相關申請案之交叉參考Cross-reference to related applications
本申請案主張於2023年9月15日提出申請之美國臨時專利申請案第63/538,599號之優先權權益，該申請案之內容特此以全文引用方式併入。This application claims priority to U.S. Provisional Patent Application No. 63/538,599, filed on September 15, 2023, the contents of which are hereby incorporated by reference in their entirety.
序列表Sequence Listing
本申請案含有命名為「50887-0047WO1.XML」之作為XML檔案以電子方式提交之序列表。XML檔案創建於2024年8月28日，大小為204,800位元組。XML檔案中之材料特此以全文引用之方式併入。This application contains a sequence listing submitted electronically as an XML file named "50887-0047WO1.XML." The XML file was created on August 28, 2024, and is 204,800 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.
本揭示內容係關於免疫學及醫學之技術領域。This disclosure relates to the technical fields of immunology and medicine.
反式活性反應DNA結合蛋白43 (Transactive response DNA binding protein 43，「TDP-43」)為主要參與RNA剪接、運輸、穩定及最終調控基因表現之核蛋白。TDP-43為含有兩個高度保守之核酸識別模體(motif)的核酸結合蛋白，且已顯示形成二聚體及寡聚體。雖然TDP-43在所有細胞類型中普遍表現，但已顯示其在神經上皮中高度表現，該神經上皮含有神經元及神經膠質之所有CNS前驅細胞。此外，已顯示TDP-43特異性結合神經元細胞中之眾多RNA。Transactive response DNA binding protein 43 (TDP-43) is a nuclear protein primarily involved in RNA splicing, transport, stabilization, and ultimately, regulation of gene expression. TDP-43 is a nucleic acid-binding protein that contains two highly conserved nucleic acid recognition motifs and has been shown to form dimers and oligomers. Although TDP-43 is ubiquitously expressed in all cell types, it has been shown to be highly expressed in the neuroepithelium, which contains all CNS progenitor cells, including neurons and neuroglia. Furthermore, TDP-43 has been shown to specifically bind to numerous RNAs in neuronal cells.
TDP-43細胞質聚集體(亦稱為包涵體(inclusion body))與若干種神經退化性疾病、病症或疾患相關。具體而言，TDP-43細胞質聚集體及/或TDP-43之錯誤折疊與神經退化性疾病相關，包括肌肉萎縮性脊髓側索硬化症(「ALS」)、額顳葉失智症(frontotemporal dementia，「FTD」或「FTLD-TDP-43」)、邊緣為主的年齡相關之TDP-43腦病(limbic-predominant age-related TDP-43 encephalopathy，「LATE」)、阿茲海默氏病、多系統蛋白病及慢性創傷性腦病。TDP-43 cytoplasmic aggregates (also known as inclusion bodies) are associated with several neurodegenerative diseases, disorders, or conditions. Specifically, TDP-43 cytoplasmic aggregates and/or misfolding of TDP-43 are associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD or "FTLD-TDP-43"), limbic-predominant age-related TDP-43 encephalopathy (LATE), Alzheimer's disease, multisystem proteinopathy, and chronic traumatic encephalopathy.
ALS為成人中最常見之運動神經元病症，最終導致控制隨意肌的運動神經元之損失。ALS每年之發病率為約1-2/100,000例且患病率為約4-6/100,000例。ALS造成上運動神經元及下運動神經元之進行性退化，通常在診斷後三至五年內導致主要歸因於呼吸衰竭之死亡。典型症狀包括肌肉僵硬、肌肉抽搐、逐漸肌肉無力及肌肉萎縮。在所有患有ALS之人中，約一半具有思考困難及/或行為症狀，且約15%患上額顳葉失智症。病理學上，在多達97%之ALS患者中可見TDP-43蛋白之異常聚集(Nguyen, HP等人, ALS Genes in the Genomic Era and their Implications for FTD,Trends in Genetics, 34(6): 404+423 (2018))。ALS is the most common motor neuron disease in adults, ultimately leading to the loss of motor neurons that control voluntary muscles. The annual incidence of ALS is approximately 1-2 cases per 100,000 people, and the prevalence is approximately 4-6 cases per 100,000 people. ALS causes progressive degeneration of upper and lower motor neurons, usually leading to death, primarily due to respiratory failure, within three to five years of diagnosis. Typical symptoms include muscle stiffness, muscle twitching, progressive muscle weakness, and muscle atrophy. Of all people with ALS, approximately half have difficulty thinking and/or behavioral symptoms, and approximately 15% develop frontotemporal dementia. Pathologically, abnormal aggregation of TDP-43 protein can be seen in up to 97% of ALS patients (Nguyen, HP et al., ALS Genes in the Genomic Era and their Implications for FTD,Trends in Genetics , 34(6): 404+423 (2018)).
目前，尚無已知的ALS治癒方法。因此，對改善患有TDP-43相關疾病(包括ALS)之患者的健康狀況(包括死亡風險及/或提高生活品質)之療法存在未滿足之需求。Currently, there is no known cure for ALS. Therefore, there is an unmet need for therapies that can improve the health outcomes (including risk of mortality and/or improve quality of life) of patients with TDP-43-related diseases, including ALS.
本揭示內容係關於包括細胞內化模組(「CIM」)及特異性結合至人類TDP-43之抗體的細胞穿透劑(「CPA」)、包含此等細胞穿透劑之組合物，及使用此等細胞穿透劑來治療TDP-43相關疾病包括肌肉萎縮性脊髓側索硬化症(ALS)之方法。The present disclosure relates to cell penetrating agents ("CPAs") comprising a cell internalization module ("CIM") and an antibody that specifically binds to human TDP-43, compositions comprising such cell penetrating agents, and methods of using such cell penetrating agents to treat TDP-43-related diseases, including amyotrophic lateral sclerosis (ALS).
因此，本文提供細胞穿透劑，其包括(i)細胞內化模組及(ii)特異性結合至43 kD之反式活性反應DNA結合蛋白(TDP-43)的抗體。Thus, provided herein are cell penetrants comprising (i) a cell internalization module and (ii) an antibody that specifically binds to 43 kD transactive DNA binding protein (TDP-43).
在一些實施例中，CIM包括細胞膜內化肽(CMIP)。在一些實施例中，CIM包括野生型M-蜘蛛毒素(M-lycotoxin)肽。在一些實施例中，CIM包括M-lycotoxin衍生物。在一些實施例中，CIM包括Penetain胺基酸序列或其衍生物。在一些實施例中，CIM包括Pepth胺基酸序列或其衍生物。在一些實施例中，CIM包括聚精胺酸胺基酸序列。在一些實施例中，CIM包括多於一個聚精胺酸胺基酸序列。在一些實施例中，CIM包括三個聚精胺酸胺基酸序列。在一些實施例中，CIM包括TAT胺基酸序列。在一些實施例中，CIM包括多於一個TAT胺基酸序列。在一些實施例中，CIM包括三個TAT胺基酸序列。In some embodiments, CIM includes a cell membrane internalization peptide (CMIP). In some embodiments, CIM includes a wild-type M-spider toxin (M-lycotoxin) peptide. In some embodiments, CIM includes an M-lycotoxin derivative. In some embodiments, CIM includes a Penetain amino acid sequence or a derivative thereof. In some embodiments, CIM includes a Pepth amino acid sequence or a derivative thereof. In some embodiments, CIM includes a polyarginine amino acid sequence. In some embodiments, CIM includes more than one polyarginine amino acid sequence. In some embodiments, CIM includes three polyarginine amino acid sequences. In some embodiments, CIM includes a TAT amino acid sequence. In some embodiments, CIM includes more than one TAT amino acid sequence. In some embodiments, CIM includes three TAT amino acid sequences.
在一些實施例中，CIM包括大環。在一些實施例中，大環由CIM中之兩個胺基酸殘基之間的共價鍵形成。在一些實施例中，大環由CIM中之兩個半胱胺酸殘基之間的二硫鍵形成。在一些實施例中，CIM包括一或多個組胺酸殘基。In some embodiments, the CIM comprises a macrocycle. In some embodiments, the macrocycle is formed by a covalent bond between two amino acid residues in the CIM. In some embodiments, the macrocycle is formed by a disulfide bond between two cysteine residues in the CIM. In some embodiments, the CIM comprises one or more histidine residues.
在一些實施例中，CIM包括具有選自以下中之一者之胺基酸序列的多肽：SEQ ID NO: 176-184、SEQ ID NO: 192及SEQ ID NO: 193。In some embodiments, the CIM comprises a polypeptide having an amino acid sequence selected from one of the following: SEQ ID NOs: 176-184, SEQ ID NO: 192, and SEQ ID NO: 193.
在一些實施例中，CIM包括一或多個間隔區。在一些實施例中，一或多個間隔區中之至少一者包括一或多個胺基酸殘基。在一些實施例中，一或多個間隔區中之至少一者包括一或多個甘胺酸殘基。在一些實施例中，一或多個間隔區中之至少一者包括選自SEQ ID NO: 200-203中之任一者的胺基酸序列。在一些實施例中，一或多個間隔區中之每一者包括選自SEQ ID NO: 200-203中之任一者的胺基酸序列。In some embodiments, the CIM comprises one or more spacers. In some embodiments, at least one of the one or more spacers comprises one or more amino acid residues. In some embodiments, at least one of the one or more spacers comprises one or more glycine residues. In some embodiments, at least one of the one or more spacers comprises an amino acid sequence selected from any one of SEQ ID NOs: 200-203. In some embodiments, each of the one or more spacers comprises an amino acid sequence selected from any one of SEQ ID NOs: 200-203.
在一些實施例中，CIM包括具有選自SEQ ID NO: 176-193中之任一者的胺基酸序列之多肽。在一些實施例中，CIM為具有選自SEQ ID NO: 176-193中之任一者的胺基酸序列之多肽。In some embodiments, the CIM comprises a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 176-193. In some embodiments, the CIM is a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 176-193.
在一些實施例中，CIM共價連接至抗體。在一些實施例中，CIM非共價連接至抗體。在一些實施例中，細胞穿透劑包括將CIM連接至抗體之連接子。在一些實施例中，連接子共價連接至CIM及抗體兩者。在一些實施例中，連接子為可裂解連接子。在一些實施例中，連接子為不可裂解連接子。在一些實施例中，連接子包括多肽。在一些實施例中，連接子包括一或多個甘胺酸殘基。在一些實施例中，連接子包括包含選自SEQ ID NO: 194-199中之任一者的胺基酸序列之多肽。在一些實施例中，連接子是包括選自SEQ ID NO: 194-199中之任一者的胺基酸序列之多肽。In some embodiments, the CIM is covalently linked to the antibody. In some embodiments, the CIM is non-covalently linked to the antibody. In some embodiments, the cell penetrant comprises a linker that links the CIM to the antibody. In some embodiments, the linker is covalently linked to both the CIM and the antibody. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker comprises a polypeptide. In some embodiments, the linker comprises one or more glycine residues. In some embodiments, the linker comprises a polypeptide comprising an amino acid sequence selected from any one of SEQ ID NOs: 194-199. In some embodiments, the linker is a polypeptide comprising an amino acid sequence selected from any one of SEQ ID NOs: 194-199.
在一些實施例中，抗體連接至CIM之C末端。在一些實施例中，抗體連接至CIM之N末端。In some embodiments, the antibody is linked to the C-terminus of CIM. In some embodiments, the antibody is linked to the N-terminus of CIM.
在一些實施例中，抗體與以下各者競爭結合至TDP-43 (例如人類TDP-43)：包括SEQ ID NO: 1之重鏈可變域及SEQ ID NO: 24之輕鏈可變域的抗體；包括SEQ ID NO: 63之重鏈可變域及SEQ ID NO: 65之輕鏈可變域的抗體；包括SEQ ID NO: 67之重鏈可變域及SEQ ID NO: 69之輕鏈可變域的抗體；包括SEQ ID NO: 71之重鏈可變域及SEQ ID NO: 73之輕鏈可變域的抗體；包括SEQ ID NO: 75之重鏈可變域及SEQ ID NO: 77之輕鏈可變域的抗體；或包括SEQ ID NO: 79之重鏈可變域及SEQ ID NO: 81之輕鏈可變域的抗體。In some embodiments, the antibody competes for binding to TDP-43 (e.g., human TDP-43) with: an antibody comprising a heavy chain variable domain of SEQ ID NO: 1 and a light chain variable domain of SEQ ID NO: 24; an antibody comprising a heavy chain variable domain of SEQ ID NO: 63 and a light chain variable domain of SEQ ID NO: 65; an antibody comprising a heavy chain variable domain of SEQ ID NO: 67 and a light chain variable domain of SEQ ID NO: 69; an antibody comprising a heavy chain variable domain of SEQ ID NO: 71 and a light chain variable domain of SEQ ID NO: 73; an antibody comprising a heavy chain variable domain of SEQ ID NO: 75 and a light chain variable domain of SEQ ID NO: 77; or an antibody comprising a heavy chain variable domain of SEQ ID NO: 79 and a light chain variable domain of SEQ ID NO: 81 light chain variable domain antibody.
在一些實施例中，抗體與以下各者結合至TDP-43 (例如人類TDP-43)上的相同表位：包括SEQ ID NO: 1之重鏈可變域及SEQ ID NO: 24之輕鏈可變域的抗體；包括SEQ ID NO: 63之重鏈可變域及SEQ ID NO: 65之輕鏈可變域的抗體；包括SEQ ID NO: 67之重鏈可變域及SEQ ID NO: 69之輕鏈可變域的抗體；包括SEQ ID NO: 71之重鏈可變域及SEQ ID NO: 73之輕鏈可變域的抗體；包括SEQ ID NO: 75之重鏈可變域及SEQ ID NO: 77之輕鏈可變域的抗體；或包括SEQ ID NO: 79之重鏈可變域及SEQ ID NO: 81之輕鏈可變域的抗體。In some embodiments, the antibody binds to the same epitope on TDP-43 (e.g., human TDP-43) as: an antibody comprising a heavy chain variable domain of SEQ ID NO: 1 and a light chain variable domain of SEQ ID NO: 24; an antibody comprising a heavy chain variable domain of SEQ ID NO: 63 and a light chain variable domain of SEQ ID NO: 65; an antibody comprising a heavy chain variable domain of SEQ ID NO: 67 and a light chain variable domain of SEQ ID NO: 69; an antibody comprising a heavy chain variable domain of SEQ ID NO: 71 and a light chain variable domain of SEQ ID NO: 73; an antibody comprising a heavy chain variable domain of SEQ ID NO: 75 and a light chain variable domain of SEQ ID NO: 77; or an antibody comprising a heavy chain variable domain of SEQ ID NO: 79 and a light chain variable domain of SEQ ID NO: 81 light chain variable domain antibody.
在一些實施例中，特異性結合至TDP-43 (例如人類TDP-43)之抗體包括小鼠抗體之三個輕鏈CDR及三個重鏈CDR，該小鼠抗體之特徵為包括SEQ ID NO: 1之重鏈可變域及包括SEQ ID NO: 24之輕鏈可變域。In some embodiments, an antibody that specifically binds to TDP-43 (e.g., human TDP-43) comprises three light chain CDRs and three heavy chain CDRs of a mouse antibody characterized by a heavy chain variable domain comprising SEQ ID NO: 1 and a light chain variable domain comprising SEQ ID NO: 24.
在一些實施例中，CDR具有選自Kabat、Chothia、Kabat/Chothia、Composite、AbM及Contact之群組的定義。In some embodiments, the CDRs have a definition selected from a group selected from Kabat, Chothia, Kabat/Chothia, Composite, AbM, and Contact.
在一些實施例中，抗體包括人源化成熟重鏈可變域，其包括：如由Kabat/Chothia Composite所定義之重鏈CDR1，其包括SEQ ID NO: 49；如由Kabat所定義之重鏈CDR2，其包括SEQ ID NO: 51；及如由Kabat或Chothia所定義之重鏈CDR3，其包括SEQ ID NO: 52；以及人源化成熟輕鏈可變域，其包括SEQ ID NO: 53-55之三個Kabat輕鏈CDR。In some embodiments, the antibody comprises a humanized mature heavy chain variable domain comprising: a heavy chain CDR1 as defined by the Kabat/Chothia Composite comprising SEQ ID NO: 49; a heavy chain CDR2 as defined by Kabat comprising SEQ ID NO: 51; and a heavy chain CDR3 as defined by Kabat or Chothia comprising SEQ ID NO: 52; and a humanized mature light chain variable domain comprising the three Kabat light chain CDRs of SEQ ID NOs: 53-55.
在一些實施例中，人源化成熟重鏈可變域包括與SEQ ID NO: 4-23中之任一者至少80%一致的序列，且人源化成熟輕鏈可變域包括與SEQ ID NO: 27-48中之任一者至少80%一致的序列。在一些實施例中，人源化成熟重鏈可變域包括與SEQ ID NO: 4-23中之任一者至少85%一致的序列，且人源化成熟輕鏈可變域包括與SEQ ID NO: 27-48中之任一者至少85%一致的序列。在一些實施例中，人源化成熟重鏈可變域包括與SEQ ID NO: 4-23中之任一者至少90%一致的序列，且人源化成熟輕鏈可變域包括與SEQ ID NO: 27-48中之任一者至少90%一致的序列。在一些實施例中，人源化成熟重鏈可變域包括與SEQ ID NO: 4-23中之任一者至少95%一致的序列，且人源化成熟輕鏈可變域包括與SEQ ID NO: 27-48中之任一者至少95%一致的序列。In some embodiments, the humanized mature heavy chain variable domain comprises a sequence that is at least 80% identical to any one of SEQ ID NOs: 4-23, and the humanized mature light chain variable domain comprises a sequence that is at least 80% identical to any one of SEQ ID NOs: 27-48. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence that is at least 85% identical to any one of SEQ ID NOs: 4-23, and the humanized mature light chain variable domain comprises a sequence that is at least 85% identical to any one of SEQ ID NOs: 27-48. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence that is at least 90% identical to any one of SEQ ID NOs: 4-23, and the humanized mature light chain variable domain comprises a sequence that is at least 90% identical to any one of SEQ ID NOs: 27-48. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence at least 95% identical to any one of SEQ ID NOs: 4-23, and the humanized mature light chain variable domain comprises a sequence at least 95% identical to any one of SEQ ID NOs: 27-48.
在一些實施例中，人源化重鏈可變域中之以下位置中的至少一個由所指定之胺基酸佔據：K19由R佔據；S35由G佔據；T40由A佔據；E42由G佔據；A49由S佔據；K43由E佔據；R44由G或D佔據；A49由S佔據；A74由S佔據；T77由S佔據；L78由A或G佔據；L80由A或G佔據；L82c由G佔據；M83由R佔據；S84由A佔據；M89由V佔據；或F91由Y佔據。In some embodiments, at least one of the following positions in the humanized heavy chain variable domain is occupied by the specified amino acid: K19 is occupied by R; S35 is occupied by G; T40 is occupied by A; E42 is occupied by G; A49 is occupied by S; K43 is occupied by E; R44 is occupied by G or D; A49 is occupied by S; A74 is occupied by S; T77 is occupied by S; L78 is occupied by A or G; L80 is occupied by A or G; L82c is occupied by G; M83 is occupied by R; S84 is occupied by A; M89 is occupied by V; or F91 is occupied by Y.
在一些實施例中，人源化重鏈可變域中之以下位置中的至少一個由所指定之胺基酸佔據：K43由E佔據；R44由G或D佔據；A49由S佔據；A74由S佔據；T77由S佔據；或F91由Y佔據。In some embodiments, at least one of the following positions in the humanized heavy chain variable domain is occupied by the specified amino acid: K43 is occupied by E; R44 is occupied by G or D; A49 is occupied by S; A74 is occupied by S; T77 is occupied by S; or F91 is occupied by Y.
在一些實施例中，人源化重鏈可變域中之以下位置中之至少一個由所指定之胺基酸佔據：S35由G佔據；L78由A或G佔據；L80由A或G佔據；或L82c由G佔據。In some embodiments, at least one of the following positions in the humanized heavy chain variable domain is occupied by the specified amino acid: S35 is occupied by G; L78 is occupied by A or G; L80 is occupied by A or G; or L82c is occupied by G.
在一些實施例中，人源化重鏈可變域之F91由Y佔據；且人源化重鏈可變域中之以下位置中之至少一個由所指定之胺基酸佔據：R44由G佔據；A49由S佔據；A74由S佔據；T77由S佔據；L78由A或G佔據；或M83由R佔據。In some embodiments, F91 of the humanized heavy chain variable domain is occupied by Y; and at least one of the following positions in the humanized heavy chain variable domain is occupied by the specified amino acid: R44 is occupied by G; A49 is occupied by S; A74 is occupied by S; T77 is occupied by S; L78 is occupied by A or G; or M83 is occupied by R.
在一些實施例中，人源化輕鏈可變域中之以下位置中的至少一個由所指定之胺基酸佔據：V3由Q佔據；L9由S佔據；D17由Q佔據；Q18由P佔據；K39由R佔據；K45由R佔據；T80由A或S佔據；L83由V佔據；L92由G或A佔據；V94由I或A佔據；A100由G、D或R佔據；或L104由V佔據。In some embodiments, at least one of the following positions in the humanized light chain variable domain is occupied by the specified amino acid: V3 is occupied by Q; L9 is occupied by S; D17 is occupied by Q; Q18 is occupied by P; K39 is occupied by R; K45 is occupied by R; T80 is occupied by A or S; L83 is occupied by V; L92 is occupied by G or A; V94 is occupied by I or A; A100 is occupied by G, D, or R; or L104 is occupied by V.
在一些實施例中，人源化輕鏈可變域中之以下位置中之至少一個由所指定之胺基酸佔據：V3由Q佔據或A100由D或R佔據。In some embodiments, at least one of the following positions in the humanized light chain variable domain is occupied by the specified amino acid: V3 is occupied by Q or A100 is occupied by D or R.
在一些實施例中，人源化輕鏈可變域中之以下位置中的至少一個由所指定之胺基酸佔據：L9由S佔據；T80由A或S佔據；L92由G或A佔據；或V94由I或A佔據。In some embodiments, at least one of the following positions in the humanized light chain variable domain is occupied by the specified amino acid: L9 is occupied by S; T80 is occupied by A or S; L92 is occupied by G or A; or V94 is occupied by I or A.
在一些實施例中，V3由Q佔據；Q18由P佔據；A100由D佔據；且人源化輕鏈可變域中之以下位置中之至少一個由所指定之胺基酸佔據：T80由A佔據或L92由A佔據。In some embodiments, V3 is occupied by Q; Q18 is occupied by P; A100 is occupied by D; and at least one of the following positions in the humanized light chain variable domain is occupied by the specified amino acid: T80 is occupied by A or L92 is occupied by A.
在一些實施例中，人源化重鏈可變域中之以下位置中的至少一個由所指定之胺基酸佔據：L5由V佔據；G44由R佔據；A49由S佔據；A74由S佔據；T77由S佔據；L78由A或G佔據；M89由V佔據，或F91由Y佔據；且人源化輕鏈可變域中之以下位置中之至少一個由所指定之胺基酸佔據：V3由Q佔據；D17由Q佔據；Q18由P佔據；K39由R佔據；K45由R佔據；T80由A佔據；L83由V佔據；L92由A佔據；A100由D佔據；或L104由V佔據。In some embodiments, at least one of the following positions in the humanized heavy chain variable domain is occupied by the specified amino acid: L5 is occupied by V; G44 is occupied by R; A49 is occupied by S; A74 is occupied by S; T77 is occupied by S; L78 is occupied by A or G; M89 is occupied by V, or F91 is occupied by Y; and the humanized At least one of the following positions in the light chain variable domain is occupied by the specified amino acid: V3 is occupied by Q; D17 is occupied by Q; Q18 is occupied by P; K39 is occupied by R; K45 is occupied by R; T80 is occupied by A; L83 is occupied by V; L92 is occupied by A; A100 is occupied by D; or L104 is occupied by V.
在一些實施例中，與包括包含SEQ ID NO: 1之重鏈可變域及包含SEQ ID NO: 24之輕鏈可變域的參考抗體相比，人源化抗體具有提高的熱穩定性。In some embodiments, the humanized antibody has improved thermal stability compared to a reference antibody comprising a heavy chain variable domain comprising SEQ ID NO: 1 and a light chain variable domain comprising SEQ ID NO: 24.
在一些實施例中，抗體包括重鏈可變域，其包括：如由Kabat/Chothia Composite所定義之重鏈CDR1，其包括SEQ ID NO: 49或SEQ ID NO: 50；如由Kabat所定義之重鏈CDR2，其包括SEQ ID NO: 51；如由Kabat或Chothia所定義之重鏈CDR3，其包括SEQ ID NO: 52；如由Kabat所定義之輕鏈CDR1，其包括SEQ ID NO: 53；如由Kabat所定義之輕鏈CDR2，其包括SEQ ID NO: 54；及如由Kabat所定義之輕鏈CDR3，其包括SEQ ID NO: 55-61中之一者。In some embodiments, the antibody comprises a heavy chain variable domain comprising: a heavy chain CDR1 as defined by the Kabat/Chothia Composite comprising SEQ ID NO: 49 or SEQ ID NO: 50; a heavy chain CDR2 as defined by Kabat comprising SEQ ID NO: 51; a heavy chain CDR3 as defined by Kabat or Chothia comprising SEQ ID NO: 52; a light chain CDR1 as defined by Kabat comprising SEQ ID NO: 53; a light chain CDR2 as defined by Kabat comprising SEQ ID NO: 54; and a light chain CDR3 as defined by Kabat comprising one of SEQ ID NOs: 55-61.
在一些實施例中，如由Kabat/Chothia Composite所定義之重鏈CDR1包括SEQ ID NO: 49；如由Kabat所定義之重鏈CDR2包括SEQ ID NO: 51；如由Kabat或Chothia所定義之重鏈CDR3包括SEQ ID NO: 52；如由Kabat所定義之輕鏈CDR1包括SEQ ID NO: 53；如由Kabat所定義之輕鏈CDR2包括SEQ ID NO: 54；且如由Kabat所定義之輕鏈CDR3包括SEQ ID NO: 55或SEQ ID NO: 61。In some embodiments, the heavy chain CDR1 as defined by the Kabat/Chothia Composite comprises SEQ ID NO: 49; the heavy chain CDR2 as defined by Kabat comprises SEQ ID NO: 51; the heavy chain CDR3 as defined by Kabat or Chothia comprises SEQ ID NO: 52; the light chain CDR1 as defined by Kabat comprises SEQ ID NO: 53; the light chain CDR2 as defined by Kabat comprises SEQ ID NO: 54; and the light chain CDR3 as defined by Kabat comprises SEQ ID NO: 55 or SEQ ID NO: 61.
在一些實施例中，重鏈可變域包括與SEQ ID NO: 4-23中之任一者至少95%一致的序列。在一些實施例中，重鏈可變域包括與以下任一者至少95%一致的序列：SEQ ID NO: 20、SEQ ID NO: 21及SEQ ID NO: 23。在一些實施例中，重鏈可變域包括與以下任一者至少98%一致的序列：SEQ ID NO: 20、SEQ ID NO: 21及SEQ ID NO: 23。在一些實施例中，重鏈可變域包括SEQ ID NO: 20之序列。在一些實施例中，重鏈可變域包括SEQ ID NO: 21之序列。在一些實施例中，重鏈可變域包括SEQ ID NO: 23之序列。In some embodiments, the heavy chain variable domain comprises a sequence that is at least 95% identical to any one of SEQ ID NOs: 4-23. In some embodiments, the heavy chain variable domain comprises a sequence that is at least 95% identical to any one of SEQ ID NOs: 20, 21, and 23. In some embodiments, the heavy chain variable domain comprises a sequence that is at least 98% identical to any one of SEQ ID NOs: 20, 21, and 23. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 20. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 21. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 23.
在一些實施例中，輕鏈可變域包括與SEQ ID NO: 27-48任一者至少95%一致的序列。在一些實施例中，輕鏈可變域包括與SEQ ID NO: 47或SEQ ID NO: 48至少95%一致的序列。在一些實施例中，輕鏈可變域包括與SEQ ID NO: 47或SEQ ID NO: 48至少98%一致的序列。在一些實施例中，輕鏈可變域包括SEQ ID NO: 47。在一些實施例中，輕鏈可變域包括SEQ ID NO: 48。In some embodiments, the light chain variable domain comprises a sequence that is at least 95% identical to any one of SEQ ID NOs: 27-48. In some embodiments, the light chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 47 or SEQ ID NO: 48. In some embodiments, the light chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 47 or SEQ ID NO: 48. In some embodiments, the light chain variable domain comprises SEQ ID NO: 47. In some embodiments, the light chain variable domain comprises SEQ ID NO: 48.
在一些實施例中，抗體包括重鏈可變域及輕鏈可變域，其包括：重鏈CDR1，其包括SEQ ID NO: 84；重鏈CDR2，其包括SEQ ID NO: 85；重鏈CDR3，其包括SEQ ID NO: 86；輕鏈CDR1，其包括SEQ ID NO: 87；輕鏈CDR2，其包括SEQ ID NO: 88；及輕鏈CDR3，其包括SEQ ID NO: 89。In some embodiments, the antibody comprises a heavy chain variable domain and a light chain variable domain comprising: a heavy chain CDR1 comprising SEQ ID NO: 84; a heavy chain CDR2 comprising SEQ ID NO: 85; a heavy chain CDR3 comprising SEQ ID NO: 86; a light chain CDR1 comprising SEQ ID NO: 87; a light chain CDR2 comprising SEQ ID NO: 88; and a light chain CDR3 comprising SEQ ID NO: 89.
在一些實施例中，重鏈可變域包含與SEQ ID NO: 63至少95%一致之序列。在一些實施例中，重鏈可變域包含與SEQ ID NO: 63至少98%一致之序列。在一些實施例中，重鏈可變域包含SEQ ID NO: 63之序列。In some embodiments, the heavy chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 63. In some embodiments, the heavy chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 63. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 63.
在一些實施例中，輕鏈可變域包含與SEQ ID NO: 65至少95%一致之序列。在一些實施例中，輕鏈可變域包含與SEQ ID NO: 65至少98%一致之序列。在一些實施例中，輕鏈可變域包含SEQ ID NO: 65之序列。In some embodiments, the light chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 65. In some embodiments, the light chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 65. In some embodiments, the light chain variable domain comprises the sequence of SEQ ID NO: 65.
在一些實施例中，抗體包括重鏈可變域及輕鏈可變域，其包括：重鏈CDR1，其包括SEQ ID NO: 90；重鏈CDR2，其包括SEQ ID NO: 91；重鏈CDR3，其包括SEQ ID NO: 92；輕鏈CDR1，其包括SEQ ID NO: 93；輕鏈CDR2，其包括SEQ ID NO: 94；及輕鏈CDR3，其包括SEQ ID NO: 95。In some embodiments, the antibody comprises a heavy chain variable domain and a light chain variable domain comprising: a heavy chain CDR1 comprising SEQ ID NO: 90; a heavy chain CDR2 comprising SEQ ID NO: 91; a heavy chain CDR3 comprising SEQ ID NO: 92; a light chain CDR1 comprising SEQ ID NO: 93; a light chain CDR2 comprising SEQ ID NO: 94; and a light chain CDR3 comprising SEQ ID NO: 95.
在一些實施例中，重鏈可變域包含與SEQ ID NO: 67至少95%一致之序列。在一些實施例中，重鏈可變域包含與SEQ ID NO: 67至少98%一致之序列。在一些實施例中，重鏈可變域包含SEQ ID NO: 67之序列。In some embodiments, the heavy chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 67. In some embodiments, the heavy chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 67. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 67.
在一些實施例中，輕鏈可變域包含與SEQ ID NO: 69至少95%一致之序列。在一些實施例中，輕鏈可變域包含與SEQ ID NO: 69至少98%一致之序列。在一些實施例中，輕鏈可變域包含SEQ ID NO: 69之序列。In some embodiments, the light chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 69. In some embodiments, the light chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 69. In some embodiments, the light chain variable domain comprises the sequence of SEQ ID NO: 69.
在一些實施例中，抗體包括重鏈可變域及輕鏈可變域，其包括：重鏈CDR1，其包括SEQ ID NO: 96；重鏈CDR2，其包括SEQ ID NO: 97；重鏈CDR3，其包括SEQ ID NO: 98；輕鏈CDR1，其包括SEQ ID NO: 99；輕鏈CDR2，其包括SEQ ID NO: 100；及輕鏈CDR3，其包括SEQ ID NO: 101。In some embodiments, the antibody comprises a heavy chain variable domain and a light chain variable domain comprising: a heavy chain CDR1 comprising SEQ ID NO: 96; a heavy chain CDR2 comprising SEQ ID NO: 97; a heavy chain CDR3 comprising SEQ ID NO: 98; a light chain CDR1 comprising SEQ ID NO: 99; a light chain CDR2 comprising SEQ ID NO: 100; and a light chain CDR3 comprising SEQ ID NO: 101.
在一些實施例中，重鏈可變域包含與SEQ ID NO: 71至少95%一致之序列。在一些實施例中，重鏈可變域包含與SEQ ID NO: 71至少98%一致之序列。在一些實施例中，重鏈可變域包含SEQ ID NO: 71之序列。In some embodiments, the heavy chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 71. In some embodiments, the heavy chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 71. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 71.
在一些實施例中，輕鏈可變域包含與SEQ ID NO: 73至少95%一致之序列。在一些實施例中，輕鏈可變域包含與SEQ ID NO: 73至少98%一致之序列。在一些實施例中，輕鏈可變域包含SEQ ID NO: 73之序列。In some embodiments, the light chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 73. In some embodiments, the light chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 73. In some embodiments, the light chain variable domain comprises the sequence of SEQ ID NO: 73.
在一些實施例中，抗體包括重鏈可變域及輕鏈可變域，其包括：重鏈CDR1，其包括SEQ ID NO: 102；重鏈CDR2，其包括SEQ ID NO: 103；重鏈CDR3，其包括SEQ ID NO: 104；輕鏈CDR1，其包括SEQ ID NO: 105；輕鏈CDR2，其包括SEQ ID NO: 106；及輕鏈CDR3，其包括SEQ ID NO: 107。In some embodiments, the antibody comprises a heavy chain variable domain and a light chain variable domain comprising: a heavy chain CDR1 comprising SEQ ID NO: 102; a heavy chain CDR2 comprising SEQ ID NO: 103; a heavy chain CDR3 comprising SEQ ID NO: 104; a light chain CDR1 comprising SEQ ID NO: 105; a light chain CDR2 comprising SEQ ID NO: 106; and a light chain CDR3 comprising SEQ ID NO: 107.
在一些實施例中，重鏈可變域包含與SEQ ID NO: 75至少95%一致之序列。在一些實施例中，重鏈可變域包含與SEQ ID NO: 75至少98%一致之序列。在一些實施例中，重鏈可變域包含SEQ ID NO: 75之序列。In some embodiments, the heavy chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 75. In some embodiments, the heavy chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 75. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 75.
在一些實施例中，輕鏈可變域包含與SEQ ID NO: 77至少95%一致之序列。在一些實施例中，輕鏈可變域包含與SEQ ID NO: 77至少98%一致之序列。在一些實施例中，輕鏈可變域包含SEQ ID NO: 77之序列。In some embodiments, the light chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 77. In some embodiments, the light chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 77. In some embodiments, the light chain variable domain comprises the sequence of SEQ ID NO: 77.
在一些實施例中，抗體包括重鏈可變域及輕鏈可變域，其包括：重鏈CDR1，其包括SEQ ID NO: 108；重鏈CDR2，其包括SEQ ID NO: 109；重鏈CDR3，其包括SEQ ID NO: 110；輕鏈CDR1，其包括SEQ ID NO: 111；輕鏈CDR2，其包括SEQ ID NO: 112；及輕鏈CDR3，其包括SEQ ID NO: 113。In some embodiments, the antibody comprises a heavy chain variable domain and a light chain variable domain comprising: a heavy chain CDR1 comprising SEQ ID NO: 108; a heavy chain CDR2 comprising SEQ ID NO: 109; a heavy chain CDR3 comprising SEQ ID NO: 110; a light chain CDR1 comprising SEQ ID NO: 111; a light chain CDR2 comprising SEQ ID NO: 112; and a light chain CDR3 comprising SEQ ID NO: 113.
在一些實施例中，重鏈可變域包含與SEQ ID NO: 79至少95%一致之序列。在一些實施例中，重鏈可變域包含與SEQ ID NO: 79至少98%一致之序列。在一些實施例中，重鏈可變域包含SEQ ID NO: 79之序列。In some embodiments, the heavy chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 79. In some embodiments, the heavy chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 79. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 79.
在一些實施例中，輕鏈可變域包含與SEQ ID NO: 81至少95%一致之序列。在一些實施例中，輕鏈可變域包含與SEQ ID NO: 81至少98%一致之序列。在一些實施例中，輕鏈可變域包含SEQ ID NO: 81之序列。In some embodiments, the light chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 81. In some embodiments, the light chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 81. In some embodiments, the light chain variable domain comprises the sequence of SEQ ID NO: 81.
在一些實施例中，抗體為人源化抗體、嵌合抗體或飾面(veneered)抗體。在一些實施例中，抗體為抗原結合抗體片段。在一些實施例中，抗原結合抗體片段為Fab片段、Fab'2片段或單鏈Fv。In some embodiments, the antibody is a humanized antibody, a chimeric antibody, or a veneered antibody. In some embodiments, the antibody is an antigen-binding antibody fragment. In some embodiments, the antigen-binding antibody fragment is a Fab fragment, aFab'2 fragment, or a single-chain Fv.
在一些實施例中，抗體為完整抗體。在一些實施例中，抗體具有人類IgG1同型。In some embodiments, the antibody is a whole antibody. In some embodiments, the antibody has a human IgG1 isotype.
在一些實施例中，重鏈可變域融合至重鏈恆定區且輕鏈可變域融合至輕鏈恆定區。In some embodiments, the heavy chain variable domain is fused to the heavy chain constant region and the light chain variable domain is fused to the light chain constant region.
在一些實施例中，重鏈恆定區為天然人類重鏈恆定區之突變體形式，其相對於天然重鏈恆定區，與Fcγ受體之結合減少。在一些實施例中，重鏈恆定區具有IgG1同型。在一些實施例中，抗體在恆定區中具有至少一個突變。在一些實施例中，至少一個突變減少恆定區之補體固定或活化。在一些實施例中，至少一個突變在按EU編號的以下一或多個位置處：241、264、265、270、296、297、318、320、322、329及331。在一些實施例中，抗體在按EU編號之位置318、320及322處具有丙胺酸。In some embodiments, the heavy chain constant region is a mutant form of a native human heavy chain constant region that has reduced binding to Fcγ receptors relative to the native heavy chain constant region. In some embodiments, the heavy chain constant region has an IgG1 isotype. In some embodiments, the antibody has at least one mutation in the constant region. In some embodiments, at least one mutation reduces complement fixation or activation of the constant region. In some embodiments, at least one mutation is at one or more of the following positions by EU numbering: 241, 264, 265, 270, 296, 297, 318, 320, 322, 329, and 331. In some embodiments, the antibody has alanine at positions 318, 320, and 322 by EU numbering.
在一些實施例中，抗體選擇性地結合至磷酸化TDP-43 (例如，磷酸化人類TDP-43)。在一些實施例中，與未磷酸化TDP-43 (例如，未磷酸化人類TDP-43)相比，抗體選擇性地結合至磷酸化TDP-43 (例如，磷酸化人類TDP-43)。在一些實施例中，與未磷酸化TDP-43 (例如，未磷酸化人類TDP-43)相比，抗體以大至少100倍之親和力結合至磷酸化TDP-43 (例如，磷酸化人類TDP-43)。在一些實施例中，與未磷酸化TDP-43 (例如，未磷酸化人類TDP-43)相比，抗體以大至少1000倍之親和力結合至磷酸化TDP-43 (例如，磷酸化人類TDP-43)。在一些實施例中，磷酸化TDP-43 (例如，磷酸化人類TDP-43)包括至少一個選自S409及S410之胺基酸殘基的磷酸化。在一些實施例中，磷酸化TDP-43 (例如，磷酸化人類TDP-43)包括S409及S410兩者之磷酸化。In some embodiments, the antibody selectively binds to phosphorylated TDP-43 (e.g., phosphorylated human TDP-43). In some embodiments, the antibody selectively binds to phosphorylated TDP-43 (e.g., phosphorylated human TDP-43) compared to unphosphorylated TDP-43 (e.g., unphosphorylated human TDP-43). In some embodiments, the antibody binds to phosphorylated TDP-43 (e.g., phosphorylated human TDP-43) with at least 100-fold greater affinity than unphosphorylated TDP-43 (e.g., unphosphorylated human TDP-43). In some embodiments, the antibody binds to phosphorylated TDP-43 (e.g., phosphorylated human TDP-43) with at least 1000-fold greater affinity than unphosphorylated TDP-43 (e.g., unphosphorylated human TDP-43). In some embodiments, phosphorylated TDP-43 (e.g., phosphorylated human TDP-43) comprises phosphorylation of at least one amino acid residue selected from S409 and S410. In some embodiments, phosphorylated TDP-43 (e.g., phosphorylated human TDP-43) comprises phosphorylation of both S409 and S410.
在一些實施例中，抗體選擇性地結合至TDP-43之細胞質聚集體(例如，人類TDP-43之細胞質聚集體)。在一些實施例中，與核TDP-43 (例如，核人類TDP-43)相比，抗體選擇性地結合至TDP-43之細胞質聚集體(例如，人類TDP-43之細胞質聚集體)。在一些實施例中，TDP-43之細胞質聚集體包括TDP-43之磷酸化聚集體(例如，人類TDP-43之磷酸化聚集體)。在一些實施例中，抗體實質上不結合未磷酸化TDP-43 (例如，未磷酸化人類TDP-43)。In some embodiments, the antibody selectively binds to cytoplasmic aggregates of TDP-43 (e.g., cytoplasmic aggregates of human TDP-43). In some embodiments, the antibody selectively binds to cytoplasmic aggregates of TDP-43 (e.g., cytoplasmic aggregates of human TDP-43) as compared to nuclear TDP-43 (e.g., nuclear human TDP-43). In some embodiments, cytoplasmic aggregates of TDP-43 include phosphorylated aggregates of TDP-43 (e.g., phosphorylated aggregates of human TDP-43). In some embodiments, the antibody does not substantially bind to unphosphorylated TDP-43 (e.g., unphosphorylated human TDP-43).
在一些實施例中，細胞穿透劑包含與選自以下任一者之序列至少95%一致的多肽序列：SEQ ID NO: 116、SEQ ID NO: 118、SEQ ID NO: 120、SEQ ID NO: 122、SEQ ID NO: 124、SEQ ID NO: 126、SEQ ID NO: 128、SEQ ID NO: 130、SEQ ID NO: 132、SEQ ID NO: 134、SEQ ID NO: 136、SEQ ID NO: 138、SEQ ID NO: 140、SEQ ID NO: 142、SEQ ID NO: 144、SEQ ID NO: 146、SEQ ID NO: 148、SEQ ID NO: 150、SEQ ID NO: 152、SEQ ID NO: 154、SEQ ID NO: 156、SEQ ID NO: 158、SEQ ID NO: 160、SEQ ID NO: 162、SEQ ID NO: 164、SEQ ID NO: 166、SEQ ID NO: 168、SEQ ID NO: 170、SEQ ID NO: 172及SEQ ID NO: 174。In some embodiments, the cell penetrant comprises a polypeptide sequence that is at least 95% identical to a sequence selected from any one of SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162. SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172 and SEQ ID NO: 174.
在一些實施例中，細胞穿透劑包含與選自以下任一者之序列至少98%一致的多肽序列：SEQ ID NO: 116、SEQ ID NO: 118、SEQ ID NO: 120、SEQ ID NO: 122、SEQ ID NO: 124、SEQ ID NO: 126、SEQ ID NO: 128、SEQ ID NO: 130、SEQ ID NO: 132、SEQ ID NO: 134、SEQ ID NO: 136、SEQ ID NO: 138、SEQ ID NO: 140、SEQ ID NO: 142、SEQ ID NO: 144、SEQ ID NO: 146、SEQ ID NO: 148、SEQ ID NO: 150、SEQ ID NO: 152、SEQ ID NO: 154、SEQ ID NO: 156、SEQ ID NO: 158、SEQ ID NO: 160、SEQ ID NO: 162、SEQ ID NO: 166、SEQ ID NO: 168、SEQ ID NO: 170、SEQ ID NO: 172及SEQ ID NO: 174。In some embodiments, the cell penetrant comprises a polypeptide sequence that is at least 98% identical to a sequence selected from any one of SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172 and SEQ ID NO: 174.
在一些實施例中，細胞穿透劑包含選自以下任一者的多肽序列：SEQ ID NO: 116、SEQ ID NO: 118、SEQ ID NO: 120、SEQ ID NO: 122、SEQ ID NO: 124、SEQ ID NO: 126、SEQ ID NO: 128、SEQ ID NO: 130、SEQ ID NO: 132、SEQ ID NO: 134、SEQ ID NO: 136、SEQ ID NO: 138、SEQ ID NO: 140、SEQ ID NO: 142、SEQ ID NO: 144、SEQ ID NO: 146、SEQ ID NO: 148、SEQ ID NO: 150、SEQ ID NO: 152、SEQ ID NO: 154、SEQ ID NO: 156、SEQ ID NO: 158、SEQ ID NO: 160、SEQ ID NO: 162、SEQ ID NO: 166、SEQ ID NO: 168、SEQ ID NO: 170、SEQ ID NO: 172及SEQ ID NO: 174。In some embodiments, the cell penetrant comprises a polypeptide sequence selected from any one of SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172 and SEQ ID NO: 174.
在一些實施例中，細胞穿透劑包含與選自以下任一者之序列至少95%一致的多肽序列：SEQ ID NO: 117、SEQ ID NO: 119、SEQ ID NO: 121、SEQ ID NO: 123、SEQ ID NO: 125、SEQ ID NO: 127、SEQ ID NO: 129、SEQ ID NO: 131、SEQ ID NO: 133、SEQ ID NO: 135、SEQ ID NO: 137、SEQ ID NO: 139、SEQ ID NO: 141、SEQ ID NO: 143、SEQ ID NO: 145、SEQ ID NO: 147、SEQ ID NO: 149、SEQ ID NO: 151、SEQ ID NO: 153、SEQ ID NO: 155、SEQ ID NO: 157、SEQ ID NO: 159、SEQ ID NO: 161、SEQ ID NO: 163、SEQ ID NO: 165、SEQ ID NO: 167、SEQ ID NO: 169、SEQ ID NO: 171、SEQ ID NO: 173及SEQ ID NO: 175。In some embodiments, the cell penetrant comprises a polypeptide sequence that is at least 95% identical to a sequence selected from any one of SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163. SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173 and SEQ ID NO: 175.
在一些實施例中，細胞穿透劑包含與選自以下任一者之序列至少98%一致的多肽序列：SEQ ID NO: 117、SEQ ID NO: 119、SEQ ID NO: 121、SEQ ID NO: 123、SEQ ID NO: 125、SEQ ID NO: 127、SEQ ID NO: 129、SEQ ID NO: 131、SEQ ID NO: 133、SEQ ID NO: 135、SEQ ID NO: 137、SEQ ID NO: 139、SEQ ID NO: 141、SEQ ID NO: 143、SEQ ID NO: 145、SEQ ID NO: 147、SEQ ID NO: 149、SEQ ID NO: 151、SEQ ID NO: 153、SEQ ID NO: 155、SEQ ID NO: 157、SEQ ID NO: 159、SEQ ID NO: 161、SEQ ID NO: 163、SEQ ID NO: 165、SEQ ID NO: 167、SEQ ID NO: 169、SEQ ID NO: 171、SEQ ID NO: 173及SEQ ID NO: 175。In some embodiments, the cell penetrant comprises a polypeptide sequence that is at least 98% identical to a sequence selected from any one of SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163. SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173 and SEQ ID NO: 175.
