tag； Note: The underline is the signal peptide sequence; the italic part is the human Fc-linker-tag;
(2)帶His標簽的人BCMA的ECD：humanBCMA-ECD-his(SEQ ID NO：2) (2) ECD of human BCMA with His tag: humanBCMA-ECD-his (SEQ ID NO: 2)
註：下劃線為信號肽序列；斜體部分為His6-linker-tag； Note: The underline is the signal peptide sequence; the italic part is His6-linker-tag;
(3)帶His標簽的食蟹猴BCMA的ECD：cynoBCMA-ECD-his(BCA-C4E3H7-1mg)(SEQ ID NO：3) (3) ECD of cynomolgus monkey BCMA with His tag: cynoBCMA-ECD-his (BCA-C4E3H7-1mg) (SEQ ID NO: 3)
註：斜體部分為linker-10his-tag； Note: The italic part is linker-10his-tag;
(4)帶人Fc標簽的食蟹猴BCMA：cynoBCMA-ECD-Fc(SEQ ID NO：4) (4) Cynomolgus monkey BCMA with human Fc tag: cynoBCMA-ECD-Fc (SEQ ID NO: 4)
註：下劃線為信號肽序列；雙下劃線為linker序列；斜體部分為human Fc-tag。 Note: The underline is the signal peptide sequence; the double underline is the linker sequence; the italic part is the human Fc-tag.
實施例2. Protein A親和層析純化帶Fc標簽的重組蛋白和鎳管柱純化帶his標簽的重組蛋白Example 2. Protein A affinity chromatography purification of recombinant protein with Fc tag and nickel column purification of recombinant protein with his tag
帶人Fc標簽的蛋白的純化：將細胞表達的BCMA上清樣品高速離心去除雜質，藉由Protein A管柱進行純化。用PBS沖洗管柱，至A280讀數降至基線。用100mM乙酸pH3.5沖提目的蛋白，用1M Tris-HCl，pH8.0中和。沖提樣品適當濃縮後換液到PBS中，將得到的蛋白經電泳，肽圖，LC-MS鑑定為正確後分裝備用。 Purification of protein with human Fc tag: The BCMA supernatant sample expressed by cells was centrifuged at high speed to remove impurities, and purified by Protein A column. Rinse the column with PBS until the A280 reading drops to baseline. The target protein was extracted with 100mM acetic acid, pH3.5, and neutralized with 1M Tris-HCl, pH8.0. After the eluting sample was properly concentrated, the liquid was changed into PBS, and the obtained protein was identified as correct by electrophoresis, peptide map, and LC-MS before being dispensed for use.
帶人his標簽的蛋白的純化：將細胞表達的BCMA上清樣品高速離心去除雜質。用含有PBS緩衝液平衡鎳管柱，沖洗2-5倍管柱體積，將上清樣品以一定流速上Ni Sepharose excel管柱。用PBS緩衝液沖洗管柱，至A280讀數降至基線，再後用PBS+10mM咪唑沖洗層析管柱，除去 非特異結合的雜蛋白，並收集流出液，最後用含有300mM咪唑的PBS溶液沖提目的蛋白，並收集沖提峰，濃縮換液，將得到的蛋白經電泳，肽圖，LC-MS鑑定為正確後分裝備用。 Purification of protein with human his tag: the supernatant sample of BCMA expressed by cells was centrifuged at high speed to remove impurities. Equilibrate the nickel column with PBS buffer, wash 2-5 times the column volume, and put the supernatant sample on the Ni Sepharose excel column at a certain flow rate. Rinse the column with PBS buffer until the A280 reading drops to the baseline, then rinse the column with PBS+10mM imidazole to remove Non-specifically bound miscellaneous proteins, and the effluent was collected, and finally the target protein was extracted with PBS solution containing 300mM imidazole, and the peaks were collected, concentrated and replaced, and the obtained protein was identified by electrophoresis, peptide map, and LC-MS as After the correct allocation of equipment for use.
實施例3. 表達重組人BCMA和食蟹猴BCMA細胞系的構建和鑑定Example 3. Construction and identification of cell lines expressing recombinant human BCMA and cynomolgus BCMA
為篩選能和細胞表面BCMA有較好的結合的抗體，本揭露構建了表達人BCMA的K562-BCMA細胞株。將人BCMA全長基因選殖到哺乳動物細胞表達載體pCDH上，用pVSV-G、pCMV-dR8.91和pCDH-humanBCMA三種質粒共同轉染HEK293T細胞(ATCC® CRL-11268)來包裝病毒，轉染48小時後，收集病毒感染K562細胞(ATCC® CCL-243)。感染72小時後藉由流式分選獲取高表達humanBCMA的K562單株細胞。本揭露還構建了表達食蟹猴的CHO-K1-cynoBCMA細胞株。將食蟹猴BCMA全長基因選殖到哺乳動物細胞表達載體pCDH上，用pVSV-G、pCMV-dR8.91和pCDH-cynoBCMA三種質粒共同轉染HEK293T細胞(ATCC® CRL-11268)來包裝病毒，轉染48小時後，收集病毒感染CHOK1細胞(ATCC® CCL-61)。感染72小時後藉由流式分選獲取高表達cynoBCMA的CHOK1單株細胞。 In order to screen antibodies that can better bind to BCMA on the cell surface, the present disclosure constructed a K562-BCMA cell line expressing human BCMA. The full-length human BCMA gene was cloned into the mammalian cell expression vector pCDH, and the three plasmids pVSV-G, pCMV-dR8.91 and pCDH-humanBCMA were co-transfected into HEK293T cells (ATCC ® CRL-11268) to package the virus, and transfected After 48 hours, virus-infected K562 cells (ATCC ® CCL-243) were harvested. After 72 hours of infection, K562 single cell line with high expression of humanBCMA was obtained by flow sorting. The disclosure also constructed a CHO-K1-cynoBCMA cell line expressing cynomolgus monkey. The full-length gene of cynomolgus monkey BCMA was cloned into the mammalian cell expression vector pCDH, and the three plasmids pVSV-G, pCMV-dR8.91 and pCDH-cynoBCMA were co-transfected into HEK293T cells (ATCC® CRL-11268) to package the virus. 48 hours after transfection, virus-infected CHOK1 cells (ATCC® CCL-61) were harvested. Seventy-two hours after infection, CHOK1 cells with high expression of cynoBCMA were obtained by flow sorting.
實施例4. 抗人BCMA融合瘤抗體的篩選和鑑定Example 4. Screening and identification of anti-human BCMA fusion tumor antibodies
本公開藉由融合瘤技術製備了針對人BCMA的單株抗體。所得抗體與人BCMA以較高的親和力特異性結合，並且可以與食蟹猴BCMA發生交叉反應；所得抗體與細胞表面的人BCMA和食蟹猴BCMA有較好的結合活性，且該結合活性不受可溶性BCMA干擾。 The disclosure prepares a monoclonal antibody against human BCMA by fusion tumor technology. The obtained antibody specifically binds to human BCMA with high affinity, and can cross-react with cynomolgus monkey BCMA; the obtained antibody has better binding activity to human BCMA and cynomolgus monkey BCMA on the cell surface, and the binding activity is not affected by Soluble BCMA interference.
將humanBCMA-ECD-his和cynoBCMA-ECD-his作為交叉免疫試劑，TiterMax® Gold Adjuvant(Sigma Cat No.T2684)與Thermo Imject® Alum(Thermo Cat No.77161)作為佐劑，交叉免疫小鼠。經過初次免疫和7次加強免疫後選擇血清中抗體滴度高小鼠10-6#(滴度625K)進行脾細胞融合。融合後根據融合瘤細胞生長密度，對融合瘤培養上清進行檢測，並篩選特異性結合細胞表面BCMA的抗體。 HumanBCMA-ECD-his and cynoBCMA-ECD-his were used as cross-immunization reagents, and TiterMax® Gold Adjuvant (Sigma Cat No.T2684) and Thermo Imject® Alum (Thermo Cat No.77161) were used as adjuvants to cross-immunize mice. After the initial immunization and 7 booster immunizations, the mouse 10-6# (titer 625K) with high antibody titer in serum was selected for splenocyte fusion. After fusion, according to the growth density of the fusion tumor cells, the culture supernatant of the fusion tumor was detected, and antibodies specifically binding to BCMA on the cell surface were screened.
篩選得到活性好的單株融合瘤細胞株4E3、33H4、3B2和78。分別收集對數生長期融合瘤細胞，用NucleoZol(MN)提取RNA(按照試劑盒說明書步驟)，並進行反轉錄(PrimeScriptTM Reverse Transcriptase，Takara,cat # 2680A)。將反轉錄得到的cDNA採用mouse Ig-Primer Set(Novagen，TB326 Rev.B 0503)進行PCR擴增後測序。4E3、33H4、3B2和78的CDR和可變區的胺基酸序列如下： The single fusion tumor cell lines 4E3, 33H4, 3B2 and 78 with good activity were screened. The fusionoma cells in the logarithmic growth phase were collected separately, RNA was extracted with NucleoZol (MN) (according to the kit instructions), and reverse transcription was performed (PrimeScript TM Reverse Transcriptase, Takara, cat # 2680A). The cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and then sequenced. The amino acid sequences of the CDRs and variable regions of 4E3, 33H4, 3B2 and 78 are as follows:
表5. BCMA抗體CDR
>4E3鼠源重鏈可變區(SEQ ID NO：29) >4E3 murine heavy chain variable region (SEQ ID NO: 29)
>4E3鼠源輕鏈可變區(SEQ ID NO：30) >4E3 murine light chain variable region (SEQ ID NO: 30)
>33H4鼠源重鏈可變區(SEQ ID NO：31) >33H4 murine heavy chain variable region (SEQ ID NO: 31)
>33H4鼠源輕鏈可變區(SEQ ID NO：32) >33H4 murine light chain variable region (SEQ ID NO: 32)
>3B2鼠源重鏈可變區(SEQ ID NO：33) >3B2 murine heavy chain variable region (SEQ ID NO: 33)
>3B2鼠源輕鏈可變區(SEQ ID NO：34) >3B2 murine light chain variable region (SEQ ID NO: 34)
>78鼠源重鏈可變區(SEQ ID NO：35) >78 murine heavy chain variable region (SEQ ID NO: 35)
>78鼠源輕鏈可變區(SEQ ID NO：36) >78 murine light chain variable region (SEQ ID NO: 36)
註：下劃線標記區為根據Kabat編號規則獲得的CDR區。 Note: The underlined region is the CDR region obtained according to the Kabat numbering rule.