在一些實施例中，細胞穿透劑包含選自以下任一者的多肽序列：SEQ ID NO: 117、SEQ ID NO: 119、SEQ ID NO: 121、SEQ ID NO: 123、SEQ ID NO: 125、SEQ ID NO: 127、SEQ ID NO: 129、SEQ ID NO: 131、SEQ ID NO: 133、SEQ ID NO: 135、SEQ ID NO: 137、SEQ ID NO: 139、SEQ ID NO: 141、SEQ ID NO: 143、SEQ ID NO: 145、SEQ ID NO: 147、SEQ ID NO: 149、SEQ ID NO: 151、SEQ ID NO: 153、SEQ ID NO: 155、SEQ ID NO: 157、SEQ ID NO: 159、SEQ ID NO: 161、SEQ ID NO: 163、SEQ ID NO: 165、SEQ ID NO: 167、SEQ ID NO: 169、SEQ ID NO: 171、SEQ ID NO: 173及SEQ ID NO: 175。In some embodiments, the cell penetrant comprises a polypeptide sequence selected from any one of SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173 and SEQ ID NO: 175.
在一些實施例中，抗體結合至治療劑、細胞毒性劑、細胞生長抑制劑、免疫調節劑、神經營養劑或神經保護劑。In some embodiments, the antibody is conjugated to a therapeutic agent, a cytotoxic agent, a cytostatic agent, an immunomodulatory agent, a neurotrophic agent, or a neuroprotective agent.
在一些實施例中，重鏈不包含C末端離胺酸殘基。In some embodiments, the heavy chain does not contain a C-terminal lysine residue.
本文亦提供醫藥組成物，其包括本文描述之任一細胞穿透劑及醫藥上可接受之載劑。Also provided herein are pharmaceutical compositions comprising any of the cell penetrating agents described herein and a pharmaceutically acceptable carrier.
本文亦提供編碼本文描述之任一細胞穿透劑之至少一部分的核酸。在一些實施例中，核酸編碼抗體之重鏈可變域及/或輕鏈可變域。在一些實施例中，核酸編碼本文描述之任一CIM。Also provided herein are nucleic acids encoding at least a portion of any of the cell penetrants described herein. In some embodiments, the nucleic acids encode a heavy chain variable domain and/or a light chain variable domain of an antibody. In some embodiments, the nucleic acids encode any of the CIMs described herein.
本文亦提供載體，其包括編碼成熟重鏈可變域及輕鏈可變域之核酸，該核酸可操作地連接至一或多個調控序列以實現本文所述細胞穿透劑中之任一者在哺乳動物細胞中之表現。Also provided herein are vectors comprising nucleic acids encoding mature heavy chain variable domains and light chain variable domains operably linked to one or more regulatory sequences to achieve expression of any of the cell penetrants described herein in mammalian cells.
在一些實施例中，一或多個調控序列包括強化子、核糖體結合位點、轉錄終止訊號及啟動子中之一或多者，視情況，其中啟動子為真核生物啟動子。在一些實施例中，核酸經密碼子最佳化以在宿主細胞中表現。In some embodiments, the one or more regulatory sequences include one or more of an enhancer, a ribosome binding site, a transcriptional termination signal, and a promoter, optionally wherein the promoter is a eukaryotic promoter. In some embodiments, the nucleic acid is codon-optimized for expression in a host cell.
本文亦提供用本文所述之任一載體轉化的宿主細胞。本文亦提供包括本文所述之核酸中之任一者的宿主細胞。Also provided herein are host cells transformed with any of the vectors described herein. Also provided herein are host cells comprising any of the nucleic acids described herein.
本文亦提供將特異性結合至TDP-43之抗體遞送至細胞中的方法，其包含使本文所述之任一細胞穿透劑與細胞接觸，由此導致至少抗體之抗原結合片段內化至細胞中。在一些實施例中，該方法進一步包含至少將抗體之抗原結合片段轉移至細胞之細胞液(cytosol)中。Also provided herein are methods for delivering an antibody that specifically binds to TDP-43 into a cell, comprising contacting a cell with any of the cell penetrating agents described herein, thereby causing internalization of at least an antigen-binding fragment of the antibody into the cell. In some embodiments, the method further comprises transferring at least the antigen-binding fragment of the antibody into the cytosol of the cell.
本文亦提供結合細胞中之細胞內TDP-43蛋白的方法，其包括使如請求項1至135中任一項之細胞穿透劑與該細胞接觸，由此導致至少抗體之抗原結合片段內化且轉移至細胞液中。Also provided herein is a method for binding intracellular TDP-43 protein in a cell, comprising contacting the cell with the cell penetrant of any one of claims 1 to 135, thereby causing internalization and translocation of at least the antigen-binding fragment of the antibody into the cytosol.
本文亦提供結合細胞中之細胞內TDP-43蛋白的方法，其包括：使如請求項1至135中任一項之細胞穿透劑與該細胞接觸，由此導致至少抗體之抗原結合片段內化且轉移至細胞液中；及使至少抗體之抗原結合片段與細胞內TDP-43蛋白結合。Also provided herein is a method for binding to an intracellular TDP-43 protein in a cell, comprising: contacting the cell with the cell of any one of claims 1 to 135, thereby causing internalization and translocation of at least an antigen-binding fragment of the antibody into the cytosol; and allowing at least the antigen-binding fragment of the antibody to bind to the intracellular TDP-43 protein.
在一些實施例中，細胞為哺乳動物細胞。在一些實施例中，接觸為活體外的。在一些實施例中，細胞係在個體中。In some embodiments, the cell is a mammalian cell. In some embodiments, the contacting is in vitro. In some embodiments, the cell is in an individual.
本文亦提供在患有TDP-43相關疾病或具有患上該疾病之風險的個體中抑制或減少TDP-43 (例如，人類TDP-43)聚集的方法，其包括向個體投予有效量之本文所述之任一細胞穿透劑，由此抑制或減少TDP-43 (例如，人類TDP-43)在個體中之聚集。Also provided herein are methods for inhibiting or reducing TDP-43 (e.g., human TDP-43) aggregation in a subject having or at risk for a TDP-43-associated disease, comprising administering to the subject an effective amount of any cell-permeable agent described herein, thereby inhibiting or reducing TDP-43 (e.g., human TDP-43) aggregation in the subject.
本文亦提供治療或實現預防個體之TDP-43相關疾病的方法，其包括投予治療有效量之本文所述之任一細胞穿透劑，由此治療或實現預防TDP-43相關疾病。Also provided herein are methods for treating or preventing a TDP-43-related disease in a subject, comprising administering a therapeutically effective amount of any cell penetrating agent described herein, thereby treating or preventing the TDP-43-related disease.
在一些實施例中，TDP-43相關疾病為肌肉萎縮性脊髓側索硬化症(ALS)、額顳葉退化(FTLD-TDP)、原發性脊髓側索硬化症及進行性肌萎縮及帕金森氏病。在一些實施例中，TDP-43相關疾病為ALS。In some embodiments, the TDP-43-associated disease is amyotrophic lateral sclerosis (ALS), frontotemporal lobe degeneration (FTLD-TDP), primary lateral sclerosis, progressive muscular dystrophy, and Parkinson's disease. In some embodiments, the TDP-43-associated disease is ALS.
本文亦提供在患有TDP-43相關疾病或具有患上該疾病之風險的個體中偵測TDP-43沉積(例如，人類TDP-43沉積)的方法，其包括向個體投予本文所述之任一細胞穿透劑，及偵測該個體中與TDP-43結合之抗體。Also provided herein are methods for detecting TDP-43 deposition (e.g., human TDP-43 deposition) in a subject having or at risk for a TDP-43-associated disease, comprising administering to the subject any of the cell-permeable agents described herein, and detecting an antibody that binds to TDP-43 in the subject.
在一些實施例中，抗體藉由靜脈內注射至個體之身體中來投予。In some embodiments, the antibody is administered by intravenous injection into the body of the individual.
本文亦提供在得自患有TDP-43相關疾病或具有患上該疾病之風險的患者獲得的樣品中偵測TDP-43的方法，其包含使該樣品與本文所述之任一細胞穿透劑接觸，及偵測針對該樣品中之TDP-43之抗體。Also provided herein are methods for detecting TDP-43 in a sample obtained from a patient having or at risk for a TDP-43-associated disease, comprising contacting the sample with any of the cell penetrating agents described herein, and detecting antibodies against TDP-43 in the sample.
在一些實施例中，抗體經標記。在一些實施例中，抗體用螢光標記、順磁性標記或放射性標記來標記。在一些實施例中，使用正電子發射斷層攝影術(PET)或單光子發射電腦斷層攝影術(SPECT)來偵測放射性標記。In some embodiments, the antibody is labeled. In some embodiments, the antibody is labeled with a fluorescent label, a paramagnetic label, or a radiolabel. In some embodiments, the radiolabel is detected using positron emission tomography (PET) or single photon emission computed tomography (SPECT).
本說明書中提及之所有出版物、專利及專利申請案均以引用方式併入本文，其程度如同每一個別出版物、專利、專利申請案或資訊項明確且個別地指示以引用方式併入一般。在以引用方式併入之出版物、專利、專利申請案及資訊項與說明書中所含之揭示內容相矛盾的範圍內，本說明書意欲取代及/或優先於任何此類矛盾材料。All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, patent application, or item of information was specifically and individually indicated as incorporated by reference. To the extent such incorporated by reference publications, patents, patent applications, and items of information conflict with the disclosure contained in this specification, this specification is intended to supersede and/or take precedence over any such conflicting material.
在用範圍來描述值時，應理解，該描述包括揭示此等範圍內之所有可能子範圍，以及落入此等範圍內之特定數值，而不管是否明確說明特定數值或特定子範圍。When values are described using ranges, it should be understood that the description includes disclosure of all possible sub-ranges within those ranges, as well as specific values falling within those ranges, regardless of whether a specific value or specific sub-range is explicitly stated.
本揭示內容提供用於將抗體遞送至細胞中之系統及方法。具體而言，本揭示內容描述細胞穿透劑，其包含細胞內化模組及特異性結合TDP-43 (例如人類TDP-43)或TDP-43聚集體(例如人類TDP-43聚集體，包括TDP-43之磷酸化聚集體)。亦提供醫藥組成物，其包括此等細胞穿透劑及醫藥上可接受之載劑、編碼此等細胞穿透劑之核酸及/或載體，及表現上述核酸及/或載體之宿主細胞。The present disclosure provides systems and methods for delivering antibodies into cells. Specifically, the present disclosure describes cell penetrating agents comprising a cell internalization module and specifically binding to TDP-43 (e.g., human TDP-43) or TDP-43 aggregates (e.g., human TDP-43 aggregates, including phosphorylated TDP-43 aggregates). Also provided are pharmaceutical compositions comprising these cell penetrating agents and a pharmaceutically acceptable carrier, nucleic acids and/or vectors encoding these cell penetrating agents, and host cells expressing the nucleic acids and/or vectors.
I.I.定義Definition
術語「抗體」包括完整抗體及其抗原結合片段。通常，片段與衍生其之完整抗體競爭特異性結合至靶標，包括單獨的重鏈、輕鏈Fab、Fab'、F(ab')2、F(ab)c、Dab、奈米抗體及Fv。片段可藉由重組DNA技術或藉由完整免疫球蛋白之酶促或化學分離來產生。術語「抗體」亦包括雙特異性或多特異性抗體及/或人源化抗體。雙特異性或雙功能或多功能抗體為具有兩個或更多個不同重鏈/輕鏈對及兩個或更多個不同結合位點之人工雜合抗體(參見，例如Songsivilai及Lachmann,Clin. Exp. Immunol., 79:315-321 (1990)；Kostelny等人,J. Immunol., 148:1547-53 (1992))。The term "antibody" includes intact antibodies and antigen-binding fragments thereof. Generally, fragments compete with the intact antibody from which they are derived for specific target binding and include individual heavy and light chain Fab, Fab', F(ab')2 , F(ab)c, Dab, nanobodies, and Fv. Fragments can be produced by recombinant DNA technology or by enzymatic or chemical isolation of intact immunoglobulins. The term "antibody" also includes bispecific or multispecific antibodies and/or humanized antibodies. Bispecific or bifunctional or multifunctional antibodies are artificial hybrid antibodies with two or more different heavy chain/light chain pairs and two or more different binding sites (see, e.g., Songsivilai and Lachmann,Clin. Exp. Immunol ., 79:315-321 (1990); Kostelnyet al. ,J. Immunol ., 148:1547-53 (1992)).
如本文所用，「細胞穿透劑」(Cell-Penetrating Agent，在本文中亦稱為「CPA」)係指能夠進入細胞(例如，活體外(in vitro)及/或活體內(in vivo)哺乳動物細胞)之劑(例如，分子及/或分子複合物)。在一些實施例中，CPA進入細胞且在由細胞內化後轉移至細胞液中。在一些實施例中，CPA包含促進由細胞內化CPA之細胞內化模組(Cell Internalizing Module，CIM)。在一些情況下，CPA包含促進CPA內化、CPA轉移至細胞液中之CIM。在一些實施例中，CPA進一步包含連接(例如，共價或非共價)至CIM之抗TDP-43抗體。在一些實施例中，CPA包含共價連接至抗TDP-43抗體(例如，經由CIM與抗TDP-43抗體之間的連接子)之CIM。在其他實施例中，CPA包含以下CIM，其非共價連接至抗TDP-43抗體(例如，經由鏈黴抗生物素蛋白-生物素相互作用)，使得CIM在相關條件(例如，血漿)下保持連接至抗TDP-43抗體。在一些實施例中，CPA進一步包含將CIM連接至抗TDP-43抗體之連接子。在各種情況下，連接子可為可裂解的或不可裂解的且/或可將CIM共價或非共價連接至抗TDP-43抗體。在一些實施例中，與不為CPA之一部分的參考抗TDP-43抗體相比，包含抗TDP-43抗體之CPA具有增強之細胞滲透。在一些實施例中，CPA包含兩種或更多種抗TDP-43抗體及/或兩種或更多種CIM (例如，包含連接至複數種CIM之樹枝狀聚合物及/或抗TDP-43抗體之CPA)。本文描述細胞穿透劑之非限制性特徵及實例。As used herein, a "cell-penetrating agent" (also referred to herein as a "CPA") refers to an agent (e.g., a molecule and/or a molecular complex) capable of entering cells (e.g., mammalian cellsin vitro and/orin vivo ). In some embodiments, the CPA enters a cell and, after internalization by the cell, is transferred to the cytosol. In some embodiments, the CPA comprises a cell internalizing module (CIM) that facilitates the internalization of the CPA by the cell. In some cases, the CPA comprises a CIM that facilitates the internalization of the CPA and transfer of the CPA to the cytosol. In some embodiments, the CPA further comprises an anti-TDP-43 antibody linked (e.g., covalently or non-covalently) to the CIM. In some embodiments, the CPA comprises a CIM covalently linked to an anti-TDP-43 antibody (e.g., via a linker between the CIM and the anti-TDP-43 antibody). In other embodiments, the CPA comprises a CIM non-covalently linked to the anti-TDP-43 antibody (e.g., via a streptavidin-biotin interaction), such that the CIM remains linked to the anti-TDP-43 antibody under relevant conditions (e.g., plasma). In some embodiments, the CPA further comprises a linker that links the CIM to the anti-TDP-43 antibody. In various instances, the linker can be cleavable or non-cleavable and/or can covalently or non-covalently link the CIM to the anti-TDP-43 antibody. In some embodiments, a CPA comprising an anti-TDP-43 antibody has enhanced cell penetration compared to a reference anti-TDP-43 antibody that is not part of the CPA. In some embodiments, the CPA comprises two or more anti-TDP-43 antibodies and/or two or more CIMs (e.g., a CPA comprising a dendrimer and/or an anti-TDP-43 antibody linked to a plurality of CIMs). Non-limiting features and examples of cell penetrants are described herein.
如本文所用，「細胞內化模組」(Cell Internalizing Module，在本文中亦稱為「CIM」)係指CPA之促進細胞內化CPA (及延伸之抗TDP-43抗體)的一部分。在各種實施例中，CIM可利用一或多個細胞內化過程(包括主動及被動細胞內化兩者)來實現內化。例示性過程包括但不限於胞吞作用(例如，受體介導之胞吞作用(RME)、吞噬作用、胞飲作用)及膜易位(例如，直接穿透及/或能量非依賴性內化)。在各種非限制性實施例中，CIM包含例如細胞膜內化肽(CMIP)、小分子配位體(例如，維生素、脂肪酸、整聯蛋白結合配位體等)、抗體之一部分(例如，結合內化細胞表面靶標之scFv部分)或誘餌受體配位體(例如，細胞介素或其衍生物)。在一些實施例中，CIM包含細胞膜內化肽(CMIP)。本文描述細胞內化模組之非限制性特徵及實例。As used herein, a "Cell Internalizing Module" (also referred to herein as a "CIM") refers to the portion of a CPA that promotes cellular internalization of the CPA (and, by extension, an anti-TDP-43 antibody). In various embodiments, the CIM can utilize one or more cellular internalization processes, including both active and passive cellular internalization, to achieve internalization. Exemplary processes include, but are not limited to, endocytosis (e.g., receptor-mediated endocytosis (RME), phagocytosis, and phagocytosis) and membrane translocation (e.g., direct penetration and/or energy-independent internalization). In various non-limiting embodiments, a CIM comprises, for example, a cell membrane internalization peptide (CMIP), a small molecule ligand (e.g., a vitamin, a fatty acid, an integrin-binding ligand, etc.), a portion of an antibody (e.g., a scFv portion that binds to an internalized cell surface target), or a ligand for a decoy receptor (e.g., a cytokine or a derivative thereof). In some embodiments, a CIM comprises a cell membrane internalization peptide (CMIP). Non-limiting features and examples of cell internalization modules are described herein.
如本文所用，「細胞膜內化肽」(Cell Membrane Internalizing Peptide，在本文中亦稱為CMIP」)為三個或更多個天然或非天然胺基酸之序列，當共價或非共價連接至抗TDP-43抗體時導致至少抗TDP-43抗體或抗TDP-43抗體之活性片段內化至哺乳動物細胞中。在各種實施例中，CMIP可利用一或多個細胞內化過程(包括主動及被動細胞內化兩者)來實現內化。例示性過程包括但不限於胞吞作用及膜易位。在一些實施例中，CMIP與細胞膜及/或細胞表面抗原相互作用，導致CPA或其一部分(例如，包含抗TDP-43抗體或其功能部分之一部分)經由內吞囊泡內化。在一些實施例中，CMIP進一步促進CPA或其部分自內吞囊泡轉移至細胞之細胞質中。在一些情況下，CMIP進一步與內吞囊泡之膜相互作用，導致囊泡破裂及CPA或其部分胞內體逃逸至細胞液中。因此，在一些實施例中，CMIP促進抗TDP-43抗體或其功能部分內化至細胞中，以及抗TDP-43抗體或其功能部分轉移至細胞液中。CMIP之非限制性實例包括例如陽離子肽(包括例如M-lycotoxin、TAT肽、pentetratin及聚精胺酸肽以及其衍生物)、兩親性肽(包括例如MPG肽、Pep-1肽、轉運蛋白肽及其衍生物)及富含脯胺酸之肽(包括例如Bac7肽及其衍生物)。在一些實施例中，CMIP包括細胞穿透肽及其衍生物，包括例如Ruseska及A. Zimmer,Internalization mechanisms of cell-penetrating peptides, BEILSTEIN J. NANOTECHOL. 202; 11:101-123中所述之彼等。本文描述CMIP之非限制性特徵及實例。As used herein, a "Cell Membrane Internalizing Peptide" (also referred to herein as a "CMIP") is a sequence of three or more natural or unnatural amino acids that, when covalently or non-covalently linked to an anti-TDP-43 antibody, results in the internalization of at least the anti-TDP-43 antibody or an active fragment of an anti-TDP-43 antibody into mammalian cells. In various embodiments, a CMIP can be internalized using one or more cellular internalization processes, including both active and passive cellular internalization. Exemplary processes include, but are not limited to, endocytosis and membrane translocation. In some embodiments, a CMIP interacts with a cell membrane and/or cell surface antigen, resulting in the internalization of a CMIP or a portion thereof (e.g., comprising an anti-TDP-43 antibody or a functional portion thereof) via endocytic vesicles. In some embodiments, CMIP further promotes the transfer of CPA or a portion thereof from endocytic vesicles into the cytoplasm of the cell. In some cases, CMIP further interacts with the membrane of the endocytic vesicle, causing vesicle rupture and endosomal escape of CPA or a portion thereof into the cytosol. Thus, in some embodiments, CMIP promotes the internalization of an anti-TDP-43 antibody or a functional portion thereof into a cell, as well as the transfer of an anti-TDP-43 antibody or a functional portion thereof into the cytosol. Non-limiting examples of CMIP include, for example, cationic peptides (including, for example, M-lycotoxin, TAT peptide, pentetratin, and polyarginine peptides and their derivatives), amphipathic peptides (including, for example, MPG peptide, Pep-1 peptide, transporter peptides and their derivatives), and proline-rich peptides (including, for example, Bac7 peptide and its derivatives). In some embodiments, CMIPs include cell-penetrating peptides and their derivatives, including those described in, for example, Ruseska and A. Zimmer,"Internalization mechanisms of cell-penetrating peptides ," BEILSTEIN J. NANOTECHOL. 202; 11: 101-123. Non-limiting features and examples of CMIPs are described herein.
術語「表位」(epitope)係指抗原上抗體所結合之位點。表位可由連續胺基酸或藉由一或多個蛋白之三級折疊來並置的不連續胺基酸形成。由連續胺基酸形成之表位(亦稱為線性表位)通常在暴露於變性溶劑時得以保留，而藉由三級折疊所形成之表位(亦稱為構形表位)通常在經變性溶劑處理時損失。表位通常包括呈獨特空間構形的至少3個，及更通常至少5個或8-10個胺基酸。確定表位之空間構形的方法包括例如x射線結晶學及二維核磁共振。參見，例如 Epitope Mapping Protocols, Methods in Molecular Biology,第66卷, Glenn E. Morris編(1996)。The term "epitope" refers to the site on an antigen to which an antibody binds. An epitope can be formed by consecutive amino acids or by non-consecutive amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed by consecutive amino acids (also called linear epitopes) are generally retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding (also called conformational epitopes) are generally lost upon treatment with denaturing solvents. An epitope typically comprises at least three, and more typically at least five or eight to ten, amino acids in a unique spatial configuration. Methods for determining the spatial configuration of an epitope include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, Methods in Molecular Biology, Vol. 66, edited by Glenn E. Morris (1996).
識別相同或重疊表位之抗體可在簡單免疫檢定中鑑別，該檢定顯示一種抗體與另一種抗體競爭結合至靶抗原之能力。抗體之表位亦可定義為結合至其抗原之抗體的X射線結晶學以鑑別接觸殘基。或者，若抗原中減少或消除一種抗體之結合的所有胺基酸突變減少或消除另一種抗體之結合，則兩種抗體具有相同的表位。若減少或消除一種抗體之結合的一些胺基酸突變減少或消除另一種抗體之結合，則兩種抗體具有重疊表位。Antibodies that recognize the same or overlapping epitopes can be identified in a simple immunoassay that shows the ability of one antibody to compete with another for binding to the target antigen. An antibody's epitope can also be defined as the contact residues identified by X-ray crystallography of the antibody bound to its antigen. Alternatively, two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other.
術語「人源化免疫球蛋白」(humanized immunoglobulin)或「人源化抗體」(humanized antibody)係指包括至少一個人源化免疫球蛋白或抗體鏈(亦即至少一個人源化輕鏈或重鏈)的免疫球蛋白或抗體。術語「人源化免疫球蛋白鏈」或「人源化抗體鏈」(亦即「人源化免疫球蛋白輕鏈」或「人源化免疫球蛋白重鏈」)係指具有可變區之免疫球蛋白或抗體鏈(亦即分別為輕鏈或重鏈)，該可變區包括實質上來自人類免疫球蛋白或抗體之可變框架區及實質上來自非人類免疫球蛋白或抗體之互補決定區(CDR) (例如至少一個CDR，較佳兩個CDR，更佳三個CDR)，且進一步包括恆定區(例如，在輕鏈之情況下為至少一個恆定區或其部分，且在重鏈之情況下較佳為三個恆定區)。術語「人源化可變區」(例如「人源化輕鏈可變區」或「人源化重鏈可變區」)係指包括實質上來自人類免疫球蛋白或抗體之可變框架區及實質上來自非人類免疫球蛋白或抗體之互補決定區(CDR)的可變區。The term "humanized immunoglobulin" or "humanized antibody" refers to an immunoglobulin or antibody that comprises at least one humanized immunoglobulin or antibody chain (i.e., at least one humanized light chain or heavy chain). The term "humanized immunoglobulin chain" or "humanized antibody chain" (i.e., "humanized immunoglobulin light chain" or "humanized immunoglobulin heavy chain") refers to an immunoglobulin or antibody chain (i.e., light chain or heavy chain, respectively) having a variable region that includes a variable framework region substantially derived from a human immunoglobulin or antibody and complementary determining regions (CDRs) substantially derived from a non-human immunoglobulin or antibody (e.g., at least one CDR, preferably two CDRs, more preferably three CDRs), and further includes a constant region (e.g., in the case of a light chain, at least one constant region or a portion thereof, and in the case of a heavy chain, preferably three constant regions). The term "humanized variable region" (e.g., "humanized light chain variable region" or "humanized heavy chain variable region") refers to a variable region that includes a variable framework region substantially derived from a human immunoglobulin or antibody and a complementary determining region (CDR) substantially derived from a non-human immunoglobulin or antibody.
抗體之間的競爭藉由其中待測抗體抑制參考抗體與共同抗原之特異性結合的檢定來確定(參見，例如Junghans等人, Cancer Res.50: 1495, 1990)。若過量的測試抗體(例如，至少2x、5x、l0x、20x或l00x)使參考抗體之結合抑制了至少50%，如在競爭性結合檢定中所量測，則測試抗體與參考抗體競爭。一些測試抗體將參考抗體之結合抑制了至少75%、90%或99%。藉由競爭檢定鑑別之抗體(競爭抗體)包括與參考抗體結合至相同表位的抗體及結合至與由參考抗體結合之表位足夠接近以發生空間位阻之相鄰表位的抗體。Competition between antibodies is determined by an assay in which a test antibody inhibits specific binding of a reference antibody to a common antigen( see, e.g., Junghanset al., Cancer Res. 50: 1495, 1990). A test antibody competes with a reference antibody if an excess of the test antibody( e.g., at least 2x, 5x, 10x, 20x, or 100x) inhibits binding of the reference antibody by at least 50%, as measured in a competitive binding assay. Some test antibodies inhibit binding of the reference antibody by at least 75%, 90%, or 99%. Antibodies identified by competition assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to adjacent epitopes that are sufficiently close to the epitope bound by the reference antibody to cause steric hindrance.
如本文所用，「連接子」(linker)係指在哺乳動物細胞中不具有催化或治療活性之化學部分，其用於共價連接兩種不同功能分子(例如，細胞內化模組及抗TDP-43抗體)。舉例而言，連接子可為約3個胺基酸至約25個胺基酸(例如約3個胺基酸至約20個或約3個胺基酸至約12個胺基酸)之肽。在其他實例中，連接子可為鍵(例如醯胺鍵、酯鍵、醚鍵及二硫鍵)。在一些實例中，連接子可包含一對親和域(例如，該對親和域之第一域可為介白素-15，且該對親和域之第二域可為介白素-15受體α之壽司域(sushi domain))。在一些實例中，連接子為甘胺酸殘基，之後為絲胺酸殘基(GS)。在一些實例中，連接子為三個連續甘胺酸殘基(例如GGG)。在一些實例中，連接子為SEQ ID NO: 194-199中之任一者。As used herein, a "linker" refers to a chemical moiety that has no catalytic or therapeutic activity in mammalian cells and is used to covalently link two different functional molecules (e.g., a cell internalization module and an anti-TDP-43 antibody). For example, a linker can be a peptide of about 3 to about 25 amino acids (e.g., about 3 to about 20 or about 3 to about 12 amino acids). In other examples, the linker can be a bond (e.g., an amide bond, an ester bond, an ether bond, and a disulfide bond). In some examples, the linker can comprise a pair of affinity domains (e.g., the first domain of the pair of affinity domains can be interleukin-15, and the second domain of the pair of affinity domains can be the sushi domain of interleukin-15 receptor α). In some instances, the linker is a glycine residue followed by a serine residue (GS). In some instances, the linker is three consecutive glycine residues (e.g., GGG). In some instances, the linker is any one of SEQ ID NOs: 194-199.
術語「醫藥上可接受」意謂載劑、稀釋劑、賦形劑或助劑與調配物之其他成分相容且對其接受者實質上無害。The term "pharmaceutically acceptable" means that the carrier, diluent, excipient or adjuvant is compatible with the other ingredients of the formulation and not substantially deleterious to the recipient thereof.
如本文所用，「M-lycotoxin衍生物」為基於起始M-蜘蛛毒素(M-lycotoxin)肽設計的包含三個或更多個胺基酸(天然或非天然)之肽。在一些實施例中，「M-lycotoxin衍生物」為與野生型M-蜘蛛毒素(M-lycotoxin)具有例如60%至99%序列同源性之多肽。本文描述M-lycotoxin衍生物之其他非限制性態樣及實例。As used herein, an "M-lycotoxin derivative" is a peptide comprising three or more amino acids (natural or non-natural) that is designed based on a starting M-lycotoxin peptide. In some embodiments, an "M-lycotoxin derivative" is a polypeptide having, for example, 60% to 99% sequence homology with wild-type M-lycotoxin. Other non-limiting aspects and examples of M-lycotoxin derivatives are described herein.
如本文所用，術語「大分子」係指分子量為至少5 kDa及/或流體動力學半徑為至少1.0 nm之分子。如本文所用，術語「大分子」包括生物分子、有機聚合物及有機金屬複合物。As used herein, the term "macromolecule" refers to a molecule having a molecular weight of at least 5 kDa and/or a hydrodynamic radius of at least 1.0 nm. As used herein, the term "macromolecule" includes biomolecules, organic polymers, and organometallic complexes.
如本文所用，「間隔子」(spacer)或「間隔區」(spacer region)係指在哺乳動物細胞中不具有催化或治療活性之胺基酸。舉例而言，間隔子可為1個胺基酸至約10個胺基酸(例如1至約8個胺基酸、1至約6個胺基酸或1至約4個胺基酸)之肽。在一些實例中，一或多個間隔區包括在細胞內化模組中。舉例而言，間隔區可分離細胞內化模組內之胺基酸，例如，間隔子可安置在CIM之第一個胺基酸之後。在一些實例中，間隔子可安置在CIM之最後的胺基酸之前。在一些實例中，CIM可具有兩個或更多個間隔區(例如，兩個間隔區、三個間隔序列、四個間隔區、五個間隔區或更多)。在一些實例中，間隔區為單個甘胺酸殘基。在一些實例中，間隔區為一對甘胺酸殘基。在一些實例中，間隔區為三個甘胺酸殘基。在一些實例中，間隔區為四個甘胺酸殘基。在一些實例中，間隔區為四個甘胺酸殘基，之後為絲胺酸殘基。在一些實例中，間隔區為SEQ ID NO: 200-203中之任一者。As used herein, a "spacer" or "spacer region" refers to an amino acid that does not have catalytic or therapeutic activity in mammalian cells. For example, a spacer can be a peptide of 1 to about 10 amino acids (e.g., 1 to about 8 amino acids, 1 to about 6 amino acids, or 1 to about 4 amino acids). In some examples, one or more spacers are included in a cell internalization module. For example, a spacer can separate amino acids within a cell internalization module, for example, a spacer can be positioned after the first amino acid of a CIM. In some examples, a spacer can be positioned before the last amino acid of a CIM. In some examples, a CIM may have two or more spacers (e.g., two spacers, three spacer sequences, four spacers, five spacers, or more). In some examples, the spacer is a single glycine residue. In some examples, the spacer is a pair of glycine residues. In some examples, the spacer is three glycine residues. In some examples, the spacer is four glycine residues. In some examples, the spacer is four glycine residues followed by a serine residue. In some examples, the spacer is any one of SEQ ID NOs: 200-203.
術語「TDP-43相關疾病」意謂至少部分地直接或間接由TDP-43聚集體之形成及/或TDP-43之錯誤定位為特徵及/或介導之疾病或病症。本文描述TDP-43相關癌症之非限制性實例。The term "TDP-43-associated disease" means a disease or disorder characterized and/or mediated, at least in part, directly or indirectly, by the formation of TDP-43 aggregates and/or mislocalization of TDP-43. Non-limiting examples of TDP-43-associated cancers are described herein.
術語「患者」或「個體」包括接受預防性或治療性治療之人類及其他哺乳動物個體(例如人類)。The term "patient" or "subject" includes humans and other mammalian subjects (e.g., humans) receiving prophylactic or therapeutic treatment.
若個體具有至少一種已知風險因素(例如遺傳、生化、家族史及情境暴露)，使具有該風險因素之個體比無該風險因素之個體具有統計學上顯著更大的患病風險，則該個體之患病風險增加。An individual is at increased risk for a disease if they have at least one known risk factor( e.g., genetic, biochemical, family history, and situational exposure) that places individuals with that risk factor at a statistically significant greater risk for the disease than individuals without that risk factor.
術語「生物樣品」係指在生物來源(例如人類或哺乳動物個體)內或可自生物來源獲得之生物材料樣品。此類樣品可為器官、細胞器、組織、組織切片、體液、外周血、血漿、血清、細胞、分子(諸如蛋白及肽)及自其衍生之任何部分或組合。術語生物樣品亦可涵蓋藉由處理樣品來衍生之任何材料。衍生材料可包括細胞或其子代。生物樣品之處理可涉及過濾、蒸餾、萃取、濃縮、固定、干擾組分之去活及其類似操作中之一或多者。The term "biological sample" refers to a sample of biological material that is within or obtainable from a biological source (e.g., a human or mammalian individual). Such samples may include organs, organelles, tissues, tissue sections, body fluids, peripheral blood, plasma, serum, cells, molecules (such as proteins and peptides), and any parts or combinations thereof derived therefrom. The term biological sample also encompasses any material derived by processing the sample. Derived material may include cells or their progeny. Processing of biological samples may involve one or more of filtration, distillation, extraction, concentration, fixation, deactivation of interfering components, and similar operations.
術語「對照樣品」係指不知道或懷疑包括TDP-43受影響區域，或至少不知道或懷疑包括給定類型之患病區域的生物樣品。對照樣品可獲自未罹患TDP-43相關疾病之個體。或者，對照樣品可獲自罹患TDP-43相關疾病之患者。此類樣品可與被認為包含TDP-43相關疾病之生物樣品同時或在不同場合獲得。生物樣品及對照樣品可獲自同一組織。較佳地，對照樣品基本上或完全由正常健康區域組成，且可用於與被認為包含TDP-43相關疾病影響區域之生物樣品進行比較。較佳地，對照樣品中之組織與生物樣品中之組織具有相同類型。較佳地，被認為在生物樣品中之TDP-43相關疾病影響細胞源自與對照樣品中之細胞類型相同的細胞類型(例如，神經元或神經膠質)。The term "control sample" refers to a biological sample that is not known or suspected to contain an area affected by TDP-43, or at least not known or suspected to contain an area of a given type of disease. A control sample can be obtained from an individual who does not suffer from a TDP-43-associated disease. Alternatively, a control sample can be obtained from a patient suffering from a TDP-43-associated disease. Such a sample can be obtained at the same time as a biological sample believed to contain a TDP-43-associated disease or at a different location. The biological sample and the control sample can be obtained from the same tissue. Preferably, the control sample consists essentially or entirely of a normal healthy area and can be used for comparison with a biological sample believed to contain an area affected by a TDP-43-associated disease. Preferably, the tissue in the control sample is of the same type as the tissue in the biological sample. Preferably, the TDP-43-associated disease-affecting cells in the biological sample are believed to be derived from the same cell type (e.g., neurons or neuroglia) as the cell type in the control sample.
出於將胺基酸取代分類為保守或非保守之目的，將胺基酸分組如下：第I組(疏水性側鏈)：Met、Ala、Val、Leu、Ile；第II組(中性親水性側鏈)：Cys、Ser、Thr；第III組(酸性側鏈)：Asp、Glu；第IV組(鹼性側鏈)：Asn、Gln、His、Lys、Arg；第V組(影響鏈取向之殘基)：Gly、Pro；及第VI組(芳族側鏈)：Trp、Tyr、Phe。保守取代涉及同一類別中的胺基酸之間的取代。非保守取代等同於將此等類別中之一者之成員交換為另一個類別之成員。For the purpose of classifying amino acid substitutions as conservative or non-conservative, amino acids are grouped as follows: Group I (hydrophobic side chain): Met, Ala, Val, Leu, Ile; Group II (neutral hydrophilic side chain): Cys, Ser, Thr; Group III (acidic side chain): Asp, Glu; Group IV (basic side chain): Asn, Gln, His, Lys, Arg; Group V (residues affecting chain orientation): Gly, Pro; and Group VI (aromatic side chain): Trp, Tyr, Phe. Conservative substitutions involve substitutions between amino acids within the same class. Non-conservative substitutions are equivalent to exchanging a member of one of these classes for a member of another class.
序列一致性百分比使用藉由Kabat編號慣例進行最大對齊之抗體序列來確定。比對後，若將本發明抗體區(例如重鏈或輕鏈之整個成熟可變區)與參考抗體之相同區進行比較，則本發明抗體區與參考抗體區之間的序列一致性百分比為本發明及參考抗體區中相同胺基酸佔據之位置數除以該兩個區之比對位置之總數(不計算空位)乘以100以轉化為百分比。Percent sequence identity is determined using antibody sequences that are maximally aligned using the Kabat numbering convention. After alignment, when comparing an antibody region of the present invention( e.g., the entire mature variable region of the heavy or light chain) to the same region of a reference antibody, the percent sequence identity between the antibody region of the present invention and the reference antibody region is calculated by dividing the number of positions occupied by the same amino acid in the antibody region of the present invention and the reference antibody region by the total number of aligned positions in the two regions (excluding gaps), multiplied by 100, to convert to a percentage.
除非自上下文中另外顯而易見，否則術語「約」涵蓋非實質性變化，諸如在規定值之標準量測誤差限度(例如SEM)內之值。Unless otherwise apparent from the context, the term "about" encompasses insubstantial variations, such as values that are within the standard measurement error limits( eg, SEM) of the stated value.
片語「實質上來自人類免疫球蛋白或抗體」意謂當出於比較目的與人類免疫球蛋白或抗體胺基序列比對時，該區與人類框架或恆定區序列享有至少80-90%、較佳90-95%、更佳95-99%一致性(亦即，局部序列一致性)，從而允許例如保守取代、共有序列取代、生殖系取代、回復突變及其類似變化。保守取代、共有序列取代、生殖系取代、回復突變及其類似變化之引入通常稱為人源化抗體或鏈之「最佳化」。片語「實質上來自非人類免疫球蛋白或抗體」或「實質上非人類」意謂具有與非人類生物體(例如非人類哺乳動物)至少80-95%、較佳90-95%、更佳96%、97%、98%或99%一致的免疫球蛋白或抗體序列。The phrase "substantially derived from a human immunoglobulin or antibody" means that, when aligned for comparison purposes with a human immunoglobulin or antibody amino acid sequence, the region shares at least 80-90%, preferably 90-95%, and even more preferably 95-99% identity (i.e., local sequence identity) with a human framework or invariant region sequence, thereby allowing for, for example, conservative substitutions, consensus substitutions, germline substitutions, backmutations, and the like. The introduction of conservative substitutions, consensus substitutions, germline substitutions, backmutations, and the like is often referred to as "optimization" of the humanized antibody or antibody. The phrase "substantially derived from a non-human immunoglobulin or antibody" or "substantially non-human" means having an immunoglobulin or antibody sequence that is at least 80-95%, preferably 90-95%, more preferably 96%, 97%, 98% or 99% identical to that of a non-human organism (e.g., a non-human mammal).
因此，人源化免疫球蛋白或抗體、或人源化免疫球蛋白或抗體鏈之所有區或殘基(可能除CDR外)與一或多個天然人類免疫球蛋白序列之相應區或殘基實質上一致。術語「相應區」(corresponding region)或「相應殘基」(corresponding residue)係指當第一序列與第二序列出於比較目的最佳地比對時，第二胺基酸或核苷酸序列上與第一胺基酸或核苷酸序列上之區或殘基佔據相同(亦即，等效)位置之區或殘基。Thus, all regions or residues of a humanized immunoglobulin or antibody, or a humanized immunoglobulin or antibody chain (possibly with the exception of the CDRs) are substantially identical to corresponding regions or residues of one or more native human immunoglobulin sequences. The term "corresponding region" or "corresponding residue" refers to a region or residue in a second amino acid or nucleotide sequence that occupies the same (i.e., equivalent) position as a region or residue in a first amino acid or nucleotide sequence when the first and second sequences are optimally aligned for comparison purposes.
II. TDP-43II. TDP-43
如本文所述，TDP-43為主要參與RNA剪接、運輸、穩定及最終調控基因表現之核蛋白。更具體而言，TDP-43為多域異質核糖核蛋白(hnRNP)。TDP-43之適當功能對於調控其結合之數百種mRNA轉錄物至關重要。TDP-43之主要功能之一為調控剪接mRNA轉錄物，然而，TDP-43亦參與RNA加工及轉運之不同機制。舉例而言，TDP-43為隱蔽外顯子包涵體之抑制劑且調控＞1,000個基因之替代性多腺苷酸化。TDP-43可在不同細胞類型(包括細胞核中之卡哈爾小體及旁斑)中形成核糖核蛋白顆粒，且募集至神經元中之mRNA轉運顆粒。如本文所述，各種神經退化性疾病與TDP-43之細胞質聚集體(例如，TDP-43相關疾病)相關。As described herein, TDP-43 is a nuclear protein that is primarily involved in RNA splicing, transport, stabilization, and ultimately, the regulation of gene expression. More specifically, TDP-43 is a multidomain heterogeneous ribonucleoprotein (hnRNP). Proper function of TDP-43 is crucial for the regulation of the hundreds of mRNA transcripts to which it binds. One of the primary functions of TDP-43 is the regulation of spliced mRNA transcripts, however, TDP-43 is also involved in different mechanisms of RNA processing and transport. For example, TDP-43 is a suppressor of cryptic exon inclusion bodies and regulates alternative polyadenylation of >1,000 genes. TDP-43 can form ribonucleoprotein granules in different cell types, including Cajal bodies and paraplaxes in the nucleus, and is recruited to mRNA transport granules in neurons. As described herein, various neurodegenerative diseases are associated with cytoplasmic aggregates of TDP-43 (e.g., TDP-43-associated diseases).
除非上下文中另外顯而易見，否則提及TDP-43意指TDP-43之天然人類形式，包括任何同功型及/或轉譯後修飾(例如磷酸化、醣基化及/或乙醯化)。人類TDP-43之胺基酸序列如下所示(SEQ ID NO: 82)：MSEYIRVTEDENDEPIEIPSEDDGTVLLSTVTAQFPGACGLRYRNPVSQCMRGVRLVEGILHAPDAGWGNLVYVVNYPKDNKRKMDETDASSAVKVKRAVQKTSDLIVLGLPWKTTEQDLKEYFSTFGEVLMVQVKKDLKTGHSKGFGFVRFTEYETQVKVMSQRHMIDGRWCDCKLPNSKQSQDEPLRSRKVFVGRCTEDMTEDELREFFSQYGDVMDVFIPKPFRAFAFVTFADDQIAQSLCGEDLIIKGISVHISNAEPKHNSNRQLERSGRFGGNPGGFGNQGGFGNSRGGGAGLGNNQGSNMGGGMNFGAFSINPAMMAAAQAALQSSWGMMGMLASQQNQSGPSGNNQNQGNMQREPNQAFGSGNNSYSGSNSGAAIGWGSASNAGSGSGFNGGFGSSMDSKSSGWGMUnless otherwise apparent from the context, reference to TDP-43 refers to the native human form of TDP-43, including any isoforms and/or post-translational modifications (e.g., phosphorylation, glycosylation, and/or acetylation). The amino acid sequence of human TDP-43 is shown below (SEQ ID NO: 82): MSEYIRVTEDENDEPIEIPSEDDGTVLLSTVTAQFPGACGLRYRNPVSQCMRGVRLVEGILHAPDAGWGNLVYVVNYPKDNKRKMDETDASSAVKVKRAVQKTSDLIVLGLPWKTTEQDLKEYFSTFGEVLMVQVKKDLKTGHSKGFGFVRFTEYETQVKVMSQRHMIDGRWCDCKLPNSKQSQDEPLRSRKVFVGRCTEDMTED ELREFFSQYGDVMDVFIPKPFRAFAFVTFADDQIAQSLCGEDLIIKGISVHISNAEPKHNSNRQLERSGRFGGNPGGFGNQGGFGNSRGGGAGLGNNQGSNMGGGMNFGAFSINPAMMAAAQAALQSSWGMMGMLASQQNQSGPSGNNQNQGNMQREPNQAFGSGNNSYSGSNSGAAIGWGSASNAGSGSGFNGGFGSSMDSKSSGWGM
TDP-43可在一或多個胺基酸處磷酸化，包括位置409及410處之絲胺酸。在一些實施例中，TDP-43可在一或多個位置處磷酸化，包括373、375、379、387、389、393、395、403、404、407、409及410 (參見，例如Gruijs da Silva, L.A.,等人, Disease-linked TDP-43 hyperphosphorylation suppresses TDP-43 condensation and aggregation,The EMBO Journal,41: e108443 (2022))。TDP-43 can be phosphorylated at one or more amino acids, including serine at positions 409 and 410. In some embodiments, TDP-43 can be phosphorylated at one or more positions, including 373, 375, 379, 387, 389, 393, 395, 403, 404, 407, 409, and 410 (see, e.g., Gruijs da Silva, LA, et al., Disease-linked TDP-43 hyperphosphorylation suppresses TDP-43 condensation and aggregation,The EMBO Journal, 41: e108443 (2022)).
除非上下文中另外顯而易見，否則提及TDP-43或其片段包括天然人類胺基酸序列，包括其同功型、突變體及對偶基因變異體。抗體或抗原結合抗體片段結合至TDP-43之能力可使用例如表面電漿子共振來確定。Unless otherwise apparent from the context, references to TDP-43 or fragments thereof include the native human amino acid sequence, including isoforms, mutants, and allelic variants thereof. The ability of an antibody or antigen-binding antibody fragment to bind to TDP-43 can be determined using, for example, surface plasmon resonance.
III.III.抗anti-TDP-43TDP-43細胞穿透劑Cell penetrants
本揭示內容提供細胞穿透劑(如本文所述)，其包括細胞內化部分及特異性結合至TDP-43 (例如，人類TDP-43)之抗體或其抗原結合片段。在一些實施例中，本文所述之抗體或抗原結合抗體片段特異性結合至磷酸化TDP-43 (例如，磷酸化人類TDP-43)。在一些實施例中，本文所述之抗體或抗原結合抗體片段特異性結合磷酸化TDP-43 (例如，磷酸化人類TDP-43)，其中SEQ ID NO: 82之位置409及/或410處的絲胺酸中之一者或兩者經磷酸化。在一些實施例中，抗體或抗原結合抗體片段結合包含SEQ ID NO: 82之位置392至位置414的胺基酸的23個胺基酸之肽(「TDP-43 (pS409/pS410)」)，其中在位置409及410處之絲胺酸經磷酸化。The present disclosure provides cell penetrating agents (as described herein) comprising a cell internalizing portion and an antibody or antigen-binding fragment thereof that specifically binds to TDP-43 (e.g., human TDP-43). In some embodiments, the antibodies or antigen-binding antibody fragments described herein specifically bind to phosphorylated TDP-43 (e.g., phosphorylated human TDP-43). In some embodiments, the antibodies or antigen-binding antibody fragments described herein specifically bind to phosphorylated TDP-43 (e.g., phosphorylated human TDP-43), wherein one or both of the serines at positions 409 and/or 410 of SEQ ID NO: 82 are phosphorylated. In some embodiments, the antibody or antigen-binding antibody fragment binds to a 23-amino acid peptide comprising amino acids from position 392 to position 414 of SEQ ID NO: 82 ("TDP-43 (pS409/pS410)"), wherein the serines at positions 409 and 410 are phosphorylated.
此外，本揭示內容係關於包括細胞內化部分及特異性結合至人類TDP-43之抗體的細胞穿透劑、包含此等細胞穿透劑之組合物，及使用此等細胞穿透劑來治療TDP-43相關疾病包括肌肉萎縮性脊髓側索硬化症(ALS)之方法。Additionally, the present disclosure relates to cell penetrating agents comprising a cell-internalizing moiety and an antibody that specifically binds to human TDP-43, compositions comprising such cell penetrating agents, and methods of using such cell penetrating agents to treat TDP-43-related diseases, including amyotrophic lateral sclerosis (ALS).
在本揭示內容通篇中，對抗TDP-43 CPA及/或抗TDP-43抗體之提及將在細胞內化及/或細胞內功能之背景下進行，但一般熟習此項技術者將理解此類提及包括完整抗TDP-43 CPA及/或抗TDP-43抗體、經化學修飾(例如，氧化、還原)之抗TDP-43 CPA及/或抗TDP-43抗體，以及其部分降解之功能片段(完整抗TDP-43 CPA、CPA片段、完整抗TDP-43抗體及/或抗TDP-43抗體片段等)。因此，對抗TDP-43 CPA或抗TDP-43抗體之內化、細胞液轉移及靶標接合之提及將包括對完整抗TDP-43 CPA及/或抗TDP-43抗體、經化學修飾之抗TDP-43 CPA及/或抗TDP-43抗體及其部分降解之功能片段之提及。舉例而言，本揭示內容之方法可指抗TDP-43抗體之細胞液之內化及/或轉移，但應理解，此類提及涵蓋完整抗TDP-43 CPA之轉移及/或抗TDP-43 CPA之功能片段，其包含抗TDP-43抗體或其一部分，以及其經化學修飾之衍生物。Throughout this disclosure, references to anti-TDP-43 CPA and/or anti-TDP-43 antibodies will be made in the context of cellular internalization and/or intracellular function, but one of ordinary skill in the art will understand that such references include intact anti-TDP-43 CPA and/or anti-TDP-43 antibodies, chemically modified (e.g., oxidized, reduced) anti-TDP-43 CPA and/or anti-TDP-43 antibodies, and partially degraded functional fragments thereof (intact anti-TDP-43 CPA, CPA fragments, intact anti-TDP-43 antibodies and/or anti-TDP-43 antibody fragments, etc.). Thus, references to internalization, cytosolic translocation, and target engagement of anti-TDP-43 CPA or anti-TDP-43 antibodies will include references to intact anti-TDP-43 CPA and/or anti-TDP-43 antibodies, chemically modified anti-TDP-43 CPA and/or anti-TDP-43 antibodies, and partially degraded functional fragments thereof. For example, the methods of the present disclosure may refer to cytosolic internalization and/or translocation of anti-TDP-43 antibodies, but it is understood that such references encompass translocation of intact anti-TDP-43 CPA and/or functional fragments of anti-TDP-43 CPA, including anti-TDP-43 antibodies or portions thereof, as well as chemically modified derivatives thereof.
本揭示內容之細胞穿透劑可利用若干生化過程中之一或多者來達成抗TDP-43抗體之內化。在各種實施例中，本揭示內容之抗TDP-43 CPA可利用被動內化、主動內化或其組合。在一些實施例中，抗TDP-43 CPA利用主動內化(例如，胞吞作用)來達成抗TDP-43抗體之細胞內遞送。The cell-penetrating agents of the present disclosure can utilize one or more of several biochemical processes to achieve internalization of the anti-TDP-43 antibody. In various embodiments, the anti-TDP-43 CPAs of the present disclosure can utilize passive internalization, active internalization, or a combination thereof. In some embodiments, the anti-TDP-43 CPA utilizes active internalization (e.g., endocytosis) to achieve intracellular delivery of the anti-TDP-43 antibody.
在各種實施例中，抗TDP-43 CPA經由胞吞作用內化至細胞中。內化後，抗TDP-43 CPA實現胞內體逃逸。在此類實施例中，抗TDP-43 CPA由此轉移至細胞液，其中抗TDP-43抗體可結合至TDP-43 (例如，磷酸-TDP-43)。在一些實施例中，抗TDP-43 CPA之胞內體逃逸係藉由CIM與胞內體膜之相互作用來達成，由此導致胞內體膜之破壞。CIM可利用一或多種方法來達成胞內體逃逸。舉例而言，CIM可包含陽離子部分(例如，帶正電荷之胺基酸、陽離子聚合物及/或寡聚物、陽離子脂質)。在一些此類實施例中，陽離子部分可與構成胞內體膜之帶負電荷之磷脂相互作用，從而破壞胞內體膜，且達成抗TDP-43 CPA之胞內體逃逸。本揭示內容之抗TDP-43 CPA亦可包含具有兩親性部分(例如，兩親性肽)之CIM。在一些此類實施例中，兩親性部分可經由與膜脂質之疏水相互作用與胞內體膜相互作用，由此破壞胞內體膜，且達成抗TDP-43 CPA之胞內體逃逸。在抗TDP-43抗體之胞內體逃逸後，抗TDP-43抗體隨後轉移至細胞液中，在細胞液中其可結合至TDP-43 (例如磷酸-TDP-43)。CPA可利用之內化機制之其他實例詳細描述於本文中。In various embodiments, anti-TDP-43 CPA is internalized into cells via endocytosis. Following internalization, the anti-TDP-43 CPA escapes endosomal folds. In such embodiments, the anti-TDP-43 CPA is thereby translocated to the cytosol, where the anti-TDP-43 antibody can bind to TDP-43 (e.g., phospho-TDP-43). In some embodiments, endosomal folds of the anti-TDP-43 CPA are achieved through interaction of the CIM with the endosomal membrane, thereby disrupting the endosomal membrane. CIMs can achieve endosomal folds by one or more methods. For example, the CIM can comprise a cationic moiety (e.g., a positively charged amino acid, a cationic polymer and/or oligomer, or a cationic lipid). In some such embodiments, the cationic moiety can interact with negatively charged phospholipids that constitute the endosomal membrane, thereby disrupting the endosomal membrane and achieving endosomal escape of the anti-TDP-43 CPA. The anti-TDP-43 CPAs of the present disclosure may also comprise a CIM having an amphiphilic moiety (e.g., an amphiphilic peptide). In some such embodiments, the amphiphilic moiety can interact with the endosomal membrane via hydrophobic interactions with membrane lipids, thereby disrupting the endosomal membrane and achieving endosomal escape of the anti-TDP-43 CPA. Following endosomal escape of the anti-TDP-43 antibody, the anti-TDP-43 antibody subsequently translocates into the cytosol, where it can bind to TDP-43 (e.g., phospho-TDP-43). Other examples of internalization mechanisms that CPA can utilize are described in detail herein.
因此，本文提供細胞穿透劑，其包括：(i)細胞內化模組(CIM)及(ii)特異性結合至TDP-43 (例如人類TDP-43)之抗體。下文描述本揭示內容之細胞穿透劑之各個態樣，包括細胞內化部分、抗TDP-43抗體及/或視情況存在之連接子的實例。一般熟習此項技術者將理解如何組合本文所揭示之各種CIM、抗TDP-43抗體及視情況存在之連接子。此類實例僅說明本揭示內容之範圍且為非限制性的。Thus, provided herein are cell penetrants comprising: (i) a cell internalization module (CIM) and (ii) an antibody that specifically binds to TDP-43 (e.g., human TDP-43). Examples of various aspects of the cell penetrants of the present disclosure, including cell internalization moieties, anti-TDP-43 antibodies, and/or optional linkers, are described below. One of ordinary skill in the art will understand how to combine the various CIMs, anti-TDP-43 antibodies, and optional linkers disclosed herein. These examples are illustrative of the scope of the present disclosure and are non-limiting.
細胞內化模組Cell internalization module(cell internalizing module)(cell internalizing module)
如本文所用，「細胞內化模組」或「CIM」為當與抗TDP-43抗體分子共價或非共價連接時，導致將至少抗TDP-43抗體或其活性片段內化至細胞中之組合物。本文描述細胞內化模組之非限制性特徵及實例。As used herein, a "cell internalization module" or "CIM" is a composition that, when covalently or non-covalently linked to an anti-TDP-43 antibody molecule, results in the internalization of at least the anti-TDP-43 antibody or an active fragment thereof into a cell. Non-limiting features and examples of cell internalization modules are described herein.
在一些實施例中，CIM為肽(例如胺基酸序列)。在此類實例中，CIM亦可稱為如本文進一步定義之細胞膜內化肽或「CMIP」。例示性細胞膜內化肽包括天然肽及其衍生物，以及合成肽。CMIP之非限制性實例包括M-lycotoxin及其衍生物、TAT及其衍生物、PEPTH、聚精胺酸序列、Penetratin、DPT-C9h、DPT-C9、Transportan、Xentry、Pep-1、Pep-7、Aurein 1.2、MTS、GFWFG、DPV1047、MPG、pVEC、ARF(1_22)、BPrPr、MAP、p28、VT5、Bac7、C105Y、PFVYLI及BR2。In some embodiments, the CIM is a peptide (e.g., an amino acid sequence). In such embodiments, the CIM may also be referred to as a cell membrane internalization peptide or "CMIP," as further defined herein. Exemplary cell membrane internalization peptides include natural peptides and their derivatives, as well as synthetic peptides. Non-limiting examples of CMIPs include M-lycotoxin and its derivatives, TAT and its derivatives, PEPTH, polyarginine sequences, Penetratin, DPT-C9h, DPT-C9, Transportan, Xentry, Pep-1, Pep-7, Aurein 1.2, MTS, GFWFG, DPV1047, MPG, pVEC, ARF(1-22), BPrPr, MAP, p28, VT5, Bac7, C105Y, PFVYLI, and BR2.
在一些實施例中，CIM為由細胞(例如哺乳動物細胞)內化之非肽部分(例如配位體)。在此類實例中，配位體可誘導抗TDP-43抗體之受體介導之內化。通常，配位體內化為受體介導之內吞過程，其中若配位體結合至細胞表面上之其同源受體蛋白，則細胞攝取細胞外分子(包括治療劑)。受體介導之內化亦包括轉胞吞作用。In some embodiments, a CIM is a non-peptide moiety (e.g., a ligand) that is internalized by a cell (e.g., a mammalian cell). In such embodiments, the ligand can induce receptor-mediated internalization of the anti-TDP-43 antibody. Typically, ligand internalization is a receptor-mediated endocytic process in which the cell takes up an extracellular molecule (including a therapeutic agent) if the ligand binds to its cognate receptor protein on the cell surface. Receptor-mediated internalization also includes transcytosis.
CIM可實現抗TDP-43抗體內化至哺乳動物細胞中。一般而言，細胞內化過程廣泛分類為胞吞作用。典型地，胞吞作用路徑可細分為兩個更廣泛類別之吞噬作用及胞飲作用。在胞飲作用期間，質膜吸收溶質，而在吞噬作用期間，細胞內化大得多之囊泡。胞飲作用通常進一步細分為巨胞飲作用、網格蛋白依賴性內吞作用(例如，受體介導之內吞作用)、小窩蛋白依賴性內吞作用及網格蛋白/小窩蛋白非依賴性內吞作用。(參見例如Marsh, M. Endocytosis, Oxford University Press (2001)；Doherty, GJ及McMahon, H.T., Mechanisms of Endocytosis,Annu. Rev. Biochem., 78:31.1-31.46 (2009)；及Xu, Y.等人, Endocytosis and membrane receptor internalization: implication of F-BAR protein Carom,Front Biosci, 22: 1439-1457 (2017)，其各自以全文引用方式併入本文中)。CIM can achieve internalization of anti-TDP-43 antibodies into mammalian cells. Generally speaking, the cellular internalization process is broadly categorized as endocytosis. Typically, the endocytosis pathway can be subdivided into two broader categories: phagocytosis and endocytosis. During endocytosis, solutes are taken up by the plasma membrane, while during phagocytosis, cells internalize much larger vesicles. Endocytosis is often further subdivided into macrophagocytosis, clathrin-dependent endocytosis (e.g., receptor-mediated endocytosis), caveolin-dependent endocytosis, and clathrin/cavolin-independent endocytosis. (See, e.g., Marsh, M. Endocytosis, Oxford University Press (2001); Doherty, GJ and McMahon, HT, Mechanisms of Endocytosis,Annu. Rev. Biochem. , 78:31.1-31.46 (2009); and Xu, Y. et al., Endocytosis and membrane receptor internalization: implication of F-BAR protein Carom,Front Biosci , 22: 1439-1457 (2017), each of which is incorporated herein by reference in its entirety).
配位體介導之內吞作用為細胞內化特定大分子之機制。在一些實例中，細胞膜(例如質膜)包括網格蛋白凹坑，網格蛋白凹坑自細胞膜突出以形成稱為網格蛋白包被之囊泡的小囊泡。此等網格蛋白包被之囊泡含有受體及結合之大分子，即配位體。接著網格蛋白包被之囊泡與早期胞內體(由位於細胞周邊之管狀延伸組成之囊泡)融合。胞內體具有促進受體自配位體解離之酸性環境(pH 6.0-6.2)。接著，將攝取之內容物分揀出以再循環至質膜或轉運至溶酶體進行降解。Ligand-mediated endocytosis is a mechanism by which cells internalize specific macromolecules. In some cases, cell membranes (e.g., the plasma membrane) contain clathrin pits that protrude from the membrane to form small vesicles called clathrin-coated vesicles. These clathrin-coated vesicles contain receptors and bound macromolecules, namely ligands. The clathrin-coated vesicles then fuse with early endosomes (vesicles composed of tubular extensions located at the cell periphery). Endosomes have an acidic environment (pH 6.0-6.2) that promotes the dissociation of receptors from their ligands. The taken-up contents are then sorted out for recycling to the plasma membrane or transported to lysosomes for degradation.
基於肽之CIM (例如CMIP)經由多種機制使抗TDP-43抗體內化。一般而言，已顯示CMIP使用如上所述之胞吞作用(例如能量依賴性內化)或直接穿透(例如易位) (能量非依賴性內化)作為兩種主要內化機制。對於直接滲透，已描述各種機制，包括地毯樣模型(膜去穩定)及孔形成模型(桶-板條)。帶正電荷之CMIP可與帶負電荷之膜組分(諸如磷脂雙層)相互作用，隨後使膜去穩定，且CMIP與抗TDP-43抗體穿過脂質雙層。研究已顯示，若干CMIP能夠根據其濃度、貨物及/或所用細胞株誘導不同攝取機制且在不同攝取機制之間轉換(參見例如Ruseska, I.及Zimmer, A., Internalization mechanisms of cell-penetrating peptides,Beilstein J Nanotechnol, 11: 101-123, (2020))。Peptide-based CIMs (e.g., CMIP) internalize anti-TDP-43 antibodies via a variety of mechanisms. Generally, CMIP has been shown to utilize either endocytosis (e.g., energy-dependent internalization) or direct penetration (e.g., translocation) (energy-independent internalization) as two major internalization mechanisms. For direct penetration, various mechanisms have been described, including the carpet-like model (membrane destabilization) and the pore-forming model (barrel-slat). Positively charged CMIP can interact with negatively charged membrane components (e.g., the phospholipid bilayer), subsequently destabilizing the membrane, and CMIP and the anti-TDP-43 antibody permeate the lipid bilayer. Studies have shown that some CMIPs can induce different uptake mechanisms and switch between different uptake mechanisms depending on their concentration, cargo, and/or cell line used (see, for example, Ruseska, I. and Zimmer, A., Internalization mechanisms of cell-penetrating peptides,Beilstein J Nanotechnol , 11: 101-123, (2020)).
一旦內化至細胞中，抗TDP-43抗體通常需要逃避胞內體路徑。一般而言，哺乳動物細胞之內吞路徑由不同的膜區室組成，該等膜區室使來自質膜之分子(即細胞穿透劑)內化且將膜結合受體再循環回表面或將內化之分子分選至各種降解路徑。內吞路徑之主要組分包括早期胞內體，其為內吞路徑之第一區室。早期胞內體通常位於細胞周邊且接受來自細胞表面之大多數類型之囊泡。該等早期胞內體具有特徵性微管囊泡結構及弱酸性pH值。早期胞內體主要為分選細胞器，其中許多內吞配位體在區室之酸性pH值中與其受體解離且再循環至細胞表面。早期胞內體亦經由跨囊泡區室分選到至後期區室(例如晚期胞內體或溶酶體)之轉胞吞路徑。Once internalized into the cell, anti-TDP-43 antibodies typically need to escape the endosomal pathway. Generally speaking, the endocytic pathway of mammalian cells is composed of distinct membrane compartments that internalize molecules (i.e., cell-permeable agents) from the plasma membrane and recycle membrane-bound receptors back to the surface or sort internalized molecules into various degradation pathways. A major component of the endocytic pathway is the early endosome, the first compartment of the endocytic pathway. Early endosomes are typically located at the cell periphery and accept most types of vesicles from the cell surface. These early endosomes have a characteristic microtubule-vesicle structure and a slightly acidic pH. Early endosomes are primarily sorting organelles, where many endocytosed ligands dissociate from their receptors in the acidic pH of this compartment and are recycled to the cell surface. Early endosomes also undergo transcytosis, a pathway that sorts them across the vesicular compartment to later compartments such as late endosomes or lysosomes.
晚期胞內體通常在至溶酶體之路徑中接受內吞材料，通常來自內吞路徑中之早期胞內體、來自生物合成路徑中之反式高爾基體網路(TGN)及來自吞噬細胞路徑中之吞噬體。晚期胞內體為酸性的(大約pH 5.5)，且通常被認為在將材料遞送至溶酶體之前介導最終分選。溶酶體為內吞路徑之最後區室。溶酶體將細胞廢物、脂肪、碳水化合物、蛋白質及其他大分子分解成簡單化合物，該等化合物作為新的細胞構建材料返回細胞質。溶酶體包括許多不同類型之水解酶，其在酸性環境(例如，大約4.8之pH)中起作用。Late endosomes typically receive endocytosed material on the pathway to lysosomes, typically from early endosomes in the endocytic pathway, from the trans-Golgi network (TGN) in the biosynthetic pathway, and from phagosomes in the phagocytic pathway. Late endosomes are acidic (approximately pH 5.5) and are generally thought to mediate final sorting before delivering material to lysosomes. Lysosomes are the final compartment of the endocytic pathway. Lysosomes break down cellular waste products, fats, carbohydrates, proteins, and other macromolecules into simple compounds that return to the cytoplasm as new cellular building blocks. Lysosomes contain many different types of hydrolases that function in an acidic environment (e.g., a pH of approximately 4.8).
在一些實施例中，CIM (如本文所定義)包括細胞膜內化肽(例如CMIP4，SEQ ID NO: 176)。在一些實施例中，CIM包括野生型M-lycotoxin肽(SEQ ID NO: 182)。在一些實施例中，CIM包括M-lycotoxin衍生物(例如，SEQ ID NO: 183)。在一些實施例中，CIM包括Penetain胺基酸序列或其衍生物(例如，SEQ ID NO: 187)。在一些實施例中，CIM包括Pepth胺基酸序列或其衍生物(例如，SEQ ID NO: 184)。在一些實施例中，CIM包括聚精胺酸胺基酸序列(例如，SEQ ID NO: 190、SEQ ID NO: 191及/或SEQ ID NO: 192)。在一些實施例中，CIM包括多於一個聚精胺酸胺基酸序列(例如，2、3、4、5個或更多個聚精胺酸胺基酸序列)。在一些實施例中，CIM包括三個聚精胺酸胺基酸序列。在一些實施例中，CIM包括TAT胺基酸序列(例如，SEQ ID NO: 181、SEQ ID NO: 186、SEQ ID NO: 189及/或SEQ ID NO: 193)。在一些實施例中，CIM包括多於一個TAT胺基酸序列或其衍生物(例如，2、3、4、5個或更多個TAT胺基酸序列或其衍生物)。在一些實施例中，CIM包括三個TAT胺基酸序列。In some embodiments, a CIM (as defined herein) comprises a cell membrane internalization peptide (e.g., CMIP4, SEQ ID NO: 176). In some embodiments, a CIM comprises a wild-type M-lycotoxin peptide (SEQ ID NO: 182). In some embodiments, a CIM comprises an M-lycotoxin derivative (e.g., SEQ ID NO: 183). In some embodiments, a CIM comprises a Penetain amino acid sequence or a derivative thereof (e.g., SEQ ID NO: 187). In some embodiments, a CIM comprises a Pepth amino acid sequence or a derivative thereof (e.g., SEQ ID NO: 184). In some embodiments, a CIM comprises a polyarginine amino acid sequence (e.g., SEQ ID NO: 190, SEQ ID NO: 191, and/or SEQ ID NO: 192). In some embodiments, CIM includes more than one polyarginine amino acid sequence (e.g., 2, 3, 4, 5 or more polyarginine amino acid sequences). In some embodiments, CIM includes three polyarginine amino acid sequences. In some embodiments, CIM includes a TAT amino acid sequence (e.g., SEQ ID NO: 181, SEQ ID NO: 186, SEQ ID NO: 189 and/or SEQ ID NO: 193). In some embodiments, CIM includes more than one TAT amino acid sequence or a derivative thereof (e.g., 2, 3, 4, 5 or more TAT amino acid sequences or derivatives thereof). In some embodiments, CIM includes three TAT amino acid sequences.
在一些實施例中，CIM包括大環。通常，大環為含有十二個或更多個原子之環的分子及離子。經典實例包括冠醚(crown ether)、杯芳烴(calixarene)、紫質(porphyrin)及環糊精(cyclodextrin)。在一些實施例中，大環由CIM之兩個胺基酸殘基之間的共價鍵形成。在一些實施例中，大環由CIM之兩個半胱胺酸殘基之間的二硫鍵形成。在一些實施例中，CIM包括一或多個組胺酸殘基。In some embodiments, a CIM comprises a macrocycle. Generally, a macrocycle is a molecule or ion containing a ring of twelve or more atoms. Classic examples include crown ethers, calixarenes, porphyrins, and cyclodextrins. In some embodiments, a macrocycle is formed by a covalent bond between two amino acid residues of a CIM. In some embodiments, a macrocycle is formed by a disulfide bond between two cysteine residues of a CIM. In some embodiments, a CIM comprises one or more histidine residues.
在一些實施例中，CIM包括具有選自SEQ ID NO: 176-193中之任一者的胺基酸序列之多肽。在一些實施例中，CIM為具有選自SEQ ID NO: 176-193中之任一者的胺基酸序列之多肽。In some embodiments, the CIM comprises a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 176-193. In some embodiments, the CIM is a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 176-193.
在一些實施例中，CIM共價連接至抗體或其抗原結合抗體片段。在一些實施例中，CIM非共價連接至抗體或其抗原結合抗體片段。在一些實施例中，細胞穿透劑包括將CIM連接至抗體之連接子。在一些實施例中，連接子共價連接至CIM及抗體兩者。在一些實施例中，連接子為可裂解連接子(例如，光可裂解連接子、化學連接子、酶可裂解連接子等)。在一些實施例中，連接子為不可裂解連接子。在一些實施例中，連接子包括多肽。在一些實施例中，連接子包括一或多個甘胺酸殘基(例如，2、3、4、5個或更多個甘胺酸殘基)。在一些實例中，連接子為甘胺酸殘基，之後為絲胺酸殘基(GS)。在一些實施例中，連接子包括包含選自SEQ ID NO: 194-199中之任一者的胺基酸序列之多肽。在一些實施例中，連接子為具有選自SEQ ID NO: 194-199中之任一者的胺基酸序列之多肽。In some embodiments, the CIM is covalently linked to an antibody or an antigen-binding antibody fragment thereof. In some embodiments, the CIM is non-covalently linked to an antibody or an antigen-binding antibody fragment thereof. In some embodiments, the cell penetrant comprises a linker that links the CIM to the antibody. In some embodiments, the linker is covalently linked to both the CIM and the antibody. In some embodiments, the linker is a cleavable linker (e.g., a photocleavable linker, a chemical linker, an enzyme-cleavable linker, etc.). In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker comprises a polypeptide. In some embodiments, the linker comprises one or more glycine residues (e.g., 2, 3, 4, 5, or more glycine residues). In some embodiments, the linker is a glycine residue followed by a serine residue (GS). In some embodiments, the linker comprises a polypeptide comprising an amino acid sequence selected from any one of SEQ ID NOs: 194-199. In some embodiments, the linker is a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 194-199.
在一些實施例中，細胞穿透劑之抗體或抗原結合抗體片段連接至CIM之C末端。在一些實施例中，細胞穿透劑之抗體或抗原結合抗體片段連接至CIM之N末端。在一些實施例中，細胞穿透劑之抗體或抗原結合抗體片段連接至CIM重鏈之C末端。在一些實施例中，細胞穿透劑之抗體或抗原結合抗體片段連接至CIM重鏈之N末端。在一些實施例中，細胞穿透劑之抗體或抗原結合抗體片段連接至CIM輕鏈之C末端。在一些實施例中，細胞穿透劑之抗體或抗原結合抗體片段連接至CIM輕鏈之N末端。In some embodiments, the cell penetrating agent antibody or antigen-binding antibody fragment is linked to the C-terminus of CIM. In some embodiments, the cell penetrating agent antibody or antigen-binding antibody fragment is linked to the N-terminus of CIM. In some embodiments, the cell penetrating agent antibody or antigen-binding antibody fragment is linked to the C-terminus of the CIM heavy chain. In some embodiments, the cell penetrating agent antibody or antigen-binding antibody fragment is linked to the N-terminus of the CIM heavy chain. In some embodiments, the cell penetrating agent antibody or antigen-binding antibody fragment is linked to the C-terminus of the CIM light chain. In some embodiments, the cell penetrating agent antibody or antigen-binding antibody fragment is linked to the N-terminus of the CIM light chain.
在一些實施例中，CIM包括一或多個間隔區。在一些實施例中，CIM不包括間隔區。舉例而言，包含選自以下之多肽胺基酸序列之CIM不包括間隔區：SEQ ID NO: 176-184、SEQ ID NO: 192及SEQ ID NO: 193。In some embodiments, the CIM comprises one or more spacers. In some embodiments, the CIM does not comprise a spacer. For example, a CIM comprising a polypeptide amino acid sequence selected from the group consisting of SEQ ID NOs: 176-184, SEQ ID NO: 192, and SEQ ID NO: 193 does not comprise a spacer.
在一些實施例中，CIM包括一或多個間隔區。在一些實施例中，一或多個間隔區中之至少一者包括一或多個胺基酸殘基。In some embodiments, the CIM comprises one or more spacers. In some embodiments, at least one of the one or more spacers comprises one or more amino acid residues.
如本文所用，間隔子係指在哺乳動物細胞中不具有催化或治療活性之胺基酸。舉例而言，間隔子可為1個胺基酸至約10個胺基酸(例如1至約8個胺基酸、1至約6個胺基酸或1至約4個胺基酸)之肽。在一些實例中，間隔區包括在細胞內化模組序列中。舉例而言，間隔區可分離細胞內化模組(CIM)內之胺基酸，例如，間隔子可安置在CIM之第一個胺基酸之後。在一些實例中，間隔子可安置在CIM之最後的胺基酸之前。在一些實例中，CIM可具有一或多個間隔區(例如，兩個間隔區、三個間隔序列、四個間隔區、五個間隔區或更多)。在一些實例中，間隔區為單個甘胺酸殘基。在一些實例中，間隔區為一對甘胺酸殘基。在一些實例中，間隔區為三個甘胺酸殘基。在一些實例中，間隔區為四個甘胺酸殘基。在一些實例中，間隔區為四個甘胺酸殘基，之後為絲胺酸殘基。在一些實施例中，一或多個間隔區中之至少一者包括選自SEQ ID NO: 200-203中之任一者的胺基酸序列。在一些實施例中，一或多個間隔區中之每一者包括選自SEQ ID NO: 200-203中之任一者的胺基酸序列。As used herein, a spacer refers to an amino acid that does not have catalytic or therapeutic activity in mammalian cells. For example, a spacer can be a peptide of 1 to about 10 amino acids (e.g., 1 to about 8 amino acids, 1 to about 6 amino acids, or 1 to about 4 amino acids). In some examples, a spacer is included in a cell internalization module sequence. For example, a spacer can separate amino acids within a cell internalization module (CIM), for example, a spacer can be placed after the first amino acid of a CIM. In some examples, a spacer can be placed before the last amino acid of a CIM. In some examples, a CIM can have one or more spacers (e.g., two spacers, three spacer sequences, four spacers, five spacers, or more). In some instances, the spacer is a single glycine residue. In some instances, the spacer is a pair of glycine residues. In some instances, the spacer is three glycine residues. In some instances, the spacer is four glycine residues. In some instances, the spacer is four glycine residues followed by a serine residue. In some embodiments, at least one of the one or more spacers comprises an amino acid sequence selected from any one of SEQ ID NOs: 200-203. In some embodiments, each of the one or more spacers comprises an amino acid sequence selected from any one of SEQ ID NOs: 200-203.
連接子connector
在本揭示內容之各個態樣中，抗TDP-43細胞穿透劑進一步包含將CIM連接至抗TDP-43抗體之連接子。本揭示內容之連接子可經由共價鍵聯或非共價鍵聯將CIM連接至抗TDP-43抗體。在一些實施例中，CIM經由連接子(亦即共價連接子)共價連接至抗TDP-43抗體。在一些實施例中，CIM經由連接子(亦即非共價連接子)非共價連接至抗TDP-43抗體。在一些實施例中，CIM共價連接至連接子。在一些實施例中，抗TDP-43抗體共價連接至連接子。在一些實施例中，連接子共價連接至CPP及抗TDP-43抗體兩者。In various aspects of the present disclosure, the anti-TDP-43 cell penetrant further comprises a linker that links the CIM to the anti-TDP-43 antibody. The linker of the present disclosure can link the CIM to the anti-TDP-43 antibody via a covalent or non-covalent linkage. In some embodiments, the CIM is covalently linked to the anti-TDP-43 antibody via a linker (i.e., a covalent linker). In some embodiments, the CIM is non-covalently linked to the anti-TDP-43 antibody via a linker (i.e., a non-covalent linker). In some embodiments, the CIM is covalently linked to the linker. In some embodiments, the anti-TDP-43 antibody is covalently linked to the linker. In some embodiments, the linker is covalently linked to both the CPP and the anti-TDP-43 antibody.
在一些實施例中，連接子為非共價連接子。非共價鍵聯可使用以非共價方式強烈相互作用(例如，藉由氫鍵結、離子鍵結、凡得瓦相互作用或其任何組合)之親和對來達成。非共價鍵聯之多個實例為此項技術中已知的。舉例而言，生物素及生物素結合劑(例如，鏈黴抗生物素蛋白)為親和對之一個實例。舉例而言，藉由將生物素連接至CPA之一側(例如，CIM)，且將生物素結合劑連接至CPA之另一側(例如，抗TDP-43抗體)，非共價鍵聯可在CIM與抗TDP-43抗體之間達成。在一些實施例中，連接子包含一對親和域(例如，該對親和域之第一域可為介白素-15，且該對親和域之第二域可為介白素-15受體α之sushi域)。In some embodiments, the linker is a non-covalent linker. Non-covalent bonding can be achieved using an affinity pair that strongly interacts non-covalently (e.g., via hydrogen bonding, ionic bonding, van der Waals interactions, or any combination thereof). Numerous examples of non-covalent bonding are known in the art. For example, biotin and a biotin-binding agent (e.g., streptavidin) are one example of an affinity pair. For example, by linking biotin to one side of a CPA (e.g., a CIM) and linking a biotin-binding agent to the other side of the CPA (e.g., an anti-TDP-43 antibody), non-covalent bonding can be achieved between the CIM and the anti-TDP-43 antibody. In some embodiments, the linker comprises a pair of affinity domains (eg, the first domain of the pair of affinity domains may be interleukin-15, and the second domain of the pair of affinity domains may be the sushi domain of interleukin-15 receptor α).
在一些實施例中，連接子為共價連接子。許多共價連接子為此項技術中已知的。例如，在一些實施例中，共價連接子包含有機連接子(例如伸烷基鏈、聚乙二醇鏈、聚丙烯醯胺、聚丙烯酸、聚乙烯醇或聚乙烯亞胺鏈)。在一些情況下，共價連接子包含未經取代或經取代之伸烷基鏈(包括例如聚乙烯醇鏈、聚丙烯醯胺鏈或聚丙烯酸鏈)。在一些情況下，共價連接子包含未經取代或經取代之伸雜烷基鏈(例如，聚乙二醇鏈或聚乙烯亞胺鏈)。在各種實施例中，連接子為直鏈連接子或分支連接子。在一些此類實施例中，分支連接子允許將兩種或更多種CIM及/或抗TDP-43抗體併入CPA (例如，樹枝狀聚合物連接子結構)中。In some embodiments, the linker is a covalent linker. Many covalent linkers are known in the art. For example, in some embodiments, the covalent linker comprises an organic linker (e.g., an alkylene chain, a polyethylene glycol chain, a polyacrylamide, polyacrylic acid, polyvinyl alcohol, or a polyethylene imine chain). In some cases, the covalent linker comprises an unsubstituted or substituted alkylene chain (including, for example, a polyvinyl alcohol chain, a polyacrylamide chain, or a polyacrylic acid chain). In some cases, the covalent linker comprises an unsubstituted or substituted heteroalkylene chain (e.g., a polyethylene glycol chain or a polyethylene imine chain). In various embodiments, the linker is a linear linker or a branched linker. In some such embodiments, a branched linker allows for the incorporation of two or more CIMs and/or anti-TDP-43 antibodies into a CPA (e.g., a dendrimer linker structure).
在一些實施例中，連接子包含胺基酸殘基。在一些實施例中，連接子包含多肽。在一些例示性實施例中，連接子可為約1個胺基酸至約50個胺基酸(例如，約1個胺基酸至約40個或約1個胺基酸至約30個胺基酸)之肽。在一些例示性實施例中，連接子可為約1個胺基酸至約25個胺基酸(例如，約1個胺基酸至約20個或約1個胺基酸至約12個胺基酸)之肽。在一些例示性實施例中，連接子可為約1個胺基酸至約10個胺基酸(例如，約1個胺基酸至約6個或約1個胺基酸至約7個胺基酸)之肽。在一些例示性實施例中，連接子可為約1個胺基酸至約5個胺基酸(例如，約1個胺基酸至約4個或約1個胺基酸至約3個胺基酸)之肽。在一些例示性實施例中，連接子可為約3個胺基酸至約20個胺基酸(例如，約3個胺基酸至約15個或約3個胺基酸至約12個胺基酸)之肽。在一些例示性實施例中，連接子可為約3個胺基酸至約10個胺基酸(例如，約3個胺基酸至約8個或約3個胺基酸至約6個胺基酸)之肽。In some embodiments, the linker comprises an amino acid residue. In some embodiments, the linker comprises a polypeptide. In some exemplary embodiments, the linker may be a peptide of about 1 amino acid to about 50 amino acids (e.g., about 1 amino acid to about 40 or about 1 amino acid to about 30 amino acids). In some exemplary embodiments, the linker may be a peptide of about 1 amino acid to about 25 amino acids (e.g., about 1 amino acid to about 20 or about 1 amino acid to about 12 amino acids). In some exemplary embodiments, the linker may be a peptide of about 1 amino acid to about 10 amino acids (e.g., about 1 amino acid to about 6 or about 1 amino acid to about 7 amino acids). In some exemplary embodiments, the linker may be a peptide of about 1 to about 5 amino acids (e.g., about 1 to about 4 or about 1 to about 3 amino acids). In some exemplary embodiments, the linker may be a peptide of about 3 to about 20 amino acids (e.g., about 3 to about 15 or about 3 to about 12 amino acids). In some exemplary embodiments, the linker may be a peptide of about 3 to about 10 amino acids (e.g., about 3 to about 8 or about 3 to about 6 amino acids).
在一些實施例中，連接子包含甘胺酸殘基。在一些實施例中，連接子包含兩個或更多個甘胺酸殘基。在一些實施例中，連接子包含兩個或更多個連續甘胺酸殘基(例如，二至三個連續甘胺酸殘基、二至四個連續甘胺酸殘基、二至五個連續甘胺酸殘基或二至六個連續甘胺酸殘基。在一些實施例中，連接子包含兩個、三個、四個、五個或六個連續甘胺酸殘基。在一些實施例中，連接子包含絲胺酸殘基。在一些實施例中，連接子包含選自GS、GGG、GGGGS、GGGSGGGS及GGGGSGGGGS之胺基酸序列。在一些實施例中，連接子包含GGGGS之胺基酸序列。在一些實施例中，連接子包含GGGSGGGS之胺基酸序列。In some embodiments, the linker comprises a glycine residue. In some embodiments, the linker comprises two or more glycine residues. In some embodiments, the linker comprises two or more consecutive glycine residues (e.g., two to three consecutive glycine residues, two to four consecutive glycine residues, two to five consecutive glycine residues, or two to six consecutive glycine residues. In some embodiments, the linker comprises two, three, four, five, or six consecutive glycine residues. In some embodiments, the linker comprises a serine residue. In some embodiments, the linker comprises an amino acid sequence selected from GS, GGG, GGGGS, GGGSGGGS, and GGGGSGGGGS. In some embodiments, the linker comprises an amino acid sequence of GGGGS. In some embodiments, the linker comprises an amino acid sequence of GGGSGGGS.
抗anti-TDP-43TDP-43抗體antibody
本揭示內容之細胞穿透劑及方法促進多種抗TDP-43抗體之細胞內化及/或細胞液釋放。在一些實施例中，抗TDP-43抗體為人源化抗體、嵌合抗體或飾面抗體(如本文所述)。互補決定區(「CDR」)可由不同系統定義。舉例而言，本文所述之CDR可選自Kabat、Chothia、Kabat/Chothia Composite、AbM及Contact之群組。The cell-penetrating agents and methods disclosed herein promote cellular internalization and/or cytosolic release of various anti-TDP-43 antibodies. In some embodiments, the anti-TDP-43 antibodies are humanized, chimeric, or veneered antibodies (as described herein). Complementary determining regions (CDRs) can be defined using various systems. For example, the CDRs described herein can be selected from the Kabat, Chothia, Kabat/Chothia Composite, AbM, and Contact groups.
在一些實施例中，細胞穿透劑之抗體包括人源化成熟重鏈可變域，其包括：如由Kabat/Chothia Composite所定義之重鏈CDR1，其包括SEQ ID NO: 49；如由Kabat所定義之重鏈CDR2，其包括SEQ ID NO: 51；及如由Kabat或Chothia所定義之重鏈CDR3，其包括SEQ ID NO: 52；以及人源化成熟輕鏈可變域，其包括SEQ ID NO: 53-55之三個Kabat輕鏈CDR。In some embodiments, the cell penetrant antibody comprises a humanized mature heavy chain variable domain comprising: a heavy chain CDR1 as defined by the Kabat/Chothia Composite comprising SEQ ID NO: 49; a heavy chain CDR2 as defined by Kabat comprising SEQ ID NO: 51; and a heavy chain CDR3 as defined by Kabat or Chothia comprising SEQ ID NO: 52; and a humanized mature light chain variable domain comprising the three Kabat light chain CDRs of SEQ ID NOs: 53-55.
在一些實施例中，抗體之人源化成熟重鏈可變域包括與SEQ ID NO: 4至23中任一者至少80%一致的序列。在一些實施例中，人源化成熟輕鏈可變域包括與SEQ ID NO: 27至48中之任一者至少80%一致的序列。在一些實施例中，抗體或其抗原結合片段包括人源化成熟重鏈可變域，其包括與SEQ ID NO: 4-23中之任一者至少80%一致的序列；及人源化成熟輕鏈可變域，其包括與SEQ ID NO: 27-48中之任一者至少80%一致的序列。In some embodiments, the humanized mature heavy chain variable domain of the antibody comprises a sequence that is at least 80% identical to any one of SEQ ID NOs: 4 to 23. In some embodiments, the humanized mature light chain variable domain comprises a sequence that is at least 80% identical to any one of SEQ ID NOs: 27 to 48. In some embodiments, the antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain comprising a sequence that is at least 80% identical to any one of SEQ ID NOs: 4-23; and a humanized mature light chain variable domain comprising a sequence that is at least 80% identical to any one of SEQ ID NOs: 27-48.
在一些實施例中，抗體之人源化成熟重鏈可變域包括與SEQ ID NO: 4至23中之任一者至少85%一致的序列。在一些實施例中，抗體之人源化成熟輕鏈可變域包括與SEQ ID NO: 27至48中之任一者至少85%一致的序列。在一些實施例中，抗體或其抗原結合片段包括人源化成熟重鏈可變域，其包括與SEQ ID NO: 4至23中之任一者至少85%一致的序列；及人源化成熟輕鏈可變域，其包括與SEQ ID NO: 27至48中之任一者至少85%一致的序列。In some embodiments, the humanized mature heavy chain variable domain of the antibody comprises a sequence that is at least 85% identical to any one of SEQ ID NOs: 4 to 23. In some embodiments, the humanized mature light chain variable domain of the antibody comprises a sequence that is at least 85% identical to any one of SEQ ID NOs: 27 to 48. In some embodiments, the antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain comprising a sequence that is at least 85% identical to any one of SEQ ID NOs: 4 to 23; and a humanized mature light chain variable domain comprising a sequence that is at least 85% identical to any one of SEQ ID NOs: 27 to 48.
在一些實施例中，抗體之人源化成熟重鏈可變域包括與SEQ ID NO: 4至23中之任一者至少90%一致的序列。在一些實施例中，抗體之人源化成熟輕鏈可變域包括與SEQ ID NO: 27至48中之任一者至少90%一致的序列。在一些實施例中，抗體或其抗原結合片段包括人源化成熟重鏈可變域，其包含與SEQ ID NO: 4至23中之任一者至少90%一致的序列；及人源化成熟輕鏈可變域，其包含與SEQ ID NO: 27至48中之任一者至少90%一致的序列。In some embodiments, the humanized mature heavy chain variable domain of the antibody comprises a sequence that is at least 90% identical to any one of SEQ ID NOs: 4 to 23. In some embodiments, the humanized mature light chain variable domain of the antibody comprises a sequence that is at least 90% identical to any one of SEQ ID NOs: 27 to 48. In some embodiments, the antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain comprising a sequence that is at least 90% identical to any one of SEQ ID NOs: 4 to 23; and a humanized mature light chain variable domain comprising a sequence that is at least 90% identical to any one of SEQ ID NOs: 27 to 48.
在一些實施例中，抗體之人源化成熟重鏈可變域包括與SEQ ID NO: 4至23中之任一者至少95%一致的序列。在一些實施例中，抗體之人源化成熟輕鏈可變域包括與SEQ ID NO: 27至48中之任一者至少95%一致的序列。在一些實施例中，抗體或其抗原結合片段包括人源化成熟重鏈可變域，其包括與SEQ ID NO: 4至23中之任一者至少95%一致的序列；及人源化成熟輕鏈可變域，其包括與SEQ ID NO: 27至48中之任一者至少95%一致的序列。In some embodiments, the humanized mature heavy chain variable domain of the antibody comprises a sequence that is at least 95% identical to any one of SEQ ID NOs: 4 to 23. In some embodiments, the humanized mature light chain variable domain of the antibody comprises a sequence that is at least 95% identical to any one of SEQ ID NOs: 27 to 48. In some embodiments, the antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain comprising a sequence that is at least 95% identical to any one of SEQ ID NOs: 4 to 23; and a humanized mature light chain variable domain comprising a sequence that is at least 95% identical to any one of SEQ ID NOs: 27 to 48.
在一些實施例中，人源化成熟重鏈可變域包括SEQ ID NO: 4至23中之一者的序列。在一些實施例中，人源化成熟重鏈可變域包括包含SEQ ID NO: 4之序列。在一些實施例中，人源化成熟重鏈可變域包括包含SEQ ID NO: 5之序列。在一些實施例中，人源化成熟重鏈可變域包括包含SEQ ID NO: 6之序列。在一些實施例中，人源化成熟重鏈可變域包括包含SEQ ID NO: 7之序列。在一些實施例中，人源化成熟重鏈可變域包括包含SEQ ID NO: 8之序列。在一些實施例中，人源化成熟重鏈可變域包括包含SEQ ID NO: 9之序列。在一些實施例中，人源化成熟重鏈可變域包括包含SEQ ID NO: 10之序列。在一些實施例中，人源化成熟重鏈可變域包括包含SEQ ID NO: 11之序列。在一些實施例中，人源化成熟重鏈可變域包括包含SEQ ID NO: 12之序列。在一些實施例中，人源化成熟重鏈可變域包括包含SEQ ID NO: 13之序列。在一些實施例中，人源化成熟重鏈可變域包括包含SEQ ID NO: 14之序列。在一些實施例中，人源化成熟重鏈可變域包括包含SEQ ID NO: 15之序列。在一些實施例中，人源化成熟重鏈可變域包括包含SEQ ID NO: 16之序列。在一些實施例中，人源化成熟重鏈可變域包括包含SEQ ID NO: 17之序列。在一些實施例中，人源化成熟重鏈可變域包括包含SEQ ID NO: 18之序列。在一些實施例中，人源化成熟重鏈可變域包括包含SEQ ID NO: 19之序列。在一些實施例中，人源化成熟重鏈可變域包括包含SEQ ID NO: 21之序列。在一些實施例中，人源化成熟重鏈可變域包括包含SEQ ID NO: 22之序列。在一些實施例中，人源化成熟重鏈可變域包括包含SEQ ID NO: 23之序列。In some embodiments, the humanized mature heavy chain variable domain comprises a sequence of one of SEQ ID NOs: 4 to 23. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence comprising SEQ ID NO: 4. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence comprising SEQ ID NO: 5. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence comprising SEQ ID NO: 6. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence comprising SEQ ID NO: 7. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence comprising SEQ ID NO: 8. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence comprising SEQ ID NO: 9. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence comprising SEQ ID NO: 10. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence comprising SEQ ID NO: 11. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence comprising SEQ ID NO: 12. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence comprising SEQ ID NO: 13. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence comprising SEQ ID NO: 14. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence comprising SEQ ID NO: 15. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence comprising SEQ ID NO: 16. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence comprising SEQ ID NO: 17. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence comprising SEQ ID NO: 18. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence comprising SEQ ID NO: 19. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence comprising SEQ ID NO: 21. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence comprising SEQ ID NO: 22. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence comprising SEQ ID NO: 23.
在一些實施例中，人源化成熟輕鏈可變域包括SEQ ID NO: 27至48中之一者的序列。在一些實施例中，人源化成熟輕鏈可變域包括包含SEQ ID NO: 27之序列。在一些實施例中，人源化成熟輕鏈可變域包括包含SEQ ID NO: 28之序列。在一些實施例中，人源化成熟輕鏈可變域包括包含SEQ ID NO: 29之序列。在一些實施例中，人源化成熟輕鏈可變域包括包含SEQ ID NO: 30之序列。在一些實施例中，人源化成熟輕鏈可變域包括包含SEQ ID NO: 31之序列。在一些實施例中，人源化成熟輕鏈可變域包括包含SEQ ID NO: 32之序列。在一些實施例中，人源化成熟輕鏈可變域包括包含SEQ ID NO: 33之序列。在一些實施例中，人源化成熟輕鏈可變域包括包含SEQ ID NO: 34之序列。在一些實施例中，人源化成熟輕鏈可變域包括包含SEQ ID NO: 35之序列。在一些實施例中，人源化成熟輕鏈可變域包括包含SEQ ID NO: 36之序列。在一些實施例中，人源化成熟輕鏈可變域包括包含SEQ ID NO: 37之序列。在一些實施例中，人源化成熟輕鏈可變域包括包含SEQ ID NO: 38之序列。在一些實施例中，人源化成熟輕鏈可變域包括包含SEQ ID NO: 39之序列。在一些實施例中，人源化成熟輕鏈可變域包括包含SEQ ID NO: 40之序列。在一些實施例中，人源化成熟輕鏈可變域包括包含SEQ ID NO: 41之序列。在一些實施例中，人源化成熟輕鏈可變域包括包含SEQ ID NO: 42之序列。在一些實施例中，人源化成熟輕鏈可變域包括包含SEQ ID NO: 43之序列。在一些實施例中，人源化成熟輕鏈可變域包括包含SEQ ID NO: 44之序列。在一些實施例中，人源化成熟輕鏈可變域包括包含SEQ ID NO: 45之序列。在一些實施例中，人源化成熟輕鏈可變域包括包含SEQ ID NO: 46之序列。在一些實施例中，人源化成熟輕鏈可變域包括包含SEQ ID NO: 47之序列。在一些實施例中，人源化成熟輕鏈可變域包括包含SEQ ID NO: 48之序列。In some embodiments, the humanized mature light chain variable domain comprises a sequence of one of SEQ ID NOs: 27 to 48. In some embodiments, the humanized mature light chain variable domain comprises a sequence comprising SEQ ID NO: 27. In some embodiments, the humanized mature light chain variable domain comprises a sequence comprising SEQ ID NO: 28. In some embodiments, the humanized mature light chain variable domain comprises a sequence comprising SEQ ID NO: 29. In some embodiments, the humanized mature light chain variable domain comprises a sequence comprising SEQ ID NO: 30. In some embodiments, the humanized mature light chain variable domain comprises a sequence comprising SEQ ID NO: 31. In some embodiments, the humanized mature light chain variable domain comprises a sequence comprising SEQ ID NO: 32. In some embodiments, the humanized mature light chain variable domain comprises a sequence comprising SEQ ID NO: 33. In some embodiments, the humanized mature light chain variable domain comprises a sequence comprising SEQ ID NO: 34. In some embodiments, the humanized mature light chain variable domain comprises a sequence comprising SEQ ID NO: 35. In some embodiments, the humanized mature light chain variable domain comprises a sequence comprising SEQ ID NO: 36. In some embodiments, the humanized mature light chain variable domain comprises a sequence comprising SEQ ID NO: 37. In some embodiments, the humanized mature light chain variable domain comprises a sequence comprising SEQ ID NO: 38. In some embodiments, the humanized mature light chain variable domain comprises a sequence comprising SEQ ID NO: 39. In some embodiments, the humanized mature light chain variable domain comprises a sequence comprising SEQ ID NO: 40. In some embodiments, the humanized mature light chain variable domain comprises a sequence comprising SEQ ID NO: 41. In some embodiments, the humanized mature light chain variable domain comprises a sequence comprising SEQ ID NO: 42. In some embodiments, the humanized mature light chain variable domain comprises a sequence comprising SEQ ID NO: 43. In some embodiments, the humanized mature light chain variable domain comprises a sequence comprising SEQ ID NO: 44. In some embodiments, the humanized mature light chain variable domain comprises a sequence comprising SEQ ID NO: 45. In some embodiments, the humanized mature light chain variable domain comprises a sequence comprising SEQ ID NO: 46. In some embodiments, the humanized mature light chain variable domain comprises a sequence comprising SEQ ID NO: 47. In some embodiments, the humanized mature light chain variable domain comprises a sequence comprising SEQ ID NO: 48.