實施例5. 抗人BCMA單株抗體的人源化設計Example 5. Humanized design of anti-human BCMA monoclonal antibody
鼠源單株抗體人源化根據本領域許多文獻公示的方法進行。簡言之，在所獲得的鼠源抗體VH/VL CDR典型結構的基礎上，從人源種系數據庫中搜索輕鏈可變區(VL)和重鏈可變區(VH)的同源序列，將鼠源抗體的CDR區移植到人源模板上，並對VL和VH的部分殘基進行突變，將鼠源抗體的恆定區替換為人恆定區，得到最終的人源化分子。 The humanization of the mouse monoclonal antibody was carried out according to the methods published in many documents in this field. Briefly, on the basis of the obtained typical structure of murine antibody VH/VL CDRs, homologous sequences of light chain variable region (VL) and heavy chain variable region (VH) were searched from human germline databases , the CDR region of the murine antibody is grafted onto the human template, and some residues of VL and VH are mutated, and the constant region of the murine antibody is replaced by a human constant region to obtain the final humanized molecule.
1. 4E3的人源FR區的選擇和回復突變。其中，重鏈可變區的FR1-3採用IGHV1-46*01，FR4採用IGHJ6*01；輕鏈可變區的FR1-3採用IGKV1-39*01，FR4採用IGKJ2*01。FR1-3的來源和回復突變見下表。 1. Selection and back mutation of the human FR region of 4E3. Among them, IGHV1-46*01 was used for FR1-3 of the heavy chain variable region, and IGHJ6*01 was used for FR4; IGKV1-39*01 was used for FR1-3 of the light chain variable region, and IGKJ2*01 was used for FR4. The sources and back mutations of FR1-3 are shown in the table below.
表6. 4E3的人源FR區選擇和回復突變
註：Graft代表鼠抗體CDR植入人種系FR區序列。示例性的，R71A表示依照Kabat編號系統，將71位R突變回A。下同。 Note: Graft represents mouse antibody CDR implanted into human germline FR region sequence. Exemplarily, R71A means that R at position 71 is mutated back to A according to the Kabat numbering system. The same below.
此外，還對抗體重鏈可變區的第1位胺基酸殘基由Q突變為E，鼠源抗體4E3人源化得到的抗體可變區具體序列如下(下劃線表示CDR，下同)： In addition, the first amino acid residue in the variable region of the heavy chain was mutated from Q to E. The specific sequence of the variable region of the antibody obtained by humanizing the murine antibody 4E3 is as follows (the underline indicates CDR, the same below):
>hu4E3H2 Graft(IGHV1-46*01)+R71A,T73K,S76T,A93V(SEQ ID NO：38) >hu4E3H2 Graft (IGHV1-46*01)+R71A, T73K, S76T, A93V (SEQ ID NO: 38)
>hu4E3H3 Graft(IGHV1-46*01)+M48I,V67A,R71A,T73K,S76T,A93V(SEQ ID NO：39) >hu4E3H3 Graft (IGHV1-46*01)+M48I, V67A, R71A, T73K, S76T, A93V (SEQ ID NO: 39)
>hu4E3L1 Graft(IGKV1-39*01)(SEQ ID NO：40) >hu4E3L1 Graft (IGKV1-39*01) (SEQ ID NO: 40)
>hu4E3L2 Graft(IGKV1-39*01)+I48V(SEQ ID NO：41) >hu4E3L2 Graft(IGKV1-39*01)+I48V(SEQ ID NO:41)
>hu4E3L3 Graft(IGKV1-39*01)+I48V,F71Y(SEQ ID NO：42) >hu4E3L3 Graft(IGKV1-39*01)+I48V,F71Y(SEQ ID NO:42)
>hu4E3L4 Graft(IGKV1-39*01)+I48V,F71Y,A43S,K45Q(SEQ ID NO：43) >hu4E3L4 Graft(IGKV1-39*01)+I48V,F71Y,A43S,K45Q(SEQ ID NO:43)
2. 33H4的人源FR區的選擇和回復突變。其中，重鏈可變區的FR1-3採用IGHV7-4-1*02，FR4採用IGHJ1*01；輕鏈可變區的FR1-3採用IGKV1-8*01或IGKV1-27*01，FR4採用IGKJ4*01。FR1-3的來源和回復突變見下表。 2. Selection and back mutation of the human FR region of 33H4. Among them, FR1-3 of the heavy chain variable region uses IGHV7-4-1*02, FR4 uses IGHJ1*01; FR1-3 of the light chain variable region uses IGKV1-8*01 or IGKV1-27*01, and FR4 uses IGKJ4*01. The sources and back mutations of FR1-3 are shown in the table below.
表7. 33H4的人源FR區選擇和回復突變
此外，還對抗體重鏈可變區的第1位胺基酸殘基由Q突變為E，鼠源抗體33H4人源化得到的抗體可變區具體序列如下： In addition, the first amino acid residue in the variable region of the heavy chain was mutated from Q to E. The specific sequence of the variable region of the antibody obtained by humanizing the murine antibody 33H4 is as follows:
>hu33H4H1 Graft(IGHV7-4-1*02)+A93L(SEQ ID NO：44) >hu33H4H1 Graft (IGHV7-4-1*02)+A93L (SEQ ID NO: 44)
>hu33H4H2 Graft(IGHV7-4-1*02)+V2I,G44V,L45F,A93L(SEQ ID NO：45) >hu33H4H2 Graft (IGHV7-4-1*02)+V2I, G44V, L45F, A93L (SEQ ID NO: 45)
>hu33H4H3 Graft(IGHV7-4-1*02)+V2I,G44V,L45F,E46K,V75A,S76N,A93L(SEQ ID NO：46) >hu33H4H3 Graft (IGHV7-4-1*02)+V2I, G44V, L45F, E46K, V75A, S76N, A93L (SEQ ID NO: 46)
>hu33H4L1 Graft(IGKV1-8*01)+A43S,G66D(SEQ ID NO：47) >hu33H4L1 Graft (IGKV1-8*01)+A43S, G66D (SEQ ID NO: 47)
>hu33H4L2 Graft(IGKV1-27*01)+V43S,G66D(SEQ ID NO：48) >hu33H4L2 Graft (IGKV1-27*01)+V43S, G66D (SEQ ID NO: 48)
3. 3B2的人源FR區的選擇和回復突變。其中，重鏈可變區的FR1-3採用IGHV3-11*01，FR4採用IGHJ1*01；輕鏈可變區的FR1-3採用IGKV1-39*01，FR4採用IGKJ2*01。FR1-3的來源和回復突變見下表。 3. Selection and back mutation of the human FR region of 3B2. Among them, IGHV3-11*01 is used for FR1-3 of the heavy chain variable region, and IGHJ1*01 is used for FR4; IGKV1-39*01 is used for FR1-3 of the light chain variable region, and IGKJ2*01 is used for FR4. The sources and back mutations of FR1-3 are shown in the table below.
表8. 3B2的人源FR區選擇和回復突變
鼠源抗體3B2人源化得到的抗體可變區具體序列如下： The specific sequence of the antibody variable region obtained by humanizing the murine antibody 3B2 is as follows:
>hu3B2H1 Graft(IGHV3-11*01)+Q1E,S30R,A93T(SEQ ID NO：49) >hu3B2H1 Graft (IGHV3-11*01)+Q1E, S30R, A93T (SEQ ID NO: 49)
>hu3B2H2 Graft(IGHV3-11*01)+Q1E,S30R,W47R,S49A,A93T(SEQ ID NO：50) >hu3B2H2 Graft (IGHV3-11*01)+Q1E, S30R, W47R, S49A, A93T (SEQ ID NO: 50)
>hu3B2L1 Graft(IGKV1-39*01)+P44V(SEQ ID NO：51) >hu3B2L1 Graft (IGKV1-39*01)+P44V (SEQ ID NO: 51)
4. 78的人源FR區的選擇和回復突變。其中，重鏈可變區的FR1-3採用IGHV2-70*01，FR4採用IGHJ6*01；輕鏈可變區的FR1-3採用IGKV1-33*01，FR4採用IGKJ2*01。FR1-3的來源和回復突變見下表。 4. Selection and back mutation of the human FR region of 78. Among them, IGHV2-70*01 was used for FR1-3 of the variable region of the heavy chain, and IGHJ6*01 was used for FR4; IGKV1-33*01 was used for FR1-3 of the variable region of the light chain, and IGKJ2*01 was used for FR4. The sources and back mutations of FR1-3 are shown in the table below.
表9. 78的人源FR區選擇和回復突變
此外，還對抗體重鏈可變區的第1位胺基酸殘基由Q突變為E，鼠源抗體78人源化得到的抗體可變區具體序列如下： In addition, the first amino acid residue in the variable region of the heavy chain was mutated from Q to E, and the specific sequence of the variable region of the antibody obtained by humanizing the murine antibody 78 is as follows:
>hu78H1 Graft(IGHV2-70*01)+F24V,S30T,T73N(SEQ ID NO：52) >hu78H1 Graft(IGHV2-70*01)+F24V,S30T,T73N(SEQ ID NO:52)
>hu78H2 Graft(IGHV2-70*01)+F24V,S30T,I37V,A49G,T73N,L80F,T89R(SEQ ID NO：53) >hu78H2 Graft(IGHV2-70*01)+F24V,S30T,I37V,A49G,T73N,L80F,T89R(SEQ ID NO:53)
>hu78L1 Graft(IGKV1-33*01)(SEQ ID NO：54) >hu78L1 Graft (IGKV1-33*01) (SEQ ID NO: 54)
實施例6. 抗BCMA/CD3雙特異性抗體的製備及鑑定Example 6. Preparation and identification of anti-BCMA/CD3 bispecific antibody
本揭露CD3結合分子可以來源於任意適宜的抗體。特別適宜的抗體描述於例如國際申請號PCT/CN2019/123548(藉由援引完整收入本文)。 The CD3 binding molecules of the present disclosure may be derived from any suitable antibody. Particularly suitable antibodies are described, for example, in International Application No. PCT/CN2019/123548 (herein incorporated by reference in its entirety).