本文所述抗體之人源化成熟重鏈可在其C末端包括離胺酸殘基。然而，在一些實施例中，人源化成熟重鏈(例如，本文所述之成熟重鏈中之任一者)不包括C末端離胺酸殘基。The humanized mature heavy chains of the antibodies described herein may include a lysine residue at their C-terminus. However, in some embodiments, the humanized mature heavy chain (e.g., any of the mature heavy chains described herein) does not include a C-terminal lysine residue.
在一些實施例中，抗體或其抗原結合片段包括人源化成熟重鏈可變域，其包括SEQ ID NO: 4至23中之一者的序列；及人源化成熟輕鏈可變域，其包括SEQ ID NO: 27至48中之一者的序列。In some embodiments, the antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain comprising the sequence of one of SEQ ID NOs: 4 to 23; and a humanized mature light chain variable domain comprising the sequence of one of SEQ ID NOs: 27 to 48.
在一些實施例中，細胞穿透劑之抗體或抗原結合片段包括SEQ ID NO: 4之人源化成熟重鏈可變域及SEQ ID NO: 27-48中之任一者的人源化成熟輕鏈可變域。在一些實施例中，細胞穿透劑之抗體或其抗原結合片段包括SEQ ID NO: 5之人源化成熟重鏈可變域及SEQ ID NO: 27至48中之任一者的人源化成熟輕鏈可變域。在一些實施例中，細胞穿透劑之抗體或其抗原結合片段包括SEQ ID NO: 6之人源化成熟重鏈可變域及SEQ ID NO: 27至48中之任一者的人源化成熟輕鏈可變域。在一些實施例中，細胞穿透劑之抗體或其抗原結合片段包括SEQ ID NO: 7之人源化成熟重鏈可變域及SEQ ID NO: 27至48中之任一者的人源化成熟輕鏈可變域。在一些實施例中，細胞穿透劑之抗體或其抗原結合片段包括SEQ ID NO: 8之人源化成熟重鏈可變域及SEQ ID NO: 27至48中之任一者的人源化成熟輕鏈可變域。在一些實施例中，細胞穿透劑之抗體或其抗原結合片段包括SEQ ID NO: 9之人源化成熟重鏈可變域及SEQ ID NO: 27至48中之任一者的人源化成熟輕鏈可變域。在一些實施例中，細胞穿透劑之抗體或其抗原結合片段包括SEQ ID NO: 10之人源化成熟重鏈可變域及SEQ ID NO: 27至48中之任一者的人源化成熟輕鏈可變域。在一些實施例中，細胞穿透劑之抗體或其抗原結合片段包括SEQ ID NO: 11之人源化成熟重鏈可變域及SEQ ID NO: 27至48中之任一者的人源化成熟輕鏈可變域。在一些實施例中，細胞穿透劑之抗體或其抗原結合片段包括SEQ ID NO: 12之人源化成熟重鏈可變域及SEQ ID NO: 27至48中之任一者的人源化成熟輕鏈可變域。在一些實施例中，細胞穿透劑之抗體或其抗原結合片段包括SEQ ID NO: 13之人源化成熟重鏈可變域及SEQ ID NO: 27至48中之任一者的人源化成熟輕鏈可變域。在一些實施例中，細胞穿透劑之抗體或其抗原結合片段包括SEQ ID NO: 14之人源化成熟重鏈可變域及SEQ ID NO: 27至48中之任一者的人源化成熟輕鏈可變域。在一些實施例中，細胞穿透劑之抗體或其抗原結合片段包括SEQ ID NO: 15之人源化成熟重鏈可變域及SEQ ID NO: 27至48中之任一者的人源化成熟輕鏈可變域。在一些實施例中，細胞穿透劑之抗體或其抗原結合片段包括SEQ ID NO: 16之人源化成熟重鏈可變域及SEQ ID NO: 27至48中之任一者的人源化成熟輕鏈可變域。在一些實施例中，細胞穿透劑之抗體或其抗原結合片段包括SEQ ID NO: 17之人源化成熟重鏈可變域及SEQ ID NO: 27至48中之任一者的人源化成熟輕鏈可變域。在一些實施例中，細胞穿透劑之抗體或其抗原結合片段包括SEQ ID NO: 18之人源化成熟重鏈可變域及SEQ ID NO: 27至48中之任一者的人源化成熟輕鏈可變域。在一些實施例中，細胞穿透劑之抗體或其抗原結合片段包括SEQ ID NO: 19之人源化成熟重鏈可變域及SEQ ID NO: 27至48中之任一者的人源化成熟輕鏈可變域。In some embodiments, the cell penetrating antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain of SEQ ID NO: 4 and a humanized mature light chain variable domain of any one of SEQ ID NOs: 27-48. In some embodiments, the cell penetrating antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain of SEQ ID NO: 5 and a humanized mature light chain variable domain of any one of SEQ ID NOs: 27-48. In some embodiments, the cell penetrating antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain of SEQ ID NO: 6 and a humanized mature light chain variable domain of any one of SEQ ID NOs: 27-48. In some embodiments, the cell penetrating antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain of SEQ ID NO: 7 and a humanized mature light chain variable domain of any one of SEQ ID NOs: 27 to 48. In some embodiments, the cell penetrating antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain of SEQ ID NO: 8 and a humanized mature light chain variable domain of any one of SEQ ID NOs: 27 to 48. In some embodiments, the cell penetrating antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain of SEQ ID NO: 9 and a humanized mature light chain variable domain of any one of SEQ ID NOs: 27 to 48. In some embodiments, the cell penetrating antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain of SEQ ID NO: 10 and a humanized mature light chain variable domain of any one of SEQ ID NOs: 27 to 48. In some embodiments, the cell penetrating antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain of SEQ ID NO: 11 and a humanized mature light chain variable domain of any one of SEQ ID NOs: 27 to 48. In some embodiments, the cell penetrating antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain of SEQ ID NO: 12 and a humanized mature light chain variable domain of any one of SEQ ID NOs: 27 to 48. In some embodiments, the cell penetrating antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain of SEQ ID NO: 13 and a humanized mature light chain variable domain of any one of SEQ ID NOs: 27 to 48. In some embodiments, the cell penetrating antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain of SEQ ID NO: 14 and a humanized mature light chain variable domain of any one of SEQ ID NOs: 27 to 48. In some embodiments, the cell penetrating antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain of SEQ ID NO: 15 and a humanized mature light chain variable domain of any one of SEQ ID NOs: 27 to 48. In some embodiments, the cell penetrating antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain of SEQ ID NO: 16 and a humanized mature light chain variable domain of any one of SEQ ID NOs: 27 to 48. In some embodiments, the cell penetrating antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain of SEQ ID NO: 17 and a humanized mature light chain variable domain of any one of SEQ ID NOs: 27 to 48. In some embodiments, the cell penetrating antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain of SEQ ID NO: 18 and a humanized mature light chain variable domain of any one of SEQ ID NOs: 27 to 48. In some embodiments, the cell penetrant antibody or antigen-binding fragment thereof comprises the humanized mature heavy chain variable domain of SEQ ID NO: 19 and the humanized mature light chain variable domain of any one of SEQ ID NOs: 27-48.
在一些實施例中，細胞穿透劑之抗體或其抗原結合片段包括SEQ ID NO: 20之人源化成熟重鏈可變域及SEQ ID NO: 27至48任一者的人源化成熟輕鏈可變域。在一些實施例中，細胞穿透劑之抗體或其抗原結合片段包括SEQ ID NO: 21之人源化成熟重鏈可變域及包括SEQ ID NO: 27至48中之任一者的人源化成熟輕鏈可變域。在一些實施例中，細胞穿透劑之抗體或其抗原結合片段包括SEQ ID NO: 22之人源化成熟重鏈可變域及包括SEQ ID NO: 27至48中之任一者的人源化成熟輕鏈可變域。在一些實施例中，細胞穿透劑之抗體或其抗原結合片段包括SEQ ID NO: 23之人源化成熟重鏈可變域及包括SEQ ID NO: 27至48中之任一者的人源化成熟輕鏈可變域。In some embodiments, the cell penetrating antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain of SEQ ID NO: 20 and a humanized mature light chain variable domain of any one of SEQ ID NOs: 27 to 48. In some embodiments, the cell penetrating antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain of SEQ ID NO: 21 and a humanized mature light chain variable domain comprising any one of SEQ ID NOs: 27 to 48. In some embodiments, the cell penetrating antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain of SEQ ID NO: 22 and a humanized mature light chain variable domain comprising any one of SEQ ID NOs: 27 to 48. In some embodiments, the cell penetrant antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain of SEQ ID NO: 23 and a humanized mature light chain variable domain comprising any one of SEQ ID NOs: 27-48.
在一些實施例中，細胞穿透劑之抗體或其抗原結合片段包括SEQ ID NO: 20之人源化成熟重鏈可變域及SEQ ID NO: 47之人源化成熟輕鏈可變域。在一些實施例中，細胞穿透劑之抗體或其抗原結合片段包括SEQ ID NO: 20之人源化成熟重鏈可變域及SEQ ID NO: 48之人源化成熟輕鏈可變域。在一些實施例中，細胞穿透劑之抗體或其抗原結合片段包括SEQ ID NO: 21之人源化成熟重鏈可變域及SEQ ID NO: 47之人源化成熟輕鏈可變域。在一些實施例中，細胞穿透劑之抗體或其抗原結合片段包括SEQ ID NO: 21之人源化成熟重鏈可變域及SEQ ID NO: 48之人源化成熟輕鏈可變域。在一些實施例中，細胞穿透劑之抗體或其抗原結合片段包括SEQ ID NO: 23之人源化成熟重鏈可變域及SEQ ID NO: 47之人源化成熟輕鏈可變域。在一些實施例中，細胞穿透劑之抗體或其抗原結合片段包括SEQ ID NO: 23之人源化成熟重鏈可變域及SEQ ID NO: 48之人源化成熟輕鏈可變域。In some embodiments, the cell penetrating antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain of SEQ ID NO: 20 and a humanized mature light chain variable domain of SEQ ID NO: 47. In some embodiments, the cell penetrating antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain of SEQ ID NO: 20 and a humanized mature light chain variable domain of SEQ ID NO: 48. In some embodiments, the cell penetrating antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain of SEQ ID NO: 21 and a humanized mature light chain variable domain of SEQ ID NO: 47. In some embodiments, the cell penetrating antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain of SEQ ID NO: 21 and a humanized mature light chain variable domain of SEQ ID NO: 48. In some embodiments, the cell penetrating antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain of SEQ ID NO: 23 and a humanized mature light chain variable domain of SEQ ID NO: 47. In some embodiments, the cell penetrating antibody or antigen-binding fragment thereof comprises a humanized mature heavy chain variable domain of SEQ ID NO: 23 and a humanized mature light chain variable domain of SEQ ID NO: 48.
在一些實施例中，人源化重鏈可變域中之以下位置中的至少一個(例如2、3、4、5、6、7、8、9、10、11、12、13、14、15、16或17個)由所指定之胺基酸佔據：K19由R佔據；S35由G佔據；T40由A佔據；E42由G佔據；A49由S佔據；K43由E佔據；R44由G或D佔據；A49由S佔據；A74由S佔據；T77由S佔據；L78由A或G佔據；L80由A或G佔據；L82c由G佔據；M83由R佔據；S84由A佔據；M89由V佔據；或F91由Y佔據。In some embodiments, at least one of the following positions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) in the humanized heavy chain variable domain is occupied by the specified amino acid: K19 is occupied by R; S35 is occupied by G; T40 is occupied by A; E42 is occupied by G ; A49 occupied by S; K43 occupied by E; R44 occupied by G or D; A49 occupied by S; A74 occupied by S; T77 occupied by S; L78 occupied by A or G; L80 occupied by A or G; L82c occupied by G; M83 occupied by R; S84 occupied by A; M89 occupied by V; or F91 occupied by Y.
在一些實施例中，人源化重鏈可變域中之以下位置中的至少一個(例如2、3、4、5或6個)由所指定之胺基酸佔據：K43由E佔據；R44由G或D佔據；A49由S佔據；A74由S佔據；T77由S佔據；或F91由Y佔據。In some embodiments, at least one (e.g., 2, 3, 4, 5, or 6) of the following positions in the humanized heavy chain variable domain is occupied by the specified amino acids: K43 is occupied by E; R44 is occupied by G or D; A49 is occupied by S; A74 is occupied by S; T77 is occupied by S; or F91 is occupied by Y.
在一些實施例中，人源化重鏈可變域中之以下位置中之至少一個(例如2、3或4個)由所指定之胺基酸佔據：S35由G佔據；L78由A或G佔據；L80由A或G佔據；或L82c由G佔據。In some embodiments, at least one (e.g., 2, 3, or 4) of the following positions in the humanized heavy chain variable domain are occupied by the specified amino acids: S35 is occupied by G; L78 is occupied by A or G; L80 is occupied by A or G; or L82c is occupied by G.
在一些實施例中，人源化重鏈可變域之F91由Y佔據；且人源化重鏈可變域中之以下位置中之至少一個(例如2、3、4、5或6個)由所指定之胺基酸佔據：R44由G佔據；A49由S佔據；A74由S佔據；T77由S佔據；L78由A或G佔據；或M83由R佔據。In some embodiments, F91 of the humanized heavy chain variable domain is occupied by Y; and at least one (e.g., 2, 3, 4, 5, or 6) of the following positions in the humanized heavy chain variable domain are occupied by the specified amino acids: R44 is occupied by G; A49 is occupied by S; A74 is occupied by S; T77 is occupied by S; L78 is occupied by A or G; or M83 is occupied by R.
在一些實施例中，人源化輕鏈可變域中之以下位置中的至少一個(例如2、3、4、5、6、7、8、9、10、11、12或13個)由所指定之胺基酸佔據：V3由Q佔據；L9由S佔據；D17由Q佔據；Q18由P佔據；K39由R佔據；K45由R佔據；T80由A或S佔據；T46由R佔據；L83由V佔據；L92由G或A佔據；V94由I或A佔據；A100由G、D或R佔據；或L104由V佔據。In some embodiments, at least one of the following positions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) in the humanized light chain variable domain is occupied by the specified amino acid: V3 is occupied by Q; L9 is occupied by S; D17 is occupied by Q; Q18 is occupied by P; K39 is occupied by R; K45 is occupied by R; T80 is occupied by A or S; T46 is occupied by R; L83 is occupied by V; L92 is occupied by G or A; V94 is occupied by I or A; A100 is occupied by G, D, or R; or L104 is occupied by V.
在一些實施例中，人源化輕鏈可變域中之以下位置中之至少一個(例如2個)由所指定之胺基酸佔據：V3由Q佔據或A100由D或R佔據。In some embodiments, at least one (e.g., two) of the following positions in the humanized light chain variable domain are occupied by the specified amino acids: V3 is occupied by Q or A100 is occupied by D or R.
在一些實施例中，人源化輕鏈可變域中之以下位置中的至少一個(例如2、3或4個)由所指定之胺基酸佔據：L9由S佔據；T80由A或S佔據；L92由G或A佔據；或V94由I或A佔據。In some embodiments, at least one (e.g., 2, 3, or 4) of the following positions in the humanized light chain variable domain are occupied by the specified amino acids: L9 is occupied by S; T80 is occupied by A or S; L92 is occupied by G or A; or V94 is occupied by I or A.
在一些實施例中，V3由Q佔據；Q18由P佔據；A100由D佔據；且人源化輕鏈可變域中之以下位置中之至少一個(例如2個)由所指定之胺基酸佔據：T80由A佔據或L92由A佔據。In some embodiments, V3 is occupied by Q; Q18 is occupied by P; A100 is occupied by D; and at least one (e.g., 2) of the following positions in the humanized light chain variable domain are occupied by the specified amino acids: T80 is occupied by A or L92 is occupied by A.
在一些實施例中，人源化重鏈可變域中之以下位置中的至少一個(例如2、3、4、5、6、7或8個)由所指定之胺基酸佔據：L5由V佔據；G44由R佔據；A49由S佔據；A74由S佔據；T77由S佔據；L78由A或G佔據；M89由V佔據，或F91由Y佔據；且人源化輕鏈可變域中之以下位置中之至少一個(例如2、3、4、5、6、7、8、9或10個)由所指定之胺基酸佔據：V3由Q佔據；D17由Q佔據；Q18由P佔據；K39由R佔據；K45由R佔據；T80由A佔據；L83由V佔據；L92由A佔據；A100由D佔據；或L104由V佔據。In some embodiments, at least one (e.g., 2, 3, 4, 5, 6, 7, or 8) of the following positions in the humanized heavy chain variable domain is occupied by the specified amino acids: L5 is occupied by V; G44 is occupied by R; A49 is occupied by S; A74 is occupied by S; T77 is occupied by S; L78 is occupied by A or G; M89 is occupied by V, or F91 is occupied by Y; and the humanized light chain may be At least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) of the following positions in the variable domain are occupied by the specified amino acid: V3 is occupied by Q; D17 is occupied by Q; Q18 is occupied by P; K39 is occupied by R; K45 is occupied by R; T80 is occupied by A; L83 is occupied by V; L92 is occupied by A; A100 is occupied by D; or L104 is occupied by V.
前述取代可賦予降低的(例如，至少1%降低、至少5%降低、至少10%降低、至少20%降低、至少30%降低、至少40%降低、至少50%降低、至少60%降低、至少70%降低、至少80%降低、至少90%降低、至少95%降低或至少99%降低的)親本抗體之免疫原性及/或提高的(例如，至少1%提高、至少10%提高、至少20%提高、至少30%提高、至少40%提高、至少50%提高、至少60%提高、至少70%提高、至少80%提高、至少90%提高或至少100%提高的)親本抗體之熱穩定性。The foregoing substitutions may confer decreased (e.g., at least 1% decreased, at least 5% decreased, at least 10% decreased, at least 20% decreased, at least 30% decreased, at least 40% decreased, at least 50% decreased, at least 60% decreased, at least 70% decreased, at least 80% decreased, at least 90% decreased, at least 95% decreased, or at least 99% decreased) immunogenicity of the parent antibody and/or increased (e.g., at least 1% increased, at least 10% increased, at least 20% increased, at least 30% increased, at least 40% increased, at least 50% increased, at least 60% increased, at least 70% increased, at least 80% increased, at least 90% increased, or at least 100% increased) thermal stability of the parent antibody.
在一些實施例中，與包括包含SEQ ID NO: 1之重鏈可變域及包含SEQ ID NO: 24之輕鏈可變域的參考抗體相比，人源化抗體具有提高的熱穩定性。在一些實施例中，與包括SEQ ID NO: 63之重鏈可變域及SEQ ID NO: 65之輕鏈可變域的參考抗體相比，人源化抗體具有提高的熱穩定性。在一些實施例中，與包括SEQ ID NO: 67之重鏈可變域及SEQ ID NO: 69之輕鏈可變域的參考抗體相比，人源化抗體具有提高的熱穩定性。在一些實施例中，與包括SEQ ID NO: 71之重鏈可變域及SEQ ID NO: 73之輕鏈可變域的參考抗體相比，人源化抗體具有提高的熱穩定性。在一些實施例中，與包括SEQ ID NO: 75之重鏈可變域及SEQ ID NO: 77之輕鏈可變域的參考抗體相比，人源化抗體具有提高的熱穩定性。在一些實施例中，與包括SEQ ID NO: 79之重鏈可變域及SEQ ID NO: 81之輕鏈可變域的參考抗體相比，人源化抗體具有提高的熱穩定性。In some embodiments, the humanized antibodies have increased thermal stability compared to a reference antibody comprising a heavy chain variable domain comprising SEQ ID NO: 1 and a light chain variable domain comprising SEQ ID NO: 24. In some embodiments, the humanized antibodies have increased thermal stability compared to a reference antibody comprising a heavy chain variable domain comprising SEQ ID NO: 63 and a light chain variable domain comprising SEQ ID NO: 65. In some embodiments, the humanized antibodies have increased thermal stability compared to a reference antibody comprising a heavy chain variable domain comprising SEQ ID NO: 67 and a light chain variable domain comprising SEQ ID NO: 69. In some embodiments, the humanized antibodies have improved thermal stability compared to a reference antibody comprising a heavy chain variable domain of SEQ ID NO: 71 and a light chain variable domain of SEQ ID NO: 73. In some embodiments, the humanized antibodies have improved thermal stability compared to a reference antibody comprising a heavy chain variable domain of SEQ ID NO: 75 and a light chain variable domain of SEQ ID NO: 77. In some embodiments, the humanized antibodies have improved thermal stability compared to a reference antibody comprising a heavy chain variable domain of SEQ ID NO: 79 and a light chain variable domain of SEQ ID NO: 81.
在一些實施例中，人源化抗體具有約55℃或更高之解鏈溫度。在一些實施例中，人源化抗體具有以下解鏈溫度：約56℃或更高、約57℃或更高、約58℃或更高、約59℃或更高、約60℃或更高、約61℃或更高、約62℃或更高、約63℃或更高、約64℃或更高、約65℃或更高、約66℃或更高、約67℃或更高、約68℃或更高、約69℃或更高、約70℃或更高、約71℃或更高、約72℃或更高、約73℃或更高、約74℃或更高、約75℃或更高、約76℃或更高、約77℃或更高、約78℃或更高、約79℃或更高、約80℃或更高、約81℃或更高、約82℃或更高、約83℃或更高、約84℃或更高、或約85℃或更高。在一些實施例中，人源化抗體具有以下解鏈溫度：至少約55℃、至少約56℃、至少約57℃、至少約58℃、至少約59℃、至少約60℃、至少約61℃、至少約62℃、至少約63℃、至少約64℃、至少約65℃、至少約66℃、至少約67℃、至少約68℃、至少約69℃、至少約70℃、至少約71℃、至少約72℃、至少約73℃、至少約74℃、至少約75℃、至少約76℃、至少約77℃、至少約78℃、至少約79℃、至少約80℃、至少約81℃、至少約82℃、至少約83℃、至少約84℃或至少約85℃。在一些實施例中，人源化抗體具有以下解鏈溫度：約55℃至約85℃、約55℃至約80℃、約55℃至約75℃、約55℃至約70℃、約55℃至約65℃、約55℃至約63℃、約55℃至約61℃、約55℃至約59℃、約55℃至約57℃、約57℃至約85℃、約57℃至約80℃、約57℃至約75℃、約57℃至約70℃、約57℃至約65℃、約57℃至約63℃、約57℃至約61℃、約57℃至約59℃、約59℃至約85℃、約59℃至約80℃、約59℃至約75℃、約59℃至約70℃、約59℃至約65℃、約59℃至約63℃、約59℃至約61℃、約61℃至約85℃、約61℃至約80℃、約61℃至約75℃、約61℃至約70℃、約61℃至約65℃、約61℃至約63℃、約63℃至約85℃、約63℃至約80℃、約63℃至約75℃、約63℃至約70℃、約63℃至約65℃、約65℃至約85℃、約65℃至約80℃、約65℃至約75℃、約65℃至約70℃、約70℃至約85℃、約70℃至約80℃、約70℃至約75℃、約75℃至約85℃、約75℃至約80℃、或約80℃至約85℃。In some embodiments, the humanized antibody has a melting temperature of about 55°C or higher. In some embodiments, the humanized antibody has a melting temperature of about 56°C or more, about 57°C or more, about 58°C or more, about 59°C or more, about 60°C or more, about 61°C or more, about 62°C or more, about 63°C or more, about 64°C or more, about 65°C or more, about 66°C or more, about 67°C or more, about 68°C or more, about 69°C or more, about 70°C or more, about 71°C or more, about 72°C or more, about 73°C or more, about 74°C or more, about 75°C or more, about 76°C or more, about 77°C or more, about 78°C or more, about 79°C or more, about 80°C or more, about 81°C or more, about 82°C or more, about 83°C or more, about 84°C or more, or about 85°C or more. In some embodiments, the humanized antibody has a melting temperature of at least about 55°C, at least about 56°C, at least about 57°C, at least about 58°C, at least about 59°C, at least about 60°C, at least about 61°C, at least about 62°C, at least about 63°C, at least about 64°C, at least about 65°C, at least about 66°C, at least about 67°C, at least about 68°C, at least about 69°C, at least about 70°C, at least about 71°C, at least about 72°C, at least about 73°C, at least about 74°C, at least about 75°C, at least about 76°C, at least about 77°C, at least about 78°C, at least about 79°C, at least about 80°C, at least about 81°C, at least about 82°C, at least about 83°C, at least about 84°C, or at least about 85°C. In some embodiments, the humanized antibodies have a melting temperature of about 55°C to about 85°C, about 55°C to about 80°C, about 55°C to about 75°C, about 55°C to about 70°C, about 55°C to about 65°C, about 55°C to about 63°C, about 55°C to about 61°C, about 55°C to about 59°C, about 55°C to about 57°C, about 57°C to about 85°C, about 57°C to about 80°C, about 57°C to about 75°C, about 57°C to about 70°C, about 57°C to about 65°C, about 57°C to about 63°C, about 57°C to about 61°C, about 57°C to about 59°C, about 59°C to about 85°C, about 59°C to about 80°C, about 59°C to about 75°C, about 59°C to about 70°C, about 59°C to about 65°C, about 59°C to about 63°C, about 59°C to about 61°C, about 61°C to about 85°C, about 61°C to about 80°C, about 61°C to about 75°C, about 61°C to about 70°C, about 61°C to about 65°C, about 61°C to about 63°C, about 63°C to about 85°C, about 63°C to about 80°C, about 63°C to about 75°C, about 63°C to about 70°C, about 63°C to about 65°C, about 65°C to about 85°C, about 65°C to about 80°C, about 65°C to about 75°C, about 65°C to about 70°C, about 70°C to about 85°C, about 70°C to about 80°C, about 70°C to about 75°C, about 75°C to about 85°C, about 75°C to about 80°C, or about 80°C to about 85°C.
本文亦提供包括CIM及特異性結合至TDP-43 (例如人類TDP-43)之抗體的細胞穿透劑，該抗體包括重鏈可變域，其包括：如由Kabat/Chothia Composite所定義之重鏈CDR1，其包括SEQ ID NO: 49或SEQ ID NO: 50；如由Kabat所定義之重鏈CDR2，其包括SEQ ID NO: 51；如由Kabat或Chothia所定義之重鏈CDR3，其包括SEQ ID NO: 52；如由Kabat所定義之輕鏈CDR1，其包括SEQ ID NO: 53；如由Kabat所定義之輕鏈CDR2，其包括SEQ ID NO: 54；及如由Kabat所定義之輕鏈CDR3，其包括SEQ ID NO: 55-61中之一者。Also provided herein are cell penetrants comprising a CIM and an antibody that specifically binds to TDP-43 (e.g., human TDP-43), the antibody comprising a heavy chain variable domain comprising: a heavy chain CDR1 as defined by the Kabat/Chothia Composite comprising SEQ ID NO: 49 or SEQ ID NO: 50; a heavy chain CDR2 as defined by Kabat comprising SEQ ID NO: 51; a heavy chain CDR3 as defined by Kabat or Chothia comprising SEQ ID NO: 52; a light chain CDR1 as defined by Kabat comprising SEQ ID NO: 53; a light chain CDR2 as defined by Kabat comprising SEQ ID NO: 54; and a light chain CDR3 as defined by Kabat comprising one of SEQ ID NOs: 55-61.
在一些實施例中，如由Kabat/Chothia Composite所定義之重鏈CDR1包括SEQ ID NO: 49；如由Kabat所定義之重鏈CDR2包括SEQ ID NO: 51；In some embodiments, the heavy chain CDR1 as defined by the Kabat/Chothia Composite comprises SEQ ID NO: 49; the heavy chain CDR2 as defined by Kabat comprises SEQ ID NO: 51;
如由Kabat或Chothia所定義之重鏈CDR3包括SEQ ID NO: 52；如由Kabat所定義之輕鏈CDR1包括SEQ ID NO: 53；如由Kabat所定義之輕鏈CDR2包括SEQ ID NO: 54；且如由Kabat所定義之輕鏈CDR3包括SEQ ID NO: 55或SEQ ID NO: 61。The heavy chain CDR3 as defined by Kabat or Chothia includes SEQ ID NO: 52; the light chain CDR1 as defined by Kabat includes SEQ ID NO: 53; the light chain CDR2 as defined by Kabat includes SEQ ID NO: 54; and the light chain CDR3 as defined by Kabat includes SEQ ID NO: 55 or SEQ ID NO: 61.
在一些實施例中，重鏈可變域包括與SEQ ID NO: 4-23中之任一者至少95% (例如，至少96%、至少97%、至少98%、至少99%)一致的序列。在一些實施例中，重鏈可變域包括與以下中之任一者至少95% (例如，至少96%、至少97%、至少98%、至少99%)一致的序列：SEQ ID NO: 20、SEQ ID NO: 21及SEQ ID NO: 23。在一些實施例中，重鏈可變域包括與以下中之任一者至少98%一致的序列：SEQ ID NO: 20、SEQ ID NO: 21及SEQ ID NO: 23。在一些實施例中，重鏈可變域包括SEQ ID NO: 20之序列。在一些實施例中，重鏈可變域包括SEQ ID NO: 21之序列。在一些實施例中，重鏈可變域包括SEQ ID NO: 23之序列。In some embodiments, the heavy chain variable domain comprises a sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) identical to any one of SEQ ID NOs: 4-23. In some embodiments, the heavy chain variable domain comprises a sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) identical to any one of SEQ ID NOs: 20, 21, and 23. In some embodiments, the heavy chain variable domain comprises a sequence that is at least 98% identical to any one of SEQ ID NOs: 20, 21, and 23. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 20. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 21. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 23.
在一些實施例中，輕鏈可變域包括與SEQ ID NO: 27-48中之任一者至少95% (例如，至少96%、至少97%、至少98%、至少99%)一致的序列。在一些實施例中，輕鏈可變域包括與SEQ ID NO: 47或SEQ ID NO: 48至少95% (例如，至少96%、至少97%、至少98%、至少99%)一致的序列。在一些實施例中，輕鏈可變域包括與SEQ ID NO: 47或SEQ ID NO: 48至少98%一致的序列。在一些實施例中，輕鏈可變域包括SEQ ID NO: 47。在一些實施例中，輕鏈可變域包括SEQ ID NO: 48。In some embodiments, the light chain variable domain comprises a sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) identical to any one of SEQ ID NOs: 27-48. In some embodiments, the light chain variable domain comprises a sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 47 or SEQ ID NO: 48. In some embodiments, the light chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 47 or SEQ ID NO: 48. In some embodiments, the light chain variable domain comprises SEQ ID NO: 47. In some embodiments, the light chain variable domain comprises SEQ ID NO: 48.
如先前所述，本文所述之人源化成熟輕鏈可變域可在其C末端包括離胺酸殘基。然而，在一些實施例中，人源化成熟輕鏈可變域(例如，本文所述之成熟輕鏈可變域中之任一者)不包括C末端離胺酸殘基。舉例而言，SEQ ID NO: 24-48、65、69、73、77或81之成熟輕鏈可變域中之任一者可缺少C末端離胺酸殘基。As previously described, the humanized mature light chain variable domains described herein may include a lysine residue at their C-terminus. However, in some embodiments, a humanized mature light chain variable domain (e.g., any of the mature light chain variable domains described herein) does not include a C-terminal lysine residue. For example, any of the mature light chain variable domains of SEQ ID NOs: 24-48, 65, 69, 73, 77, or 81 may lack a C-terminal lysine residue.
在一些實施例中，細胞穿透劑之抗體為人源化抗體、嵌合抗體或飾面抗體。在一些實施例中，抗體為抗原結合抗體片段。在一些實施例中，抗原結合抗體片段為Fab片段、Fab'2片段或單鏈Fv。In some embodiments, the antibody of the cell penetrant is a humanized antibody, a chimeric antibody, or a masked antibody. In some embodiments, the antibody is an antigen-binding antibody fragment. In some embodiments, the antigen-binding antibody fragment is a Fab fragment, aFab'2 fragment, or a single-chain Fv.
在一些實施例中，細胞穿透劑之抗體為完整抗體。在一些實施例中，抗體具有人類IgG1同型。In some embodiments, the antibody of the cell penetrant is a whole antibody. In some embodiments, the antibody has a human IgG1 isotype.
在一些實施例中，重鏈可變域融合至重鏈恆定區(例如，本文所述之任何重鏈恆定區)且輕鏈可變域融合至輕鏈恆定區(例如，本文所述之任何輕鏈恆定區)。In some embodiments, the heavy chain variable domain is fused to a heavy chain constant region (e.g., any heavy chain constant region described herein) and the light chain variable domain is fused to a light chain constant region (e.g., any light chain constant region described herein).
在一些實施例中，重鏈恆定區為天然人類重鏈恆定區之突變體形式，其相對於天然重鏈恆定區，與Fc…受體之結合減少。在一些實施例中，重鏈恆定區具有IgG1同型。在一些實施例中，抗體在恆定區中具有至少一個突變。在一些實施例中，至少一個突變減少恆定區之補體固定或活化。在一些實施例中，至少一個突變在按EU編號的以下一或多個(例如2、3、4、5、6、7、8、9、10或11個)位置處：241、264、265、270、296、297、318、320、322、329及331。在一些實施例中，抗體在按EU編號之位置318、320及322處具有丙胺酸。In some embodiments, the heavy chain constant region is a mutant form of a native human heavy chain constant region that has reduced binding to Fc receptors relative to the native heavy chain constant region. In some embodiments, the heavy chain constant region has an IgG1 isotype. In some embodiments, the antibody has at least one mutation in the constant region. In some embodiments, at least one mutation reduces complement fixation or activation of the constant region. In some embodiments, at least one mutation is at one or more of the following positions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) by EU numbering: 241, 264, 265, 270, 296, 297, 318, 320, 322, 329, and 331. In some embodiments, the antibody has alanine at positions 318, 320, and 322 by EU numbering.
在一些實施例中，細胞穿透劑之抗體或抗原結合抗體片段選擇性地結合至磷酸化TDP-43 (例如，磷酸化人類TDP-43)。在一些實施例中，與未磷酸化TDP-43 (例如，未磷酸化人類TDP-43)相比，細胞穿透劑之抗體或抗原結合抗體片段選擇性地結合至磷酸化TDP-43 (例如，磷酸化人類TDP-43)。In some embodiments, the antibody or antigen-binding antibody fragment of the cell penetrating agent selectively binds to phosphorylated TDP-43 (e.g., phosphorylated human TDP-43). In some embodiments, the antibody or antigen-binding antibody fragment of the cell penetrating agent selectively binds to phosphorylated TDP-43 (e.g., phosphorylated human TDP-43) as compared to unphosphorylated TDP-43 (e.g., unphosphorylated human TDP-43).
在一些實施例中，細胞穿透劑之抗體或抗原結合抗體片段以比未磷酸化TDP-43 (例如，未磷酸化人類TDP-43)高(例如，至少10倍、至少20倍、至少30倍、至少40倍、至少50倍、至少100倍、至少150倍、至少200倍、至少250倍、至少300倍、至少350倍、至少400倍、至少450倍、至少500倍、至少550倍、至少600倍、至少650倍、至少700倍、至少750倍、至少800倍、至少850倍、至少900倍、至少950倍、至少1,000倍、至少1,050倍、至少1,100倍、至少1,150倍、至少1,200倍、至少1,250倍、至少1,300倍、至少1,350倍、至少1,400倍、至少1,450倍、或至少1,500倍、或約10倍至約1,500倍、約10倍至1,400倍、約10倍至約1,300倍、約10倍至約1,200倍、約10倍至約1,100倍、約10倍至約1,000倍、約10倍至約900倍、約10倍至約800倍、約10倍至約700倍、約10倍至約600倍、約10倍至約500倍、約10倍至約400倍、約10倍至約300倍、約10倍至約200倍、約10倍至約100倍、約10倍至約50倍、約50倍至約1,500倍、約50倍至1,400倍、約50倍至約1,300倍、約50倍至約1,200倍、約50倍至約1,100倍、約50倍至約1,000倍、約50倍至約900倍、約50倍至約800倍、約50倍至約700倍、約50倍至約600倍、約50倍至約500倍、約50倍至約400倍、約50倍至約300倍、約50倍至約200倍、約50倍至約100倍、約100倍至約1,500倍、約100倍至1,400倍、約100倍至約1,300倍、約100倍至約1,200倍、約100倍至約1,100倍、約100倍至約1,000倍、約100倍至約900倍、約100倍至約800倍、約100倍至約700倍、約100倍至約600倍、約100倍至約500倍、約100倍至約400倍、約100倍至約300倍、約100倍至約200倍、約200倍至約1,500倍、約200倍至1,400倍、約200倍至約1,300倍、約200倍至約1,200倍、約200倍至約1,100倍、約200倍至約1,000倍、約200倍至約900倍、約200倍至約800倍、約200倍至約700倍、約200倍至約600倍、約200倍至約500倍、約200倍至約400倍、約200倍至約300倍、約500倍至約1,500倍、約500倍至1,400倍、約500倍至約1,300倍、約500倍至約1,200倍、約500倍至約1,100倍、約500倍至約1,000倍、約500倍至約900倍、約500倍至約800倍、約500倍至約700倍、約500倍至約600倍、約800倍至約1,500倍、約800倍至1,400倍、約800倍至約1,300倍、約800倍至約1,200倍、約800倍至約1,100倍、約800倍至約1,000倍、約800倍至約900倍、約1,000倍至約1,500倍、約1,000倍至1,400倍、約1,000倍至約1,300倍、約1,000倍至約1,200倍、或約1,000倍至約1,100倍)的親和力結合至磷酸化TDP-43 (例如，磷酸化人類TDP-43)。In some embodiments, the antibody or antigen-binding antibody fragment of the cell penetrant has a higher (e.g., at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 300-fold, at least 350-fold, at least 400-fold, at least 450-fold, at least 500-fold, at least 550-fold, at least 600-fold, at least 650-fold, at least 700-fold, at least 750-fold, at least 800-fold, at least 850-fold) than unphosphorylated TDP-43 (e.g., unphosphorylated human TDP-43). , at least 900-fold, at least 950-fold, at least 1,000-fold, at least 1,050-fold, at least 1,100-fold, at least 1,150-fold, at least 1,200-fold, at least 1,250-fold, at least 1,300-fold, at least 1,350-fold, at least 1,400-fold, at least 1,450-fold, or at least 1,500-fold, or about 10-fold to about 1,500-fold, about 10-fold to about 1,400-fold, about 10-fold to about 1,300-fold, about 10-fold to about 1,200-fold 0 times, about 10 times to about 1,100 times, about 10 times to about 1,000 times, about 10 times to about 900 times, about 10 times to about 800 times, about 10 times to about 700 times, about 10 times to about 600 times, about 10 times to about 500 times, about 10 times to about 400 times, about 10 times to about 300 times, about 10 times to about 200 times, about 10 times to about 100 times, about 10 times to about 50 times, about 50 times to about 1,500 times, about 50 times to 1,400 times, about 50 times to about 1,300 times, about 50 times to about 1,200 times, about 50 times to about 1,100 times, about 50 times to about 1,000 times, about 50 times to about 900 times, about 50 times to about 800 times, about 50 times to about 700 times, about 50 times to about 600 times, about 50 times to about 500 times, about 50 times to about 400 times, about 50 times to about 300 times, about 50 times to about 200 times, about 50 times to about 100 times, about 100 times to about 1,500 times , about 100 times to about 1,400 times, about 100 times to about 1,300 times, about 100 times to about 1,200 times, about 100 times to about 1,100 times, about 100 times to about 1,000 times, about 100 times to about 900 times, about 100 times to about 800 times, about 100 times to about 700 times, about 100 times to about 600 times, about 100 times to about 500 times, about 100 times to about 400 times, about 100 times to about 300 times, about 100 times to about 20 0 times, about 200 times to about 1,500 times, about 200 times to 1,400 times, about 200 times to about 1,300 times, about 200 times to about 1,200 times, about 200 times to about 1,100 times, about 200 times to about 1,000 times, about 200 times to about 900 times, about 200 times to about 800 times, about 200 times to about 700 times, about 200 times to about 600 times, about 200 times to about 500 times, about 200 times to about 400 times, about 200 times to about about 300 times, about 500 times to about 1,500 times, about 500 times to 1,400 times, about 500 times to about 1,300 times, about 500 times to about 1,200 times, about 500 times to about 1,100 times, about 500 times to about 1,000 times, about 500 times to about 900 times, about 500 times to about 800 times, about 500 times to about 700 times, about 500 times to about 600 times, about 800 times to about 1,500 times, about 800 times to 1,400 times, In some embodiments, the protein binds to phosphorylated TDP-43 (e.g., phosphorylated human TDP-43) with an affinity of about 800-fold to about 1,300-fold, about 800-fold to about 1,200-fold, about 800-fold to about 1,100-fold, about 800-fold to about 1,000-fold, about 800-fold to about 900-fold, about 1,000-fold to about 1,500-fold, about 1,000-fold to 1,400-fold, about 1,000-fold to about 1,300-fold, about 1,000-fold to about 1,200-fold, or about 1,000-fold to about 1,100-fold).
在一些實施例中，磷酸化TDP-43 (例如磷酸化人類TDP-43)包括至少一個選自S409及S410之胺基酸殘基的磷酸化。在一些實施例中，磷酸化TDP-43 (例如，磷酸化人類TDP-43)包括S409及S410兩者之磷酸化。In some embodiments, phosphorylated TDP-43 (e.g., phosphorylated human TDP-43) comprises phosphorylation of at least one amino acid residue selected from S409 and S410. In some embodiments, phosphorylated TDP-43 (e.g., phosphorylated human TDP-43) comprises phosphorylation of both S409 and S410.
在一些實施例中，細胞穿透劑之抗體或其片段選擇性地結合至TDP-43之細胞質聚集體(例如，人類TDP-43之細胞質聚集體)。在一些實施例中，與核TDP-43 (例如，核人類TDP-43)相比，細胞穿透劑之抗體或其片段選擇性地結合至TDP-43之細胞質聚集體(例如，人類TDP-43之細胞質聚集體)。在一些實施例中，TDP-43之細胞質聚集體(例如，人類TDP-43之細胞質聚集體)包括TDP-43之磷酸化聚集體。在一些實施例中，細胞穿透劑之抗體或其片段實質上不結合未磷酸化TDP-43 (例如，未磷酸化人類TDP-43)。In some embodiments, the cell penetrant antibody or fragment thereof selectively binds to cytoplasmic aggregates of TDP-43 (e.g., cytoplasmic aggregates of human TDP-43). In some embodiments, the cell penetrant antibody or fragment thereof selectively binds to cytoplasmic aggregates of TDP-43 (e.g., cytoplasmic aggregates of human TDP-43) as compared to nuclear TDP-43 (e.g., nuclear human TDP-43). In some embodiments, the cytoplasmic aggregates of TDP-43 (e.g., cytoplasmic aggregates of human TDP-43) include phosphorylated aggregates of TDP-43. In some embodiments, the cell penetrant antibody or fragment thereof does not substantially bind to unphosphorylated TDP-43 (e.g., unphosphorylated human TDP-43).
存在於細胞穿透劑中之重鏈及輕鏈可變區可連接至人類恆定區之至少一部分。恆定區之選擇部分地取決於是否需要抗體結合物依賴性細胞介導之細胞毒性、抗體結合物依賴性細胞吞噬作用及/或補體依賴性細胞毒性。舉例而言，人類同型IgG1及IgG3具有補體依賴性細胞毒性，而人類同型IgG2及IgG4則不具有。人類IgG1及IgG3亦誘導比人類IgG2及IgG4更強之細胞介導之效應功能。輕鏈恆定區可為λ或κ。恆定區之編號慣例包括EU編號(Edelman, G.M.等人,Proc. Natl. Acad. Sci. U.S.A.63:78-85 (1969))、Kabat編號(Kabat,Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, MD, 1991、IMGT獨特編號(Lefranc M.-P.等人, IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig superfamily C-like domains,Dev. Comp. Immunol.29:185- 203 (2005)及IMGT外顯子編號(Lefranc，同上)。The heavy and light chain variable regions present in the cell penetrant can be linked to at least a portion of a human constant region. The choice of constant region depends in part on whether antibody-bound cell-mediated cytotoxicity, antibody-bound cell-mediated phagocytosis, and/or complement-dependent cytotoxicity are desired. For example, human isotypes IgG1 and IgG3 exhibit complement-dependent cytotoxicity, while human isotypes IgG2 and IgG4 do not. Human IgG1 and IgG3 also induce stronger cell-mediated effector functions than human IgG2 and IgG4. The light chain constant region can be either λ or κ. Conventional numbers for constant regions include EU numbering (Edelman, GM et al.,Proc. Natl. Acad. Sci. USA 63:78-85 (1969)), Kabat numbering (Kabat,Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1991), IMGT unique numbering (Lefranc M.-P. et al., IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig superfamily C-like domains,Dev. Comp. Immunol. 29:185-203 (2005)), and IMGT exon numbering (Lefranc, supra).