本揭露雙特異性抗體中的抗CD3臂的CDR和可變區序列如下所示： The CDR and variable region sequences of the anti-CD3 arm in the disclosed bispecific antibody are as follows:
表10. CD3臂的CDR
可變區具體序列如下： The specific sequence of the variable region is as follows:
>S107E-VH(SEQ ID NO：63) >S107E-VH (SEQ ID NO: 63)
>S107E-VL(SEQ ID NO：64) >S107E-VL (SEQ ID NO: 64)
>6164-VH(SEQ ID NO：65) >6164-VH (SEQ ID NO: 65)
>6164-VL(SEQ ID NO：66) >6164-VL (SEQ ID NO: 66)
將人源化的抗BCMA抗體可變區分別與抗CD3的抗體的S107E和6164的可變區以及IgG1突變體IgG1(AA)(L234A/L235A)組合。各形成幾種不同形式的雙特異性抗體，分別為Format-11，Format-11-6164，Format-14和Format-15。 The variable regions of the humanized anti-BCMA antibody were combined with the variable regions of S107E and 6164 of the anti-CD3 antibody and the IgG1 mutant IgG 1 (AA) (L234A/L235A), respectively. Each forms several different formats of bispecific antibodies, namely Format-11, Format-11-6164, Format-14 and Format-15.
Format-11為對稱結構分子，包含兩條相同的重鏈(鏈1)和兩條相同的輕鏈(鏈2)。 Format-11 is a molecule with a symmetrical structure, comprising two identical heavy chains (chain 1) and two identical light chains (chain 2).
重鏈為：VH(anti-BCMA)-IgG1(CH1)-VH(S107E)-linker1-VL(S107E)-linker2-IgG1(AA)Fc； The heavy chain is: VH(anti-BCMA)-IgG 1 (CH1)-VH(S107E)-linker1-VL(S107E)-linker2-IgG 1 (AA)Fc;
輕鏈為VL(anti-BCMA)-CL，其示意圖如圖1A所示。 The light chain is VL(anti-BCMA)-CL, the schematic diagram of which is shown in Figure 1A.
Format-11-6164為對稱結構分子，包含兩條相同的重鏈和兩條相同的輕鏈。 Format-11-6164 is a molecule with symmetrical structure, containing two identical heavy chains and two identical light chains.
重鏈為：VH(anti-BCMA)-IgG1(CH1)-VH(6164)-linker1-VL(6164)-1inker2-IgG1(AA)Fc； The heavy chain is: VH(anti-BCMA)-IgG 1 (CH1)-VH(6164)-linker1-VL(6164)-linker2-IgG 1 (AA)Fc;
輕鏈為VL(anti-BCMA)-CL，其示意圖如圖1A所示。 The light chain is VL(anti-BCMA)-CL, the schematic diagram of which is shown in Figure 1A.
其中， in,
>IgG1(CH1)(SEQ ID NO：67) > IgG 1 (CH1) (SEQ ID NO: 67)
>CL(SEQ ID NO：68) >CL (SEQ ID NO: 68)
>IgG1(AA)Fc(SEQ ID NO：69) > IgG 1 (AA) Fc (SEQ ID NO: 69)
Format-14為非對稱結構分子，完整分子共四條鏈，四條鏈均不相同，鏈1為：VH(anti-BCMA)-IgG1(CH1)-IgG1Fc(Hole)，鏈2為：VL(anti-BCMA)-CL，鏈3為T-Knob：VH(S107E)-linker3-Titin-IgG1Fc(Knob)；鏈4為VL-Ob：VL(S107E)-linker3-Obscurin，如圖1B所示(Ob代表Obscurin)。 Format-14 is an asymmetric structure molecule. The complete molecule has four chains, all of which are different. Chain 1 is: VH(anti-BCMA)-IgG 1 (CH1)-IgG 1 Fc(Hole), chain 2 is: VL (anti-BCMA)-CL, chain 3 is T-Knob: VH(S107E)-linker3-Titin-IgG 1 Fc(Knob); chain 4 is VL-Ob: VL(S107E)-linker3-Obscurin, as shown in Figure 1B shown (Ob stands for Obscurin).
其中， in,
>T-Knob：VH(S107E)-linker3-Titin-IgG1Fc(Knob)(SEQ ID NO：70) >T-Knob: VH(S107E)-linker3-Titin-IgG 1 Fc(Knob) (SEQ ID NO:70)
>VL-Ob：VL(S107E)-linker3-Obscurin(SEQ ID NO：71) >VL-Ob: VL(S107E)-linker3-Obscurin (SEQ ID NO:71)
Format-15為非對稱結構分子，完整分子共四條鏈，四條鏈均不相同，鏈1為：VH(anti-BCMA)-IgG1(CH1)-IgG1Fc(Knob，E356D，M358L)，鏈2為：VL(anti-BCMA)-CL，鏈3為Ob-Hole；鏈4為VL-Titin，其示意圖如圖1C所示(Ob代表Obscurin)。 Format-15 is an asymmetric structure molecule. The complete molecule has four chains, all of which are different. Chain 1 is: VH(anti-BCMA)-IgG 1 (CH1)-IgG 1 Fc (Knob, E356D, M358L), chain 2 is: VL(anti-BCMA)-CL, chain 3 is Ob-Hole; chain 4 is VL-Titin, and its schematic diagram is shown in Figure 1C (Ob stands for Obscurin).
>Ob-Hole：VH(S107E)-linker3-Obscurin-IgG1Fc(Hole，E356D，M358L)(SEQ ID NO：167) >Ob-Hole: VH(S107E)-linker3-Obscurin-IgG 1 Fc (Hole, E356D, M358L) (SEQ ID NO: 167)
>VL-Titin：VL(S107E)-linker3-Titin(SEQ ID NO：168) >VL-Titin: VL(S107E)-linker3-Titin (SEQ ID NO: 168)
註：單下劃線標記區為根據Kabat編號規則獲得的CD3結合結構域的CDR區，雙下劃線標記區為Titin或Obscurin序列，斜體為恆定區。 Note: The single underlined region is the CDR region of the CD3 binding domain obtained according to the Kabat numbering rule, the double underlined region is the Titin or Obscurin sequence, and the italicized constant region.
>IgG1Fc(Knob)(SEQ ID NO：153) > IgG 1 Fc (Knob) (SEQ ID NO: 153)
>IgG1Fc(Hole)(SEQ ID NO：154) >IgG 1 Fc (Hole) (SEQ ID NO: 154)
>IgG1Fc-1(Knob，E356D，M358L)(SEQ ID NO：169) > IgG 1 Fc-1 (Knob, E356D, M358L) (SEQ ID NO: 169)
>IgG1Fc-1(Hole，E356D，M358L)(SEQ ID NO：170) > IgG 1 Fc-1 (Hole, E356D, M358L) (SEQ ID NO: 170)
基於4E3、33H4、3B2和78的人源化抗體胺基酸序列，分別構建了如下所示的雙特異性抗體。 Based on the amino acid sequences of humanized antibodies 4E3, 33H4, 3B2 and 78, the bispecific antibodies shown below were respectively constructed.
表11. 本揭露的雙特異性抗體
表11中，4E3-11-1中的hu4E3H1-11表示採用hu4E3H1作為VH(anti-BCMA)，結構為Format-11；4E3-11-6164-1中的hu4E3H2-11-6164代表表示hu4E3H2作為VH(anti-BCMA)，結構為Format-11-6164，以此類推。具體胺基酸序列如下： In Table 11, hu4E3H1-11 in 4E3-11-1 means that hu4E3H1 is used as VH (anti-BCMA), and the structure is Format-11; hu4E3H2-11-6164 in 4E3-11-6164-1 means that hu4E3H2 is used as VH (anti-BCMA), the structure is Format-11-6164, and so on. The specific amino acid sequence is as follows:
>hu4E3L1-11(SEQ ID NO：72) >hu4E3L1-11 (SEQ ID NO: 72)
>hu4E3L2-11(SEQ ID NO：73) >hu4E3L2-11 (SEQ ID NO: 73)
>hu4E3L3-11(SEQ ID NO：74) >hu4E3L3-11 (SEQ ID NO: 74)
>hu4E3L4-11(SEQ ID NO：75) >hu4E3L4-11 (SEQ ID NO: 75)
>hu4E3H1-11(SEQ ID NO：76) >hu4E3H1-11 (SEQ ID NO: 76)
>hu4E3H2-11(SEQ ID NO：77) >hu4E3H2-11 (SEQ ID NO: 77)
>hu4E3H3-11(SEQ ID NO：78) >hu4E3H3-11 (SEQ ID NO: 78)
>hu4E3H1-11-6164(SEQ ID NO：79) >hu4E3H1-11-6164 (SEQ ID NO: 79)
>hu4E3H2-11-6164(SEQ ID NO：80) >hu4E3H2-11-6164 (SEQ ID NO: 80)
>hu4E3H3-11-6164(SEQ ID NO：81) >hu4E3H3-11-6164 (SEQ ID NO: 81)
>hu33H4L1-11(SEQ ID NO：82) >hu33H4L1-11 (SEQ ID NO: 82)
>hu33H4L2-11(SEQ ID NO：83) >hu33H4L2-11 (SEQ ID NO: 83)
>hu33H4H1-11(SEQ ID NO：84) >hu33H4H1-11 (SEQ ID NO: 84)
>hu33H4H2-11(SEQ ID NO：85) >hu33H4H2-11 (SEQ ID NO: 85)
>hu33H4H3-11(SEQ ID NO：86) >hu33H4H3-11 (SEQ ID NO: 86)
>hu33H4H2-15(SEQ ID NO：171) >hu33H4H2-15 (SEQ ID NO: 171)
>hu3B2L1-11(SEQ ID NO：87) >hu3B2L1-11 (SEQ ID NO: 87)
>hu3B2H1-11(SEQ ID NO：88) >hu3B2H1-11 (SEQ ID NO: 88)
>hu3B2H2-11(SEQ ID NO：89) >hu3B2H2-11 (SEQ ID NO: 89)
>hu3B2H1-11-6164(SEQ ID NO：90) >hu3B2H1-11-6164 (SEQ ID NO: 90)
>hu3B2H2-11-6164(SEQ ID NO：91) >hu3B2H2-11-6164 (SEQ ID NO: 91)
>hu3B2H1-14(SEQ ID NO：92) >hu3B2H1-14 (SEQ ID NO: 92)
>hu3B2H2-14(SEQ ID NO：93) >hu3B2H2-14 (SEQ ID NO: 93)
>hu3B2H2-15(SEQ ID NO：172) >hu3B2H2-15 (SEQ ID NO: 172)
>hu78L1-11(SEQ ID NO：94) >hu78L1-11 (SEQ ID NO: 94)
>hu78H1-11(SEQ ID NO：95) >hu78H1-11 (SEQ ID NO: 95)
>hu78H1-11-6164(SEQ ID NO：96) >hu78H1-11-6164 (SEQ ID NO: 96)
>hu78H2-11-6164(SEQ ID NO：97) >hu78H2-11-6164 (SEQ ID NO: 97)
本揭露所用的陽性對照分子AMGEN701和REGN5458。 The positive control molecules used in this disclosure are AMGEN701 and REGN5458.