輕鏈及/或重鏈之胺基或羧基端之一或若干個胺基酸(諸如重鏈之C末端離胺酸)可在一定比例或全部分子中缺失或衍生化。可在恆定區中進行取代以降低或增強效應功能，諸如補體介導之細胞毒性或ADCC(參見，例如Winter等人，美國專利第5,624,821號；Tso等人，美國專利第5,834,597號；及Lazar等人,Proc. Natl. Acad. Sci. USA103:4005 (2006))，或延長人類中之半衰期(參見，例如Hinton等人,J. Biol. Chem.279:6213 ( 2004))。例示性取代包括位置250處之Gln及/或位置428處之Leu (EU編號在此段落中用於恆定區)，用於延長抗體之半衰期。在位置234、235、236及/或237中之任一者或所有處之取代降低對Fey受體、尤其FcγRI受體之親和力(參見，例如美國專利第 6,624,821號)。人類IgG 1之位置234、235及237處之丙胺酸取代可用於降低效應功能。一些抗體在人類IgG 1之位置234、235及237處具有丙胺酸取代以降低效應功能。視情況，人類IgG2中之位置234、236及/或237經丙胺酸取代且位置235經麩醯胺酸取代(參見，例如美國專利第5,624,821號)。在一些抗體中，使用人類IgG 1在由EU編號的位置241、264、265、270、296、297、322、329及331中之一或多者(例如，2、3、4、5、6、7、8或9)處的突變。在一些抗體中，使用人類IgG1在由EU編號的位置318、320及322中之一或多者(例如2或3)處的突變。在一些抗體中，位置234及/或235經丙胺酸取代及/或位置329經甘胺酸取代。在一些抗體中，位置234及235經丙胺酸取代。在一些抗體中，同型為人類IgG2或IgG4。One or more amino acids at the amino or carboxyl termini of the light and/or heavy chains (e.g., the C-terminal lysine of the heavy chain) may be deleted or derivatized in a certain proportion or the entire molecule. Substitutions may be made in the constant region to reduce or enhance effector functions, such as complement-mediated cytotoxicity or ADCC( see, e.g., Winter et al., U.S. Patent No. 5,624,821; Tso et al., U.S. Patent No. 5,834,597; and Lazar et al.,Proc. Natl. Acad. Sci. USA 103:4005 (2006)), or to extend half-life in humans( see, e.g., Hinton et al.,J. Biol. Chem. 279:6213 (2004)). Exemplary substitutions include Gln at position 250 and/or Leu at position 428 (EU numbering is used in this paragraph for the constant region), which are used to extend the half-life of the antibody. Substitutions at any or all of positions 234, 235, 236, and/or 237 reduce affinity for Fcγ receptors, particularly FcγRI receptors( see, e.g., U.S. Patent No. 6,624,821). Alanine substitutions at positions 234, 235, and 237 of human IgG1 can be used to reduce effector function. Some antibodies have alanine substitutions at positions 234, 235, and 237 of human IgG1 to reduce effector function. Optionally, positions 234, 236, and/or 237 in human IgG2 are substituted with alanine and position 235 is substituted with glutamine( see, e.g., U.S. Patent No. 5,624,821). In some antibodies, mutations at one or more of positions 241, 264, 265, 270, 296, 297, 322, 329, and 331 (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) of human IgG1 by EU numbering are used. In some antibodies, mutations at one or more of positions 318, 320, and 322 (e.g., 2 or 3) of human IgG1 by EU numbering are used. In some antibodies, positions 234 and/or 235 are substituted with alanine and/or position 329 is substituted with glycine. In some antibodies, positions 234 and 235 are substituted with alanine. In some antibodies, the isotype is human IgG2 or IgG4.
抗體可表現為含有兩條輕鏈及兩條重鏈之四聚體、單獨之重鏈、輕鏈、Fab、Fab'、F(ab')2及Fv，或表現為其中重鏈及輕鏈成熟可變域經由間隔子連接之單鏈抗體。Antibodies can be expressed as tetramers containing two light chains and two heavy chains, as individual heavy chains, light chains, Fab, Fab', F(ab')2 , and Fv, or as single-chain antibodies in which the heavy and light chain mature variable domains are linked by a spacer.
人類恆定區顯示不同個體之間的同種異型變異及同族同種異型變異，亦即，恆定區在一或多個多態性位置處可在不同個體中不同。同族同種異型與同種異型之不同之處在於識別同族同種異型之血清結合至一或多種其他同型之非多態性區域。因此，舉例而言，另一重鏈恆定區為具有或不具有C末端離胺酸之IgG1 Glm3。對人類恆定區之提及包括具有任何天然同種異型或佔據天然同種異型中之位置的殘基之任何排列的恆定區。例示性抗TDP-43細胞穿透劑Human constant regions show allotypic variation and cognate isotypic variation between different individuals, that is, constant regions can differ in different individuals at one or more polymorphic positions. Cognate isotypes differ from allotypes in that sera that recognize a cognate isotype bind to non-polymorphic regions of one or more other isotypes. Thus, for example, another heavy chain constant region is IgG1 Glm3 with or without a C-terminal lysine. Reference to a human constant region includes constant regions having any natural isotype or any arrangement of residues occupying positions in natural isotypes.Exemplary anti-TDP-43cell penetrants
本文已揭示細胞穿透劑之各種組分，包括抗TDP-43抗體、連接子及細胞內化部分(例如CMIP)。一般熟習此項技術者將理解如何組合此等特徵以獲得功能性抗TDP-43細胞穿透劑。本文亦描述具有CMIP、視情況存在之連接子及作為單一多肽(亦即融合蛋白)表現之抗TDP-43抗體之輕鏈及/或重鏈的細胞穿透劑。Various components of cell penetrants have been disclosed herein, including an anti-TDP-43 antibody, a linker, and a cell internalization moiety (e.g., CMIP). One skilled in the art will understand how to combine these features to obtain a functional anti-TDP-43 cell penetrant. Also described herein are cell penetrants comprising a CMIP, optionally a linker, and the light and/or heavy chains of an anti-TDP-43 antibody expressed as a single polypeptide (i.e., a fusion protein).
在一些實施例中，細胞穿透劑包含與選自以下中之任一者之序列至少95%一致的多肽序列：SEQ ID NO: 116、SEQ ID NO: 118、SEQ ID NO: 120、SEQ ID NO: 122、SEQ ID NO: 124、SEQ ID NO: 126、SEQ ID NO: 128、SEQ ID NO: 130、SEQ ID NO: 132、SEQ ID NO: 134、SEQ ID NO: 136、SEQ ID NO: 138、SEQ ID NO: 140、SEQ ID NO: 142、SEQ ID NO: 144、SEQ ID NO: 146、SEQ ID NO: 148、SEQ ID NO: 150、SEQ ID NO: 152、SEQ ID NO: 154、SEQ ID NO: 156、SEQ ID NO: 158、SEQ ID NO: 160、SEQ ID NO: 162、SEQ ID NO: 166、SEQ ID NO: 168、SEQ ID NO: 170、SEQ ID NO: 172及SEQ ID NO: 174。In some embodiments, the cell penetrant comprises a polypeptide sequence that is at least 95% identical to a sequence selected from any one of SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172 and SEQ ID NO: 174.
在一些實施例中，細胞穿透劑包含與選自以下中之任一者之序列至少98%一致的多肽序列：SEQ ID NO: 116、SEQ ID NO: 118、SEQ ID NO: 120、SEQ ID NO: 122、SEQ ID NO: 124、SEQ ID NO: 126、SEQ ID NO: 128、SEQ ID NO: 130、SEQ ID NO: 132、SEQ ID NO: 134、SEQ ID NO: 136、SEQ ID NO: 138、SEQ ID NO: 140、SEQ ID NO: 142、SEQ ID NO: 144、SEQ ID NO: 146、SEQ ID NO: 148、SEQ ID NO: 150、SEQ ID NO: 152、SEQ ID NO: 154、SEQ ID NO: 156、SEQ ID NO: 158、SEQ ID NO: 160、SEQ ID NO: 162、SEQ ID NO: 166、SEQ ID NO: 168、SEQ ID NO: 170、SEQ ID NO: 172及SEQ ID NO: 174。In some embodiments, the cell penetrant comprises a polypeptide sequence that is at least 98% identical to a sequence selected from any one of SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172 and SEQ ID NO: 174.
在一些實施例中，細胞穿透劑包含與選自以下中之任一者之序列至少98%一致的多肽序列：SEQ ID NO: 116、SEQ ID NO: 118、SEQ ID NO: 120、SEQ ID NO: 122、SEQ ID NO: 124、SEQ ID NO: 126、SEQ ID NO: 128、SEQ ID NO: 130、SEQ ID NO: 132、SEQ ID NO: 134、SEQ ID NO: 136、SEQ ID NO: 138、SEQ ID NO: 140、SEQ ID NO: 142、SEQ ID NO: 144、SEQ ID NO: 146、SEQ ID NO: 148、SEQ ID NO: 150、SEQ ID NO: 152、SEQ ID NO: 154、SEQ ID NO: 156、SEQ ID NO: 158、SEQ ID NO: 160、SEQ ID NO: 162、SEQ ID NO: 166、SEQ ID NO: 168、SEQ ID NO: 170、SEQ ID NO: 172及SEQ ID NO: 174。In some embodiments, the cell penetrant comprises a polypeptide sequence that is at least 98% identical to a sequence selected from any one of SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172 and SEQ ID NO: 174.
在一些實施例中，細胞穿透劑包含與選自以下中之任一者之序列至少95%一致的多肽序列：SEQ ID NO: 117、SEQ ID NO: 119、SEQ ID NO: 121、SEQ ID NO: 123、SEQ ID NO: 125、SEQ ID NO: 127、SEQ ID NO: 129、SEQ ID NO: 131、SEQ ID NO: 133、SEQ ID NO: 135、SEQ ID NO: 137、SEQ ID NO: 139、SEQ ID NO: 141、SEQ ID NO: 143、SEQ ID NO: 145、SEQ ID NO: 147、SEQ ID NO: 149、SEQ ID NO: 151、SEQ ID NO: 153、SEQ ID NO: 155、SEQ ID NO: 157、SEQ ID NO: 159、SEQ ID NO: 161、SEQ ID NO: 163、SEQ ID NO: 165、SEQ ID NO: 167、SEQ ID NO: 169、SEQ ID NO: 171、SEQ ID NO: 173及SEQ ID NO: 175。In some embodiments, the cell penetrant comprises a polypeptide sequence that is at least 95% identical to a sequence selected from any one of SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163. SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173 and SEQ ID NO: 175.
在一些實施例中，細胞穿透劑包含與選自以下中之任一者之序列至少98%一致的多肽序列：SEQ ID NO: 117、SEQ ID NO: 119、SEQ ID NO: 121、SEQ ID NO: 123、SEQ ID NO: 125、SEQ ID NO: 127、SEQ ID NO: 129、SEQ ID NO: 131、SEQ ID NO: 133、SEQ ID NO: 135、SEQ ID NO: 137、SEQ ID NO: 139、SEQ ID NO: 141、SEQ ID NO: 143、SEQ ID NO: 145、SEQ ID NO: 147、SEQ ID NO: 149、SEQ ID NO: 151、SEQ ID NO: 153、SEQ ID NO: 155、SEQ ID NO: 157、SEQ ID NO: 159、SEQ ID NO: 161、SEQ ID NO: 163、SEQ ID NO: 165、SEQ ID NO: 167、SEQ ID NO: 169、SEQ ID NO: 171、SEQ ID NO: 173及SEQ ID NO: 175。In some embodiments, the cell penetrant comprises a polypeptide sequence that is at least 98% identical to a sequence selected from any one of SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163. SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173 and SEQ ID NO: 175.
在一些實施例中，細胞穿透劑包含選自以下中之任一者的多肽序列：SEQ ID NO: 117、SEQ ID NO: 119、SEQ ID NO: 121、SEQ ID NO: 123、SEQ ID NO: 125、SEQ ID NO: 127、SEQ ID NO: 129、SEQ ID NO: 131、SEQ ID NO: 133、SEQ ID NO: 135、SEQ ID NO: 137、SEQ ID NO: 139、SEQ ID NO: 141、SEQ ID NO: 143、SEQ ID NO: 145、SEQ ID NO: 147、SEQ ID NO: 149、SEQ ID NO: 151、SEQ ID NO: 153、SEQ ID NO: 155、SEQ ID NO: 157、SEQ ID NO: 159、SEQ ID NO: 161、SEQ ID NO: 163、SEQ ID NO: 165、SEQ ID NO: 167、SEQ ID NO: 169、SEQ ID NO: 171、SEQ ID NO: 173及SEQ ID NO: 175。In some embodiments, the cell penetrant comprises a polypeptide sequence selected from any one of SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173 and SEQ ID NO: 175.
在一些實施例中，細胞穿透劑包含含有抗體之重鏈之第一多肽序列及含有抗體之輕鏈之第二多肽。在一些實施例中，第一多肽及/或第二多肽包含CMIP及視情況存在之連接子。在一些實施例中，第一多肽係選自以下中之任一者：SEQ ID NO: 116、SEQ ID NO: 118、SEQ ID NO: 120、SEQ ID NO: 122、SEQ ID NO: 124、SEQ ID NO: 126、SEQ ID NO: 128、SEQ ID NO: 130、SEQ ID NO: 132、SEQ ID NO: 134、SEQ ID NO: 136、SEQ ID NO: 138、SEQ ID NO: 140、SEQ ID NO: 142、SEQ ID NO: 144、SEQ ID NO: 146、SEQ ID NO: 148、SEQ ID NO: 150、SEQ ID NO: 152、SEQ ID NO: 154、SEQ ID NO: 156、SEQ ID NO: 158、SEQ ID NO: 160、SEQ ID NO: 162、SEQ ID NO: 166、SEQ ID NO: 168、SEQ ID NO: 170、SEQ ID NO: 172及SEQ ID NO: 174；且第二多肽係選自以下中之任一者：SEQ ID NO: 117、SEQ ID NO: 119、SEQ ID NO: 121、SEQ ID NO: 123、SEQ ID NO: 125、SEQ ID NO: 127、SEQ ID NO: 129、SEQ ID NO: 131、SEQ ID NO: 133、SEQ ID NO: 135、SEQ ID NO: 137、SEQ ID NO: 139、SEQ ID NO: 141、SEQ ID NO: 143、SEQ ID NO: 145、SEQ ID NO: 147、SEQ ID NO: 149、SEQ ID NO: 151、SEQ ID NO: 153、SEQ ID NO: 155、SEQ ID NO: 157、SEQ ID NO: 159、SEQ ID NO: 161、SEQ ID NO: 163、SEQ ID NO: 165、SEQ ID NO: 167、SEQ ID NO: 169、SEQ ID NO: 171、SEQ ID NO: 173及SEQ ID NO: 175。In some embodiments, the cell penetrant comprises a first polypeptide sequence comprising the heavy chain of an antibody and a second polypeptide sequence comprising the light chain of an antibody. In some embodiments, the first polypeptide and/or the second polypeptide comprises CMIP and, optionally, a linker. In some embodiments, the first polypeptide is selected from any one of the following: SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174 NO: 168, SEQ ID NO: 170, SEQ ID NO: 172 and SEQ ID NO: 174; and the second polypeptide is selected from any one of the following: SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 160 NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173 and SEQ ID NO: 175.
在一些實施例中，細胞穿透劑包含第一多肽及第二多肽，進一步地其中：第一多肽包含與SEQ ID NO: 116至少95%一致之序列且第二多肽包含與SEQ ID NO: 117至少95%一致之序列；第一多肽包含與SEQ ID NO: 118至少95%一致之序列且第二多肽包含與SEQ ID NO: 119至少95%一致之序列；第一多肽包含與SEQ ID NO: 120至少95%一致之序列且第二多肽包含與SEQ ID NO: 121至少95%一致之序列；第一多肽包含與SEQ ID NO: 122至少95%一致之序列且第二多肽包含與SEQ ID NO: 123至少95%一致之序列；第一多肽包含與SEQ ID NO: 124至少95%一致之序列且第二多肽包含與SEQ ID NO: 125至少95%一致之序列；第一多肽包含與SEQ ID NO: 126至少95%一致之序列且第二多肽包含與SEQ ID NO: 127至少95%一致之序列；第一多肽包含與SEQ ID NO: 128至少95%一致之序列且第二多肽包含與SEQ ID NO: 129至少95%一致之序列；第一多肽包含與SEQ ID NO: 130至少95%一致之序列且第二多肽包含與SEQ ID NO: 131至少95%一致之序列；第一多肽包含與SEQ ID NO: 132至少95%一致之序列且第二多肽包含與SEQ ID NO: 133至少95%一致之序列；第一多肽包含與SEQ ID NO: 134至少95%一致之序列且第二多肽包含與SEQ ID NO: 135至少95%一致之序列；第一多肽包含與SEQ ID NO: 136至少95%一致之序列且第二多肽包含與SEQ ID NO: 137至少95%一致之序列；第一多肽包含與SEQ ID NO: 138至少95%一致之序列且第二多肽包含與SEQ ID NO: 139至少95%一致之序列；第一多肽包含與SEQ ID NO: 140至少95%一致之序列且第二多肽包含與SEQ ID NO: 141至少95%一致之序列；第一多肽包含與SEQ ID NO: 142至少95%一致之序列且第二多肽包含與SEQ ID NO: 143至少95%一致之序列；第一多肽包含與SEQ ID NO: 144至少95%一致之序列且第二多肽包含與SEQ ID NO: 145至少95%一致之序列；第一多肽包含與SEQ ID NO: 146至少95%一致之序列且第二多肽包含與SEQ ID NO: 147至少95%一致之序列；第一多肽包含與SEQ ID NO: 148至少95%一致之序列且第二多肽包含與SEQ ID NO: 149至少95%一致之序列；第一多肽包含與SEQ ID NO: 150至少95%一致之序列且第二多肽包含與SEQ ID NO: 151至少95%一致之序列；第一多肽包含與SEQ ID NO: 152至少95%一致之序列且第二多肽包含與SEQ ID NO: 153至少95%一致之序列；第一多肽包含與SEQ ID NO: 154至少95%一致之序列且第二多肽包含與SEQ ID NO: 155至少95%一致之序列；第一多肽包含與SEQ ID NO: 156至少95%一致之序列且第二多肽包含與SEQ ID NO: 157至少95%一致之序列；第一多肽包含與SEQ ID NO: 158至少95%一致之序列且第二多肽包含與SEQ ID NO: 159至少95%一致之序列；第一多肽包含與SEQ ID NO: 160至少95%一致之序列且第二多肽包含與SEQ ID NO: 161至少95%一致之序列；第一多肽包含與SEQ ID NO: 162至少95%一致之序列且第二多肽包含與SEQ ID NO: 163至少95%一致之序列；第一多肽包含與SEQ ID NO: 164至少95%一致之序列且第二多肽包含與SEQ ID NO: 165至少95%一致之序列；第一多肽包含與SEQ ID NO: 166至少95%一致之序列且第二多肽包含與SEQ ID NO: 167至少95%一致之序列；第一多肽包含與SEQ ID NO: 168至少95%一致之序列且第二多肽包含與SEQ ID NO: 169至少95%一致之序列；第一多肽包含與SEQ ID NO: 170至少95%一致之序列且第二多肽包含與SEQ ID NO: 171至少95%一致之序列；第一多肽包含與SEQ ID NO: 172至少95%一致之序列且第二多肽包含與SEQ ID NO: 173至少95%一致之序列；或第一多肽包含與SEQ ID NO: 174至少95%一致之序列且第二多肽包含與SEQ ID NO: 175至少95%一致之序列。In some embodiments, the cell penetrating agent comprises a first polypeptide and a second polypeptide, further wherein: the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 116 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 117; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 118 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 119; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 120 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 121; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 122 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 123; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 124 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 125; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 126 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 127; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 128 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 129; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 130 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 131; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 132 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 133; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 134 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 135; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 136 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 137; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 138 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 139; the first polypeptide comprising a sequence at least 95% identical to SEQ ID NO: 138 and the second polypeptide comprising a sequence at least 95% identical to SEQ ID NO: 139; the first polypeptide comprising a sequence at least 95% identical to SEQ ID NO: 140 and the second polypeptide comprising a sequence at least 95% identical to SEQ ID NO: 141; the first polypeptide comprising a sequence at least 95% identical to SEQ ID NO: 142 and the second polypeptide comprising a sequence at least 95% identical to SEQ ID NO: 143; the first polypeptide comprising a sequence at least 95% identical to SEQ ID NO: 144 and the second polypeptide comprising a sequence at least 95% identical to SEQ ID NO: 145; the first polypeptide comprising a sequence at least 95% identical to SEQ ID NO: 146 and the second polypeptide comprising a sequence at least 95% identical to SEQ ID NO: 147; the first polypeptide comprising a sequence at least 95% identical to SEQ ID NO: 148 and the second polypeptide comprising a sequence at least 95% identical to SEQ ID NO: 149; the first polypeptide comprising a sequence at least 95% identical to SEQ ID NO: 150 150 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 151; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 152 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 153; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 154 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 155; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 156 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 157; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 158 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 159; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 160 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 161; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 162 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 163; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 164 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 165; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 166 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 167; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 168 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 169; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 170 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 171; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 172 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 173; or the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: The first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 174 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 175.
在一些實施例中，細胞穿透劑包含第一多肽及第二多肽，其中：第一多肽包含SEQ ID NO: 116且第二多肽包含SEQ ID NO: 117；第一多肽包含SEQ ID NO: 118且第二多肽包含SEQ ID NO: 119；第一多肽包含SEQ ID NO: 120且第二多肽包含SEQ ID NO: 121；第一多肽包含SEQ ID NO: 122且第二多肽包含SEQ ID NO: 123；第一多肽包含SEQ ID NO: 124且第二多肽包含SEQ ID NO: 125；第一多肽包含SEQ ID NO: 126且第二多肽包含SEQ ID NO: 127；第一多肽包含SEQ ID NO: 128且第二多肽包含SEQ ID NO: 129；第一多肽包含SEQ ID NO: 130且第二多肽包含SEQ ID NO: 131；第一多肽包含SEQ ID NO: 132且第二多肽包含SEQ ID NO: 133；第一多肽包含SEQ ID NO: 134且第二多肽包含SEQ ID NO: 135；第一多肽包含SEQ ID NO: 136且第二多肽包含SEQ ID NO: 137；第一多肽包含SEQ ID NO: 138且第二多肽包含SEQ ID NO: 139；第一多肽包含SEQ ID NO: 140且第二多肽包含SEQ ID NO: 141；第一多肽包含SEQ ID NO: 142且第二多肽包含SEQ ID NO: 143；第一多肽包含SEQ ID NO: 144且第二多肽包含SEQ ID NO: 145；第一多肽包含SEQ ID NO: 146且第二多肽包含SEQ ID NO: 147；第一多肽包含SEQ ID NO: 148且第二多肽包含SEQ ID NO: 149；第一多肽包含SEQ ID NO: 150且第二多肽包含SEQ ID NO: 151；第一多肽包含SEQ ID NO: 152且第二多肽包含SEQ ID NO: 153；第一多肽包含SEQ ID NO: 154且第二多肽包含SEQ ID NO: 155；第一多肽包含SEQ ID NO: 156且第二多肽包含SEQ ID NO: 157；第一多肽包含SEQ ID NO: 158且第二多肽包含SEQ ID NO: 159；第一多肽包含SEQ ID NO: 160且第二多肽包含SEQ ID NO: 161；第一多肽包含SEQ ID NO: 162且第二多肽包含SEQ ID NO: 163；第一多肽包含SEQ ID NO: 164且第二多肽包含SEQ ID NO: 165；第一多肽包含SEQ ID NO: 166且第二多肽包含SEQ ID NO: 167；第一多肽包含SEQ ID NO: 168且第二多肽包含SEQ ID NO: 169；第一多肽包含SEQ ID NO: 170且第二多肽包含SEQ ID NO: 171；第一多肽包含SEQ ID NO: 172且第二多肽包含SEQ ID NO: 173；或第一多肽包含SEQ ID NO: 174且第二多肽包含SEQ ID NO: 175。In some embodiments, the cell penetrant comprises a first polypeptide and a second polypeptide, wherein: the first polypeptide comprises SEQ ID NO: 116 and the second polypeptide comprises SEQ ID NO: 117; the first polypeptide comprises SEQ ID NO: 118 and the second polypeptide comprises SEQ ID NO: 119; the first polypeptide comprises SEQ ID NO: 120 and the second polypeptide comprises SEQ ID NO: 121; the first polypeptide comprises SEQ ID NO: 122 and the second polypeptide comprises SEQ ID NO: 123; the first polypeptide comprises SEQ ID NO: 124 and the second polypeptide comprises SEQ ID NO: 125; the first polypeptide comprises SEQ ID NO: 126 and the second polypeptide comprises SEQ ID NO: 127; the first polypeptide comprises SEQ ID NO: 128 and the second polypeptide comprises SEQ ID NO: 129; the first polypeptide comprises SEQ ID NO: 130 and the second polypeptide comprises SEQ ID NO: 131; the first polypeptide comprises SEQ ID NO: 132 and the second polypeptide comprises SEQ ID NO: 133; the first polypeptide comprises SEQ ID NO: 134. NO: 134 and the second polypeptide comprises SEQ ID NO: 135; the first polypeptide comprises SEQ ID NO: 136 and the second polypeptide comprises SEQ ID NO: 137; the first polypeptide comprises SEQ ID NO: 138 and the second polypeptide comprises SEQ ID NO: 139; the first polypeptide comprises SEQ ID NO: 140 and the second polypeptide comprises SEQ ID NO: 141; the first polypeptide comprises SEQ ID NO: 142 and the second polypeptide comprises SEQ ID NO: 143; the first polypeptide comprises SEQ ID NO: 144 and the second polypeptide comprises SEQ ID NO: 145; the first polypeptide comprises SEQ ID NO: 146 and the second polypeptide comprises SEQ ID NO: 147; the first polypeptide comprises SEQ ID NO: 148 and the second polypeptide comprises SEQ ID NO: 149; the first polypeptide comprises SEQ ID NO: 150 and the second polypeptide comprises SEQ ID NO: 151; the first polypeptide comprises SEQ ID NO: 152 and the second polypeptide comprises SEQ ID NO: 153; the first polypeptide comprises SEQ ID NO: 154 and the second polypeptide comprises SEQ ID NO: 155; the first polypeptide comprises SEQ ID NO: 156 and the second polypeptide comprises SEQ ID NO: 157; the first polypeptide comprises SEQ ID NO: 158 and the second polypeptide comprises SEQ ID NO: 159; the first polypeptide comprises SEQ ID NO: 160 and the second polypeptide comprises SEQ ID NO: 161; the first polypeptide comprises SEQ ID NO: 162 and the second polypeptide comprises SEQ ID NO: 163; the first polypeptide comprises SEQ ID NO: 164 and the second polypeptide comprises SEQ ID NO: 165; the first polypeptide comprises SEQ ID NO: 166 and the second polypeptide comprises SEQ ID NO: 167; the first polypeptide comprises SEQ ID NO: 168 and the second polypeptide comprises SEQ ID NO: 169; the first polypeptide comprises SEQ ID NO: 170 and the second polypeptide comprises SEQ ID NO: 171; the first polypeptide comprises SEQ ID NO: 172 and the second polypeptide comprises SEQ ID NO: 173; or the first polypeptide comprises SEQ ID NO: 174 and the second polypeptide comprises SEQ ID NO: 175.
在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 116之第一多肽及含有SEQ ID NO: 117之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 118之第一多肽及含有SEQ ID NO: 119之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 120之第一多肽及含有SEQ ID NO: 121之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 122之第一多肽及含有SEQ ID NO: 123之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 124之第一多肽及含有SEQ ID NO: 125之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 126之第一多肽及含有SEQ ID NO: 127之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 128之第一多肽及含有SEQ ID NO: 129之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 130之第一多肽及含有SEQ ID NO: 131之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 132之第一多肽及含有SEQ ID NO: 133之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 134之第一多肽及含有SEQ ID NO: 135之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 136之第一多肽及含有SEQ ID NO: 137之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 138之第一多肽及含有SEQ ID NO: 139之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 140之第一多肽及含有SEQ ID NO: 141之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 142之第一多肽及含有SEQ ID NO: 143之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 144之第一多肽及含有SEQ ID NO: 145之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 146之第一多肽及含有SEQ ID NO: 147之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 148之第一多肽及含有SEQ ID NO: 149之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 150之第一多肽及含有SEQ ID NO: 151之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 152之第一多肽及含有SEQ ID NO: 153之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 154之第一多肽及含有SEQ ID NO: 155之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 156之第一多肽及含有SEQ ID NO: 157之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 158之第一多肽及含有SEQ ID NO: 159之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 160之第一多肽及含有SEQ ID NO: 161之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 162之第一多肽及含有SEQ ID NO: 163之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 164之第一多肽及含有SEQ ID NO: 165之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 166之第一多肽及含有SEQ ID NO: 167之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 168之第一多肽及含有SEQ ID NO: 169之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 170之第一多肽及含有SEQ ID NO: 171之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 172之第一多肽及含有SEQ ID NO: 173之第二多肽。在一些實施例中，細胞穿透劑包含含有SEQ ID NO: 174之第一多肽及含有SEQ ID NO: 175之第二多肽。In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 116 and a second polypeptide comprising SEQ ID NO: 117. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 118 and a second polypeptide comprising SEQ ID NO: 119. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 120 and a second polypeptide comprising SEQ ID NO: 121. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 122 and a second polypeptide comprising SEQ ID NO: 123. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 124 and a second polypeptide comprising SEQ ID NO: 125. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 126 and a second polypeptide comprising SEQ ID NO: 127. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 128 and a second polypeptide comprising SEQ ID NO: 129. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 130 and a second polypeptide comprising SEQ ID NO: 131. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 132 and a second polypeptide comprising SEQ ID NO: 133. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 134 and a second polypeptide comprising SEQ ID NO: 135. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 136 and a second polypeptide comprising SEQ ID NO: 137. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 138 and a second polypeptide comprising SEQ ID NO: 139. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 140 and a second polypeptide comprising SEQ ID NO: 141. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 142 and a second polypeptide comprising SEQ ID NO: 143. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 144 and a second polypeptide comprising SEQ ID NO: 145. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 146 and a second polypeptide comprising SEQ ID NO: 147. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 148 and a second polypeptide comprising SEQ ID NO: 149. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 150 and a second polypeptide comprising SEQ ID NO: 151. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 152 and a second polypeptide comprising SEQ ID NO: 153. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 154 and a second polypeptide comprising SEQ ID NO: 155. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 156 and a second polypeptide comprising SEQ ID NO: 157. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 158 and a second polypeptide comprising SEQ ID NO: 159. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 160 and a second polypeptide comprising SEQ ID NO: 161. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 162 and a second polypeptide comprising SEQ ID NO: 163. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 164 and a second polypeptide comprising SEQ ID NO: 165. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 166 and a second polypeptide comprising SEQ ID NO: 167. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 168 and a second polypeptide comprising SEQ ID NO: 169. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 170 and a second polypeptide comprising SEQ ID NO: 171. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 172 and a second polypeptide comprising SEQ ID NO: 173. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 174 and a second polypeptide comprising SEQ ID NO: 175.
細胞穿透劑亦可以編碼細胞穿透劑或存在於細胞穿透劑內之抗體之核酸的形式投予。若重鏈與輕鏈皆存在，則該等鏈較佳連接為單鏈抗體。The cell penetrant can also be administered in the form of a nucleic acid encoding the cell penetrant or an antibody present within the cell penetrant. If both heavy and light chains are present, the chains are preferably linked as a single-chain antibody.
IV.IV.核酸、載體及宿主細胞Nucleic acids, vectors, and host cells
本揭示內容提供編碼本文所述之任一細胞穿透劑之至少一部分的核酸。舉例而言，除本文所述之任何重鏈及/或輕鏈以外，核酸亦可編碼本文所述之任何CIM、連接子及/或間隔子。在一些實施例中，核酸編碼第一多肽，其包含特異性結合至TDP-43之抗體的重鏈，該第一多肽包含以下中之任一者：SEQ ID NO: 116、SEQ ID NO: 118、SEQ ID NO: 120、SEQ ID NO: 122、SEQ ID NO: 124、SEQ ID NO: 126、SEQ ID NO: 128、SEQ ID NO: 130、SEQ ID NO: 132、SEQ ID NO: 134、SEQ ID NO: 136、SEQ ID NO: 138、SEQ ID NO: 140、SEQ ID NO: 142、SEQ ID NO: 144、SEQ ID NO: 146、SEQ ID NO: 148、SEQ ID NO: 150、SEQ ID NO: 152、SEQ ID NO: 154、SEQ ID NO: 156、SEQ ID NO: 158、SEQ ID NO: 160、SEQ ID NO: 162、SEQ ID NO: 166、SEQ ID NO: 168、SEQ ID NO: 170、SEQ ID NO: 172及SEQ ID NO: 174。在一些實施例中，核酸編碼第二多肽，其包含特異性結合至TDP-43之抗體的輕鏈，該第二多肽包含以下中之任一者：SEQ ID NO: 117、SEQ ID NO: 119、SEQ ID NO: 121、SEQ ID NO: 123、SEQ ID NO: 125、SEQ ID NO: 127、SEQ ID NO: 129、SEQ ID NO: 131、SEQ ID NO: 133、SEQ ID NO: 135、SEQ ID NO: 137、SEQ ID NO: 139、SEQ ID NO: 141、SEQ ID NO: 143、SEQ ID NO: 145、SEQ ID NO: 147、SEQ ID NO: 149、SEQ ID NO: 151、SEQ ID NO: 153、SEQ ID NO: 155、SEQ ID NO: 157、SEQ ID NO: 159、SEQ ID NO: 161、SEQ ID NO: 163、SEQ ID NO: 165、SEQ ID NO: 167、SEQ ID NO: 169、SEQ ID NO: 171、SEQ ID NO: 173及SEQ ID NO: 175。The present disclosure provides nucleic acids encoding at least a portion of any of the cell penetrants described herein. For example, in addition to any heavy and/or light chains described herein, the nucleic acids can also encode any of the CIMs, linkers, and/or spacers described herein. In some embodiments, the nucleic acid encodes a first polypeptide comprising a heavy chain of an antibody that specifically binds to TDP-43, the first polypeptide comprising any one of SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205 ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172 and SEQ ID NO: 174. In some embodiments, the nucleic acid encodes a second polypeptide comprising a light chain of an antibody that specifically binds to TDP-43, the second polypeptide comprising any one of SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170 ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173 and SEQ ID NO: 175.
在一些實施例中，核酸編碼第一多肽，其包含特異性結合至TDP-43之抗體的重鏈，該第一多肽包含以下中之任一者：SEQ ID NO: 116、SEQ ID NO: 118、SEQ ID NO: 120、SEQ ID NO: 122、SEQ ID NO: 124、SEQ ID NO: 126、SEQ ID NO: 128、SEQ ID NO: 130、SEQ ID NO: 132、SEQ ID NO: 134、SEQ ID NO: 136、SEQ ID NO: 138、SEQ ID NO: 140、SEQ ID NO: 142、SEQ ID NO: 144、SEQ ID NO: 146、SEQ ID NO: 148、SEQ ID NO: 150、SEQ ID NO: 152、SEQ ID NO: 154、SEQ ID NO: 156、SEQ ID NO: 158、SEQ ID NO: 160、SEQ ID NO: 162、SEQ ID NO: 166、SEQ ID NO: 168、SEQ ID NO: 170、SEQ ID NO: 172及SEQ ID NO: 174；且核酸核酸編碼第二多肽，其包含特異性結合至TDP-43之抗體的輕鏈，該第二多肽包含以下中之任一者：SEQ ID NO: 117、SEQ ID NO: 119、SEQ ID NO: 121、SEQ ID NO: 123、SEQ ID NO: 125、SEQ ID NO: 127、SEQ ID NO: 129、SEQ ID NO: 131、SEQ ID NO: 133、SEQ ID NO: 135、SEQ ID NO: 137、SEQ ID NO: 139、SEQ ID NO: 141、SEQ ID NO: 143、SEQ ID NO: 145、SEQ ID NO: 147、SEQ ID NO: 149、SEQ ID NO: 151、SEQ ID NO: 153、SEQ ID NO: 155、SEQ ID NO: 157、SEQ ID NO: 159、SEQ ID NO: 161、SEQ ID NO: 163、SEQ ID NO: 165、SEQ ID NO: 167、SEQ ID NO: 169、SEQ ID NO: 171、SEQ ID NO: 173及SEQ ID NO: 175。In some embodiments, the nucleic acid encodes a first polypeptide comprising a heavy chain of an antibody that specifically binds to TDP-43, the first polypeptide comprising any one of SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205 SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, and SEQ ID NO: 174; and the nucleic acid encodes a second polypeptide comprising a light chain of an antibody that specifically binds to TDP-43, the second polypeptide comprising any one of SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147. SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173 and SEQ ID NO: 175.
在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 116之第一多肽及含有SEQ ID NO: 117之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 118之第一多肽及含有SEQ ID NO: 119之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 120之第一多肽及含有SEQ ID NO: 121之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 122之第一多肽及含有SEQ ID NO: 123之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 124之第一多肽及含有SEQ ID NO: 125之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 126之第一多肽及含有SEQ ID NO: 127之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 128之第一多肽及含有SEQ ID NO: 129之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 130之第一多肽及含有SEQ ID NO: 131之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 132之第一多肽及含有SEQ ID NO: 133之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 134之第一多肽及含有SEQ ID NO: 135之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 136之第一多肽及含有SEQ ID NO: 137之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 138之第一多肽及含有SEQ ID NO: 139之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 140之第一多肽及含有SEQ ID NO: 141之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 142之第一多肽及含有SEQ ID NO: 143之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 144之第一多肽及含有SEQ ID NO: 145之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 146之第一多肽及含有SEQ ID NO: 147之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 148之第一多肽及含有SEQ ID NO: 149之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 150之第一多肽及含有SEQ ID NO: 151之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 152之第一多肽及含有SEQ ID NO: 153之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 154之第一多肽及含有SEQ ID NO: 155之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 156之第一多肽及含有SEQ ID NO: 157之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 158之第一多肽及含有SEQ ID NO: 159之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 160之第一多肽及含有SEQ ID NO: 161之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 162之第一多肽及含有SEQ ID NO: 163之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 164之第一多肽及含有SEQ ID NO: 165之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 166之第一多肽及含有SEQ ID NO: 167之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 168之第一多肽及含有SEQ ID NO: 169之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 170之第一多肽及含有SEQ ID NO: 171之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 172之第一多肽及含有SEQ ID NO: 173之第二多肽。在一些實施例中，核酸編碼細胞穿透劑，其包含含有SEQ ID NO: 174之第一多肽及含有SEQ ID NO: 175之第二多肽。In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 116 and a second polypeptide comprising SEQ ID NO: 117. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 118 and a second polypeptide comprising SEQ ID NO: 119. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 120 and a second polypeptide comprising SEQ ID NO: 121. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 122 and a second polypeptide comprising SEQ ID NO: 123. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 124 and a second polypeptide comprising SEQ ID NO: 125. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 126 and a second polypeptide comprising SEQ ID NO: 127. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 128 and a second polypeptide comprising SEQ ID NO: 129. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 130 and a second polypeptide comprising SEQ ID NO: 131. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 132 and a second polypeptide comprising SEQ ID NO: 133. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 134 and a second polypeptide comprising SEQ ID NO: 135. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 136 and a second polypeptide comprising SEQ ID NO: 137. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 138 and a second polypeptide comprising SEQ ID NO: 139. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 140 and a second polypeptide comprising SEQ ID NO: 141. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 142 and a second polypeptide comprising SEQ ID NO: 143. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 144 and a second polypeptide comprising SEQ ID NO: 145. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 146 and a second polypeptide comprising SEQ ID NO: 147. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 148 and a second polypeptide comprising SEQ ID NO: 149. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 150 and a second polypeptide comprising SEQ ID NO: 151. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 152 and a second polypeptide comprising SEQ ID NO: 153. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 154 and a second polypeptide comprising SEQ ID NO: 155. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 156 and a second polypeptide comprising SEQ ID NO: 157. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 158 and a second polypeptide comprising SEQ ID NO: 159. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 160 and a second polypeptide comprising SEQ ID NO: 161. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 162 and a second polypeptide comprising SEQ ID NO: 163. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 164 and a second polypeptide comprising SEQ ID NO: 165. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 166 and a second polypeptide comprising SEQ ID NO: 167. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 168 and a second polypeptide comprising SEQ ID NO: 169. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 170 and a second polypeptide comprising SEQ ID NO: 171. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 172 and a second polypeptide comprising SEQ ID NO: 173. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 174 and a second polypeptide comprising SEQ ID NO: 175.
本揭示內容進一步提供編碼本文所述抗體中之任一者的重鏈可變域及/或輕鏈可變域中之任一者的核酸。舉例而言，核酸可編碼包含SEQ ID NO: 4-23中之任一者的重鏈可變域及/或包含SEQ ID NO: 27-48中之任一者的輕鏈可變域。視情況，此類核酸進一步編碼訊號肽且可與連接至恆定區之訊號肽一起表現。核酸之編碼序列可與調控序列可操作地連接以確保編碼序列(諸如啟動子、強化子、核糖體結合位點、轉錄終止訊號及其類似物)之表現。編碼重鏈及輕鏈之核酸可以分離形式存在或可選殖至一或多個載體中。核酸可藉由例如固態合成或重疊寡核苷酸之PCR來合成。編碼重鏈及輕鏈之核酸可例如在表現載體內接合為一個連續核酸，或可為單獨的，例如各自選殖至其自身之表現載體中。在一些實施例中，核酸經密碼子最佳化以在宿主細胞中表現。The present disclosure further provides nucleic acids encoding any of the heavy chain variable domains and/or light chain variable domains of any of the antibodies described herein. For example, the nucleic acid may encode a heavy chain variable domain comprising any of SEQ ID NOs: 4-23 and/or a light chain variable domain comprising any of SEQ ID NOs: 27-48. Optionally, such nucleic acids further encode a signal peptide and may be expressed together with the signal peptide linked to a constant region. The coding sequence of the nucleic acid may be operably linked to a regulatory sequence to ensure expression of the coding sequence (e.g., a promoter, enhancer, ribosome binding site, transcriptional termination signal, and the like). The nucleic acids encoding the heavy and light chains may be present in isolated form or may be cloned into one or more vectors. Nucleic acids can be synthesized, for example, by solid-state synthesis or PCR using overlapping oligonucleotides. Nucleic acids encoding the heavy and light chains can be joined into a single continuous nucleic acid, for example within an expression vector, or can be separate, for example, each cloned into its own expression vector. In some embodiments, the nucleic acids are codon-optimized for expression in host cells.
已知多種使用抗體表現細胞株(例如融合瘤)產生嵌合及人源化抗體之方法。例如，抗體之免疫球蛋白可變區可使用熟知方法選殖及測序。在一種方法中，重鏈可變VH區藉由RT-PCR使用自融合瘤細胞製備之mRNA來選殖。對包含轉譯起始密碼子之VH區前導肽採用共有引子作為5'引子及g2b恆定區特異性3'引子。例示性引子描述於Schenk等人之美國專利公開案US 2005/0009150(以下簡稱「Schenk」)中。可比較來自多個獨立衍生之純系的序列以確保在擴增期間不引入變化。VH區之序列亦可藉由對藉由5' RACE RT-PCR方法及3' g2b特異性引子獲得之VH片段進行測序來確定或確認。A variety of methods are known for producing chimeric and humanized antibodies using antibody-expressing cell lines(e.g., hybridomas). For example, the immunoglobulin variable region of the antibody can be cloned and sequenced using well-known methods. In one method, the rechain variable VH region is cloned by RT-PCR using mRNA prepared from hybridoma cells. A common primer is used as a 5' primer and a g2b constant region-specific 3' primer for the VH region leader peptide containing the translation start codon. Exemplary primers are described in U.S. Patent Publication No. US 2005/0009150 by Schenk et al. (hereinafter referred to as "Schenk"). Sequences from multiple independently derived clones can be compared to ensure that no changes were introduced during the amplification period. The sequence of the VH region can also be determined or confirmed by sequencing a VH fragment obtained by 5' RACE RT-PCR using a 3' g2b-specific primer.
輕鏈可變VL區可以類似方式選殖。在一種方法中，共有引子組經設計用於使用經設計以與包含轉譯起始密碼子之VL區雜交的5'引子及對V-J接合區下游之Ck區具有特異性的3'引子來擴增VL區。在第二種方法中，採用5’RACE RT-PCR方法來選殖VL編碼cDNA。例示性引子描述於Schenk，同上中。接著將經選殖序列與編碼人類(或其他非人類物種)恆定區之序列組合。Light chain variable VL regions can be cloned in a similar manner. In one approach, a consensus primer set is designed to amplify the VL region using a 5' primer designed to hybridize with the VL region containing the translation start codon and a 3' primer specific for the Ck region downstream of the V-J junction. In a second approach, a 5' RACE RT-PCR method is employed to clone VL-encoding cDNAs. Exemplary primers are described in Schenk, supra. The cloned sequence is then combined with sequences encoding constant regions from humans (or other non-human species).
本文亦提供載體，其包括可操作地連接至一或多個調控序列以實現本文所述之任一細胞穿透劑在哺乳動物細胞中之表現的本文所述之任何核酸。Also provided herein are vectors comprising any of the nucleic acids described herein operably linked to one or more regulatory sequences to achieve expression of any of the cell penetrating agents described herein in mammalian cells.
本文亦提供載體，其包括編碼成熟重鏈可變域(例如，本文所述之重鏈可變域中之任一者)及輕鏈可變域(例如，本文所述之輕鏈可變域中之任一者)的核酸，該核酸可操作地連接至一或多個調控序列以實現本文所述之抗體或抗原結合片段中之任一者在哺乳動物細胞中之表現。Also provided herein are vectors comprising nucleic acids encoding a mature heavy chain variable domain (e.g., any of the heavy chain variable domains described herein) and a light chain variable domain (e.g., any of the light chain variable domains described herein) operably linked to one or more regulatory sequences to enable expression of any of the antibodies or antigen-binding fragments described herein in mammalian cells.
在一種方法中，重鏈及輕鏈可變區經再工程化以編碼各別VDJ或VJ接合下游之剪接供體序列，且選殖至哺乳動物表現載體中，諸如用於重鏈之pCMV-hyl及用於輕鏈之pCMV-Mcl。此等載體將人類…l及Ck恆定區編碼為所插入可變區卡匣下游之外顯子片段。序列驗證後，可將重鏈及輕鏈表現載體共轉染至CHO細胞中以產生嵌合抗體。條件培養基在轉染後48小時加以收集並藉由西方墨點分析檢定抗體產生或藉由ELISA檢定抗原結合。將嵌合抗體如上所述地人源化。In one approach, the heavy and light chain variable regions are reengineered to encode splice donor sequences downstream of the respective VDJ or VJ junctions and cloned into mammalian expression vectors, such as pCMV-hyl for the heavy chain and pCMV-Mcl for the light chain. These vectors encode the human Ck and Ck constant regions as exonic fragments downstream of the inserted variable region cassettes. After sequence verification, the heavy and light chain expression vectors can be co-transfected into CHO cells to produce chimeric antibodies. Conditioned media are collected 48 hours after transfection and assayed for antibody production by Western blot analysis or antigen binding by ELISA. Chimeric antibodies are humanized as described above.
嵌合抗體、飾面抗體、人源化抗體及人類抗體通常藉由重組表現來產生。重組多核苷酸構建體通常包括可操作地連接至抗體鏈之編碼序列的表現控制序列，包括天然地相關或異源表現之控制元件，諸如啟動子。表現控制序列可為能夠轉化或轉染真核或原核宿主細胞的載體中之啟動子系統。一旦載體已併入適當宿主中，則將宿主維持在適於核苷酸序列之高水準表現以及交叉反應抗體之收集及純化的條件下。Chimeric, masked, humanized, and human antibodies are typically produced by recombinant expression. Recombinant polynucleotide constructs typically include expression control sequences, including naturally occurring or heterologous expression control elements, such as promoters, operably linked to the coding sequence of the antibody chain. The expression control sequences can be promoter systems in vectors capable of transforming or transfecting eukaryotic or prokaryotic host cells. Once the vector has been incorporated into an appropriate host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and for the collection and purification of cross-reactive antibodies.
因此，本文提供用本文所述之載體中之任一者轉化的宿主細胞。本文亦提供包括本文所述之核酸中之任一者的宿主細胞。Thus, provided herein are host cells transformed with any of the vectors described herein. Also provided herein are host cells comprising any of the nucleic acids described herein.
表現載體通常可作為游離基因組或作為宿主染色體DNA之組成部分在宿主生物體中複製。通常，表現載體含有選擇標記，例如胺芐青黴素抗性或潮黴素抗性，以允許偵測經所需DNA序列轉化之彼等細胞。Expression vectors are typically replicated in the host organism as episomes or as an integral part of the host chromosomal DNA. Typically, expression vectors contain a selectable marker, such as ampicillin resistance or hygromycin resistance, to allow detection of cells transformed with the desired DNA sequence.
大腸桿菌為可用於表現抗體，尤其抗體片段的一種原核宿主。微生物，諸如酵母，亦可用於表現。酵母屬為具有合適載體之酵母宿主，該等載體根據需要具有表現控制序列、複製起點、終止序列及其類似序列。典型啟動子包括3-磷酸甘油酸激酶及其他糖酵解酶。可誘導酵母啟動子尤其包括來自醇脫氫酶、異細胞色素C及負責麥芽糖及半乳糖利用之酶的啟動子。Escherichia coli is a prokaryotic host useful for expressing antibodies, particularly antibody fragments. Microorganisms, such as yeast, can also be used for expression. Saccharomyces spp.are yeast hosts with suitable vectors containing expression control sequences, origins of replication, terminator sequences, and the like, as needed. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, those from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization.
哺乳動物細胞可用於表現編碼免疫球蛋白或其片段之核苷酸區段。參見Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987)。已開發出許多能夠分泌完整異源蛋白之合適的宿主細胞株，且包括CHO細胞株、各種COS細胞株、HeLa細胞、HEK293細胞、L細胞及非抗體產生性骨髓瘤，包括Sp2/0及NS0。細胞可為非人類的。此等細胞之表現載體可包括表現控制序列，諸如複製起點、啟動子、強化子(Queen等人, Immunol. Rev.89:49 (1986))、以及必需的處理資訊位點，諸如核糖體結合位點、RNA剪接位點、多腺苷酸化位點及轉錄終止子序列。表現控制序列可包括源自內源基因、巨細胞病毒、SV 40、腺病毒、牛乳頭狀瘤病毒及其類似物之啟動子。參見Co等人,J. Immunol.148: 1149 (1992)。在一些實施例中，啟動子為真核生物啟動子。Mammalian cells can be used to express nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones (VCH Publishers, NY, 1987). Many suitable host cell lines capable of secreting intact heterologous proteins have been developed, including CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myelomas, including Sp2/0 and NS0. The cells may be non-human. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queenet al., Immunol. Rev. 89:49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. Expression control sequences can include promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papilloma virus, and the like. See Co et al.,J. Immunol. 148:1149 (1992). In some embodiments, the promoter is a eukaryotic promoter.
或者，可將抗體編碼序列併入轉殖基因中，以引入轉殖基因動物之基因體中且隨後在轉殖基因動物之乳汁中表現(參見，例如美國專利第5,741,957號；美國專利第5,304,489號；及美國專利第Alternatively, the antibody coding sequence can be incorporated into a transgene for introduction into the genome of a transgenic animal and subsequently expressed in the milk of the transgenic animal( see, e.g., U.S. Patent Nos. 5,741,957; 5,304,489; and 6,304,489).
5,849,992號)。合適的轉殖基因包括與來自乳腺特異性基因(諸如酪蛋白或β乳球蛋白)之啟動子及強化子可操作地連接之輕鏈及/或重鏈的編碼序列。5,849,992). Suitable transgenes include light and/or heavy chain coding sequences operably linked to a promoter and enhancer from a mammary gland-specific gene, such as casein or beta lactoglobulin.
含有所關注DNA區段的載體可藉由取決於細胞宿主之類型的方法來轉移至宿主細胞中。舉例而言，氯化鈣轉染通常用於原核細胞，而磷酸鈣處理、電穿孔、脂質轉染、基因槍或基於病毒之轉染可用於其他細胞宿主。用於轉化哺乳動物細胞之其他方法包括使用聚凝胺、原生質體融合、脂質體、電穿孔及微注射。為產生轉殖基因動物，可將反式基因顯微注射至受精卵母細胞中或可併入胚胎幹細胞之基因體中，且將此類細胞之核轉移至去核卵母細胞中。Vectors containing the DNA segment of interest can be transferred into host cells by methods that depend on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, gene guns, or viral-based transfection can be used for other cellular hosts. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection. To produce transgenic animals, genes can be microinjected in trans into fertilized oocytes or can be incorporated into the genome of embryonic stem cells, and the nuclei of these cells are transferred into enucleated oocytes.
將編碼抗體重鏈及輕鏈之載體引入細胞培養物中後，可針對無血清培養基中之生長生產率及產物品質篩選細胞池。然後，頂部產生細胞池可經受基於FACS之單細胞選殖以產生單株細胞株。可使用高於50 pg或100 pg/細胞/天之比生產率，其對應於大於7.5 g/L培養物之產物效價。亦可測試由單細胞殖株產生之抗體的濁度、過濾性質、PAGE、IEF、UV掃描、HPSEC、碳水化合物-寡醣定位、質譜及結合檢定，諸如ELISA或Biacore。接著可將所選純系儲存於多個小瓶中且冷凍儲存以供後續使用。After introducing vectors encoding the heavy and light chains of the antibodies into cell cultures, the cell pool can be screened for growth productivity and product quality in serum-free medium. The top producer cell pool can then undergo FACS-based single-cell selection to generate individual cell lines. Specific productivity of greater than 50 pg or 100 pg/cell/day can be used, corresponding to product titers greater than 7.5 g/L of culture. Antibodies produced by single cell lines can also be tested for turbidity, filterability, PAGE, IEF, UV scanning, HPSEC, carbohydrate-oligosaccharide mapping, mass spectrometry, and binding assays such as ELISA or Biacore. The selected pure lines can then be stored in multiple vials and frozen for later use.
一旦表現，可根據此項技術之標準程序純化抗體，包括蛋白A捕獲、HPLC純化、管柱層析、凝膠電泳及類似方法(一般參見Scopes,Protein Purification(Springer-Verlag, NY, 1982))。Once expressed, the antibody can be purified according to standard procedures of the technology, including protein A capture, HPLC purification, column chromatography, gel electrophoresis, and similar methods (see generally Scopes,Protein Purification (Springer-Verlag, NY, 1982)).
可採用商業生產抗體之方法，包括密碼子最佳化、啟動子之選擇、轉錄元件之選擇、終止子之選擇、無血清單細胞選殖、細胞庫、使用選擇標記擴增拷貝數、CHO終止子、或改善蛋白質效價(參見，例如美國專利第5,786,464號；美國專利第6,114,148號；US 6,063,598；美國專利第7,569,339號；W02004/050884；W02008/012142；W02008/012142；W02005/019442；W02008/107388；W02009/027471；及美國專利第5,888,809號)。Methods for commercial antibody production can be used, including codon optimization, promoter selection, transcription element selection, terminator selection, serum-free single cell selection, cell banking, use of selectable markers to increase copy number, CHO terminators, or improving protein titer (see, e.g., U.S. Patent Nos. 5,786,464; 6,114,148; U.S. Patent Nos. 5,786,464 and 6,114,148). 6,063,598; U.S. Patent No. 7,569,339; WO2004/050884; WO2008/012142; WO2008/012142; WO2005/019442; WO2008/107388; WO2009/027471; and U.S. Patent No. 5,888,809).
DNA可以裸形式(亦即，不含膠體或囊封材料)遞送。或者，可使用多種病毒載體系統，包括反轉錄病毒系統(參見，例如Lawrie及Tumin, Cur. Opin. Genet. Develop. 3, 102-109 (1993))；腺病毒載體(參見，例如Bett等人, J. Virol. 67, 591 1 (1993))；腺相關病毒載體(參見，例如Zhou等人, J. Exp. Med. 179, 1867 (1994))、來自痘家族(包括痘瘡病毒及禽痘病毒)之病毒載體、來自α病毒屬之病毒載體，諸如衍生自辛德比(Sindbis)及Semliki森林病毒之彼等病毒載體(參見，例如Dubensky等人, J. Virol. 70, 508-519 (1996))、委內瑞拉馬腦炎病毒(參見美國專利第5,643,576號)及棒狀病毒，諸如水皰性口炎病毒(參見WO 96/34625)及乳頭瘤病毒(Ohe等人,Human Gene Therapy6:325-333 (1995)；Woo等人, WO 94/12629 and Xiao & Brandsma,Nucleic Acids. Res.24:2630-2622 (1996))。The DNA can be delivered in naked form (i.e., without a colloid or encapsulating material). Alternatively, a variety of viral vector systems can be used, including retroviral systems (see, e.g., Lawrie and Tumin, Cur. Opin. Genet. Develop. 3, 102-109 (1993)); adenoviral vectors (see, e.g., Bett et al., J. Virol. 67, 591 1 (1993)); adeno-associated viral vectors (see, e.g., Zhou et al., J. Exp. Med. 179, 1867 (1994)), viral vectors from the pox family (including poxvirus and fowlpox virus), viral vectors from the alphavirus genus, such as those derived from Sindbis and Semliki forest viruses (see, e.g., Dubensky et al., J. Virol. 70, 508-519 (1996)), Venezuelan equine encephalitis virus (see U.S. Patent No. 5,643,576), and rhabdoviruses such as stomatitis virus (see WO 96/34625), and papillomaviruses (Ohe et al.,Human Gene Therapy 6:325-333 (1995); Woo et al., WO 94/12629 and Xiao & Brandsma,Nucleic Acids. Res. 24:2630-2622 (1996)).
編碼免疫原之DNA或含有該免疫原之載體可包裝至脂質體中。合適的脂質及相關類似物描述於美國專利第5,208,036號、美國專利第5,264,618號、美國專利第5,279,833號及美國專利第5,283,185號中。載體及編碼免疫原之DNA亦可吸附至微粒載體或與微粒載體締合，微粒載劑之實例包括聚甲基丙烯酸甲酯聚合物及聚丙交酯及聚(丙交酯-共-乙交酯) (參見，例如McGee等人,J. Micro Encap.1996)。DNA encoding the immunogen or a vector containing the immunogen can be packaged into liposomes. Suitable lipids and related analogs are described in U.S. Patent Nos. 5,208,036, 5,264,618, 5,279,833, and 5,283,185. The vector and DNA encoding the immunogen can also be adsorbed onto or conjugated to microparticle carriers. Examples of microparticle carriers include polymethyl methacrylate polymers and polylactide and poly(lactide-co-glycolide) (see, e.g., McGee et al.,J. Micro Encap. 1996).
V.V.額外結合物Additional binders
特異性結合至抗原諸如TDP-43 (例如人類TDP-43)之結合抗體及抗原結合抗體片段可用於偵測TDP-43之存在；監測及評估用於治療經診斷患有肌肉萎縮性脊髓側索硬化症(ALS)、額顳葉退化(FTLD-TDP)、原發性脊髓側索硬化症及進行性肌萎縮及帕金森氏病之患者的治療劑之功效；抑制或減少TDP-43之聚集；減少或清除TDP-43聚集體；穩定TDP-43之無毒構形；或治療或實現患者之肌肉萎縮性脊髓側索硬化症(ALS)、額顳葉退化(FTLD-TDP)、原發性脊髓側索硬化症及進行性肌萎縮及帕金森氏病之預防。Specific binding to antigens such as TDP-43 Antibodies and antigen-binding antibody fragments that bind to an antigen (e.g., human TDP-43) can be used to detect the presence of TDP-43; monitor and evaluate the efficacy of therapeutic agents used to treat patients diagnosed with amyotrophic lateral sclerosis (ALS), frontotemporal degeneration (FTLD-TDP), primary lateral sclerosis, and progressive muscular dystrophy and Parkinson's disease; inhibit or reduce TDP-43 aggregation; reduce or eliminate TDP-43 aggregates; stabilize the non-toxic conformation of TDP-43; or treat or achieve prevention of amyotrophic lateral sclerosis (ALS), frontotemporal degeneration (FTLD-TDP), primary lateral sclerosis, and progressive muscular dystrophy and Parkinson's disease in patients.
本文所述之細胞穿透劑可進一步與其他治療部分、其他蛋白、其他抗體及/或可偵測標記結合。參見WO 03/057838；美國專利第8,455,622號。此類治療部分可為可用於治療、對抗、改善、預防或改良患者之不想要的疾患或疾病(諸如肌肉萎縮性脊髓側索硬化症(ALS)、額顳葉退化(FTLD-TDP)、原發性脊髓側索硬化症、及進行性肌萎縮及帕金森氏病)的任何劑。The cell-penetrating agents described herein can be further conjugated to other therapeutic moieties, other proteins, other antibodies, and/or detectable markers. See WO 03/057838; U.S. Patent No. 8,455,622. Such therapeutic moieties can be any agent useful for treating, combating, ameliorating, preventing, or modifying an unwanted condition or disease in a patient, such as amyotrophic lateral sclerosis (ALS), frontotemporal lobe degeneration (FTLD-TDP), primary lateral sclerosis, and progressive muscular dystrophy and Parkinson's disease.
結合之治療部分可包括細胞毒性劑、細胞生長抑制劑、神經營養劑、神經保護劑、放射治療劑、免疫調節劑或促進或提高抗體活性之任何生物活性劑。細胞毒性劑可為對細胞有毒之任何劑。細胞生長抑制劑可為抑制細胞增殖之任何劑。神經營養劑可為促進神經元維持、生長或分化之任何劑，包括化學劑或蛋白質劑。神經保護劑可為保護神經元免於急性損傷或退化過程之劑，包括化學劑或蛋白質劑。免疫調節劑可為刺激或抑制免疫反應之發展或維持之任何劑。放射治療劑可為發射輻射之任何分子或化合物。若此類治療部分偶合至TDP-43特異性抗體或抗原結合抗體片段，諸如本文所述之抗體及抗原結合抗體片段，則與正常細胞相比，偶合的治療部分將對TDP-43相關疾病受累細胞具有特異性親和力。The conjugated therapeutic moiety may include a cytotoxic agent, a cytostatic agent, a neurotrophic agent, a neuroprotective agent, a radiotherapeutic agent, an immunomodulatory agent, or any biologically active agent that promotes or enhances antibody activity. A cytotoxic agent may be any agent that is toxic to cells. A cytostatic agent may be any agent that inhibits cell proliferation. A neurotrophic agent may be any agent that promotes neuron maintenance, growth, or differentiation, including chemical agents or protein agents. A neuroprotective agent may be any agent that protects neurons from acute damage or degenerative processes, including chemical agents or protein agents. An immunomodulatory agent may be any agent that stimulates or inhibits the development or maintenance of an immune response. A radiotherapeutic agent can be any molecule or compound that emits radiation. If such a therapeutic moiety is conjugated to a TDP-43-specific antibody or antigen-binding antibody fragment, such as those described herein, the conjugated therapeutic moiety will have a specific affinity for cells affected by a TDP-43-associated disease compared to normal cells.
因此，投予此等進一步結合之抗體或結合之抗原結合抗體片段將直接靶向細胞，對周圍正常健康組織之損傷最小。此可特別適用於毒性太大而無法單獨投予之治療部分。另外，可使用較小量之治療部分。Thus, administration of these further conjugated antibodies or conjugated antigen-binding antibody fragments will directly target cells with minimal damage to surrounding normal healthy tissue. This is particularly useful for therapeutic moieties that are too toxic to administer alone. Additionally, smaller amounts of the therapeutic moiety can be used.
一些此類細胞穿透劑可連接至放射性同位素。放射性同位素之實例包括例如釔90(90Y)、銦111(111In)、1311、99mTc、放射性銀-111、放射性銀-199及鉍213。放射性同位素與抗體或抗原結合抗體片段之鍵聯可用習知雙功能螯合物進行。對於放射性銀-111及放射性銀-199鍵聯，可使用基於硫之連接子。參見Hazra等人,Cell Biophys.24-25:1-7 (1994)。銀放射性同位素之鍵聯可涉及用抗壞血酸還原免疫球蛋白。對於放射性同位素(諸如111In及90Y)，可使用替伊莫單抗(ibritumomab tiuxetan)且將與此類同位素反應以分別形成111In-替伊莫單抗及90Y-替伊莫單抗。參見Witzig,Cancer Chemother. Pharmacol.,48 (增刊l):S91-S95 (2001)。Some of these cell penetrants can be linked to radioactive isotopes. Examples of radioactive isotopes include, for example, yttrium-90 (90Y), indium-111 (111In),1311,99mTc , radiosilver-111, radiosilver-199, and bismuth-213 . Conjugation of the radioisotope to the antibody or antigen-binding antibody fragment can be accomplished using known bifunctional chelates. For radiosilver-111 and radiosilver-199 conjugation, sulfur-based linkers can be used. See Hazra et al.,Cell Biophys. 24-25:1-7 (1994). Conjugation of the silver radioisotope can involve reduction of the immunoglobulin with ascorbic acid. For radioactive isotopes such as 111In and 90Y, ibritumomab tiuxetan can be used and will react with these isotopes to form 111In-ibritumomab tiuxetan and 90Y-ibritumomab tiuxetan, respectively. See Witzig,Cancer Chemother. Pharmacol., 48 (Suppl 1): S91-S95 (2001).
一些此類抗體或抗原結合抗體片段可連接至其他治療部分。此類治療部分可為例如細胞毒性的、細胞生長抑制的、免疫調節的、神經營養的或神經保護的。舉例而言，抗體及抗原結合抗體片段可與毒性化學治療藥物(諸如美登素(maytansine)、格爾德黴素(geldanamycin))、微管蛋白抑制劑(諸如微管蛋白結合劑(例如澳瑞他汀))或小溝區結合劑(諸如卡奇黴素(calicheamicin))結合。其他代表性治療部分包括已知可用於治療、管理或改善肌肉萎縮性脊髓側索硬化症(ALS)、額顳葉退化(FTLD-TDP)、原發性脊髓側索硬化症、及進行性肌萎縮及帕金森氏病之劑。Some of these antibodies or antigen-binding antibody fragments can be linked to other therapeutic moieties. Such therapeutic moieties can be, for example, cytotoxic, cytostatic, immunomodulatory, neurotrophic, or neuroprotective. For example, antibodies and antigen-binding antibody fragments can be conjugated to toxic chemotherapeutic drugs (e.g., maytansine, geldanamycin), tubulin inhibitors (e.g., tubulin-binding agents(e.g. , auristatin)), or sulcus-binding agents (e.g., calicheamicin). Other representative therapeutic components include agents known to treat, manage or ameliorate amyotrophic lateral sclerosis (ALS), frontotemporal degeneration (FTLD-TDP), primary lateral sclerosis, and progressive muscular dystrophy and Parkinson's disease.
抗體或抗原結合抗體片段亦可與可偵測標記偶合。此類抗體及抗原結合抗體片段可用於例如診斷ALS、FTLD-TDP、原發性脊髓側索硬化症、及進行性肌萎縮及帕金森氏病。可偶合或連接至抗體或抗原結合抗體片段之代表性可偵測標記包括各種酶，諸如辣根過氧化物酶、鹼性磷酸酶、β半乳糖苷酶或乙醯膽鹼酯酶；輔基，諸如鏈黴抗生物素蛋白/生物素及抗生物素蛋白/生物素；螢光材料，諸如繖形酮、螢光素、異硫氰酸螢光素、若丹明、二氯三嗪基胺螢光素、丹磺醯氯或藻紅蛋白；發光材料，諸如魯米諾(luminol)；生物發光材料，諸如螢光素酶、螢光素及水母發光蛋白；放射性材料，諸如放射性銀-111、放射性銀-199、鉍213、碘(131I、125I、123I、121I)、碳(14C)、硫(5S)、氚(3H)、銦(115In113In112In111In)、鍀(99Tc)、鉈(201Ti)、鎵(68Ga、67Ga)、鈀(103Pd)、鉬(99Mo)、氙(133Xe)、氟(18F)、153Sm、177Lu、159Gd、149Pm、140La、175Yb、166Ho、90Y、47Sc、186Re、188Re、142Pr、105Rh、97Ru、68Ge、57Co、65ZN、85SR、32P、153Gd、169Yb、51CR、54Mn、75Se、113Sn及117Sn；使用各種正電子發射斷層攝影術之正電子發射金屬；非放射性順磁性金屬離子；以及經放射性標記或與特定放射性同位素結合之分子。The antibodies or antigen-binding antibody fragments can also be coupled to a detectable label. Such antibodies and antigen-binding antibody fragments can be used, for example, to diagnose ALS, FTLD-TDP, primary lateral sclerosis, and progressive muscular dystrophy and Parkinson's disease. Representative detectable labels that can be coupled or linked to the antibody or antigen-binding antibody fragment include enzymes such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholine esterase; cofactors such as streptavidin/biotin and avidin/biotin; fluorescent materials such as fluorescein, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials such as luminol; bioluminescent materials such as luciferase, fluorescein, and aequorin; radioactive materials such as radiosilver-111, radiosilver-199, bismuth-213 , iodine (131 I、125 I、123 I、121 I)、Carbon (14 C)、Sulfur (5 S)、Tritium (3 H)、Indium (115 In113 In112 In111 In)、Tesla (99 Tc)、Boc (201 Ti)、Gallium (68 Ga、67 Ga)、Palladium (103 Pd)、Molybdenum (99 Mo)、Xenon (133 Xe)、Fluorine (18 F)、153 Sm)、 177 Lu)、159 Gd)、149 Pm)、140 La)、175 Yb)、166 Ho)、90 Y)、47 Sc)、186 Re)、 188Re )、142 Pr)、105 Rh)、97 Ru)、68 Ge)、57 Co) 、65 Zn)、85 SR,32 P,153 Gd,169 Yb,51 CR,54 Mn,75 Se,113 Sn, and117 Sn; positron-emitting metals used in various positron emission tomography techniques; non-radioactive paramagnetic metal ions; and molecules that are radiolabeled or bound to specific radioisotopes.
治療部分、其他蛋白質、其他抗體及/或可偵測標記可直接或經由中間物(例如，連接子)間接地偶合或結合至本發明之抗體或抗原結合抗體片段。參見，例如Arnon等人, 「Monoclonal Antibodies For Immunotargeting of Drugs in Cancer Therapy,」 in Monoclonal Antibodies And Cancer Therapy, Reisfeld等人(編), 第243-56頁(Alan R. Liss, Inc. 1985)；Hellstrom等人, 「Antibodies For Drug Delivery,」in Controlled Drug Delivery (第2版), Robinson等人(編), 第623-53頁(Marcel Dekker, Inc. 1987)；Thorpe, 「Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review,」 in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera等人(編), 第475-506頁(1985)；「Analysis, Results, and Future Prospective of The Therapeutic Use of Radio labeled Antibody in Cancer Therapy,」 in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin等人(編), 第303-16頁(Academic Press 1985)；及Thorpe等人,Immunol. Rev.,62:119-58 (1982)。合適的連接子包括例如可裂解及不可裂解連接子。可採用在酸性或還原條件下，在暴露於特定蛋白酶時或在其他限定條件下釋放偶合的治療部分、蛋白質、抗體及/或可偵測標記之不同連接子。Therapeutic moieties, other proteins, other antibodies, and/or detectable labels can be coupled or conjugated to the antibodies or antigen-binding antibody fragments of the invention directly or indirectly through an intermediary(e.g., a linker). See, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting of Drugs in Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd ed.), Robinson et al. (eds.), Pages 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review," in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (Eds.), pages 475-506 (1985); "Analysis, Results, and Future Prospective of The Therapeutic Use of Radio labeled Antibody in Cancer Therapy," in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985); and Thorpe et al.,Immunol. Rev., 62:119-58 (1982). Suitable linkers include, for example, cleavable and non-cleavable linkers. Different linkers can be used that release the coupled therapeutic moiety, protein, antibody, and/or detectable label under acidic or reducing conditions, upon exposure to a specific protease, or under other defined conditions.
在一些實施例中，細胞穿透劑亦與如本文所述之治療劑、細胞毒性劑、細胞生長抑制劑、免疫調節劑、神經營養劑或神經保護劑結合。舉例而言，存在於細胞穿透劑中之抗體可與治療部分(諸如細胞毒性劑、放射治療劑、免疫調節劑或第二抗體)偶合(亦即，結合) (例如，以形成抗體異源結合物)。代表性治療部分包括已知可用於治療、管理或改善TDP-43相關疾病或TDP-43相關疾病之症狀的劑。In some embodiments, the cell penetrating agent is also conjugated to a therapeutic agent, cytotoxic agent, cell growth inhibitory agent, immunomodulatory agent, neurotrophic agent, or neuroprotective agent as described herein. For example, the antibody present in the cell penetrating agent can be coupled (i.e., conjugated) to a therapeutic moiety (e.g., a cytotoxic agent, a radiotherapeutic agent, an immunomodulatory agent, or a second antibody) (e.g. , to form an antibody heteroconjugate). Representative therapeutic moieties include agents known to treat, manage, or ameliorate TDP-43-associated diseases or symptoms of TDP-43-associated diseases.
治療部分及/或可偵測物質可使用此項技術中已知的技術經由中間物(例如，連接子)直接偶合或結合至本文所述之鼠類、嵌合或人源化抗體中之任一者。參見，例如Arnon等人, 「Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,」 in Monoclonal Antibodies And Cancer Therapy, Reisfeld等人(編), 第243-56頁(Alan R. Liss, Inc. 1985)；Hellstrom等人, 「Antibodies For Drug Delivery,」 in Controlled Drug Delivery (第2版), Robinson等人(編), 第623-53頁(Marcel Dekker, Inc. 1987); Thorpe, 「Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,」 in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera等人(編), 第475-506頁(1985); 「Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy,」 in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin等人(編), 第303-16頁(Academic Press 1985)及Thorpe等人, Immunol. Rev., 1982, 62:119-58。The therapeutic moiety and/or detectable substance can be coupled directly or conjugated to any of the murine, chimeric, or humanized antibodies described herein via an intermediate (e.g. , a linker) using techniques known in the art. See, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd ed.), Robinson et al. (eds.), Pages 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pages 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985) and Thorpe et al., Immunol. Rev., 1982, 62:119-58.
所揭示之調配物中使用的細胞穿透劑亦包括鼠類、嵌合或人源化13D3抗體之經修飾形式，其相對於相應未經修飾之抗體具有延長的活體內半衰期。此類經修飾形式可例如藉由醣基化、乙醯化、聚乙二醇化、磷酸化、醯胺化、藉由已知保護/阻斷基團之衍生化、蛋白水解裂解、與細胞配位體或其他蛋白質之鍵聯等來製備。作為一個實例，用於延長抗體半衰期之代表性方法描述於PCT國際公開案第WO 02/060919號中。Cell penetrants used in the disclosed formulations also include modified forms of murine, chimeric, or humanized 13D3 antibodies that have an extendedin vivo half-life relative to the corresponding unmodified antibody. Such modified forms can be prepared, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, orlinkage to cellular ligands or other proteins. As an example, representative methods for extending antibody half-life are described in PCT International Publication No. WO 02/060919.
VI.VI.醫藥組成物及產物Pharmaceutical compositions and products
本揭示內容亦提供醫藥組成物及產物。因此，本文提供醫藥組成物，其包括本文描述之任一細胞穿透劑及醫藥上可接受之載劑。The present disclosure also provides pharmaceutical compositions and products. Thus, provided herein are pharmaceutical compositions comprising any of the cell penetrating agents described herein and a pharmaceutically acceptable carrier.
用於非經腸投予之醫藥組成物較佳為無菌的且實質上等滲且在GMP條件下製造。醫藥組成物可以單位劑型(亦即，單次投予之劑量)提供。醫藥組成物可使用一或多種生理學上可接受之載劑、稀釋劑、賦形劑或助劑來調配。調配物視所選投予途徑而定。對於注射，可將細胞穿透劑調配於水溶液中，較佳調配於生理相容之緩衝液，諸如漢克氏溶液(Hank's solution)、林格氏溶液(Ringer's solution)或生理鹽水或乙酸鹽緩衝液(以減少注射部位之不適)中。溶液可含有調配劑，諸如懸浮劑、穩定劑及/或分散劑。或者，細胞穿透劑可呈凍乾形式，用於在使用前用合適的媒劑(例如無菌無熱原水)構成。Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under GMP conditions. The pharmaceutical compositions can be provided in unit dose form (i.e., a single dose). The pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the selected route of administration. For injection, the cell penetrant can be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or saline or acetate buffer (to reduce injection site discomfort). The solution may contain formulatory agents such as suspending agents, stabilizers and/or dispersing agents. Alternatively, the cell penetrating agent may be in lyophilized form for constitution with a suitable vehicle (e.g., sterile, pyrogen-free water) before use.
本文所述之抗TDP-43細胞穿透劑可存在於任何醫藥上可接受之賦形劑或載劑中。舉例而言，本文所述之抗TDP-43細胞穿透劑可存在於緩衝液中。緩衝液可具有約6至約7之pH值。通常，調配物為無菌的，例如，如藉由使用0.2 μm或0.22 μm過濾器進行無菌過濾來實現。本文所揭示之調配物在冷凍及解凍後通常亦係穩定的。The anti-TDP-43 cell penetrants described herein can be present in any pharmaceutically acceptable formulation or carrier. For example, the anti-TDP-43 cell penetrants described herein can be present in a buffer. The buffer can have a pH of about 6 to about 7. Typically, the formulation is sterile, for example, by sterile filtration using a 0.2 μm or 0.22 μm filter. The formulations disclosed herein are also generally stable after freezing and thawing.
在一些實施例中，可能需要在離體或活體外方法中使用包含本文所述之任一細胞穿透劑的醫藥組成物。舉例而言，該方法可用於非診斷及/或非治療目的。在此類情況下，使已自患者取出之樣品(諸如細胞、組織及/或器官)暴露於包含本文所述之任一細胞穿透劑的醫藥組成物。In some embodiments, it may be desirable to use a pharmaceutical composition comprising any of the cell penetrating agents described herein inan in vitro orex vivo method. For example, such methods may be used for non-diagnostic and/or non-therapeutic purposes. In such cases, a sample (e.g., cells, tissues, and/or organs) removed from a patient is exposed to a pharmaceutical composition comprising any of the cell penetrating agents described herein.
在預防性應用中，將細胞穿透劑(例如，或編碼本文所述之任一細胞穿透劑的核酸或載體)或其醫藥組成物在有效降低TDP-43相關疾病之至少一種徵像或症狀的風險、減輕其嚴重程度或延遲其發作的方案(投予劑量、頻率及途徑)中向疑似或處於諸如ALS、FTLD-TDP、原發性脊髓側索硬化症、及進行性肌萎縮及帕金森氏病之疾病的風險下之患者投予。具體而言，該方案較佳有效地抑制或延遲腦中之TDP-43聚集體(例如人類TDP-43聚集體)，及/或抑制或延遲其毒性作用及/或抑制/或延遲行為缺陷之發展。In prophylactic applications, a cell-penetrating agent (e.g., or a nucleic acid or vector encoding any of the cell-penetrating agents described herein) or a pharmaceutical composition thereof is administered to a patient suspected of or at risk for a disease such as ALS, FTLD-TDP, primary lateral sclerosis, and progressive muscular dystrophy and Parkinson's disease, in a regimen (dose, frequency, and route of administration) that is effective to reduce the risk, lessen the severity, or delay the onset of at least one sign or symptom of a TDP-43-related disease. Specifically, the regimen is preferably effective in inhibiting or delaying the formation of TDP-43 aggregates (e.g., human TDP-43 aggregates) in the brain and/or inhibiting or delaying their toxic effects and/or inhibiting or delaying the development of behavioral deficits.
在治療性應用中，將細胞穿透劑在有效改善或至少抑制疾病(例如ALS)之至少一種徵像或症狀的進一步惡化之方案(投予劑量、頻率及途徑)中向疑似或已患有該疾病之患者投予。具體而言，該方案較佳有效地減少或至少抑制TDP-43 (例如人類TDP-43)及/或由其形成之聚集體的細胞質水準之進一步增加、相關毒性及/或行為缺陷。In therapeutic applications, a cell-penetrating agent is administered to a patient suspected of or already suffering from a disease (e.g., ALS) in a regimen (dosage, frequency, and route of administration) that is effective to ameliorate or at least inhibit further progression of at least one sign or symptom of the disease. Specifically, the regimen is preferably effective in reducing or at least inhibiting further increases in cytoplasmic levels of TDP-43 (e.g., human TDP-43) and/or aggregates thereof, associated toxicities, and/or behavioral defects.
若個別經治療之患者達成的結果比未藉由本文所揭示之方法治療的可比較患者之對照群體中的平均結果更有利，則認為方案在治療上或預防上有效。A regimen is considered therapeutically or prophylactically effective if the outcome achieved by an individual treated patient is more favorable than the average outcome in a control population of comparable patients not treated by the methods disclosed herein.
VII.VII.治療方案Treatment options
如本文所用，術語「治療(treat)」及「治療(treatment)」係指減輕或改善與疾病相關之一或多種症狀或效應，預防、抑制或延遲疾病之一或多種症狀或效應之發作，減輕疾病之一或多種症狀或效應之嚴重程度或頻率，及/或增加或趨向於如本文所述之期望結果。As used herein, the terms "treat" and "treatment" refer to the alleviation or amelioration of one or more symptoms or effects associated with a disease, the prevention, inhibition, or delay of the onset of one or more symptoms or effects of a disease, the reduction of the severity or frequency of one or more symptoms or effects of a disease, and/or the increase or tendency toward a desired outcome as described herein.
本文所揭示之治療之期望結果根據TDP-43相關疾病及患者概況而變化，且對於熟習此項技術者而言可容易確定。期望結果包括患者健康狀況之改善。通常，期望結果包括可量測指標，諸如病理性ALS、FTLD-TDP、原發性脊髓側索硬化症、及進行性肌萎縮及帕金森氏病之減少或清除。The desired outcome of the treatments disclosed herein varies depending on the TDP-43-related disease and the patient's profile and can be readily determined by one skilled in the art. Desired outcomes include improvement in the patient's health status. Typically, desired outcomes include a reduction or elimination of measurable indicators, such as pathological ALS, FTLD-TDP, primary lateral sclerosis, and progressive muscular dystrophy and Parkinson's disease.
本文提供將特異性結合至TDP-43之抗體遞送至細胞中的方法，其包含使本文所述之任一細胞穿透劑與細胞接觸，由此導致至少抗體之抗原結合片段內化至細胞中。在一些實施例中，該方法包括至少將抗體之抗原結合片段轉移至細胞之細胞液中。Provided herein are methods for delivering an antibody that specifically binds to TDP-43 into a cell, comprising contacting the cell with any of the cell-penetrating agents described herein, thereby causing internalization of at least an antigen-binding fragment of the antibody into the cell. In some embodiments, the method comprises transferring at least the antigen-binding fragment of the antibody into the cytosol of the cell.
本文亦提供結合細胞中之細胞內TDP-43蛋白的方法，其包括使如請求項1至135中任一項之細胞穿透劑與該細胞接觸，由此導致至少抗體之抗原結合片段內化且轉移至細胞液中。Also provided herein is a method for binding intracellular TDP-43 protein in a cell, comprising contacting the cell with the cell penetrant of any one of claims 1 to 135, thereby causing internalization and translocation of at least the antigen-binding fragment of the antibody into the cytosol.
本文亦提供結合細胞中之細胞內TDP-43蛋白的方法，其包括：使如請求項1至135中任一項之細胞穿透劑與該細胞接觸，由此導致至少抗體之抗原結合片段內化且轉移至細胞液中；及使至少抗體之抗原結合片段與細胞內TDP-43蛋白結合。Also provided herein is a method for binding to an intracellular TDP-43 protein in a cell, comprising: contacting the cell with the cell of any one of claims 1 to 135, thereby causing internalization and translocation of at least an antigen-binding fragment of the antibody into the cytosol; and allowing at least the antigen-binding fragment of the antibody to bind to the intracellular TDP-43 protein.
此外，本文提供抑制或減少患有TDP-43相關疾病或具有患上該疾病之風險的個體中之TDP-43 (例如人類TDP-43)聚集的方法，其包括向個體投予有效量之本文所述之任一細胞穿透劑，由此抑制或減少TDP-43在個體(亦即患者)中之聚集。Furthermore, provided herein are methods for inhibiting or reducing TDP-43 (e.g., human TDP-43) aggregation in an individual having or at risk of developing a TDP-43-associated disease, comprising administering to the individual an effective amount of any cell-permeable agent described herein, thereby inhibiting or reducing TDP-43 aggregation in the individual (i.e., patient).
本文亦提供治療或實現預防個體之TDP-43相關疾病的方法，其包括投予治療有效量之本文所述之任一細胞穿透劑，由此治療或實現預防TDP-43相關疾病。Also provided herein are methods for treating or preventing a TDP-43-related disease in a subject, comprising administering a therapeutically effective amount of any cell penetrating agent described herein, thereby treating or preventing the TDP-43-related disease.
在一些實施例中，TDP-43相關疾病為ALS、FTLD-TDP、原發性脊髓側索硬化症、及進行性肌萎縮及帕金森氏病。在一些實施例中，TDP-43相關疾病為ALS。In some embodiments, the TDP-43-associated disease is ALS, FTLD-TDP, primary lateral sclerosis, and progressive muscular dystrophy and Parkinson's disease. In some embodiments, the TDP-43-associated disease is ALS.
本文亦提供偵測患有TDP-43相關疾病或具有患上該疾病之風險的個體中之TDP-43沉積(例如，人類TDP-43沉積)的方法，其包括向個體投予本文所述之任一細胞穿透劑，及偵測該個體中與TDP-43結合之抗體。Also provided herein are methods for detecting TDP-43 deposition (e.g., human TDP-43 deposition) in a subject having or at risk for a TDP-43-associated disease, comprising administering to the subject any of the cell-permeable agents described herein, and detecting an antibody that binds to TDP-43 in the subject.
在一些實施例中，細胞穿透劑藉由靜脈內注射至個體之身體中來投予。在一些實施例中，細胞穿透劑或細胞穿透劑中之抗體經標記。在一些實施例中，細胞穿透劑用螢光標記、順磁性標記或放射性標記來標記。在一些實施例中，使用正電子發射斷層攝影術(PET)或單光子發射電腦斷層攝影術(SPECT)來偵測放射性標記。In some embodiments, the cell penetrating agent is administered by intravenous injection into the body of the individual. In some embodiments, the cell penetrating agent or the antibody in the cell penetrating agent is labeled. In some embodiments, the cell penetrating agent is labeled with a fluorescent label, a paramagnetic label, or a radioactive label. In some embodiments, the radioactive label is detected using positron emission tomography (PET) or single photon emission computed tomography (SPECT).
細胞穿透劑以有效方案投予，該有效方案意謂延遲正在治療之病症的至少一種徵像或症狀之發作、降低嚴重程度、抑制進一步惡化及/或改善其狀況之劑量、投予途徑及投予頻率。若患者已患有病症，則該方案可稱為治療有效方案。若患者相對於一般群體處於升高的病症之風險下但尚未經歷症狀，則該方案可稱為預防有效方案。在一些情況下，相對於個別患者之歷史對照或過去經歷，可在同一患者中觀察到治療或預防功效。在其他情況下，相對於未經治療患者之對照群體，治療或預防功效可在經治療患者群體中在臨床前或臨床試驗中得到證明。The cell-penetrating agent is administered at an effective regimen, meaning a dose, route of administration, and frequency of administration that delays the onset of, reduces the severity of, inhibits further progression of, and/or improves at least one sign or symptom of the condition being treated. If the patient already has the condition, the regimen may be considered therapeutically effective. If the patient is at increased risk for the condition relative to the general population but has not yet experienced symptoms, the regimen may be considered prophylactically effective. In some cases, therapeutic or prophylactic efficacy may be observed in the same patient relative to historical controls or past experience. In other cases, therapeutic or preventive efficacy may be demonstrated preclinically or in clinical trials in a population of treated patients compared to a control population of untreated patients.
投予可為非經腸、靜脈內、經口、皮下、動脈內、顱內、鞘內、腹膜內、局部、鼻內或肌肉內。一些細胞穿透劑可藉由靜脈內或皮下投予來投予至全身循環中。Administration can be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Some cell penetrating agents can be administered to the systemic circulation by intravenous or subcutaneous administration.
細胞穿透劑調配物可以約0.5 mg/kg至約30 mg/kg宿主體重之劑量範圍靜脈內或皮下投予。舉例而言，劑量可為約0.5 mg/kg體重、約1.0 mg/kg、約1.5 mg/kg、約2.0 mg/kg、約4.0 mg/kg、約5.0 mg/kg、約8.0 mg/kg、約10 mg/kg、約15 mg/kg、約16 mg/kg、約20 mg/kg、約24 mg/kg、約25 mg/kg或約30 mg/kg體重。劑量亦可根據體表面積自約0.5 mg/m2至約500 mg/m2(例如0.5、5、10、50、100、250或500 mg/m2)投予。對於靜脈內給藥，將足以達成個體患者之期望劑量之量的細胞穿透劑調配物自一或多個小瓶轉移至一或多個含有液體(例如鹽水)之靜脈內袋中且向該患者投予。The cell penetrant formulation can be administered intravenously or subcutaneously in a dosage range of about 0.5 mg/kg to about 30 mg/kg of host body weight. For example, the dosage can be about 0.5 mg/kg body weight, about 1.0 mg/kg, about 1.5 mg/kg, about 2.0 mg/kg, about 4.0 mg/kg, about 5.0 mg/kg, about 8.0 mg/kg, about 10 mg/kg, about 15 mg/kg, about 16 mg/kg, about 20 mg/kg, about 24 mg/kg, about 25 mg/kg, or about 30 mg/kg body weight. The dosage can also be administered based on body surface area, ranging from about 0.5 mg/m2 to about 500 mg/m2 (e.g., 0.5, 5, 10, 50, 100, 250, or 500 mg/m2 ). For intravenous administration, an amount of the cell penetrating agent formulation sufficient to achieve the desired dose for an individual patient is transferred from one or more vials into one or more intravenous bags containing a liquid (e.g., saline) and administered to the patient.
細胞穿透劑通常在多個場合投予。投予頻率視細胞穿透劑在循環中之半衰期、患者之狀況及投予途徑等因素而定。頻率可為每天、每週、每月、每季度或以響應於患者狀況之變化或所治療病症之或進展的不規則間隔。Cell-penetrating agents are typically administered on multiple occasions. The frequency of administration depends on factors such as the half-life of the cell-penetrating agent in the circulation, the patient's condition, and the route of administration. The frequency may be daily, weekly, monthly, quarterly, or at irregular intervals in response to changes in the patient's condition or the progression of the condition being treated.
例示性治療方案需要每兩週投予一次、每月投予一次或每3至6個月投予一次。所投予之劑量數目視病症為急性抑或慢性及病症對治療之反應而定。給藥頻率可根據患者中抗體調配物之藥物動力學概況來調整。舉例而言，細胞穿透劑之半衰期可保證兩週之給藥頻率。在本文揭示之一些實施例中，向患者投予細胞穿透劑持續至少8個月、至少9個月、至少10個月、至少11個月、至少12個月、5年、10年或患者終生。Exemplary treatment regimens entail administration every two weeks, monthly, or every three to six months. The number of doses administered depends on whether the condition is acute or chronic and the condition's response to treatment. The frequency of dosing can be adjusted based on the pharmacokinetic profile of the antibody formulation in the patient. For example, the half-life of the cell penetrating agent may warrant a two-week dosing frequency. In some embodiments disclosed herein, the cell penetrating agent is administered to the patient for at least eight months, at least nine months, at least ten months, at least eleven months, at least twelve months, five years, ten years, or for the patient's lifetime.
正常水準之TDP-43 (例如人類TDP-43)可在尚未診斷患有特定TDP-43相關疾病(例如ALS)且不視為處於患上此類疾病之較高風險下的一般群體中的個體之代表性樣品(例如，50歲以下無病個體之代表性樣品)之腦中確定。或者，若根據本發明方法，在已知發展TDP-43聚集體(例如人類TDP-43聚集體)之腦區域中的PET訊號與來自已知通常不會形成此類沉積物之腦區域的訊號無差異(在量測精度內)，則可在個別患者中識別出正常水準。個體中升高之水準可藉由與正常水準(例如外部平均值及標準偏差之方差)的比較或與未知與沉積物相關之區域相比，僅僅來自與TDP-43聚集體(例如人類TDP-43聚集體)相關之腦區域中超出實驗誤差的升高訊號來識別。出於比較個體與群體中TDP-43聚集體(例如人類TDP-43聚集體)之水準之目的，TDP-43聚集體較佳應在腦之相同區域中測定，此等區域包括已知其中形成與特定疾病(例如ALS)相關之TDP-43聚集體的至少一個區域(例如在細胞質中)。Normal levels of TDP-43 (e.g., human TDP-43) can be determined in the brain of a representative sample of individuals from the general population who have not been diagnosed with a particular TDP-43-related disease (e.g., ALS) and are not considered to be at increased risk for developing such a disease (e.g., a representative sample of disease-free individuals under the age of 50). Alternatively, normal levels can be identified in an individual patient if, according to the methods of the invention, the PET signal in a brain region known to develop TDP-43 aggregates (e.g., human TDP-43 aggregates) is indistinguishable (within the measurement accuracy) from the signal in a brain region known not to normally form such deposits. Elevated levels in an individual can be identified by elevated signals beyond experimental error in brain regions associated with TDP-43 aggregates (e.g., human TDP-43 aggregates) compared to normal levels(e.g., variance of the external mean and standard deviation) or compared to regions not known to be associated with deposits. For the purpose of comparing the levels of TDP-43 aggregates (e.g., human TDP-43 aggregates) in an individual and a population, TDP-43 aggregates should preferably be measured in the same regions of the brain, including at least one region (e.g., in the cytoplasm) where TDP-43 aggregates associated with a particular disease (e.g., ALS) are known to form.
具有升高水準之TDP-43聚集體(例如人類TDP-43聚集體)之患者為開始免疫療法之候選者。在開始免疫療法後，TDP-43聚集體(例如人類TDP-43聚集體)之水準降低可首先被視為治療具有期望效果的指示。所觀察到之降低可例如在基線值之1-100%、1-50%或1-25%之範圍內。此類效果可在已知其中形成沉積物的腦之一或多個區域中量測，或可自此類區域之平均值量測。治療之總效果可藉由將相對於基線之減少百分比與TDP-43聚集體(例如人類TDP-43聚集體)之增加相加來近似，否則在普通未經治療之患者中會發生此類增加。Patients with elevated levels of TDP-43 aggregates (e.g., human TDP-43 aggregates) are candidates for initiating immunotherapy. After initiating immunotherapy, a decrease in the level of TDP-43 aggregates (e.g., human TDP-43 aggregates) can initially be considered an indication that the treatment is having the desired effect. The observed decrease can, for example, be in the range of 1-100%, 1-50%, or 1-25% of the baseline value. Such an effect can be measured in one or more regions of the brain where deposits are known to form, or can be measured from an average of such regions. The overall effect of the treatment can be approximated by adding the percent decrease relative to baseline to the increase in TDP-43 aggregates (e.g., human TDP-43 aggregates) that would otherwise occur in an ordinary untreated patient.
將TDP-43聚集體(例如，人類TDP-43聚集體)維持在大致恆定之水準或TDP-43聚集體(例如，人類TDP-43聚集體)之甚至小幅增加亦可指示對治療之反應，儘管是次最佳反應。可將此類反應與患有未接受治療之特定疾病(例如ALS)之患者的TDP-43聚集體(例如人類TDP-43聚集體)水準之時程進行比較，以確定免疫療法是否具有抑制TDP-43聚集體(例如，人類TDP-43聚集體)進一步增加之效果。Maintaining TDP-43 aggregates (e.g., human TDP-43 aggregates) at a roughly constant level or even a small increase in TDP-43 aggregates (e.g., human TDP-43 aggregates) can indicate a response to treatment, albeit a suboptimal response. Such a response can be compared to the course of TDP-43 aggregate levels (e.g., human TDP-43 aggregates) in patients with a particular disease (e.g., ALS) who have not received treatment to determine whether the immunotherapy has the effect of inhibiting further increases in TDP-43 aggregates (e.g., human TDP-43 aggregates).
VIII.VIII.套組Set
本揭示內容進一步提供套組(例如容器)，其包含本文所述之任一細胞穿透劑及相關材料，諸如使用說明書(例如包裝插頁)。使用說明書可含有例如關於投予細胞穿透劑及視情況存在的一或多種其他劑之說明書。細胞穿透劑之容器可為單位劑量、散裝包裝(例如多劑量包裝)或次單位劑量。The present disclosure further provides kits (e.g., containers) comprising any of the cell penetrating agents described herein and related materials, such as instructions for use (e.g., package inserts). The instructions for use may include, for example, instructions for administering the cell penetrating agent and, optionally, one or more other agents. The container of the cell penetrating agent may be a unit dose, bulk packaging (e.g., multi-dose packaging), or sub-unit dose.
IX.IX.偵測方法Detection method
在一些態樣中，本揭示內容之細胞穿透劑進一步提供偵測樣品中之TDP-43的方法。舉例而言，在一些實施例中，本揭示內容提供偵測樣品中之TDP-43的方法，其包含使本揭示內容之細胞穿透劑與樣品接觸及偵測細胞穿透劑或抗體與TDP-43之結合。舉例而言，此類方法可為離體或活體外方法。在一些實施例中，樣品為源自個體(例如，人類個體)之生物樣品。在一些實施例中，個體為人類。在一些實施例中，個體為患有TDP-43相關疾病或處於患有該疾病之風險下的患者。在此類情況下，使已自患者移除之樣品(諸如細胞、組織及/或器官)暴露於本文所述之抗體或抗原結合片段。在一些實施例中，樣品包含源自患者之細胞，且在投予本文所述之抗體或抗原結合片段之前溶解細胞。In some aspects, the cell penetrating agents of the present disclosure further provide methods for detecting TDP-43 in a sample. For example, in some embodiments, the present disclosure provides methods for detecting TDP-43 in a sample, comprising contacting a cell penetrating agent of the present disclosure with the sample and detecting binding of the cell penetrating agent or antibody to TDP-43. For example, such methods can bein vitro orex vivo methods. In some embodiments, the sample is a biological sample derived from an individual (e.g., a human individual). In some embodiments, the individual is a human. In some embodiments, the individual is a patient suffering from or at risk for a TDP-43-related disease. In such cases, a sample (such as cells, tissues, and/or organs) removed from a patient is exposed to an antibody or antigen-binding fragment described herein. In some embodiments, the sample comprises cells derived from the patient, and the cells are lysed prior to administration of the antibody or antigen-binding fragment described herein.