AMGEN701(SEQ ID NO：158)，序列來源：WO2017134134 AMGEN701 (SEQ ID NO: 158), sequence source: WO2017134134
REGN5458序列來源：專利WO2020018830 REGN5458 sequence source: patent WO2020018830
鏈1：BCMA重鏈(SEQ ID NO：159) Chain 1: BCMA heavy chain (SEQ ID NO: 159)
鏈2：CD3重鏈(SEQ ID NO：160) Chain 2: CD3 heavy chain (SEQ ID NO: 160)
鏈3/鏈4：BCMA輕/CD3輕鏈(SEQ ID NO：161) Chain 3/Strand 4: BCMA light/CD3 light chain (SEQ ID NO: 161)
實施例7. 含有Titin-T鏈/Obscurin-O鏈的抗原結合分子Example 7. Antigen-binding molecules containing Titin-T chain/Obscurin-O chain
本揭露的Titin鏈/Obscurin鏈可以來源於任意適宜的多肽，包括來源於PCT/CN2021/070832及其公開文本(藉由援引完整收入本文)和CN202110527339.7及將其作為優先權文件的專利(藉由援引完整收入本文)中的多肽。構建雙特異性抗體，其中CL為PCT/CN2021/070832中的kappa輕鏈恆定區，Titin鏈和Obscurin鏈的胺基酸序列見表3-1和表3-2，連接子序列包括GGGGS(SEQ ID NO：157)、ASTKG(SEQ ID NO：173)或RTVAS(SEQ ID NO：174)，本實施例中的Fc1、Fc2和CH1的胺基酸序列分別如SEQ ID NO：153、SEQ ID NO：154和SEQ ID NO：67所示。 The Titin chain/Obscurin chain of the present disclosure can be derived from any suitable polypeptide, including PCT/CN2021/070832 and its publications (incorporated herein by reference) and CN202110527339.7 and its patents as priority documents ( The polypeptides herein are incorporated by reference in their entirety). Construct a bispecific antibody, wherein CL is the kappa light chain constant region in PCT/CN2021/070832, the amino acid sequences of Titin chain and Obscurin chain are shown in Table 3-1 and Table 3-2, and the linker sequence includes GGGGS (SEQ ID NO: 157), ASTKG (SEQ ID NO: 173) or RTVAS (SEQ ID NO: 174), the amino acid sequences of Fc1, Fc2 and CH1 in this example are respectively as SEQ ID NO: 153, SEQ ID NO : 154 and SEQ ID NO: 67.
7.1 DI雙特異性抗體7.1 DI bispecific antibody
參照PCT/CN2021/070832實施例5，構建抗hNGF和hRANKL的DI雙特異性抗體：DI-2至DI-20，其包含如下所述的第一重鏈、第二重鏈、第一輕鏈和第二輕鏈： Referring to Example 5 of PCT/CN2021/070832, construct DI bispecific antibodies against hNGF and hRANKL: DI-2 to DI-20, which comprise the first heavy chain, the second heavy chain, and the first light chain as described below and the second light chain:
第一重鏈：從N端到C端依次為：[VH1-I]-[連接子1]-[Obscurin鏈]-[Fc2]， The first heavy chain: from N-terminal to C-terminal: [VH1-I]-[Linker 1]-[Obscurin chain]-[Fc2],
第一輕鏈：從N端到C端依次為：[VL1-I]-[連接子2]-[Titin鏈]， The first light chain: from N-terminal to C-terminal: [VL1-I]-[Linker 2]-[Titin chain],
第二重鏈：從N端到C端依次為：[VH2-D]-[CH1]-[Fc1]，和 Second heavy chain: from N-terminus to C-terminus: [VH2-D]-[CH1]-[Fc1], and
第二輕鏈：從N端到C端依次為：[VL2-D]-[CL]； The second light chain: from N-terminal to C-terminal: [VL2-D]-[CL];
其中，VH1-I和VL1-I分別為PCT/CN2021/070832中I0的重鏈可變區和輕鏈可變區，VH2-D和VL2-D分別為PCT/CN2021/070832中D0的 重鏈可變區和輕鏈可變區。本實施例中DI雙特異性抗體中Obscurin-O鏈、Titin-T鏈、連接子1、連接子2結構見下表。 Among them, VH1-I and VL1-I are the heavy chain variable region and the light chain variable region of I0 in PCT/CN2021/070832, respectively, and VH2-D and VL2-D are the D0 in PCT/CN2021/070832. Heavy chain variable region and light chain variable region. The structures of Obscurin-O chain, Titin-T chain, linker 1 and linker 2 in the DI bispecific antibody in this example are shown in the table below.
表i-1. DI雙特異性抗體中Obscurin-O鏈/Titin-T鏈和連接子對應表
註：表格中Titin鏈和Obscurin鏈的編號見表3-1和表3-2。 Note: See Table 3-1 and Table 3-2 for the numbers of Titin chains and Obscurin chains in the table.
採用PCT/CN2021/070832的測試例4中的方法檢測DI-2至DI-20雙特異性抗體與其抗原的結合活性。對抗體進行熱穩定性研究。研究方法：用PBS將抗體的濃度稀釋至5mg/mL，採用高通量微分掃描螢光儀(UNCHAINED，規格型號：Unit)測定其熱穩定性(上樣量9μL；參數設置：Start Temp 20℃；Incubation 0s；Rate 0.3℃/min；Plate Hold 5s；End Temp 95℃)。實驗結果表明，改造後的雙特異性抗體對抗原的結合活性沒有顯著變化；並且，與DI-2相比，DI-4至DI-8、DI-10至DI-16、DI-20的Tm1(℃)、Tonset(℃)有明顯的提升，雙特異性抗體的熱穩定性更優。 The method in Test Example 4 of PCT/CN2021/070832 was used to detect the binding activity of DI-2 to DI-20 bispecific antibodies and their antigens. Thermostability studies were performed on antibodies. Research method: Dilute the concentration of the antibody to 5 mg/mL with PBS, and measure its thermal stability using a high-throughput differential scanning fluorometer (UNCHAINED, specification model: Unit) (sample volume 9 μL; parameter setting: Start Temp 20°C ; Incubation 0s; Rate 0.3℃/min; Plate Hold 5s; End Temp 95℃). The experimental results showed that the antigen-binding activity of the engineered bispecific antibody did not change significantly; and, compared with DI-2, the Tm1 of DI-4 to DI-8, DI-10 to DI-16, and DI-20 (°C) and Tonset (°C) have been significantly improved, and the thermal stability of the bispecific antibody is better.
表i-2. DI雙特異性抗體的結合活性檢測
表i-3. DI雙特異性抗體的熱穩定性實驗結果
採用10mM乙酸，pH5.5，9%蔗糖的緩衝液配製含DI雙特異性抗體的溶液，將溶液置於40℃恆溫箱中孵育四週，結束後將抗體濃度濃縮至孵育開始時濃度，觀察溶液沉澱情況。實驗結果表明，DI-2雙特異性抗體組溶液出現沉澱，DI-3至DI-7相比DI-2具有更好的穩定性。 Use 10mM acetic acid, pH 5.5, 9% sucrose buffer to prepare a solution containing DI bispecific antibody, place the solution in a 40°C incubator and incubate for four weeks, then concentrate the antibody concentration to the concentration at the beginning of the incubation, and observe the solution Precipitation condition. The experimental results showed that the solution of DI-2 bispecific antibody group precipitated, and DI-3 to DI-7 had better stability than DI-2.
表i-8. DI雙特異性抗體的沉澱
7.2 PL雙特異性抗體7.2 PL bispecific antibody
構建抗hPDL1和hCTLA4的PL雙特異性抗體：PL-1至PL-19，其包含如下所述的第一重鏈、第二重鏈、第一輕鏈和第二輕鏈： Construction of PL bispecific antibodies against hPDL1 and hCTLA4: PL-1 to PL-19 comprising a first heavy chain, a second heavy chain, a first light chain and a second light chain as follows:
第一重鏈：從N端到C端依次為：[VH1-P]-[連接子1]-[Obscurin鏈]-[Fc1]， The first heavy chain: from N-terminal to C-terminal: [VH1-P]-[Linker 1]-[Obscurin chain]-[Fc1],
第一輕鏈：從N端到C端依次為：[VL1-P]-[連接子2]-[Titin鏈]， The first light chain: from N-terminal to C-terminal: [VL1-P]-[Linker 2]-[Titin chain],
第二重鏈：從N端到C端依次為：[VH2-L]-[CH1]-[Fc2]，和 Second heavy chain: from N-terminus to C-terminus: [VH2-L]-[CH1]-[Fc2], and
第二輕鏈：從N端到C端依次為：[VL2-L]-[CL]； The second light chain: from N-terminal to C-terminal: [VL2-L]-[CL];
其中，VH1-P和VL1-P分別為WO2020177733A1中h1831K抗體的重鏈可變區和輕鏈可變區，VH1-L和VL1-L的胺基酸序列如下所示。 Wherein, VH1-P and VL1-P are the heavy chain variable region and light chain variable region of the h1831K antibody in WO2020177733A1 respectively, and the amino acid sequences of VH1-L and VL1-L are as follows.
>VH2-L(SEQ ID NO：175) >VH2-L (SEQ ID NO: 175)
>VL2-L(SEQ ID NO：176) >VL2-L (SEQ ID NO: 176)
本實施例中PL雙特異性抗體中Obscurin-O鏈、Titin-T鏈、連接子1、連接子2結構見下表。 The structures of Obscurin-O chain, Titin-T chain, linker 1 and linker 2 in the PL bispecific antibody in this example are shown in the table below.
表i-4. PL雙特異性抗體中Obscurin-O鏈/Titin-T鏈和連接子對應表
註：表格中Titin鏈和Obscurin鏈的編號見表3-1和表3-2。 Note: See Table 3-1 and Table 3-2 for the numbers of Titin chains and Obscurin chains in the table.
參照PCT/CN2021/070832中測試例4中的ELISA方法檢測PL雙特異性抗體的結合活性，其中hPDL1、hCTLA4抗原購自：Sino biology。對抗體進行熱穩定性研究。方法：用PBS將抗體的濃度稀釋至1.4-3mg/mL，採用高通量微分掃描螢光儀(UNCHAINED，規格型號：Unit)測定其熱穩定性(上樣量9μL；參數設置：Start Temp 20℃；Incubation 0s；Rate 0.3℃/min；Plate Hold 5s；End Temp 95℃)。實驗結果表明，PL雙特異性抗體對抗原仍具有良好的結合活性；並且，與PL-1相比，PL-2至PL-19的Tm1(℃)、Tagg 266(℃)、Tonset(℃)有明顯的提升，雙特異性抗體的熱穩定性更優。 Refer to the ELISA method in Test Example 4 in PCT/CN2021/070832 to detect the binding activity of the PL bispecific antibody, wherein the hPDL1 and hCTLA4 antigens were purchased from: Sino biology. Thermostability studies were performed on antibodies. Method: Dilute the concentration of the antibody to 1.4-3 mg/mL with PBS, and measure its thermal stability using a high-throughput differential scanning fluorometer (UNCHAINED, specification model: Unit) (sample volume 9 μL; parameter setting: Start Temp 20 ℃; Incubation 0s; Rate 0.3℃/min; Plate Hold 5s; End Temp 95℃). The experimental results show that the PL bispecific antibody still has good binding activity to the antigen; and, compared with PL-1, the Tm1(°C), Tagg 266(°C), Tonset(°C) of PL-2 to PL-19 There is a significant improvement, and the thermal stability of the bispecific antibody is better.
表i-5. PL雙特異性抗體的結合活性檢測
表i-6. PL雙特異性抗體的熱穩定性實驗結果
7.3 HJ雙特異性抗體7.3 HJ bispecific antibody
構建抗hIL5和hTSLP的HJ雙特異性抗體：HJ-3至HJ11，其包含如下所述的第一重鏈、第二重鏈、第一輕鏈和第二輕鏈： Construction of HJ bispecific antibodies against hIL5 and hTSLP: HJ-3 to HJ11 comprising a first heavy chain, a second heavy chain, a first light chain and a second light chain as follows:
第一重鏈：從N端到C端依次為：[VH1-H]-[連接子1]-[Titin鏈]-[Fc1]， The first heavy chain: from N-terminal to C-terminal: [VH1-H]-[Linker 1]-[Titin chain]-[Fc1],
第一輕鏈：從N端到C端依次為：[VL1-H]-[連接子2]-[Obscurin鏈]， The first light chain: from N-terminal to C-terminal: [VL1-H]-[Linker 2]-[Obscurin chain],
第二重鏈：從N端到C端依次為：[VH2-J]-[CH1]-[Fc2]，和 Second heavy chain: from N-terminal to C-terminal: [VH2-J]-[CH1]-[Fc2], and
第二輕鏈：從N端到C端依次為：[VL2-J]-[CL]； The second light chain: from N-terminal to C-terminal: [VL2-J]-[CL];
其中，VH1-H和VL1-H分別為PCT/CN2021/070832中H0的重鏈可變區和輕鏈可變區，VH2-J和VL2-J分別為PCT/CN2021/070832中J1的 重鏈可變區和輕鏈可變區。本實施例中HJ雙特異性抗體中Obscurin-O鏈、Titin-T鏈、連接子1、連接子2結構見下表。 Among them, VH1-H and VL1-H are the heavy chain variable region and light chain variable region of H0 in PCT/CN2021/070832, respectively, and VH2-J and VL2-J are the J1 in PCT/CN2021/070832. Heavy chain variable region and light chain variable region. The structures of Obscurin-O chain, Titin-T chain, linker 1 and linker 2 in the HJ bispecific antibody in this example are shown in the table below.
表i-7. HJ雙特異性抗體中Obscurin-O鏈/Titin-T鏈和連接子對應表
參照PCT/CN2021/070832中測試例4中的方法檢測HJ雙特異性抗體的抗原結合活性。對抗體的熱穩定性進行研究，方法：用10mM乙酸pH5.5、9%蔗糖的緩衝液配製HJ雙特異性抗體稀釋溶液，然後藉由超濾濃縮的方法將雙特異性抗體濃縮，獲得不同濃度的HJ雙特異性抗體溶液(HJ雙特異性抗體的濃度見表13-2)，然後將濃縮溶液置於40℃恆溫箱中孵育，第0天(也即40℃孵育開始前，D0)，第7天(40℃孵育第7天，D7)，第14天(40℃孵育第14天，D14)，第21天(40℃孵育第21天，D21)和第28天(40℃孵育第28天，D28)檢測樣品的SEC純度，40℃孵育28天後，馬上取樣檢測樣品CE-SDS純度。實驗結果表明，本揭露構建的HJ雙特異性抗體對抗原的結合活性沒有顯著變化；並且，與HJ-3相比，HJ-5至HJ-11雙特異性抗體的熱穩定性更優。
The antigen-binding activity of the HJ bispecific antibody was detected by referring to the method in Test Example 4 in PCT/CN2021/070832. The thermal stability of the antibody was studied. The method: prepare the HJ bispecific antibody dilution solution with a buffer solution of 10mM acetic acid pH5.5 and 9% sucrose, and then concentrate the bispecific antibody by ultrafiltration to obtain different concentration of the HJ bispecific antibody solution (see Table 13-2 for the concentration of the HJ bispecific antibody), and then place the concentrated solution in a 40°C incubator for incubation on day 0 (that is, before the start of incubation at 40°C, D0). , day 7 (40°C incubation day 7, D7), day 14 (40°
表i-8. PL雙特異性抗體的結合活性檢測
表i-9. HJ雙特異性抗體加速穩定性實驗結果
測試例test case
測試例1. FACS測定BCMA/CD3雙特異性抗體對BCMA的親和力Test example 1. FACS determination of the affinity of BCMA/CD3 bispecific antibody to BCMA
為測試本揭露BCMA/CD3雙特異性抗體與BCMA的結合能力，本測試例用流式細胞術方法檢測了BCMA/CD3雙特異性抗體與過表 達人BCMA的穩轉細胞株K562-humanBCMA-High(K562)和過表達食蟹猴BCMA的穩轉細胞株CHOK1-cynoBCMA-High(CHOK1)的結合能力。 In order to test the binding ability of the disclosed BCMA/CD3 bispecific antibody to BCMA, this test example uses flow cytometry to detect the BCMA/CD3 bispecific antibody and overexpressed The binding ability of human BCMA-transformed cell line K562-humanBCMA-High (K562) and cynomolgus monkey BCMA-overexpressed stable cell line CHOK1-cynoBCMA-High (CHOK1).
具體如下：上述細胞培養於10% FBS的IMDM的培養基中，放置於37℃，5% CO2培養箱中，培養2天，按每孔細胞數1×105個將細胞加至細胞板中，300g離心5min，1% BSA洗一次。抗體梯度稀釋8個濃度，按每孔100μL加入細胞板中，4℃孵育1小時，1% BSA洗一次，每孔加入100μL APC-Goat Anti-human IgG Fc螢光二抗稀釋液(1：400)，4℃孵育1個小時。1% BSA洗板三次，每孔加入100μL PBS讀板。各抗體與相應細胞結合的EC50(nM)如下表所示。 The details are as follows: the above-mentioned cells were cultured in 10% FBS IMDM medium, placed in a 5% CO 2 incubator at 37°C, cultured for 2 days, and the number of cells per well was 1×10 5 cells were added to the cell plate , centrifuged at 300g for 5min, washed once with 1% BSA. The antibody was diluted to 8 concentrations, 100 μL per well was added to the cell plate, incubated at 4°C for 1 hour, washed once with 1% BSA, and 100 μL of APC-Goat Anti-human IgG Fc fluorescent secondary antibody dilution (1:400) was added to each well , and incubated at 4°C for 1 hour. The plate was washed three times with 1% BSA, and 100 μL PBS was added to each well to read the plate. The EC50 (nM) of each antibody binding to the corresponding cells is shown in the table below.