實例Example
已包括下列實例以說明本文所揭示之模式。下列實例之某些態樣係根據本發明共同發明者發現或預期在本文所揭示之實踐中運作良好的技術及程序來描述。根據本揭示內容及熟習此項技術者之一般水準，熟習此項技術者應瞭解，下列實例僅意欲為例示性的，且可在不脫離本揭示內容之範疇的情況下採用多種變化、修改及變更。The following examples are included to illustrate the modes disclosed herein. Certain aspects of the following examples are described based on techniques and procedures discovered or expected by the co-inventors of the present invention to work well in the practice of the present invention. Based on this disclosure and the level of skill in the art, those skilled in the art will appreciate that the following examples are intended to be illustrative only and that numerous variations, modifications, and alterations may be made without departing from the scope of this disclosure.
實例Example1.1.人源化抗Humanized antibodyTDP-43TDP-43抗體antibody
自鼠類單株抗體13D3生成人源化抗TDP-43抗體。分別地，圖1顯示重鏈可變域之經註明之版本且圖2顯示輕鏈可變域之經註解之版本。重鏈可變域及輕鏈可變域序列皆顯示天然訊號肽、可變域及部分恆定域。部分重鏈恆定域區為IgG2a小鼠恆定域。部分輕鏈恆定域為小鼠κ恆定域。對本文所述之胺基酸取代之提及係指Kabat編號系統(參見，例如Kabat E.A.等人,Sequences of Proteins of Immunological Interest(第5版). Bethesda, MD: National Institutes of Health (1991))。Humanized anti-TDP-43 antibodies were generated from the murine monoclonal antibody 13D3. Figure 1 shows an annotated version of the heavy chain variable domain and Figure 2 shows an annotated version of the light chain variable domain, respectively. Both the heavy chain variable domain and light chain variable domain sequences show the native signal peptide, variable domain, and portion of the constant domain. The portion of the heavy chain constant domain region is the IgG2a mouse constant domain. The portion of the light chain constant domain is the mouse kappa constant domain. References to amino acid substitutions described herein refer to the Kabat numbering system (see, e.g., Kabat EA et al.,Sequences of Proteins of Immunological Interest (5th ed.). Bethesda, MD: National Institutes of Health (1991)).
簡言之，蛋白質序列在Protein Data Bank (PDB)資料庫中鑑別(參見Deshpande等人, The RCSB Protein Data Bank: a redesigned query system and relational database based on the mmCIF schema,Nucleic Acids Research, 33:D233-D237 (2005))以尋找將提供13D3之類似結構模型的結構。抗體Fab PDB代碼「5BK5」之晶體結構，其為人類生殖系抗體。基於其與13D3 VH及VK之總體序列相似性、解析品質(3.0 Å)，針對VH及VK結構兩者選擇5BK5，且其為互補決定區(CDR)環保留相同的正則結構(Scally等人, Crystal structure of anti-cirumsporozoite protein 663 germline antibody; Direct deposit to PDB (2017))。Briefly, protein sequences were identified in the Protein Data Bank (PDB) database (see Deshpande et al., The RCSB Protein Data Bank: a redesigned query system and relational database based on the mmCIF schema,Nucleic Acids Research , 33: D233-D237 (2005)) to search for structures that would provide a similar structural model to 13D3. The crystal structure of the antibody Fab with PDB code "5BK5," a human germline antibody, was obtained. 5BK5 was selected for both the VH and VK structures based on its overall sequence similarity to the 13D3 VH and VK, its resolution quality (3.0 Å), and the fact that it retains the same canonical structure for the complementarity-determining region (CDR) loops (Scally et al., Crystal structure of anti-cirumsporozoite protein 663 germline antibody; Direct deposit to PDB (2017)).
另外，由於5BK5抗體為人類生殖系衍生抗體且對於可變重鏈域具有屬於人類生殖系IGHV3-48’03 (SEQ ID NO: 2)的相同正則類別及對於可變輕鏈域具有屬於人類生殖系IGKV2-30*02 (SEQ ID NO: 25)的相同正則類別，因此使用此等序列作為人類受體框架。In addition, since the 5BK5 antibody is a human germline-derived antibody and has the same canonical class as the human germline IGHV3-48'03 (SEQ ID NO: 2) for the variable heavy chain domain and the same canonical class as the human germline IGKV2-30*02 (SEQ ID NO: 25) for the variable light chain domain, these sequences were used as human acceptor frameworks.
因此，選擇5BK5 VH及5BK5 VL之框架區(framework region)作為13D3之CDR的受體(acceptor)序列。建立移植至VH及VL之各別人類框架上的13D3 CDR模型，且用作進一步回復突變以提高結合特異性且降低免疫原性之指導。Therefore, the framework regions of 5BK5 VH and 5BK5 VL were selected as acceptor sequences for the 13D3 CDRs. A 13D3 CDR model grafted onto the respective human frameworks of VH and VL was constructed and used as a guide for further backmutations to improve binding specificity and reduce immunogenicity.
更具體而言，由5BK5 VH人類框架及13D3 CDR組成之胺基酸序列命名為hu13D3VHv1d (SEQ ID NO: 20)，且由5BK5 VL人類框架及13D3 VL CDR組成之胺基酸序列命名為hu13D3VLv1d (SEQ ID NO: 47)。More specifically, the amino acid sequence consisting of the 5BK5 VH human framework and 13D3 CDRs was named hu13D3VHv1d (SEQ ID NO: 20), and the amino acid sequence consisting of the 5BK5 VL human framework and 13D3 VL CDRs was named hu13D3VLv1d (SEQ ID NO: 47).
hu13D3VH及hu13D3VL之額外形式經設計以使得能夠評估各種框架殘基對抗原結合、熱穩定性、可開發性(例如，去胺、氧化、N-醣基化、蛋白水解及聚集)及免疫原性之貢獻。考慮取代之位置基於多種因素，包括：定義正則CDR構形之位置(參見Martin, A.C.R., Protein sequence and structure analysis of antibody variable domains, In: Kontermann R and Dübel S (編).Antibody Engineering. Heidelberg, Germany: Springer International Publishing AG (2010))；游標區內的位置(參見Foote J.及Winter, G., Antibody framework residues affecting the conformation of the hypervariable loops.J Mol Biol. 224(2):487-99 (1992))；定位於VH/VL域界面處的位置(參見Léger O.J.P.及Saldanha, J. Preparation of recombinant antibodies from immune rodent spleens and the design of their humanization by CDR grafting. In: Shepherd P and Dean C (編).Monoclonal Antibodies: A Practical Approach. Oxford, UK: Oxford University Press (2000))；易於進行轉譯後修飾(諸如醣基化或焦麩胺酸化)之位置；根據移植至VH及VL框架上的13D3 CDR模型，由經預測與CDR相互作用之殘基佔據的位置；及/或由經測序之人類抗體中罕見的殘基佔據之位置，其中親代鼠類13D3殘基或其他殘基在人類抗體譜系中要普遍得多。Additional forms of hu13D3VH and hu13D3VL were designed to enable evaluation of the contribution of various framework residues to antigen binding, thermal stability, developability (e.g., deamination, oxidation, N-glycosylation, proteolysis, and aggregation), and immunogenicity. The positions considered for substitution are based on a variety of factors, including: positions that define the canonical CDR conformation (see Martin, ACR, Protein sequence and structure analysis of antibody variable domains, In: Kontermann R and Dübel S (eds.).Antibody Engineering . Heidelberg, Germany: Springer International Publishing AG (2010)); positions within the vernier region (see Foote J. and Winter, G., Antibody framework residues affecting the conformation of the hypervariable loops.J Mol Biol . 224(2):487-99 (1992)); positions located at the VH/VL domain interface (see Léger OJP and Saldanha, J. Preparation of recombinant antibodies from immune rodent spleens and the design of their humanization by CDR grafting. In: Shepherd P and Dean C (eds.).Monoclonal Antibodies: A Practical Approach . Oxford, UK: Oxford University Press (2000)); positions that are susceptible to post-translational modifications (such as glycosylation or pyroglutamination); positions occupied by residues predicted to interact with the CDRs based on the 13D3 CDR models grafted onto the VH and VL frameworks; and/or positions occupied by residues that are rare in sequenced human antibodies, where the parental murine 13D3 residue or other residues are much more prevalent in the human antibody repertoire.
以下為人源化13D3抗體之概述。The following is an overview of the humanized 13D3 antibody.
重鏈可變域Relinked variable domain
hu13D3VHvd1 (SEQ ID NO: 20)由移植至5BK5 VH之框架上的13D3-VH之CDR-H1、CDR-H2及CDR-H3環組成且在定義Chothia正則類別之關鍵位置處恢復所有框架取代，該等取代為游標區之一部分，且定位於VH/VL域界面或有助於結構穩定性。Hu13D3VHvd1包括以下取代，該等取代為生殖系抗體之回復突變且在以下位置處最常見：位置L5V及T77S。hu13D3VHvd1 (SEQ ID NO: 20) consists of the CDR-H1, CDR-H2, and CDR-H3 loops of 13D3-VH grafted onto the 5BK5 VH framework and restores all framework substitutions at key positions that define the Chothia canonical class, are part of the vernier region, are located at the VH/VL domain interface, or contribute to structural stability. Hu13D3VHvd1 includes the following substitutions, which are germline antibody reversion mutations and are most common at the following positions: position L5V and T77S.
hu13D3VHv2d (SEQ ID NO: 21)包括以下取代：L78A。如免疫表位資料庫(「IEDB」)分析所指示，位置78處之白胺酸具有免疫原性。因此，去免疫分析預測在位置78處具有丙胺酸取代之免疫原性降低。hu13D3VHv2d (SEQ ID NO: 21) includes the following substitution: L78A. Leucine at position 78 is immunogenic, as indicated by Immune Epitope Database (IEDB) analysis. Therefore, deimmunization analysis predicted reduced immunogenicity with an alanine substitution at position 78.
Hu13D3VHv3d (SEQ ID NO: 22)包括以下取代：G44R、S49A及S74A。位置44處之精胺酸與可變輕鏈域中進行之G100D (甘胺酸至天冬胺酸)取代形成從頭接觸，從而加強重鏈可變域:輕鏈可變域界面。具體而言，位置44處之精胺酸與G100D形成氫鍵及鹽橋，且亦與可變輕鏈域中之F98形成氫鍵。位置49為游標區殘基，且自絲胺酸至丙胺酸之取代為回復突變，以評估對CDR構形及結合之影響。最後，位置74處絲胺酸至丙胺酸之取代為生殖系取代。Hu13D3VHv3d (SEQ ID NO: 22) includes the following substitutions: G44R, S49A, and S74A. The arginine at position 44 forms ade novo contact with the G100D (glycine to aspartic acid) substitution made in the variable light domain, thereby strengthening the heavy chain variable domain:light chain variable domain interface. Specifically, the arginine at position 44 forms a hydrogen bond and a salt bridge with G100D and also forms a hydrogen bond with F98 in the variable light domain. Position 49 is a vernier residue, and the serine to alanine substitution was a back mutation to assess its effects on CDR conformation and binding. Finally, the serine to alanine substitution at position 74 is a germline substitution.
Hu13D3VHv4d (SEQ ID NO: 23)包括在嘗試組合之hu1353VHv1d、hu1353VHv2d及hu1353VHv3d中進行之各種取代。Hu13D3VHv4d (SEQ ID NO: 23) includes various substitutions made in the attempted combinations of hu1353VHv1d, hu1353VHv2d, and hu1353VHv3d.
輕鏈可變域Light chain variable domain
hu13D3VLv1d (SEQ ID NO: 47)由移植至5BK5 VL之框架上的13D3VL之CDR-L1、CDR-L2及CDR-L3環組成並且在定義Chothia正則類別之關鍵位置處恢復所有框架取代，該等取代為游標區之一部分，且定位於VH/VL域界面。Hu1353VLv1d包括以下取代：V3Q、P15L、E17Q、L38Q、K39R、Q100D及L104V。hu13D3VLv1d (SEQ ID NO: 47) consists of the CDR-L1, CDR-L2, and CDR-L3 loops of 13D3VL grafted onto the 5BK5VL framework and restores all framework substitutions at key positions defining the Chothia canonical class, which are part of the vernier region and located at the VH/VL domain interface. Hu1353VLv1d includes the following substitutions: V3Q, P15L, E17Q, L38Q, K39R, Q100D, and L104V.
在位置3處用纈胺酸取代麩醯胺酸會降低輕鏈可變域之免疫原性。在位置15處用白胺酸取代脯胺酸為生殖系取代。在位置17處用麩醯胺酸取代麩胺酸為罕見取代。位置17具有顯著的表面暴露，由此在蛋白質表面產生負電荷斑塊。取代為麩醯胺酸減少抗體表面正斑塊。由於小鼠結構模型中之麩醯胺酸在輕鏈可變域內形成股間接觸以維持結構構形，因此在位置38處用麩醯胺酸取代白胺酸。相比之下，白胺酸殘基不能形成相同的接觸，因此，此回復突變提高構形穩定性。在位置39處用精胺酸取代離胺酸藉由與相鄰殘基形成離胺酸殘基所不能的額外接觸來提高構形穩定性。輕鏈可變域中之位置100位於重鏈可變域與輕鏈可變域界面處，然而，不與麩醯胺酸殘基形成鏈間接觸。在重鏈可變域中之位置44處取代為天冬胺酸以及同時取代精胺酸產生更強的從頭接觸，且因此提高抗體之熱穩定性。預測用纈胺酸取代位置104之白胺酸會降低免疫原性。Substituting glutamine for valine at position 3 reduces the immunogenicity of the light chain variable domain. Substituting leucine for proline at position 15 is a germline substitution. Substituting glutamine for glutamine at position 17 is a rare substitution. Position 17 is significantly surface exposed, resulting in a negatively charged patch on the protein surface. Substituting glutamine for glutamine reduces the positive patch on the antibody surface. Because glutamine in mouse structural models forms interstrand contacts within the light chain variable domain to maintain structural conformation, leucine was substituted with glutamine at position 38. In contrast, the leucine residue does not form the same contacts, so this reversion mutation improves conformational stability. Substituting lysine for arginine at position 39 improves conformational stability by forming additional contacts with adjacent residues that lysine residues cannot. Position 100 in the light chain variable domain is located at the interface between the heavy and light chain variable domains, but does not form interchain contacts with glutamine residues. Substitution of aspartic acid at position 44 in the heavy chain variable domain, along with arginine, creates stronger de novo contacts and thus improves the thermal stability of the antibody. Substitution of leucine at position 104 with valine is predicted to reduce immunogenicity.
Hu13D3VLv2d (SEQ ID NO: 48)由上文hu13D3VLv1d中所述之上述取代組成，但亦包括以下取代：L92A。預測在位置92處之白胺酸具有免疫原性，因此，在位置92處用丙胺酸之取代降低可變輕鏈域之免疫原性。Hu13D3VLv2d (SEQ ID NO: 48) consists of the above substitutions described above for hu13D3VLv1d, but also includes the following substitution: L92A. Leucine at position 92 is predicted to be immunogenic, therefore, substitution of alanine at position 92 reduces the immunogenicity of the variable light domain.
下表1及表2分別顯示與人源化形式相比之13D3可變重鏈域序列比對及可變輕鏈域序列比對。Tables 1 and 2 below show the sequence alignment of the 13D3 variable heavy chain domain and variable light chain domain, respectively, compared to the humanized version.
表1：13D3人源化可變重鏈域序列比對
表2：13D3人源化可變輕鏈序列比對
單獨地，對於Vk，選擇具有NCBI登錄碼ABC66863 (Shringer等人, 2006)之人類κ輕鏈(SEQ ID NO: 26)。此κ輕鏈具有CDR-L1及L2相同之正則類別，且根據IMGT慣例，屬於人類生殖系IGKV2-30*02 (SEQ ID NO: 25)。對於VH，選擇人類Ig重鏈AEX28899 (SEQ ID NO: 3) (GenBank: AEX28899) (Bowers等人, 2014)，同樣具有相同的正則類別且屬於人類生殖系IGHV3-48'03。其為Kabat人類重亞組3之成員。Separately, for Vk, a human kappa light chain (SEQ ID NO: 26) with NCBI accession code ABC66863 (Shringer et al., 2006) was selected. This kappa light chain has the same canonical class for CDRs L1 and L2 and, according to IMGT convention, belongs to the human germline IGKV2-30*02 (SEQ ID NO: 25). For VH, human Ig heavy chain AEX28899 (SEQ ID NO: 3) (GenBank: AEX28899) (Bowers et al., 2014) was selected, also having the same canonical class and belonging to the human germline IGHV3-48'03. It is a member of Kabat human subgroup 3.
AEX28899及ABC66863抗體為人類生殖系衍生抗體，其具有相同的正則類別且對於重鏈可變域，屬於人類生殖系IGHV3-48’03 (SEQ ID NO: 2)，而對於輕鏈可變域，則屬於人類生殖系IGHV3-48*03 (SEQ ID NO: 25)。因此，AEX28899重鏈可變域及ABC66863輕鏈可變域序列用作13D3之CDR的人類受體框架。建立移植至VH及VL之各別人類框架上的13D3 CDR模型且用作進一步回復突變之指導。AEX28899 and ABC66863 antibodies are human germline-derived antibodies with the same canonical class and are derived from human germline IGHV3-48'03 (SEQ ID NO: 2) for the heavy chain variable domain and human germline IGHV3-48*03 (SEQ ID NO: 25) for the light chain variable domain. Therefore, the AEX28899 heavy chain variable domain and ABC66863 light chain variable domain sequences were used as human acceptor frameworks for the 13D3 CDRs. A 13D3 CDR model was constructed, grafted onto the respective human frameworks for VH and VL, and used as a guide for further backmutation.
AEX28899重鏈可變域及ABC66863輕鏈可變域序列之人源化形式用作13D3之CDR的人類受體框架，且經設計以使得能夠評估各種框架殘基對抗原結合、熱穩定性、可開發性(例如去胺、氧化、N-醣基化、蛋白水解及聚集)及免疫原性之貢獻。考慮取代之位置基於多種因素，包括：定義正則CDR構形之位置(參見Martin, A.C.R., Protein sequence and structure analysis of antibody variable domains, In: Kontermann R and Dübel S (編).Antibody Engineering. Heidelberg, Germany: Springer International Publishing AG (2010))；游標區內的位置(參見Foote J.及Winter, G., Antibody framework residues affecting the conformation of the hypervariable loops.J Mol Biol. 224(2):487-99 (1992))；定位於VH/VL域界面處的位置(參見Léger OJP及Saldanha, J). Preparation of recombinant antibodies from immune rodent spleens and the design of their humanization by CDR grafting. In: Shepherd P and Dean C (編).Monoclonal Antibodies: A Practical Approach. Oxford, UK: Oxford University Press (2000))；易於進行轉譯後修飾(諸如醣基化或焦麩胺酸化)之位置；根據移植至VH及VL框架上的13D3 CDR模型，由經預測與CDR相互作用之殘基佔據的位置；及/或由經測序之人類抗體中罕見的殘基佔據之位置，其中親代鼠類13D3殘基或其他殘基在人類抗體譜系中要普遍得多。Humanized versions of the AEX28899 heavy chain variable domain and ABC66863 light chain variable domain sequences were used as human acceptor frameworks for the CDRs of 13D3 and were designed to enable assessment of the contribution of various framework residues to antigen binding, thermal stability, developability (e.g., deamination, oxidation, N-glycosylation, proteolysis, and aggregation), and immunogenicity. The positions considered for substitution are based on a variety of factors, including: positions that define the canonical CDR conformation (see Martin, ACR, Protein sequence and structure analysis of antibody variable domains, In: Kontermann R and Dübel S (eds.).Antibody Engineering . Heidelberg, Germany: Springer International Publishing AG (2010)); positions within the vernier region (see Foote J. and Winter, G., Antibody framework residues affecting the conformation of the hypervariable loops.J Mol Biol . 224(2):487-99 (1992)); positions located at the VH/VL domain interface (see Léger OJP and Saldanha, J. Preparation of recombinant antibodies from immune rodent spleens and the design of their humanization by CDR grafting. In: Shepherd P and Dean C (eds.).Monoclonal Antibodies: A Practical Approach . Oxford, UK: Oxford University Press). (2000)); positions that are susceptible to post-translational modifications (such as glycosylation or pyroglutamination); positions occupied by residues predicted to interact with the CDRs based on the 13D3 CDR models grafted onto the VH and VL frameworks; and/or positions occupied by residues that are rare in sequenced human antibodies, where the parental murine 13D3 residue or other residues are much more prevalent in the human antibody repertoire.
以下為SEQ ID NO: 3中之VH域取代的概述：R19K：小鼠抗體結構中之K19與SEQ ID NO: 3之W79產生π陽離子相互作用；G44R：在此位置處用Arg取代Gly潛在地建立與輕鏈之接觸，從而提高抗體穩定性；S77T：蘇胺酸降低免疫原性；L78A：預測白胺酸具有免疫原性，因此用丙胺酸之取代降低預測的免疫原性；L78G：甘胺酸亦降低預測的免疫原性；L80A：自該位置移除白胺酸降低免疫原性；L80G：類似於丙胺酸，位置80處之甘胺酸降低免疫原性；L82cG：甘胺酸潛在地降低重鏈免疫原性；及R83M：為可賦予抗體穩定性之回復突變。The following is a summary of the VH domain substitutions in SEQ ID NO: 3: R19K: K19 in the mouse antibody structure and SEQ ID NO: W79 of 3 generates a π-cation-ion interaction; G44R: Substitution of Gly with Arg at this position potentially establishes a contact with the light chain, thereby improving antibody stability; S77T: Threonine reduces immunogenicity; L78A: Leucine is predicted to be immunogenic, so substitution with alanine reduces the predicted immunogenicity; L78G: Glycine also reduces the predicted immunogenicity; L80A: Removal of leucine from this position reduces immunogenicity; L80G: Similar to alanine, glycine at position 80 reduces immunogenicity; L82cG: Glycine potentially reduces heavy chain immunogenicity; and R83M: is a reversion mutation that confers antibody stability.
以下為SEQ ID NO: 26中之VL域取代的概述：I2V為游標區殘基，保留纈胺酸以保持CDR構形；L9S：預測白胺酸具有免疫原性，用絲胺酸之取代降低預測的免疫原性；P18Q：小鼠抗體模型麩醯胺酸與K74形成H鍵(LC)，從而穩定鏈間相互作用；R46L：為界面及游標區殘基，因此保留Leu；A80S：絲胺酸降低預測的免疫原性；L92G：L92為CDR殘基且經預測具有免疫原性，甘胺酸殘基經預測降低免疫原性；V94I：預測在位置94處之纈胺酸具有低水準之免疫原性，預測異白胺酸降低免疫原性；及V94A：丙胺酸為經預測降低免疫原性之替代性取代。The following is a summary of the VL domain substitutions in SEQ ID NO: 26: I2V is a residual residue in the vernier region, and valine is retained to maintain the CDR conformation; L9S: Leucine is predicted to be immunogenic, and substitution with serine reduces the predicted immunogenicity; P18Q: Glutamic acid in the mouse antibody model forms an H bond (LC) with K74, thereby stabilizing the interchain interaction; R46L: is an interface and vernier region residual, so it is retained. Leu; A80S: serine reduces predicted immunogenicity; L92G: L92 is a CDR residue and is predicted to be immunogenic, and the glycine residue is predicted to reduce immunogenicity; V94I: valine at position 94 is predicted to have a low level of immunogenicity, and isoleucine is predicted to reduce immunogenicity; and V94A: alanine is an alternative substitution predicted to reduce immunogenicity.
實例Example2.2.人源化Humanization13D313D3抗體變異體之表徵Characterization of antibody variants
如本文所論述，在許多神經退化性疾病且尤其ALS中發現TDP-43之細胞質聚集體。通常，在細胞質聚集體中發現之個別TDP-43蛋白在位置409及/或410處之絲胺酸殘基處經磷酸化。建立結合篩選檢定以確定13D3及其人源化變異體是否將選擇性地結合至在細胞質聚集體中發現的磷酸化TDP-43。As discussed herein, cytoplasmic aggregates of TDP-43 are found in many neurodegenerative diseases, particularly ALS. Typically, individual TDP-43 proteins found in cytoplasmic aggregates are phosphorylated at the serine residues at positions 409 and/or 410. A binding screening assay was established to determine whether 13D3 and its humanized variants would selectively bind to phosphorylated TDP-43 found in cytoplasmic aggregates.
將約50 RU之生物素-磷酸TDP43蛋白與NeutrAvidin一起固定於CM3晶片上。使各種人源化13D3抗體及嵌合13D3抗體流過晶片且分析與磷酸化TDP43蛋白之結合。具體而言，對hu13D3H9L8、hu13D3H10L6、hu13D3H10L7、hu13D3H10L8及hu13D3H9L7進行分析，且顯示與嵌合13D3抗體類似的與磷酸TDP43之結合(圖3)。類似地，測試hu13D3Hd1Ld1、hu13D3Hd2Ld2、hu13D3Hd3Ld1、hu13D3HD4LD1、hu13D3HD1LD2、hu13D3HD2LD2、hu13D3HD3LD2及hu13D3HD4LD2與磷酸TDP43之結合。各變異體顯示出與嵌合13D3抗體類似的與磷酸TDP43之結合(圖4)。Approximately 50 RU of biotin-phospho-TDP43 protein was immobilized on a CM3 chip along with NeutrAvidin. Various humanized and chimeric 13D3 antibodies were flowed over the chip and analyzed for binding to phosphorylated TDP43 protein. Specifically, hu13D3H9L8, hu13D3H10L6, hu13D3H10L7, hu13D3H10L8, and hu13D3H9L7 were analyzed and showed similar binding to phospho-TDP43 as the chimeric 13D3 antibodies (Figure 3). Similarly, hu13D3Hd1Ld1, hu13D3Hd2Ld2, hu13D3Hd3Ld1, hu13D3HD4LD1, hu13D3HD1LD2, hu13D3HD2LD2, hu13D3HD3LD2, and hu13D3HD4LD2 were tested for binding to phospho-TDP43. Each variant showed similar binding to phospho-TDP43 as the chimeric 13D3 antibody ( FIG. 4 ).
在另一實驗中，將抗體固定於蛋白A晶片(約2000 RU)上，且使在位置409及410處磷酸化之23個胺基酸之TDP-43肽(「TDP-43肽(pS409/pS410)」)以介於1 nM與100 nM之間的濃度流過晶片(SEQ ID NO: 83)。作為對照，亦在12 nM與1 μM之間測試在位置409或410處未磷酸化的23個胺基酸之TDP-43肽。具體而言，人源化形式hu13D3H5L2展示與各種濃度(1.2345 nM；3.703 nM；11.111 nM；33.33 nM；及100 nM)之TDP-43肽(pS409/pS410)的1:1結合，類似於13D3鼠類抗體(圖5)。此外，hu13D3H5L2在12.345 nM、37.03 nM、111.111 nM、333.333 nM及1 μM下未顯示對非磷酸化22個胺基酸之TDP-43肽的親和力(資料未顯示)。In another experiment, the antibody was immobilized on a Protein A chip (approximately 2000 RU), and a 23-amino acid TDP-43 peptide phosphorylated at positions 409 and 410 ("TDP-43 peptide (pS409/pS410)") was flowed over the chip at concentrations between 1 nM and 100 nM (SEQ ID NO: 83). As a control, a 23-amino acid TDP-43 peptide not phosphorylated at positions 409 or 410 was also tested between 12 nM and 1 μM. Specifically, the humanized form, hu13D3H5L2, exhibited 1:1 binding to the TDP-43 peptide (pS409/pS410) at various concentrations (1.2345 nM; 3.703 nM; 11.111 nM; 33.33 nM; and 100 nM), similar to the 13D3 murine antibody (Figure 5). Furthermore, hu13D3H5L2 showed no affinity for the non-phosphorylated 22-amino acid TDP-43 peptide at 12.345 nM, 37.03 nM, 111.111 nM, 333.333 nM, and 1 μM (data not shown).
亦評估抗體之熱穩定性。用表徵蛋白質或其他生物分子(例如抗體或其抗原結合片段)之穩定性的不同掃描量熱法進行熱穩定性分析。在pH 7.4下在1xPBS中測試2.66 mM (0.4 mg/mL)抗體。測試之溫度範圍為25℃至100℃。The thermal stability of the antibody was also assessed. Thermal stability analysis was performed using various scanning calorimetric methods used to characterize the stability of proteins or other biomolecules (e.g., antibodies or antigen-binding fragments thereof). Antibodies were tested at 2.66 mM (0.4 mg/mL) in 1x PBS at pH 7.4. The temperature range of the assay was 25°C to 100°C.
嵌合13D3及其人源化形式之結合資料總結於表3及表4中。表3及表4亦總結熱穩定性資料及免疫原性資料。The binding data for chimeric 13D3 and its humanized forms are summarized in Tables 3 and 4. Tables 3 and 4 also summarize the thermal stability data and immunogenicity data.
表3：人源化抗TDP-43度量
表4：人源化13D3抗體度量
總之，表3及表4中所示之資料顯示相對於小鼠13D3抗體具有低預測的免疫原性評分(下文進一步詳細解釋)之例示性人源化抗體。資料進一步展示人源化抗體之高產率及解鏈溫度(例如，類似於小鼠13D3抗體)。參見，例如表4中所示之抗體h13D3Hd1-Ld1、h13D3Hd1-Ld2及h13D3Hd2-Ld1。此外，資料亦展示與小鼠13D3抗體具有相似結合度量之人源化抗體。In summary, the data presented in Tables 3 and 4 demonstrate exemplary humanized antibodies with low predicted immunogenicity scores (explained in further detail below) relative to the mouse 13D3 antibody. The data further demonstrate high yields and melting temperatures (e.g., similar to those of the mouse 13D3 antibody) of the humanized antibodies. See, for example, the antibodies h13D3Hd1-Ld1, h13D3Hd1-Ld2, and h13D3Hd2-Ld1 shown in Table 4. Furthermore, the data demonstrate humanized antibodies with similar binding metrics to the mouse 13D3 antibody.
人源化抗體之電腦模擬免疫原性分析In silico immunogenicity analysis of humanized antibodies
表3及表4中所示之免疫原性評分係自兩個不同軟體程式計算之免疫原性預測值：Immune Epitope Database (IEDB)及EpiQuest。IEDB免疫原性分析工具由National Institute of Allergy and Infectious Diseases主辦。The immunogenicity scores shown in Tables 3 and 4 are immunogenicity predictions calculated using two different software programs: the Immune Epitope Database (IEDB) and EpiQuest. The IEDB immunogenicity analysis tool is hosted by the National Institute of Allergy and Infectious Diseases.
對於IEDB，所用免疫原性預測方法係基於蛋白質序列內的肽與主要組織相容性類別(MHC) II類之預測的潛在結合。該程式鑑別蛋白質序列內之潛在免疫原性區域。MHC II類工具使用人類群體中MHC II類之廣譜對偶基因(亦即26個參考對偶基因)預測免疫原性區域。軟體生成一系列15個殘基之肽，其在10個殘基上重疊。預測各生成的15個殘基之肽結合至26個參考對偶基因。For the IEDB, the immunogenicity prediction method used is based on the predicted potential binding of peptides within protein sequences to major histocompatibility class (MHC) class II. The program identifies potentially immunogenic regions within protein sequences. The MHC Class II tool predicts immunogenic regions using a broad spectrum of MHC Class II alleles in the human population (i.e., 26 reference alleles). The software generates a series of 15-residue peptides that overlap by 10 residues. Each generated 15-residue peptide is predicted to bind to one of the 26 reference alleles.
對於各肽，藉由將各肽之評分與選自SWISSPROT資料庫之五百萬個隨機15個殘基之肽的評分進行比較來生成三種方法(組合文庫、SMM_align及Sturniolo)中之各者的百分等級。經調整之百分等級為基於肽長度之頻率調整的百分等級。低數字百分等級指示高親和力。接著使用三種方法之中值百分等級來生成共同方法之等級。默認情況下，預測結果經折疊以僅顯示百分等級及經調整之等級。將最大中值百分等級臨限值設定為20。For each peptide, a percentile rank for each of the three methods (combinatorial library, SMM_align, and Sturniolo) was generated by comparing the score of each peptide with the scores of five million random 15-residue peptides selected from the SWISSPROT database. The adjusted percentile rank is the percentile rank adjusted for frequency based on peptide length. Low percentile ranks indicate high affinity. The median percentile rank of the three methods was then used to generate a common method rank. By default, the prediction results are folded to display only the percentile rank and the adjusted rank. The maximum median percentile rank threshold was set to 20.
EpiQuest工具T-Scanner根據細胞毒性T淋巴球(CTL)表位預測的免疫顯性對其進行分類。表位之免疫顯性定義為其在與各別細胞介素之靶標殺傷或釋放相關之功能檢定中的相對強度。此等參數指示T-表位之功能。此程式經設計以根據其免疫顯性分析及分選自靶細胞溶析之CTL肽表位。通常，僅少數結合於MHC I類之肽表位具有實際的功能活性。CTL (T)表位之相對強度由其與MHC I類及T細胞受體(TCR) (在MHCI之情形中)之結合強度來定義。該算法偵測肽表位之結構及組成特徵，該等特徵使其能夠引發對TCR之高親和力。此分析為單倍型特異性的，且EpiQuest T-scanner具有用於分析HLAA2及H2kB單倍型結合肽之基質。The EpiQuest tool T-Scanner classifies cytotoxic T lymphocyte (CTL) epitopes according to their predicted immunodominance. The immunodominance of an epitope is defined as its relative strength in functional assays related to target killing or release of the respective interleukin. These parameters indicate the functionality of the T-epitope. This program is designed to analyze and sort CTL peptide epitopes lysed from target cells based on their immunodominance. Generally, only a few peptide epitopes that bind to MHC class I have actual functional activity. The relative strength of a CTL (T) epitope is defined by its binding strength to MHC class I and the T cell receptor (TCR) (in the case of MHC I). The algorithm detects the structural and compositional characteristics of a peptide epitope that enable it to elicit a high affinity for the TCR. The assay is haplotype-specific, and the EpiQuest T-scanner has matrices for analyzing HLA-A2 and H2kB haplotype-binding peptides.
實例Example3.3.在existFTPFTP腦組織及模型系統中抗Antibodies in brain tissue and model systemsTDP-43TDP-43抗體與Antibodies andTDP-43TDP-43之磷酸化細胞質聚集體的結合Binding of phosphorylated cytoplasmic aggregates
圖6A至圖6C顯示額顳葉失智症(「FTD」)腦組織(圖6A)及健康腦組織(圖6C)。圖6B為圖6A之插圖，顯示13D3抗體與磷酸化TDP-43 FTD相關之神經元細胞質聚集體的共定位。資料表明，13D3抗體特異性結合至FTD腦組織中之細胞質聚集體，但在健康腦組織中則沒有。類似地，圖7A-C各自顯示TDP-43蛋白病之rNLS8 dox可抑制模型中的13D3特異性結合細胞質聚集體。Figures 6A to 6C show frontotemporal dementia (FTD) brain tissue (Figure 6A) and healthy brain tissue (Figure 6C). Figure 6B is an inset to Figure 6A, showing colocalization of the 13D3 antibody with neuronal cytoplasmic aggregates associated with FTD of phosphorylated TDP-43. The data demonstrate that the 13D3 antibody specifically binds to cytoplasmic aggregates in FTD brain tissue, but not in healthy brain tissue. Similarly, Figures 7A-C each demonstrate that 13D3 specifically binds to cytoplasmic aggregates in the rNLS8 dox-inhibitory model of TDP-43 proteinopathy.
實例Example4.4.在經轉染之In the transfectedHEKHEK細胞中抗Cell neutralizing antibodiesTDP-43TDP-43抗體與Antibodies andTDP-43TDP-43之磷酸化細胞質聚集體及核Phosphorylated cytoplasmic aggregates and nuclearTDP-43TDP-43聚集體的結合Aggregate binding
圖8A顯示用GFP-2a-TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)]或僅GFP (2a為在表現時釋放TDP-43之自裂解肽)瞬時轉染的HEK細胞之共焦顯微圖像。上圖顯示GFP、細胞核(灰色)及pTDP-43 (白色)之染色。下圖僅顯示pTDP-43染色，該染色不存在於單獨GFP對照轉染中。圖8B (左)為展示經GFP-2a-TDP43或單獨GFP轉染之HEK細胞的細胞計數之圖。資料證明兩個群體之間的細胞計數大致相等。此外，圖8B (右)顯示經GFP-2a-TDP43或單獨GFP轉染之HEK細胞的pTDP-43病灶計數。資料表明TDP-43病灶僅在經GFP-2a-TDP43轉染之HEK細胞中形成。圖8C顯示用市售抗體或本揭示內容之抗體(包括13D3、13C13及2D4)進行之TDP-43染色。用抗體處理細胞24小時，且24小時後，將細胞與100 ug/ml抗體一起再培育24小時。接著將細胞洗滌，固定/透化，且用抗pTDP-43抗體染色，繼之用AF647結合之抗小鼠二級抗體染色。藉由高含量成像(Operetta系統)用40x水物鏡對染色細胞進行成像。用Harmony軟體進行定量分析。Figure 8A shows confocal micrographs of HEK cells transiently transfected with GFP-2a-TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)] or GFP alone (2a is a self-cleaving peptide that releases TDP-43 upon expression). The upper panel shows staining for GFP, nuclei (gray), and pTDP-43 (white). The lower panel shows staining for pTDP-43 alone, which is absent in the GFP alone control transfection. Figure 8B (left) shows a graph showing cell counts for HEK cells transfected with GFP-2a-TDP43 or GFP alone. The data demonstrate that cell counts were approximately equivalent between the two populations. In addition, Figure 8B (right) shows pTDP-43 foci counts in HEK cells transfected with GFP-2a-TDP43 or GFP alone. The data demonstrate that TDP-43 foci form exclusively in HEK cells transfected with GFP-2a-TDP43. Figure 8C shows TDP-43 staining using commercially available antibodies or antibodies of the present disclosure, including 13D3, 13C13, and 2D4. Cells were treated with antibodies for 24 hours, and 24 hours later, cells were incubated with 100 μg/ml of antibody for an additional 24 hours. Cells were then washed, fixed/permeabilized, and stained with anti-pTDP-43 antibodies, followed by staining with AF647-conjugated anti-mouse secondary antibodies. Stained cells were imaged using high-content imaging (Operetta system) with a 40x objective lens. Quantitative analysis was performed using Harmony software.
抗體13D3、13C13及2D4偵測HEK細胞中過表現之錯誤定位的TDP-43。頂行顯示用GFP-2a-TDP-43之轉染，其中磷酸化TDP43不包括核定位訊號(亦即，磷酸化TDP43保留在細胞質中)，且底行顯示用僅GFP構築體之轉染。資料表明TDP-43病灶僅在經GFP-2a-TDP-43轉染之HEK細胞中形成。Antibodies 13D3, 13C13, and 2D4 detect overexpressed, mislocalized TDP-43 in HEK cells. The top row shows transfection with GFP-2a-TDP-43, in which phosphorylated TDP43 does not include a nuclear localization signal (i.e., phosphorylated TDP43 remains in the cytoplasm), and the bottom row shows transfection with a GFP-only construct. The data demonstrate that TDP-43 foci form exclusively in HEK cells transfected with GFP-2a-TDP-43.
檢定中包括各種對照抗體，包括pTDP-43 (Cosmo)+、pTDP-43 (1D3)+、總TDP-43 (PT)及3B12 (ED)+。對照抗體驗證細胞質內之TDP-43聚集。類似地測試抗體13D3、13C13及2D4，且展示與磷酸化TDP-43之細胞質聚集體的類似結合。Various control antibodies were included in the assay, including pTDP-43 (Cosmo)+, pTDP-43 (1D3)+, total TDP-43 (PT), and 3B12 (ED)+. The control antibodies validated TDP-43 aggregation in the cytoplasm. Antibodies 13D3, 13C13, and 2D4 were similarly tested and demonstrated similar binding to cytoplasmic aggregates of phosphorylated TDP-43.
實例Example5.5.人源化抗Humanized antibodyTDP-43TDP-43細胞穿透劑之特徵Characteristics of cell penetrants
如先前實例3中所述評估抗TDP-43細胞穿透劑之結合資料、免疫原性及熱穩定性。包含鼠類13D3及其人源化形式之細胞穿透劑的結合資料、免疫原性評分及熱穩定性資料匯總於表5及表6中。Binding data, immunogenicity, and thermal stability of anti-TDP-43 cell penetrants were evaluated as previously described in Example 3. Binding data, immunogenicity scores, and thermal stability data for cell penetrants comprising murine 13D3 and its humanized form are summarized in Tables 5 and 6.
表5：抗TDP-43 CPA之穩定性、IHC及標靶接合資料
表6：抗TDP-43 CPA之結合資料(二價分析物模式)
如表5中之資料所證明，本揭示內容之抗TDP-43 CPA為高度穩定的，具有與相應抗體相當之穩定性。舉例而言，表5中之抗TDP-43 CPA具有69.9℃至77.5℃之解鏈溫度且以高產率表示。此外，抗TDP-43 CPA藉由免疫組織化學(IHC)分析及細胞檢定證明靶標接合。表6提供例示性抗TDP-43 CPA之結合資料，顯示該等CPA以亞奈莫耳KD值結合磷酸-TDP-43。因此，表5及表6中之資料表明，本揭示內容之抗TDP-43 CPA保留與親代抗體之磷酸-TDP-43之強結合、熱穩定性及活體外靶標接合。As demonstrated by the data in Table 5, the anti-TDP-43 CPAs of the present disclosure are highly stable, possessing stability comparable to that of the corresponding antibodies. For example, the anti-TDP-43 CPAs in Table 5 have melting temperatures ranging from 69.9°C to 77.5°C and are expressed in high yields. Furthermore, the anti-TDP-43 CPAs demonstrated target engagement by immunohistochemistry (IHC) analysis and cellular assays. Table 6 provides binding data for exemplary anti-TDP-43 CPAs, demonstrating that these CPAs bind to phospho-TDP-43 with subnanomolar KD values. Thus, the data in Tables 5 and 6 demonstrate that the anti-TDP-43 CPAs of the present disclosure retain the strong binding, thermal stability, and in vitro target engagement of the parental antibodies to phospho-TDP-43.
實例Example6.6.用抗Use antiTDP-43TDP-43細胞穿透劑轉染之細胞中Cells transfected with cell penetrantsTDP-43TDP-43聚集體之內化及清除Internalization and clearance of aggregates
圖9至圖22顯示本揭示內容之抗TDP-43細胞穿透劑之內化及/或靶標接合。在圖9至圖22中，提及「M-Lyco」、「Lycotoxin」、「ML」及諸如此類係指包含連接至13D3抗體(鼠類、嵌合或人源化)之L17E_M-lycotoxin的CPA。在圖9-22中，對其他CIM及/或CMIP (例如，「CMIP」、「CMIP4」、「cTAT」、「PEPTH」等)之提及係指包含連接至13D3抗體(鼠類、嵌合或人源化)之相應CIM之CPA。除非另有說明(例如藉由HC)，否則CIM經由輕鏈之c末端連接至13D3抗體。Figures 9 to 22 illustrate internalization and/or target engagement of the anti-TDP-43 cell penetrants of the present disclosure. In Figures 9 to 22, references to "M-Lyco," "Lycotoxin," "ML," and the like refer to CPAs comprising L17E_M-lycotoxin linked to a 13D3 antibody (murine, chimeric, or humanized). In Figures 9-22, references to other CIMs and/or CMIPs (e.g., "CMIP," "CMIP4," "cTAT," "PEPTH," etc.) refer to CPAs comprising the corresponding CIM linked to a 13D3 antibody (murine, chimeric, or humanized). Unless otherwise indicated (e.g., via an HC), the CIM is linked to the 13D3 antibody via the C-terminus of the light chain.
用GFP-2a-TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)]瞬時轉染HEK細胞。24小時後，將細胞與100 ug/ml包括細胞內化部分及抗TDP-43抗體之細胞穿透劑一起培育4小時。接著將細胞洗滌，固定/透化，繼之用AF647結合之抗小鼠二級抗體。藉由高含量成像(Operetta系統)用40x水物鏡對染色細胞進行成像。用Harmony軟體進行定量分析。具體而言，圖9A顯示在與包括不同CIM (亦即，TAT、M-Lycotoxin_L17E (LC)、M-Lycotoxin_L17E (HC)、PEPTH (HC))之m13D3 CPA一起培育後或在各種對照條件(亦即未標記、同型及媒劑)下，CPA陽性細胞之百分比。HC (重鏈)及LC (輕鏈)指示CIM之位置。圖9中「M-Lycotoxin」之標記係指包含在m13D3抗體之輕鏈(LC)或重鏈(HC)之C末端連接M-Lycotoxin_L17E的CPA。HEK cells were transiently transfected with GFP-2a-TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)]. Twenty-four hours later, cells were incubated with 100 μg/ml of a cell permeabilizer containing a cell-internalizing moiety and an anti-TDP-43 antibody for 4 hours. Cells were then washed, fixed/permeabilized, and then treated with an AF647-conjugated anti-mouse secondary antibody. Stained cells were imaged using high-content imaging (Operetta system) at a 40x objective lens. Quantitative analysis was performed using Harmony software. Specifically, Figure 9A shows the percentage of CPA-positive cells after incubation with m13D3 CPA containing different CIMs (i.e., TAT, M-Lycotoxin_L17E (LC), M-Lycotoxin_L17E (HC), PEPTH (HC)) or under various control conditions (i.e., unlabeled, isotype, and vehicle). HC (heavy chain) and LC (light chain) indicate the location of the CIMs. The "M-Lycotoxin" designation in Figure 9 refers to CPA containing M-Lycotoxin_L17E linked to the C-terminus of the light chain (LC) or heavy chain (HC) of the m13D3 antibody.
資料表明，與裸抗體(「未標記」)及媒劑對照相比，m13D3 CPA之內化增加。圖9B顯示與m13D3 CPA一起培育後各細胞之CPA陽性斑點數目。資料表明，與裸抗體及媒劑對照相比，m13D3 CPA之pTDP-43病灶偵測增加。The data demonstrate increased internalization of m13D3 CPA compared to naked antibody ("unlabeled") and vehicle controls. Figure 9B shows the number of CPA-positive puncta per cell after incubation with m13D3 CPA. The data demonstrate increased detection of pTDP-43 foci with m13D3 CPA compared to naked antibody and vehicle controls.
圖10A至圖10E為顯示用各種m13D3 CPA轉染之HEK細胞之結果的圖。簡言之，用GFP-2a-TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)]或無質體(例如，未轉染)作為陰性對照瞬時轉染HEK細胞。24小時後，將細胞與100 ug/ml抗體(例如CPA)一起再培育24小時。接著將細胞洗滌，固定/透化，且用抗pTDP-43抗體染色，繼之用AF647結合之抗小鼠二級抗體染色。藉由高含量成像(Operetta系統)用40x水物鏡對染色細胞進行成像。用Harmony軟體進行定量分析。Figures 10A to 10E show the results of HEK cells transfected with various m13D3 CPAs. Briefly, HEK cells were transiently transfected with GFP-2a-TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)] or plasmid-free (e.g., untransfected) as a negative control. After 24 hours, cells were incubated with 100 μg/ml of antibody (e.g., CPA) for an additional 24 hours. Cells were then washed, fixed/permeabilized, and stained with anti-pTDP-43 antibodies, followed by AF647-conjugated anti-mouse secondary antibodies. Stained cells were imaged using high-content imaging (Operetta system) at a 40x microscope. Quantitative analysis was performed using Harmony software.