表12. 抗體結合BCMA的能力測定
結果顯示，採用本揭露所篩選的BCMA抗體構建成不同的BCMA-CD3雙抗皆有很好結合膜表面BCMA的能力，且與CynoBCMA有較好的交叉結合活性。 The results show that the different BCMA-CD3 double antibodies constructed using the BCMA antibodies screened in this disclosure all have a good ability to bind to BCMA on the membrane surface, and have good cross-binding activity with CynoBCMA.
測試例2. 本揭露的抗體結合膜BCMA受可溶性BCMA影響的程度Test Example 2. The degree to which the antibody binding membrane BCMA of the present disclosure is affected by soluble BCMA
多發性骨髓瘤患者血液中存在的高濃度可溶性BCMA(平均30ng/mL)會干擾BCMA-CD3雙特異性抗體對膜表面BCMA的特異性結 合。本測試例測試了高濃度可溶性BCMA的存在是否影響本揭露的雙特異性抗體結合膜表面的BCMA抗原。 The high concentration of soluble BCMA (average 30 ng/mL) present in the blood of patients with multiple myeloma interferes with the specific binding of BCMA-CD3 bispecific antibodies to BCMA on the membrane surface. combine. This test example tests whether the presence of high concentration of soluble BCMA affects the binding of the disclosed bispecific antibody to the BCMA antigen on the membrane surface.
取活率(viability)
表13. 抗體結合膜表面BCMA的能力測定
測試例3. 本揭露的雙特異性抗體體外激活T細胞的活性Test example 3. The bispecific antibody of the present disclosure activates the activity of T cells in vitro
本測試例研究了本揭露所述抗體對T細胞的激活能力，用表達BCMA的細胞系U266B和不表達BCMA的CHOK1作為靶細胞，來檢測雙特異性抗體對Jurkat-LuciaTM NFAT細胞的激活能力。 This test example studies the activation ability of the antibody described in this disclosure on T cells, using the cell line U266B expressing BCMA and CHOK1 not expressing BCMA as target cells to detect the activation ability of the bispecific antibody on Jurkat-Lucia TM NFAT cells .
Jurkat-LuciaTM NFAT細胞收集離心，使用1640+10%FBS的培養基重新懸浮計數，調整細胞數為1×106個/mL，每孔加入50μL(5×104/孔)。靶細胞U266B或者CHOK1收集離心後，使用培養基1640+10%FBS重新懸浮計數，調整細胞數為8×105個/mL，每孔加入25μL，E：T分別為2.5：1。雙特異性抗體用1640+10%FBS的培養基稀釋，起始濃度為80nM，5倍稀釋生成9個梯度後，每孔加入25μL溶液。然後將處理好的細胞放在37℃，5% CO2的培養箱培養5h後，取出培養板，按1：1的比例加入100μL QUANTI-LucTM Gold，室溫孵育5min，用酶標儀檢測luminescence的讀值，使用Graphpad Prism 5對數據進行處理分析。 Jurkat-Lucia TM NFAT cells were collected and centrifuged, resuspended in 1640+10% FBS medium for counting, adjusted the number of cells to 1×10 6 cells/mL, and added 50 μL to each well (5×10 4 /well). After the target cells U266B or CHOK1 were collected and centrifuged, they were resuspended and counted using medium 1640+10% FBS, and the number of cells was adjusted to 8×10 5 cells/mL. Add 25 μL to each well, and the E:T ratio was 2.5:1. The bispecific antibody was diluted with 1640+10% FBS medium, and the initial concentration was 80nM. After 5-fold dilution to generate 9 gradients, 25 μL of the solution was added to each well. Then place the treated cells in an incubator at 37°C and 5% CO 2 for 5 hours, take out the culture plate, add 100 μL QUANTI-Luc TM Gold at a ratio of 1:1, incubate at room temperature for 5 minutes, and detect with a microplate reader The reading value of luminescence was processed and analyzed using Graphpad Prism 5.
結果顯示，在靶細胞U266B存在下，本揭露的雙特異性抗體可以特異性激活Jurkat-LuciaTM NFAT細胞，激活能力與AMGEN701相比顯著提高；而在不表達BCMA的CHOK1細胞上，本揭露的雙特異性抗體不激活Jurkat-LuciaTM NFAT細胞，證明本揭露的雙特異性抗體激活T細胞的能力是其靶點BCMA依賴的。 The results show that in the presence of target cell U266B, the bispecific antibody disclosed herein can specifically activate Jurkat-Lucia TM NFAT cells, and the activation ability is significantly improved compared with AMGEN701; while on CHOK1 cells that do not express BCMA, the disclosed bispecific antibody The bispecific antibody does not activate Jurkat-Lucia TM NFAT cells, which proves that the ability of the disclosed bispecific antibody to activate T cells is dependent on its target BCMA.
表14. 抗體體外激活T細胞的活性
註：“--”表示陰性。 Note: "--" means negative.
測試例4. 本揭露雙特異性抗體的體外細胞毒活性Test Example 4. In vitro cytotoxic activity of the disclosed bispecific antibody
本測試例研究了本揭露的雙特異性抗體作為T細胞銜接分子(adapter)對腫瘤細胞的殺傷活性。用表達BCMA的細胞系RPMI8226，H929、和不表達BCMA的CHOK1，作為靶細胞來檢測本揭露的雙特異性抗體的靶點特異性細胞毒活性。 This test example studies the killing activity of the disclosed bispecific antibody as a T cell adapter molecule (adapter) on tumor cells. The cell lines RPMI8226, H929 expressing BCMA, and CHOK1 not expressing BCMA were used as target cells to detect the target-specific cytotoxic activity of the bispecific antibody disclosed herein.
新鮮PBMC(購自軒峰生物公司)300g離心10min，棄去上清，用無酚紅1640+4%FBS重新懸浮，再次離心後重新懸浮、計數，調整細胞數為1.5×106個/mL，每孔加入50μL。收集靶細胞，1000rpm離心3min，重新懸浮、計數，調整細胞數為3×105個/mL，每孔加入25μL，E：T為10：1。抗體用無酚紅完全培養基稀釋，起始濃度為400nM(4×終濃度)，10倍稀釋9個梯度，每孔加入25μL。然後將細胞放在37℃，5% CO2的培養箱培養48h。檢測前，在只有靶細胞的其中兩個孔中吸出10μL培養基，然後加入10μL Lysis Solution(10×)，裂解45min後取出培養板，1000rpm離心3min，吸取50μL上清於新的96孔板(#3590)中，按1：1的比例加入溶解好的50μL CytoTox 96® Reagent，室溫孵育0.5h後 加入50μL終止液，用FlexStation 3(Molecular Devices)檢測吸收光(490nm)。 Centrifuge fresh PBMC (purchased from Xuanfeng Biological Company) at 300g for 10min, discard the supernatant, resuspend with phenol red-free 1640+4%FBS, resuspend and count after centrifugation again, and adjust the cell number to 1.5× 106 /mL , add 50 μL to each well. Collect target cells, centrifuge at 1000rpm for 3min, resuspend and count, adjust the number of cells to 3×10 5 cells/mL, add 25 μL to each well, and E:T is 10:1. The antibody was diluted with phenol red-free complete medium, the initial concentration was 400nM (4×final concentration), 10-fold diluted 9 gradients, and 25 μL was added to each well. The cells were then placed in an incubator at 37°C with 5% CO 2 for 48 hours. Before detection, aspirate 10 μL of medium in two wells with only target cells, then add 10 μL Lysis Solution (10×), lyse for 45 minutes, take out the culture plate, centrifuge at 1000 rpm for 3 minutes, and pipette 50 μL of supernatant into a new 96-well plate (# 3590), add 50 μL of dissolved CytoTox 96 ® Reagent at a ratio of 1:1, incubate at room temperature for 0.5 h, add 50 μL of stop solution, and use FlexStation 3 (Molecular Devices) to detect the absorption light (490 nm).
表15-1. 抗體對RPMI8266的細胞毒活性
表15-2. 抗體對H929的細胞毒活性
表15-3. 抗體對CHOK1的細胞毒活性
結果顯示，本揭露的雙特異性抗體的細胞毒活性強於AMGEN701；並且，本揭露的雙特異性抗體的細胞毒活性是BCMA靶點特異性的，在沒有BCMA表達的CHOK1細胞系上表現出更弱的細胞毒活性，提示本揭露的雙特異性抗體有更好的安全性。 The results show that the cytotoxic activity of the disclosed bispecific antibody is stronger than that of AMGEN701; and, the cytotoxic activity of the disclosed bispecific antibody is BCMA target-specific, and it is shown on the CHOK1 cell line without BCMA expression Weaker cytotoxic activity suggests that the bispecific antibody disclosed herein has better safety.
測試例5. 可溶性BCMA對本揭露抗體的體外細胞毒活性的影響Test Example 5. Effect of Soluble BCMA on the In Vitro Cytotoxic Activity of the Antibody of the Disclosure
本揭露研究了可溶性BCMA對BCMA/CD3雙特異性抗體體外腫瘤細胞殺傷活性的影響。 This disclosure studies the effect of soluble BCMA on the tumor cell killing activity of BCMA/CD3 bispecific antibody in vitro.
採用表達BCMA的細胞系U266B作為靶細胞，在細胞毒活性測試時，額外添加了可溶性BCMA。取新鮮PBMC(購自軒峰生物公司)，300g離心10min，棄去上清，用無酚紅1640+4%FBS重新懸浮，再次離心後重新懸浮、計數，調整細胞數為1.5×106個/mL，每孔加入50μL細胞混懸液。收集靶細胞U266B，1000rpm離心3min，重新懸浮、計數，調整細胞數為3×105個/mL，每孔加入25μL細胞混懸液，確保E：T為10： 1。抗體用含有可溶性BCMA的無酚紅完全培養基稀釋，起始濃度都是400nM(4×終濃度)，10倍稀釋9個梯度，每孔加入25μL，可溶性BCMA的終濃度是30ng/mL。然後將細胞放在37℃，5% CO2的培養箱培養48h。檢測前，在只含有靶細胞的兩個孔中吸出10μL培養基，然後加入10μL Lysis Solution(10×)，裂解45min後取出培養板，1000rpm離心3min，吸取50μL上清於新的96孔板(#3590)中，按1：1的比例加入溶解好的50μL CytoTox 96® Reagent，室溫孵育0.5h後加入50μL Stop solution，用FlexStation 3(Molecular Devices)檢測吸收光(490nm)。 The BCMA-expressing cell line U266B was used as target cells, and soluble BCMA was additionally added in the cytotoxic activity test. Take fresh PBMC (purchased from Xuanfeng Biological Company), centrifuge at 300g for 10min, discard the supernatant, resuspend with phenol red-free 1640+4%FBS, centrifuge again, resuspend and count, and adjust the number of cells to 1.5× 106 /mL, add 50 μL of cell suspension to each well. Collect target cells U266B, centrifuge at 1000rpm for 3min, resuspend and count, adjust the number of cells to 3×10 5 cells/mL, add 25 μL of cell suspension to each well, and ensure that E:T is 10:1. The antibody was diluted with phenol red-free complete medium containing soluble BCMA, the initial concentration was 400nM (4×final concentration), 10-fold diluted 9 gradients, 25 μL was added to each well, and the final concentration of soluble BCMA was 30ng/mL. The cells were then placed in an incubator at 37°C with 5% CO 2 for 48 hours. Before detection, aspirate 10 μL of the medium in the two wells containing only target cells, then add 10 μL Lysis Solution (10×), lyse for 45 minutes, take out the culture plate, centrifuge at 1000 rpm for 3 minutes, and pipette 50 μL of the supernatant into a new 96-well plate (# 3590), add dissolved 50 μL CytoTox 96® Reagent at a ratio of 1:1, incubate at room temperature for 0.5 h, then add 50 μL Stop solution, and use FlexStation 3 (Molecular Devices) to detect absorbance (490 nm).
表16. 可溶性BCMA對抗體細胞殺傷活性的影響
結果顯示，本揭露的雙特異性抗體的細胞毒活性受可溶性BCMA影響較小，與沒有可溶性BCMA存在的條件下，30ng/mL的可溶性BCMA僅使細胞毒作用EC50變至原來的4-9倍，而AMGEN701卻增加到原來的20.5倍；REGN5458受可溶性BCMA的影響與本揭露的雙特異性抗體相當，但細胞殺傷活性顯著低於本揭露的雙特異性抗體。 The results show that the cytotoxic activity of the disclosed bispecific antibody is less affected by soluble BCMA, and 30 ng/mL soluble BCMA only changes the cytotoxicity EC50 to the original 4-9 times under the condition that there is no soluble BCMA , while AMGEN701 increased to 20.5 times; REGN5458 was affected by soluble BCMA comparable to the bispecific antibody of the present disclosure, but the cell killing activity was significantly lower than that of the bispecific antibody of the present disclosure.
測試例6. BCMA/CD3雙特異性抗體的細胞因子釋放水平Test Example 6. Cytokine Release Level of BCMA/CD3 Bispecific Antibody
CD3 T細胞銜接分子會引起細胞因子風暴。因此開發CD3 T細胞銜接分子時，需要能將細胞因子(尤其是和藥效無關但會引起副作用的因子IL6)，保持在較低水平。本測試例研究了本揭露的雙特異性抗體的細胞因子IFNγ和IL6釋放水平。 CD3 T cell engager molecules cause a cytokine storm. Therefore, when developing CD3 T cell engagement molecules, it is necessary to keep cytokines (especially the factor IL6 that has nothing to do with drug efficacy but can cause side effects) at a low level. This test case studies the release levels of cytokines IFNγ and IL6 of the disclosed bispecific antibody.
新鮮PBMC(購自軒峰生物公司)300g離心10min，棄去上清，用無酚紅1640+4%FBS重新懸浮，再次離心後重新懸浮、計數，調整細胞數為1.5×106個/mL，每孔加入50μL。收集靶細胞U266B或H929，1000rpm離心3min，重新懸浮、計數，調整細胞數為3×105個/mL，每孔加入25μL，E：T為10：1。抗體用無酚紅完全培養基稀釋，起始濃度為400nM(4×終濃度)，梯度稀釋，每孔加入25μL。然後將細胞放在37℃，5% CO2的培養箱培養48h備用。 Centrifuge fresh PBMC (purchased from Xuanfeng Biological Company) at 300g for 10min, discard the supernatant, resuspend with phenol red-free 1640+4%FBS, resuspend and count after centrifugation again, and adjust the cell number to 1.5× 106 /mL , add 50 μL to each well. Collect target cells U266B or H929, centrifuge at 1000rpm for 3min, resuspend and count, adjust the number of cells to 3×10 5 cells/mL, add 25 μL to each well, and E:T is 10:1. The antibody was diluted with phenol red-free complete medium, the initial concentration was 400nM (4×final concentration), serially diluted, and 25 μL was added to each well. The cells were then cultured in an incubator at 37°C and 5% CO 2 for 48 hours.
IL6的檢測採用ELISA方法。將上述細胞樣品1000rpm離心3min，收集50μL的細胞上清用標本通用稀釋液稀釋6倍，IL6標準品亦稀釋6倍，在檢測板中加入稀釋好的樣品或者不同濃度標準品(100μL/孔)，用封板膠紙封住反應孔，37℃孵育90min後洗板5次，加入生物素化抗體工作液(100μL/孔)，用新封板膠紙封住反應孔，37℃孵育60分鐘後洗板5次，加入酶結合物工作液(100μL/孔)，用新封板膠紙封住反應孔，37℃孵育30分鐘後洗板5次，加入顯色受質(TMB)100μL/孔，避光37℃孵育8分鐘，加入反應終止液100μL/孔，混勻後即刻測量OD450值(3分鐘內)。 IL6 was detected by ELISA method. Centrifuge the above cell sample at 1000rpm for 3min, collect 50μL of the cell supernatant and dilute it 6 times with the sample universal diluent, the IL6 standard is also diluted 6 times, and add the diluted sample or different concentration standard to the assay plate (100μL/well) , seal the reaction wells with sealing tape, incubate at 37°C for 90 minutes, wash the plate 5 times, add biotinylated antibody working solution (100 μL/well), seal the reaction wells with new sealing tape, and incubate at 37°C for 60 minutes After that, wash the plate 5 times, add enzyme conjugate working solution (100 μL/well), seal the reaction well with new sealing tape, incubate at 37°C for 30 minutes, wash the plate 5 times, add chromogenic substrate (TMB) 100 μL/well Wells were incubated in the dark at 37°C for 8 minutes, 100 μL/well of reaction termination solution was added, and the OD450 value was measured immediately after mixing (within 3 minutes).
IFNγ的檢測採用HTRF方法。將上述細胞樣品1000rpm離心3min，收集50μL的細胞上清，檢測試劑盒平衡至常溫，使用檢測緩衝液稀釋試劑盒中的兩個檢測抗體(20倍稀釋)。取16μL 20倍稀釋的樣本以及IFNγ標準品於384孔板中，加入4μL稀釋好的相對應的檢測抗體；貼上密封材料，震盪混勻，1000rpm離心1分鐘，常溫孵育過夜後，1000rpm離心1分鐘，取掉密封材料，使用PHERAstar多功能酶標儀讀取665nm和620nm的吸收值，使用Graphpad Prism 5對數據進行處理分析。 The detection of IFNγ adopts HTRF method. The above cell samples were centrifuged at 1000 rpm for 3 min, 50 μL of cell supernatant was collected, the detection kit was equilibrated to room temperature, and the detection buffer was used to dilute the two detection antibodies in the kit (20-fold dilution). Take 16 μL of the 20-fold diluted sample and IFNγ standard in a 384-well plate, add 4 μL of the corresponding diluted detection antibody; paste the sealing material, shake and mix, centrifuge at 1000rpm for 1 minute, incubate overnight at room temperature, and centrifuge at 1000rpm for 1 minute. Minutes, remove the sealing material, use the PHERAstar multifunctional microplate reader to read the absorbance values at 665nm and 620nm, and use Graphpad Prism 5 to process and analyze the data.
表17-1. U266B細胞毒活性實驗中的IL-6釋放
表17-2. U266B細胞毒活性實驗中的IFNγ的釋放
表17-3. H929細胞毒活性實驗中的IFNγ的釋放
結果顯示，本揭露的雙特異性抗體釋放IL6和IFNγ的水平低於AMGEN701，提示本揭露的雙特異性抗體具有更好的安全性。 The results showed that the level of IL6 and IFNγ released by the disclosed bispecific antibody was lower than that of AMGEN701, suggesting that the disclosed bispecific antibody has better safety.
體內活性生物學評價Biological evaluation of in vivo activity
測試例7. BCMA/CD3雙特異性抗體在NCI-H929皮下移植瘤模型中的藥效Test Example 7. The efficacy of BCMA/CD3 bispecific antibody in the NCI-H929 subcutaneous xenograft model
本揭露使用人骨髓瘤NCI-H929細胞人PBMC NOG小鼠異種移植瘤模型，對BCMA&CD3雙抗在抗腫瘤活性方面進行評價。 This disclosure uses human myeloma NCI-H929 cell human PBMC NOG mouse xenograft tumor model to evaluate the anti-tumor activity of BCMA&CD3 double antibody.
NOG小鼠，雌性，8-10週齡，購自北京維通利華實驗動物技術有限公司。 NOG mice, female, 8-10 weeks old, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.
人多發性骨髓瘤細胞株NCI-H929購自上海名勁生物科技有限公司。 Human multiple myeloma cell line NCI-H929 was purchased from Shanghai Mingjin Biotechnology Co., Ltd.
將NCI-H929細胞加入含10% FBS和0.05mM 2-巰基乙醇的RPMI-1640培養基，並於在5% CO2 37℃的飽和濕度培養箱中培養。收集對數生長期NCI-H929細胞，重新懸浮於含50% Matrigel的RPMI-1640培養基中，調整細胞濃度至2×107/mL。在無菌條件下，接種0.1mL細胞 懸液至小鼠右側背部皮下，接種濃度為2×106/0.1mL/鼠。腫瘤細胞接種後1天，復蘇液氮凍存的hPBMC，培養於含10% HIFBS(FBS，56℃ 30min)的PRMI-1640培養基中，在含5% CO2的37℃培養箱中孵育6h。收集孵育後hPBMC，重新懸浮於PBS緩衝液中，調整細胞濃度至2.5×107/mL。在無菌條件下，腹腔注射0.2mL細胞懸液至小鼠體內，注射濃度為5×106/0.2mL/鼠。在平均腫瘤體積達到100mm3左右時，將動物按腫瘤體積隨機分組，使各組腫瘤體積差異小於均值的10%。分組當日記為D0，並按照動物體重開始給藥。分組後立即給藥，每週2次腹腔給藥，連續給藥4次(IP,BIW×2)。第17天(D17)處死動物，取瘤稱重並拍照記錄。 NCI-H929 cells were added to RPMI-1640 medium containing 10% FBS and 0.05mM 2-mercaptoethanol, and cultured in a saturated humidity incubator at 5% CO 2 at 37°C. Collect NCI-H929 cells in the logarithmic growth phase, resuspend in RPMI-1640 medium containing 50% Matrigel, and adjust the cell concentration to 2×10 7 /mL. Under sterile conditions, inoculate 0.1 mL of the cell suspension subcutaneously on the right back of the mouse, with an inoculation concentration of 2×10 6 /0.1 mL/mouse. One day after tumor cell inoculation, hPBMCs cryopreserved in liquid nitrogen were resuscitated, cultured in PRMI-1640 medium containing 10% HIFBS (FBS, 56°C for 30 min), and incubated for 6 h in a 37°C incubator containing 5% CO 2 . After incubation, hPBMC were collected, resuspended in PBS buffer, and the cell concentration was adjusted to 2.5×10 7 /mL. Under sterile conditions, intraperitoneally inject 0.2mL of the cell suspension into mice, and the injection concentration is 5×10 6 /0.2mL/mouse. When the average tumor volume reached about 100 mm 3 , the animals were randomly divided into groups according to the tumor volume, so that the difference in tumor volume among the groups was less than 10% of the average value. The day of grouping was recorded as D0, and the administration was started according to the body weight of the animals. Immediately after the grouping, intraperitoneal administration was administered twice a week for 4 consecutive administrations (IP, BIW×2). On the 17th day (D17), the animals were sacrificed, and the tumors were weighed and photographed for record.
抑瘤率(%)=1-T/C(%)。T/C(%)=(T-T0)/(C-C0)×100%，其中T、C為實驗結束時治療組和對照組的腫瘤體積或腫瘤重量；T0、C0為實驗開始時的腫瘤體積或腫瘤重量。 Tumor inhibition rate (%)=1-T/C (%). T/C(%)=(T-T0)/(C-C0)×100%, where T and C are the tumor volume or tumor weight of the treatment group and the control group at the end of the experiment; T0 and C0 are the tumor weight at the beginning of the experiment Tumor volume or tumor weight.
表18. 抗體在NCI-H929皮下移植瘤模型中的藥效
實驗結果顯示，本揭露的4E3-11-1在更低的給藥劑量條件下，體內抑制腫瘤生長的活性高於對應劑量的AMGEN701和REGN5458。實驗期間未觀測到藥物相關動物死亡及其他明顯藥物相關毒副反應。 The experimental results show that the 4E3-11-1 disclosed in the present disclosure has a lower tumor growth inhibitory activity than the corresponding doses of AMGEN701 and REGN5458 under the condition of lower dosage. During the experiment, no drug-related animal death and other obvious drug-related toxic and side effects were observed.
測試例8. BCMA/CD3雙特異性抗體在RPMI-8226原位瘤模型的藥效Test Example 8. The efficacy of BCMA/CD3 bispecific antibody in RPMI-8226 orthotopic tumor model
本實驗採用NDG小鼠藉由尾靜脈接種人骨髓瘤RPMI-8226-lucG細胞建立原位腫瘤模型，分別給予BCMA-CD3雙特異性抗體藉由測量給藥後荷瘤小鼠的生物發光信號值(Total Flux)和體重(BW)，評價BCMA-CD3雙特異性抗體對人骨髓瘤RPMI-8226-lucG小鼠原位移植瘤的療效。 In this experiment, NDG mice were used to inoculate human myeloma RPMI-8226-lucG cells into the tail vein to establish an orthotopic tumor model, and the BCMA-CD3 bispecific antibody was administered to measure the bioluminescent signal value of the tumor-bearing mice after administration. (Total Flux) and body weight (BW), to evaluate the efficacy of BCMA-CD3 bispecific antibody on orthotopic transplantation of human myeloma RPMI-8226-lucG mice.
將RPMI-8226-lucG細胞以5×106個/200μL/鼠尾靜脈接種於NDG小鼠(購自百奧賽圖實驗動物有限公司)體內。腫瘤細胞接種14天後將兩名供體新鮮分離的PBMCs以1：1比例混合，以4×106個/100μL/鼠注射到小鼠腹腔。腫瘤細胞接種18天後，每隻小鼠腹腔注射生物發光受質(15mg/mL)，按照10mL/kg體積注射，藉由異氟烷麻醉，注射10分鐘後藉由小動物成像系統拍照成像。去除體重、生物發光信號值過大和過小的，按生物發光信號將小鼠隨機分組，每組6隻，並將分組當天定義為該實驗D0、D1開始腹腔注射各抗體，每週2次，共給藥6次(表19)。每週拍照成像2次，稱體重，記錄數據。以下抑瘤率以D21的數據進行計算。 RPMI-8226-lucG cells were inoculated into NDG mice (purchased from Biocytogen Laboratory Animal Co., Ltd.) at 5×10 6 cells/200 μL/rat tail vein. Fourteen days after tumor cell inoculation, freshly isolated PBMCs from two donors were mixed at a ratio of 1:1, and injected into the abdominal cavity of mice at 4×10 6 /100 μL/mouse. Eighteen days after tumor cell inoculation, each mouse was intraperitoneally injected with a bioluminescence substrate (15 mg/mL) at a volume of 10 mL/kg, anesthetized with isoflurane, and photographed by a small animal imaging system 10 minutes after the injection. The mice whose body weight and bioluminescence signal value were too large and too small were removed, and the mice were randomly divided into groups according to the bioluminescence signal, with 6 mice in each group, and the day of grouping was defined as D0 and D1 of the experiment. Administration was given 6 times (Table 19). Photographs and images were taken twice a week, body weight was weighed, and data was recorded. The following tumor inhibition rate is calculated based on the data of D21.
抑瘤率(%)=1-T/C(%)。T/C(%)=(T-T0)/(C-C0)×100%，其中T、C為實驗結束時治療組和對照組的生物發光信號值；T0、C0為實驗開始時的生物發光信號值。 Tumor inhibition rate (%)=1-T/C (%). T/C(%)=(T-T0)/(C-C0)×100%, where T and C are the bioluminescent signal values of the treatment group and the control group at the end of the experiment; T0 and C0 are the bioluminescent signal values at the beginning of the experiment Luminous signal value.
表19. 抗體在RPMI-8226原位瘤模型中的藥效
實驗結果顯示，4E3-11-1與4E3-11-6164-5的抑瘤率達100%以上，3B2-11-6164-2也表現出較強的抑瘤效果；而REGN5458沒有表現出藥效。 The experimental results showed that the tumor inhibition rates of 4E3-11-1 and 4E3-11-6164-5 were over 100%, and 3B2-11-6164-2 also showed a strong tumor inhibitory effect; while REGN5458 did not show drug efficacy .
將RPMI-8226-lucG細胞以5×106個/200μL/鼠尾靜脈接種於NDG小鼠體內。腫瘤細胞接種14天後，每隻小鼠腹腔注射生物發光受質(15mg/mL)，按照10mL/kg體積注射，藉由異氟烷麻醉，注射10分鐘後藉由小動物成像系統拍照成像。去除體重過小的、生物發光信號值過大和過小的，按生物發光信號將小鼠隨機分為Vehicle(PBS)與33H4-15-1(0.2mpk)2組，每組9隻。分組後第2天，將一名供體新鮮分離的PBMCs以4.5×106個/鼠注射到小鼠腹腔。分組後第5天開始腹腔注射抗體，並將該天定義為該實驗D0，每週1次，共給藥2次(表20)。每週拍照成像2次，稱體重，記錄數據。以下抑瘤率以D14的數據進行計算。 RPMI-8226-lucG cells were inoculated into NDG mice at 5×10 6 cells/200 μL/mouse tail vein. Fourteen days after tumor cell inoculation, each mouse was intraperitoneally injected with a bioluminescence substrate (15 mg/mL) at a volume of 10 mL/kg, anesthetized with isoflurane, and photographed by a small animal imaging system 10 minutes after the injection. Remove the underweight, too large and too small bioluminescence signal value, according to the bioluminescence signal, the mice were randomly divided into Vehicle (PBS) and 33H4-15-1 (0.2mpk) 2 groups, with 9 mice in each group. On the second day after grouping, freshly isolated PBMCs from one donor were injected into the abdominal cavity of mice at 4.5×10 6 /mouse. The antibody was injected intraperitoneally on the 5th day after grouping, and this day was defined as D0 of the experiment, administered once a week and administered twice in total (Table 20). Photographs and images were taken twice a week, body weight was weighed, and data was recorded. The following tumor inhibition rate is calculated based on the data of D14.
表20. 抗體在RPMI-8226原位瘤模型中的藥效
雖然為了清楚的理解，已經借助於圖式和實例詳細描述了上述發明，但是描述和實例不應當解釋為限制本揭露的範圍。本文中引用的所有專利和科學文獻的公開內容藉由引用完整地清楚結合。 While the foregoing invention has been described in detail by means of drawings and examples for clarity of understanding, the description and examples should not be construed as limiting the scope of the disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.
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