圖10A顯示各孔面積之pTDP-43病灶數目。圖10中「M-Lycotoxin」之標記係指包含在m13D3抗體之輕鏈之C末端連接M-Lycotoxin_L17E的CPA。資料表明，用不同m13D3 CPA處理之細胞與用未標記之m13D3 (例如，無細胞內化模組)處理之細胞相比具有較低各孔面積之病灶數目。如所預期，在未轉染細胞中未觀察到病灶。圖10B顯示pTDP-43病灶之平均聚焦強度。資料表明，用不同m13D3 CPA處理之細胞與用未標記之m13D3抗體處理之細胞相比具有較低平均聚焦強度。如所預期，在未轉染細胞中觀察到極低之聚焦強度。圖10C顯示所有測試細胞群體(亦即，用不同m13D3 CPA轉染或未轉染細胞)之各孔之一致細胞計數。圖10D顯示藉由細胞計數正規化之pTDP-43病灶數目。資料表明，用不同m13D3 CPA處理之細胞與用未標記之m13D3處理之細胞相比具有較低各細胞之病灶數目。如所預期，在未轉染細胞中未觀察到病灶。圖10E顯示p-TDP-43病灶之平均病灶面積，表明用不同m13D3 CPA處理之細胞與用未標記之m13D3處理之細胞相比具有較低之平均面積強度；在未轉染細胞中觀察到極低之聚焦強度Figure 10A shows the number of pTDP-43 foci per well area. The label "M-Lycotoxin" in Figure 10 refers to the CPA containing M-Lycotoxin_L17E linked to the C-terminus of the light chain of the m13D3 antibody. The data show that cells treated with different m13D3 CPAs have a lower number of foci per well area than cells treated with unlabeled m13D3 (e.g., without a cell internalization module). As expected, no foci were observed in untransfected cells. Figure 10B shows the average focus intensity of pTDP-43 foci. The data show that cells treated with different m13D3 CPAs have a lower average focus intensity than cells treated with the unlabeled m13D3 antibody. As expected, very low focus intensity was observed in untransfected cells. Figure 10C shows consistent cell counts across wells for all tested cell populations (i.e., cells transfected with different m13D3 CPAs or untransfected). Figure 10D shows the number of pTDP-43 foci normalized to cell counts. The data demonstrate that cells treated with different m13D3 CPAs have a lower number of foci per cell compared to cells treated with unlabeled m13D3. As expected, no foci were observed in untransfected cells. Figure 10E shows the average area of p-TDP-43 foci, indicating that cells treated with different m13D3 CPAs have lower average area intensities than cells treated with unlabeled m13D3; very low focal intensities were observed in untransfected cells.
圖11A顯示各孔面積之pTDP-43病灶數目。資料表明，用不同m13D3 CPA (亦即，TAT (HC)、L17E M-Lycotoxin (LC)、L17E M-lycotoxin (HC)及PEPTH (HC))處理之細胞與用未標記之m13D3處理之細胞相比具有較低的各孔面積之病灶數目。圖11中「M-Lycotoxin」之標記係指包含在m13D3抗體之輕鏈(LC)或重鏈(HC)之C末端連接M-Lycotoxin_L17E的CPA。圖11B顯示在所有測試細胞群體中各孔之一致細胞計數。圖11C顯示藉由細胞計數正規化之pTDP-43病灶數目，此表明用不同m13D3 CPA處理之細胞與用未標記之m13D3處理之細胞相比具有較低各細胞之病灶數目。如所預期，在未轉染細胞中未觀察到病灶。圖11D顯示pTDP-43病灶之平均病灶面積。資料表明用不同m13D3 CPA處理之細胞與用未標記之m13D3處理之細胞相比具有較低之平均面積強度；在未轉染細胞中觀察到極低之聚焦強度Figure 11A shows the number of pTDP-43 foci per well area. The data demonstrate that cells treated with different m13D3 CPAs (i.e., TAT (HC), L17E M-Lycotoxin (LC), L17E M-lycotoxin (HC), and PEPTH (HC)) exhibit lower foci counts per well area compared to cells treated with unlabeled m13D3. The "M-Lycotoxin" designation in Figure 11 refers to a CPA containing M-Lycotoxin_L17E linked to the C-terminus of either the light chain (LC) or heavy chain (HC) of the m13D3 antibody. Figure 11B shows consistent cell counts across wells across all tested cell populations. Figure 11C shows the number of pTDP-43 foci normalized by cell count, indicating that cells treated with different m13D3 CPAs had a lower number of foci per cell compared to cells treated with unlabeled m13D3. As expected, no foci were observed in untransfected cells. Figure 11D shows the mean foci area of pTDP-43 foci. The data indicate that cells treated with different m13D3 CPAs had a lower mean area intensity than cells treated with unlabeled m13D3; very low focus intensity was observed in untransfected cells.
圖12A至圖12D為顯示在圖11A-D中所述之相同實驗條件下與不同濃度之m13D3 M-Lycotoxin [17E] CPA一起培育之細胞之結果的圖。圖11中「M-Lycotoxin」之標記係指包含在m13D3抗體之輕鏈(LC)或重鏈(HC)之C末端連接M-Lycotoxin_L17E的CPA。另外，細胞內化模組位於m13D3抗體之重鏈或輕鏈上。亦將細胞與未標記之m13D3、IgG同型對照及PBS對照一起培育。將未轉染HEK細胞用作陰性對照。Figures 12A to 12D show the results of cells incubated with varying concentrations of m13D3 M-Lycotoxin [17E] CPA under the same experimental conditions as those described in Figures 11A-D. The "M-Lycotoxin" label in Figure 11 refers to CPA containing M-Lycotoxin_L17E linked to the C-terminus of the light chain (LC) or heavy chain (HC) of the m13D3 antibody. Additionally, the cellular internalization module is located on the heavy or light chain of the m13D3 antibody. Cells were also incubated with unlabeled m13D3, an IgG isotype control, and a PBS control. Untransfected HEK cells were used as a negative control.
圖12A顯示各孔面積之pTDP-43總病灶面積。資料顯示經m13D3 M-Lycotoxin_L17E CPA處理之細胞之總病灶面積之濃度依賴性減小。圖12中「M-Lycotoxin」之標記係指包含在m13D3抗體之輕鏈(LC)或重鏈(HC)之C末端連接M-Lycotoxin_L17E的CPA。相比之下，與用m13D3 m-Lycotoxin [L17E] CPA處理之細胞相比，用13D3抗體、IgG同型對照及PBS處理之細胞顯示出更高水準之病灶。未轉染之細胞無病灶(資料未顯示)。圖12B顯示在所有測試細胞群體中各孔之一致細胞計數，且經m13D3 M-lycotoxinCPA轉染之細胞之細胞活力無濃度依賴性降低。圖12C顯示pTDP-43病灶計數，表明m13D3 M-Lycotoxin CPA之病灶計數呈濃度依賴性增加，其中病灶數目顯著高於對照。圖12D顯示pTDP-43平均病灶大小，表明m13D3 M-Lycotoxin CPA之平均病灶大小呈濃度依賴性減小，與對照相比，平均病灶大小顯著降低。總之，圖12A-D表明m13D3 M-Lycotoxin CPA以濃度依賴性方式干擾病灶聚集。Figure 12A shows the total pTDP-43 foci area per well. The data show a concentration-dependent reduction in total foci area in cells treated with m13D3 M-Lycotoxin_L17E CPA. The "M-Lycotoxin" designation in Figure 12 refers to CPA containing M-Lycotoxin_L17E linked to the C-terminus of the light chain (LC) or heavy chain (HC) of the m13D3 antibody. In contrast, cells treated with the 13D3 antibody, an IgG isotype control, and PBS exhibited higher levels of foci compared to cells treated with m13D3 m-Lycotoxin [L17E] CPA. Untransfected cells showed no foci (data not shown). Figure 12B shows consistent cell counts across wells across all tested cell populations, with no concentration-dependent reduction in cell viability observed in cells transfected with m13D3 M-lycotoxin CPA. Figure 12C shows pTDP-43 foci counts, demonstrating a concentration-dependent increase in foci counts with m13D3 M-lycotoxin CPA, with the number of foci significantly higher than controls. Figure 12D shows mean pTDP-43 foci size, demonstrating a concentration-dependent decrease in mean foci size with m13D3 M-lycotoxin CPA, with a significant decrease compared to controls. In summary, Figures 12A-D demonstrate that m13D3 M-lycotoxin CPA interferes with foci aggregation in a concentration-dependent manner.
圖13顯示與m13D3抗體或M-Lycotoxin m13D3 CPA一起培育24小時且進行XTT代謝檢定以評估細胞活力之未轉染之HEK細胞(左)或經GFP-2a-TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)]轉染(右)之圖。圖13中「M-lycotoxin」之標記係指包含在m13D3抗體之輕鏈(LC)之C末端連接M-Lycotoxin_L17E的CPA。資料顯示所有測試群體之細胞死亡無顯著差異(如藉由細胞死亡百分比所量測)，此表明CPA (m13D3 M-Lycotoxin [L17E])不會誘導顯著細胞毒性。Figure 13 shows images of untransfected HEK cells (left) or transfected with GFP-2a-TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)] (right) that were incubated with the m13D3 antibody or M-Lycotoxin m13D3 CPA for 24 hours and subjected to an XTT metabolism assay to assess cell viability. The "M-lycotoxin" designation in Figure 13 refers to CPA containing M-Lycotoxin_L17E linked to the C-terminus of the light chain (LC) of the m13D3 antibody. The data showed no significant differences in cell death among all tested populations (as measured by percent cell death), indicating that CPA (m13D3 M-Lycotoxin [L17E]) does not induce significant cytotoxicity.
總之，圖10至圖13表明本揭示內容之抗TDP-43細胞穿透劑由細胞內化且可有效地接合細胞內TDP-43。In summary, Figures 10 to 13 demonstrate that the anti-TDP-43 cell penetrants of the present disclosure are internalized by cells and can effectively bind to intracellular TDP-43.
實例Example7.7.用抗Use antiTDP-43TDP-43細胞穿透劑對Cell penetrantsTDP-43TDP-43之磷酸化細胞質聚集體之內化及清除Internalization and clearance of phosphorylated cytoplasmic aggregates
圖14A至圖14E為顯示與不同m13D3 CPA及未標記之m13D3抗體一起培育之細胞的圖。將未轉染HEK細胞用作陰性對照。圖14A-E中之資料係用上文圖11A至圖11D中所述之相同實驗條件生成。Figures 14A to 14E are graphs showing cells incubated with different m13D3 CPAs and unlabeled m13D3 antibodies. Untransfected HEK cells were used as negative controls. The data in Figures 14A-E were generated using the same experimental conditions described above for Figures 11A to 11D.
更具體而言，圖14A顯示各孔面積之pTDP-43病灶之總和。圖14中「ML-13D3」之標記係指包含在m13D3抗體之輕鏈(LC)之C末端連接M-Lycotoxin_L17E的CPA。圖14中「CMIP1-5」之標記係指包含在m13D3抗體之輕鏈(LC)之C末端連接之CMIP1、CMIP2、CMIP3、CMIP4或CMIP5的CPA。資料表明，用不同m13D3 CPA (M-Lyco_L17E、CMIP1、CMIP2、CMIP3、CMIP4、CMIP5)處理之細胞與用未標記之m13D3處理之細胞相比具有較低各孔面積之病灶數目。如所預期，在未轉染細胞中未觀察到病灶。圖14B顯示pTDP-43病灶之平均聚焦強度。資料表明，用不同m13D3 CPA處理之細胞與用未標記之m13D3抗體處理之細胞相比具有較低平均聚焦強度，且在未轉染細胞中觀察到極低之聚焦強度。圖14C顯示在所有測試細胞群體中各孔之一致細胞計數。圖14D顯示p-TDP-43病灶之數目(藉由細胞計數正規化)。資料表明，用不同m13D3 CPA處理之細胞與用未標記之m13D3抗體處理之細胞相比具有較高各細胞之病灶數目抗體。如所預期，在未轉染細胞中未觀察到病灶。最後，圖14E顯示pTDP-43病灶之平均病灶面積。資料表明用不同m13D3 CPA處理之細胞與用未標記之m13D3處理之細胞相比具有較低之平均面積強度；在未轉染細胞中觀察到極低之聚焦強度。More specifically, Figure 14A shows the sum of pTDP-43 foci per well area. The "ML-13D3" label in Figure 14 refers to a CPA containing M-Lycotoxin_L17E linked to the C-terminus of the light chain (LC) of the m13D3 antibody. The "CMIP1-5" label in Figure 14 refers to a CPA containing CMIP1, CMIP2, CMIP3, CMIP4, or CMIP5 linked to the C-terminus of the light chain (LC) of the m13D3 antibody. The data demonstrate that cells treated with different m13D3 CPAs (M-Lyco_L17E, CMIP1, CMIP2, CMIP3, CMIP4, and CMIP5) exhibited a lower number of foci per well area than cells treated with unlabeled m13D3. As expected, no foci were observed in untransfected cells. Figure 14B shows the mean focus intensity of pTDP-43 foci. The data show that cells treated with different m13D3 CPAs have lower mean focus intensity than cells treated with unlabeled m13D3 antibody, and very low focus intensity was observed in untransfected cells. Figure 14C shows consistent cell counts in each well across all tested cell populations. Figure 14D shows the number of p-TDP-43 foci (normalized by cell count). The data show that cells treated with different m13D3 CPAs have a higher number of foci per cell than cells treated with unlabeled m13D3 antibody. As expected, no foci were observed in untransfected cells. Finally, Figure 14E shows the mean area of pTDP-43 foci. The data show that cells treated with different m13D3 CPAs had lower mean area intensities compared to cells treated with unlabeled m13D3; very low focal intensities were observed in untransfected cells.
圖15A至圖15D為顯示將細胞與不同濃度之m13D3 m-Lycotoxin [L17E] CPA或m13D3 CMIP4 CPA一起培育；將細胞與未標記之m13D3或IgG同型對照一起培育；未轉染之HEK細胞用作陰性對照之結果的圖。圖15A-D中之資料係用上文圖11A-D中所述之相同實驗條件生成。圖15中「13D3-ML」之標記係指包含在m13D3抗體之輕鏈(LC)之C末端連接M-Lycotoxin_L17E的CPA。圖15及圖16中「13D3-CMIP4」之標記係指包含在m13D3抗體之輕鏈(LC)之C末端連接之CMIP4的CPA。Figures 15A to 15D show the results of incubating cells with varying concentrations of m13D3 m-Lycotoxin [L17E] CPA or m13D3 CMIP4 CPA; incubating cells with unlabeled m13D3 or an IgG isotype control; and using untransfected HEK cells as a negative control. The data in Figures 15A-D were generated using the same experimental conditions as described above for Figures 11A-D. The "13D3-ML" designation in Figure 15 refers to CPA containing M-Lycotoxin_L17E linked to the C-terminus of the light chain (LC) of the m13D3 antibody. The "13D3-CMIP4" designation in Figures 15 and 16 refers to CPA containing CMIP4 linked to the C-terminus of the light chain (LC) of the m13D3 antibody.
圖15A顯示病灶p-TDP-43之總數，表明用13D3 m-Lyco及13D3 CMIP4 CPA處理之細胞的總病灶面積呈濃度依賴性減小。相比之下，用13D3抗體及同型處理之細胞顯示更高水準之病灶。未轉染之細胞未顯示病灶。圖15B顯示在所有測試細胞群體中各孔之一致細胞計數，且對於m13D3 m-Lycotoxin CPA之細胞活力無濃度依賴性降低。圖15C顯示藉由細胞計數正規化之p-TDP-43病灶數目，表明13D3 m-Lycotoxin及13D3 CMIP4 CPA之病灶計數呈濃度依賴性增加，其中病灶數目顯著高於對照。圖15D顯示p-TDP-43平均病灶面積，表明13D3 m-Lycotoxin及13D3 CMIP4 CPA之平均病灶面積呈濃度依賴性減小，與對照相比，平均病灶面積顯著降低。因此，圖15A至圖15D表明13D3 m-Lycotoxin及13D3 CMIP4 CPA以濃度依賴性方式干擾病灶聚集。Figure 15A shows the total number of p-TDP-43 foci, demonstrating a concentration-dependent reduction in total foci area in cells treated with 13D3 m-Lyco and 13D3 CMIP4 CPA. In contrast, cells treated with the 13D3 antibody and isotype displayed higher levels of foci. Untransfected cells showed no foci. Figure 15B demonstrates consistent cell counts across wells for all tested cell populations, with no concentration-dependent reduction in cell viability in response to m13D3 m-Lycotoxin CPA. Figure 15C shows the number of p-TDP-43 foci normalized by cell counts, demonstrating that 13D3 m-Lycotoxin and 13D3 CMIP4 CPA increased foci counts in a concentration-dependent manner, with the number of foci significantly higher than that of the control. Figure 15D shows the mean p-TDP-43 foci area, demonstrating that 13D3 m-Lycotoxin and 13D3 CMIP4 CPA decreased the mean foci area in a concentration-dependent manner, with a significant decrease compared to the control. Therefore, Figures 15A to 15D demonstrate that 13D3 m-Lycotoxin and 13D3 CMIP4 CPA interfere with foci aggregation in a concentration-dependent manner.
為獲得圖16A至圖16E中所提供之資料，將細胞與m13D3 CMIP4 CPA及未標記之m13D3抗體一起在乙酸鹽緩衝液或PBS中培育。圖16A至圖16E中之資料係用上文圖11A-D中所述之相同實驗條件生成。To obtain the data presented in Figures 16A to 16E, cells were incubated with m13D3 CMIP4 CPA and unlabeled m13D3 antibody in acetate buffer or PBS. The data in Figures 16A to 16E were generated using the same experimental conditions described above for Figures 11A-D.
更具體而言，圖16A顯示各孔面積之pTDP-43病灶，表明用m13D3 CMIP4 CPA處理之細胞與用未標記之m13D3處理之細胞相比，各孔面積之病灶數目較低；在PBS與乙酸鹽之間未觀察到細胞穿透活性之變化。圖16B顯示pTDP-43病灶之平均聚焦強度，表明用m13D3 CMIP4 CPA處理之細胞與用未標記之m13D3處理之細胞相比具有較低平均聚焦強度；在PBS與乙酸鹽之間未觀察到細胞穿透劑活性之變化。圖16C顯示在所有測試細胞群體中各孔之一致細胞計數。圖16D顯示藉由細胞計數正規化之p-TDP-43病灶數目，表明用m13D3 CMIP4 CPA處理之細胞與用未標記之m13D3處理之彼等細胞相比具有更高的各細胞之病灶數目；未轉染之細胞未觀察到病灶。圖16E顯示p-TDP-43病灶之平均病灶面積，表明用m13D3 CMIP4 CPA處理之細胞與用未標記之m13D3處理之細胞相比具有較低之平均面積強度More specifically, Figure 16A shows pTDP-43 foci per well area, demonstrating that cells treated with m13D3 CMIP4 CPA had a lower number of foci per well area than cells treated with unlabeled m13D3; no difference in cell penetrant activity was observed between PBS and acetate. Figure 16B shows the mean focus intensity of pTDP-43 foci, demonstrating that cells treated with m13D3 CMIP4 CPA had a lower mean focus intensity than cells treated with unlabeled m13D3; no difference in cell penetrant activity was observed between PBS and acetate. Figure 16C shows consistent cell counts across wells across all tested cell populations. Figure 16D shows the number of p-TDP-43 foci normalized by cell counting, indicating that cells treated with m13D3 CMIP4 CPA had a higher number of foci per cell compared to those treated with unlabeled m13D3; no foci were observed in untransfected cells. Figure 16E shows the average foci area of p-TDP-43 foci, indicating that cells treated with m13D3 CMIP4 CPA had a lower average area intensity than cells treated with unlabeled m13D3.
實例Example8.8.使用新穎細胞內化模組及嵌合抗Using novel cell internalization modules and chimeric antibodiesTDP-43TDP-43抗體對Antibody pairTDP-43TDP-43之磷酸化細胞質聚集體之內化及清除Internalization and clearance of phosphorylated cytoplasmic aggregates
用GFP-2a-TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)] (2a為自裂解肽，其在表現後釋放TDP-43)瞬時轉染HEK細胞。24小時後，將細胞與100 ug/ml抗體(例如CPA)一起再培育24小時。接著將細胞洗滌，固定/透化，且用抗pTDP-43抗體染色，繼之用AF647結合之抗小鼠二級抗體及AF594結合之抗人類二級抗體染色。藉由高含量成像(Operetta系統)用40x水物鏡對染色細胞進行成像。用Harmony軟體進行定量分析。HEK cells were transiently transfected with GFP-2a-TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)] (2a is a self-cleaving peptide that releases TDP-43 upon expression). Twenty-four hours later, cells were incubated with 100 μg/ml of an antibody (e.g., CPA) for an additional 24 hours. Cells were then washed, fixed/permeabilized, and stained with an anti-pTDP-43 antibody, followed by an anti-mouse secondary antibody conjugated to AF647 and an anti-human secondary antibody conjugated to AF594. Stained cells were imaged using high-content imaging (Operetta system) at a 40x objective lens. Quantitative analysis was performed using Harmony software.
圖17A顯示用未標記之ch13D3抗體(左)及ch13D3 m-Lycotoxin_L17E CPA (右)處理之細胞的共焦顯微圖像。病灶以白色顯示，13D3抗體以淺灰色顯示，且細胞核以深灰色顯示。白色箭頭指示與13D3抗體共定位之說明性病灶。圖17A表明經ch13D3 m-Lycotoxin CPA處理之細胞之pTDP43病灶與13D3抗體之間的顯著共定位，對於經ch13D3抗體處理之細胞觀察到極少之共定位。圖17B為顯示與ch13D3抗體共定位之pTDP43之百分比的圖。資料表明，與用ch13D3抗體處理之細胞的共定位(約15-20%)相比，用ch13D3 m-Lycotoxin CPA處理之細胞的共定位(約50-60%)顯著更大。總之，圖17A至圖17B表明本揭示內容之抗TDP-43 CPA由細胞內化且結合至細胞內p-TDP-43。Figure 17A shows confocal micrographs of cells treated with unlabeled ch13D3 antibody (left ) and ch13D3 m-Lycotoxin_L17E CPA (right ). Foci are shown in white, 13D3 antibody in light gray, and cell nuclei in dark gray. White arrows indicate illustrative foci colocalizing with the 13D3 antibody. Figure 17A demonstrates significant colocalization between pTDP43 foci and the 13D3 antibody in cells treated with ch13D3 m-Lycotoxin CPA, while minimal colocalization was observed in cells treated with the ch13D3 antibody. Figure 17B is a graph showing the percentage of pTDP43 colocalizing with the ch13D3 antibody. The data showed that the colocalization of cells treated with ch13D3 m-Lycotoxin CPA was significantly greater (approximately 50-60%) compared to the colocalization of cells treated with ch13D3 antibody (approximately 15-20%). In summary, Figures 17A and 17B demonstrate that the anti-TDP-43 CPA of the present disclosure is internalized by cells and binds to intracellular p-TDP-43.
圖18A至圖18D為顯示本揭示內容之抗TDP-43 CPA ((亦即，嵌合13D3 CPA (ch13D3 m-Lyco CPA、具有LALA突變之ch13D3 CMIP4、具有H310-H435Q突變之ch13D3 CMIP4)、未標記之ch13D3抗體及hIgG同型對照)由細胞內化且結合至細胞內p-TDP-43 (亦即，如本文所述經GFP-2a-TDP43轉染之細胞)。圖18(全)中「M-Lyco」之標記係指包含在m13D3抗體之輕鏈(LC)之C末端連接M-Lycotoxin_L17E的CPA。Figures 18A-18D show that anti-TDP-43 CPAs of the present disclosure (i.e., chimeric 13D3 CPAs (ch13D3 m-Lyco CPA, ch13D3 CMIP4 with the LALA mutation, ch13D3 CMIP4 with the H310-H435Q mutation), untagged ch13D3 antibody, and hIgG isotype control) are internalized by cells and bind to intracellular p-TDP-43 (i.e., cells transfected with GFP-2a-TDP43 as described herein). The "M-Lyco" label in Figure 18 (all) refers to CPA comprising M-Lycotoxin_L17E linked to the C-terminus of the light chain (LC) of the m13D3 antibody.
圖18A提供顯示使用共焦(左)及非共焦(右)顯微鏡，對於ch13D3 CPA以及ch13D3抗體、同型及PBS對照，與ch13D3抗體共定位之p-TDP-43之百分比的圖。圖18A顯示與用ch13D3抗體處理之細胞的共定位(約15-20%)相比，用各種ch13D3 CPA處理之細胞的共定位(約50-60%)顯著更大。用同型對照及PBS處理之細胞未表現出共定位。圖18B提供顯示各種ch13D3 CPA以及ch13D3抗體、同型及PBS對照之各細胞之13D3抗體斑點之平均數目的圖。圖18B表明，與對照相比，ch13D3 CPA產生顯著更多之各細胞之13D3斑點(約各細胞2.5-3個斑點)。圖18C提供顯示對於各種ch13D3 CPA以及ch13D3抗體、同型及PBS對照之13D3抗體斑點的平均斑點大小。圖18C顯示ch13D3 CPA導致各細胞之13D3斑點顯著小於對照。圖18D提供顯示對於各種ch13D3 CPA以及ch13D3抗體、同型及PBS對照之13D3抗體斑點的平均斑點大小。圖18D顯示ch13D3 CPA導致各細胞之13D3斑點顯著小於對照(針對斑點強度校正)。Figure 18A provides a graph showing the percentage of p-TDP-43 colocalized with the ch13D3 antibody for ch13D3 CPA and ch13D3 antibody, isotype, and PBS controls using confocal (left) and non-confocal (right) microscopy. Figure 18A shows that colocalization was significantly greater in cells treated with each ch13D3 CPA (approximately 50-60%) compared to the colocalization in cells treated with the ch13D3 antibody (approximately 15-20%). Cells treated with the isotype control and PBS did not show colocalization. Figure 18B provides a graph showing the average number of 13D3 antibody spots per cell for each ch13D3 CPA and ch13D3 antibody, isotype, and PBS controls. Figure 18B shows that ch13D3 CPA produced significantly more 13D3 puncta per cell compared to the control (approximately 2.5-3 puncta per cell). Figure 18C provides a graph showing the average puncta size of 13D3 antibody puncta for each ch13D3 CPA and ch13D3 antibody, isotype, and PBS control. Figure 18C shows that ch13D3 CPA resulted in significantly smaller 13D3 puncta per cell compared to the control. Figure 18D provides a graph showing the average puncta size of 13D3 antibody puncta for each ch13D3 CPA and ch13D3 antibody, isotype, and PBS control. Figure 18D shows that ch13D3 CPA resulted in significantly smaller 13D3 puncta per cell compared to the control (corrected for puncta intensity).
因此，圖18A至圖18D表明本揭示內容之抗TDP-43 CPA由細胞內化且結合細胞內p-TDP-43。Therefore, Figures 18A to 18D demonstrate that the anti-TDP-43 CPA of the present disclosure is internalized by cells and binds to intracellular p-TDP-43.
實例Example9.9.抗anti-TDP-43TDP-43細胞穿透劑對Cell penetrantsTDP-43TDP-43之磷酸化細胞質聚集體之內化及清除Internalization and clearance of phosphorylated cytoplasmic aggregates
圖19A至圖19D為顯示pTDP-43之磷酸化細胞質聚集體(亦即，用如本文所述之GFP-2a-TDP43轉染之細胞)與新穎細胞內化模組及人源化抗TDP-43抗體之內化及共定位的圖。圖19A至圖19D所示之資料係在與上文圖11A-D中所述之相同實驗條件下生成。圖19(全)中「ch13D3-ML」之標記係指包含在m13D3抗體之輕鏈(LC)之C末端連接M-Lycotoxin_L17E的CPA。Figures 19A to 19D are graphs showing the internalization and colocalization of phosphorylated cytoplasmic aggregates of pTDP-43 (i.e., cells transfected with GFP-2a-TDP43 as described herein) with the emerging cell internalization module and a humanized anti-TDP-43 antibody. The data shown in Figures 19A to 19D were generated under the same experimental conditions as described above for Figures 11A-D. The "ch13D3-ML" label in Figure 19 (all) refers to CPA containing M-Lycotoxin_L17E linked to the C-terminus of the light chain (LC) of the m13D3 antibody.
圖19A提供顯示人源化13D3 CPA以及ch13D3及h13D3抗體對照之與人源化13D3抗體共定位之pTDP43之百分比的圖。圖19A顯示與用h13D3抗體處理之細胞的共定位(約40%)相比，用各種h13D3 CPA處理之細胞的共定位(約60-70%)顯著更大。圖19B顯示在所有測試細胞群體中各孔之一致細胞計數。圖19C為顯示在用人源化13D3 CPA以及IgG同型、ch13D3及h13D3抗體對照處理之細胞中觀察到的與人源化13D3抗體共定位之pTDP43之各孔13D3斑點面積的圖。圖19C顯示用本揭示內容之人源化13D3 CPA處理之細胞的總共定位面積顯著增加。圖19D提供顯示在用人源化13D3 CPA以及IgG同型、ch13D3及h13D3抗體對照處理之細胞中觀察到的各細胞之13D3共定位斑點數目的圖。圖19D顯示用本揭示內容之人源化13D3 CPA處理之細胞的共定位斑點數目顯著增加。Figure 19A provides a graph showing the percentage of pTDP43 colocalizing with the humanized 13D3 antibody for humanized 13D3 CPA and ch13D3 and h13D3 antibody controls. Figure 19A shows significantly greater colocalization (approximately 60-70%) in cells treated with various h13D3 CPAs compared to the colocalization in cells treated with the h13D3 antibody (approximately 40%). Figure 19B shows consistent cell counts across wells across all tested cell populations. Figure 19C is a graph showing the area of 13D3 spots per well of pTDP43 colocalizing with the humanized 13D3 antibody observed in cells treated with humanized 13D3 CPA and IgG isotype, ch13D3, and h13D3 antibody controls. Figure 19C shows that the total colocalization area was significantly increased in cells treated with the humanized 13D3 CPA of the present disclosure. Figure 19D provides a graph showing the number of 13D3 colocalized puncta observed per cell in cells treated with humanized 13D3 CPA and IgG isotype, ch13D3, and h13D3 antibody controls. Figure 19D shows that the number of colocalized puncta was significantly increased in cells treated with the humanized 13D3 CPA of the present disclosure.
總之，圖19A至圖D中之資料顯示pTDP-43之磷酸化細胞質聚集體與新穎細胞內化模組及人源化抗TDP-43抗體之內化及共定位。In summary, the data in Figures 19A to D show that phosphorylated cytoplasmic aggregates of pTDP-43 are internalized and colocalized with the novel cell internalization module and the humanized anti-TDP-43 antibody.
實例Example10.10.抗anti-TDP-43TDP-43細胞穿透劑對神經膠質母細胞瘤細胞中Effects of cell penetrants on glioblastoma cellsTDP-43TDP-43之磷酸化細胞質聚集體之內化及清除Internalization and clearance of phosphorylated cytoplasmic aggregates
圖20A至圖20C為顯示在具有包括細胞內化模組及抗TDP-43抗體之CPA之膠質母細胞瘤細胞中TDP-43之磷酸化細胞質聚集體(亦即，用如本文所述之GFP-2a-TDP43轉染之細胞)之內化及共定位的圖。圖20A-C中「M-lycotoxin」之標記係指包含在m13D3抗體之輕鏈(LC)之C末端連接M-Lycotoxin_L17E的CPA。簡言之，用GFP-2a-TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)]瞬時轉染U251細胞。24小時後，將細胞與100 ug/ml抗體(例如CPA)一起再培育24小時。接著將細胞洗滌，固定/透化，且用抗pTDP-43抗體染色，繼之用AF647結合之抗小鼠二級抗體染色。藉由高含量成像(Operetta系統)用40x水物鏡對染色細胞進行成像。用Harmony軟體進行定量分析。Figures 20A-20C are graphs showing the internalization and colocalization of phosphorylated cytoplasmic aggregates of TDP-43 in glioblastoma cells transfected with a CPA containing a cell internalization module and an anti-TDP-43 antibody (i.e., cells transfected with GFP-2a-TDP43 as described herein). The "M-lycotoxin" label in Figures 20A-C refers to a CPA containing M-Lycotoxin_L17E linked to the C-terminus of the light chain (LC) of the m13D3 antibody. Briefly, U251 cells were transiently transfected with GFP-2a-TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)]. After 24 hours, cells were incubated with 100 μg/ml of antibody (e.g., CPA) for an additional 24 hours. Cells were then washed, fixed/permeabilized, and stained with an anti-pTDP-43 antibody, followed by an AF647-conjugated anti-mouse secondary antibody. Stained cells were imaged using high-content imaging (Operetta system) at a 40x objective lens. Quantitative analysis was performed using Harmony software.
更具體而言，圖20A為顯示用m13D3 m-Lycotoxin CPA或單獨的m13D3抗體(例如，未標記)處理之U251神經膠質母細胞瘤細胞之總病灶面積的圖。資料表明，與用m13D3抗體處理之細胞相比，用m13D3 m-Lycotoxin CPA處理之神經膠質母細胞瘤細胞中總病灶面積之減少顯著減少。圖20B為顯示用m13D3 m-Lycotoxin CPA或單獨的m13D3抗體(例如，未標記)處理之U251神經膠質母細胞瘤細胞之平均病灶大小的圖。資料表明，與用m13D3抗體處理之細胞相比，用m13D3 m-Lycotoxin CPA處理之神經膠質母細胞瘤細胞中平均病灶大小之減少顯著減少。圖20C為顯示用m13D3 m-Lycotoxin CPA或m13D3抗體處理之U251神經膠質母細胞瘤細胞之總病灶計數的圖。資料表明，與用m13D3抗體處理之細胞相比，用m13D3 m-Lycotoxin CPA處理之神經膠質母細胞瘤細胞中總病灶計數之增加顯著增加。More specifically, Figure 20A is a graph showing the total lesion area in U251 glioblastoma cells treated with m13D3 m-Lycotoxin CPA or the m13D3 antibody alone (e.g., unlabeled). The data demonstrate that the reduction in total lesion area in glioblastoma cells treated with m13D3 m-Lycotoxin CPA was significantly reduced compared to cells treated with the m13D3 antibody. Figure 20B is a graph showing the mean lesion size in U251 glioblastoma cells treated with m13D3 m-Lycotoxin CPA or the m13D3 antibody alone (e.g., unlabeled). The data demonstrate that the mean lesion size in glioblastoma cells treated with m13D3 m-Lycotoxin CPA was significantly reduced compared to cells treated with the m13D3 antibody. Figure 20C is a graph showing the total lesion counts in U251 glioblastoma cells treated with m13D3 m-Lycotoxin CPA or the m13D3 antibody. The data demonstrate that the total lesion counts in glioblastoma cells treated with m13D3 m-Lycotoxin CPA were significantly increased compared to cells treated with the m13D3 antibody.
因此，圖20A至圖20C表明本揭示內容之抗TDP-43 CPA由結合至細胞內p-TDP-43且因此破壞TDP-43聚集之神經膠質母細胞瘤細胞內化。Thus, Figures 20A to 20C demonstrate that the anti-TDP-43 CPA of the present disclosure is internalized by glioblastoma cells, binding to intracellular p-TDP-43 and thereby disrupting TDP-43 aggregation.
實例Example11.11.抗anti-TDP-43TDP-43細胞穿透劑對大鼠皮質神經元細胞中Effects of cell penetrants on rat cortical neuronsTDP-43TDP-43之磷酸化細胞質聚集體之內化及清除Internalization and clearance of phosphorylated cytoplasmic aggregates
圖21A至圖21B及圖22A至22F為顯示包括細胞內化模組及抗TDP-43抗體之CPA內化及磷酸化細胞質聚集體共定位於原代大鼠皮質神經元中的圖。簡言之，將原代大鼠皮質神經元細胞(DIV15)與50 ug/ml抗體一起培育2小時。接著將細胞洗滌，固定/透化，且用抗EEA1抗體染色，繼之用AF596結合之抗兔二級抗體及AF647結合之抗小鼠二級抗體染色。藉由高含量成像(Operetta系統)用40x水物鏡對染色細胞進行成像。用Harmony軟體進行定量分析。Figures 21A-21B and 22A-22F show colocalization of CPA internalization and phosphorylated cytoplasmic aggregates in primary rat cortical neurons, including a cell internalization module and an anti-TDP-43 antibody. Briefly, primary rat cortical neuron cells (DIV 15) were incubated with 50 μg/ml of antibody for 2 hours. The cells were then washed, fixed/permeabilized, and stained with an anti-EEA1 antibody, followed by an anti-rabbit secondary antibody conjugated to AF596 and an anti-mouse secondary antibody conjugated to AF647. Stained cells were imaged using high-content imaging (Operetta system) at a 40x objective lens. Quantitative analysis was performed using Harmony software.
具體而言，圖21A顯示用IgG同型對照(左上圖)、m13D3 (右上圖)、m13D3 m-Lycotoxin CPA (左下圖)及m13D3CMIP4 CPA (右下圖)處理之原代大鼠皮質神經元細胞的圖像。圖21A顯示m13D3 CPA之顯著內化(由細胞內之白色斑點描繪，下圖)及同型對照之極少內化或無內化(上圖)。圖21B為顯示各孔之總病灶面積的圖，其表明同型對照及13D3抗體之極少或無內化以及m13D3 m-Lycotoxin_L17E CPA CPA及m13D3 CMIP4 CPA (標記為13D3-ML)之實質內化。Specifically, Figure 21A shows images of primary rat cortical neurons treated with an IgG isotype control (upper left panel), m13D3 (upper right panel), m13D3 m-Lycotoxin CPA (lower left panel), and m13D3 CMIP4 CPA (lower right panel). Figure 21A shows significant internalization of m13D3 CPA (depicted by white spots within the cells, lower panel) and minimal or no internalization of the isotype control (upper panel). Figure 21B is a graph showing the total lesion area for each well, demonstrating minimal or no internalization of the isotype control and 13D3 antibodies, as well as substantial internalization of m13D3 m-Lycotoxin_L17E CPA and m13D3 CMIP4 CPA (labeled 13D3-ML).
圖22A為顯示與hIgG同型對照、m13D3抗體、m13D3 m-Lycotoxin CPA或m13D3 CMIP4 CPA一起培育之DIV15大鼠神經元細胞之各細胞之13D3斑點總數的圖。圖22A表明用m13D3-C末端-LC-M-lycotoxin_L17E CPA (在圖22中標記為「M-Lyco」)及m13D3-C末端-LC-M CMIP4 CPA (在圖22(全)中標記為「CMIP」)，而非用IgG同型對照或m13D3抗體處理之細胞。Figure 22A is a graph showing the total number of 13D3 puncta per cell in DIV15 rat neurons incubated with a hIgG isotype control, the m13D3 antibody, m13D3 m-Lycotoxin CPA, or m13D3 CMIP4 CPA. Figure 22A shows cells treated with m13D3-C-terminal-LC-M-lycotoxin_L17E CPA (labeled "M-Lyco" in Figure 22 ) and m13D3-C-terminal-LC-M CMIP4 CPA (labeled "CMIP" in Figure 22 (whole)), but not with the IgG isotype control or the m13D3 antibody.
圖22B為顯示與hIgG同型對照、m13D3抗體、m13D3 m-Lyco CPA或m13D3 CMIP4 CPA一起培育之DIV15大鼠神經元細胞之各孔13D3斑點總面積的圖。圖22B表明與用IgG同型對照或m13D3抗體處理之細胞相比，用m13D3 m-Lycotoxin CPA及m13D3 CMIP4 CPA處理之細胞的各孔總斑點面積更高。Figure 22B is a graph showing the total 13D3 plaque area per well of DIV15 rat neuronal cells incubated with a hIgG isotype control, the m13D3 antibody, m13D3 m-Lyco CPA, or m13D3 CMIP4 CPA. Figure 22B demonstrates that the total plaque area per well was higher in cells treated with m13D3 m-Lycotoxin CPA and m13D3 CMIP4 CPA compared to cells treated with the IgG isotype control or the m13D3 antibody.
圖22C為顯示與hIgG同型對照、m13D3抗體、m13D3 m-Lycotoxin CPA或m13D3 CMIP4 CPA一起培育之DIV15大鼠神經元細胞中13D3斑點之平均斑點大小的圖。圖22C表明與用IgG同型對照或m13D3抗體處理之細胞相比，用m13D3 m-Lycotoxin CPA及m13D3 CMIP4 CPA處理之細胞的平均斑點大小更大。Figure 22C is a graph showing the average 13D3 puncta size in DIV15 rat neurons incubated with a hIgG isotype control, the m13D3 antibody, m13D3 m-Lycotoxin CPA, or m13D3 CMIP4 CPA. Figure 22C demonstrates that the average puncta size is larger in cells treated with m13D3 m-Lycotoxin CPA and m13D3 CMIP4 CPA compared to cells treated with the IgG isotype control or the m13D3 antibody.
圖22D為顯示與hIgG同型對照、m13D3抗體、m13D3 m-Lycotoxin CPA或m13D3 CMIP4 CPA一起培育之DIV15大鼠神經元細胞中13D3斑點之斑點積分強度的圖。圖22D表明與用IgG同型對照或m13D3抗體處理之細胞相比，用m13D3 m-Lycotoxin CPA及m13D3 CMIP4 CPA處理之細胞的斑點積分強度更高。圖22E為顯示與hIgG同型對照、m13D3抗體、m13D3 m-Lycotoxin CPA或m13D3 CMIP4 CPA一起培育之DIV15大鼠神經元細胞中與EEA1 (早期胞內體抗原1)共定位之13D3斑點之百分比的圖。圖22E表明在用m13D3 m-Lycotoxin CPA及m13D3 CMIP4 CPA處理之細胞中，與EEA1共定位之13D3斑點為約10-20%，對於用IgG同型對照或m13D3抗體處理之細胞未觀察到共定位。圖22F顯示在所有測試細胞群體中各孔之一致細胞計數。總之，圖21A-D及圖22A-F顯示本揭示內容之細胞內化模組由原代大鼠皮質神經元內化。Figure 22D is a graph showing the integrated intensity of 13D3 puncta in DIV15 rat neurons incubated with a hIgG isotype control, the m13D3 antibody, m13D3 m-Lycotoxin CPA, or m13D3 CMIP4 CPA. Figure 22D demonstrates that the integrated intensity of puncta was higher in cells treated with m13D3 m-Lycotoxin CPA and m13D3 CMIP4 CPA than in cells treated with the IgG isotype control or the m13D3 antibody. Figure 22E is a graph showing the percentage of 13D3 puncta colocalizing with EEA1 (early endosomal antigen 1) in DIV15 rat neurons incubated with a hIgG isotype control, the m13D3 antibody, m13D3 m-Lycotoxin CPA, or m13D3 CMIP4 CPA. Figure 22E demonstrates that approximately 10-20% of 13D3 puncta colocalized with EEA1 in cells treated with m13D3 m-Lycotoxin CPA and m13D3 CMIP4 CPA, while no colocalization was observed in cells treated with the IgG isotype control or the m13D3 antibody. Figure 22F shows consistent cell counts across wells across all tested cell populations. In summary, Figures 21A-D and 22A-F show that the cell internalization module of the present disclosure is internalized by primary rat cortical neurons.
參考文獻Bowers, E., et al., Decreased Mutation Frequencies among Immunoglobulin G Variable Region Genes during Viremic HIV-1 Infection,PLoS ONE, 9(1): E81913 (2014). Foote J and Winter G., Antibody framework residues affecting the conformation of the hypervariable loops.J. Mol. Biol. 224(2):487-99 (1992). Kabat EA, Wu TT, Foeller C, Perry HM, Gottesman KS,Sequences of Proteins of Immunological Interest(5thedition). Bethesda, MD: National Institutes of Health (1991). Piechotta A, Parthier C, Kleinschmidt M, Gnoth K, Pillot T, Lues I, Demuth HU, Schilling S, Rahfeld JU, and Stubbs MT, Structural and functional analyses of pyroglutamate-amyloid-β-specific antibodies as a basis for Alzheimer immunotherapy.J Biol Chem. 2017 Jul 28; 292(30): 12713–12724 (2017). Wedemayer, G.J., Patten, P.A., Wang, L.H., Schultz, P.G., Stevens, R.C., Structural insights into the evolution of an antibody combining site. Science 276: 1665-1669 (1997). Cooper, L.J., Shikhman, A.R., Glass, D.D., Kangisser, D., Cunningham, M.W. and Greenspan, N.S., Role of heavy chain constant domains in antibody-antigen interaction. Apparent specificity differences among streptococcal IgG antibodies expressing identical variable domains,J. Immunol.150(6): 2231-2242 (1993) Léger OJP and Saldanha J., Preparation of recombinant antibodies from immune rodent spleens and the design of their humanisation by CDR grafting, In: Shepherd P and Dean C (eds).Monoclonal Antibodies: A Practical Approach. Oxford, UK: Oxford University Press (2000). Martin ACR, Protein sequence and structure analysis of antibody variable domains. In: Kontermann R and Dübel S (eds).Antibody Engineering. Heidelberg, Germany: Springer International Publishing AG (2010). Jimenez-Gomez,G., Gomez-Perales, J.L., Ramos-Amaya,A., Gonzalez-Garcia,I., Campos-Caro,A. and Brieva, J.A., Modulated selection of IGHV gene somatic hypermutation during systemic maturation of human plasma cells,J. Leukoc. Biol.87 (3), 523-530 (2010). Scally, SW, Bosch A, Triller G, Wardemann H, Julien J.P., Crystal structure of anti-cirumsporozoite protein 663 germline antibody. Direct deposit to PDB (2017). Shriner, A.K., et al., Analysis of the young and elderly variable gene repertoire in response to pneumococcal polysaccharides using a reconstituted SCID mouse model,Vaccine, 24(49-50): 7159-7166 (2006).References Bowers, E., et al., Decreased Mutation Frequencies among Immunoglobulin G Variable Region Genes during Viremic HIV-1 Infection,PLoS ONE , 9(1): E81913 (2014). Foote J and Winter G., Antibody framework residues affecting the conformation of the hypervariable loops.J. Mol. Biol . 224(2):487-99 (1992). Kabat EA, Wu TT, Foeller C, Perry HM, Gottesman KS,Sequences of Proteins of Immunological Interest (5th edition). Bethesda, MD: National Institutes of Health (1991). Piechotta A, Parthier C, Kleinschmidt M, Gnoth K, Pillot T, Lues I, Demuth HU, Schilling S, Rahfeld JU, and Stubbs MT, Structural and functional analyzes of pyroglutamate-amyloid-β-specific antibodies as a basis for Alzheimer immunotherapy. J Biol Chem. 2017 Jul 28; 292(30): 12713–12724 (2017). Wedemayer, GJ, Patten, PA, Wang, LH, Schultz, PG, Stevens, RC, Structural insights into the evolution of an antibody combining site. Science 276: 1665-1669 (1997). Cooper, LJ, Shikhman, AR, Glass, DD, Kangisser, D., Cunningham, MW and Greenspan, NS, Role of heavy chain constant domains in antibody-antigen interaction. Apparent specificity differences among streptococcal IgG antibodies expressing identical variable domains,J. Immunol. 150(6): 2231-2242 (1993) Léger OJP and Saldanha J., Preparation of recombinant antibodies from immune rodent spleens and the design of their humanization by CDR grafting, In: Shepherd P and Dean C (eds).Monoclonal Antibodies: A Practical Approach . Oxford, UK: Oxford University Press (2000). Martin ACR, Protein sequence and structure analysis of antibody variable domains. In: Kontermann R and Dübel S (eds).Antibody Engineering . Heidelberg, Germany: Springer International Publishing AG (2010). Jimenez-Gomez, G., Gomez-Perales, JL, Ramos-Amaya, A., Gonzalez-Garcia, I., Campos-Caro, A. and Brieva, JA, Modulated selection of IGHV gene somatic hypermutation during systemic maturation of human plasma cells,J. Leukoc. Biol. 87 (3), 523-530 (2010). Scally, SW, Bosch A, Triller G, Wardemann H, Julien JP, Crystal structure of anti-cirumsporozoite protein 663 germline antibody. Direct deposit to PDB (2017). Shriner, AK, et al., Analysis of the young and elderly variable gene repertoire in response to pneumococcal polysaccharides using a reconstituted SCID mouse model,Vaccine , 24(49-50): 7159-7166 (2006).
序列sequence附錄Appendix
可變重鏈參考序列 SEQ ID NO: 1 m13D3-VH EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSA SEQ ID NO: 2 IGHV3-48*03 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARYFDYWGQGTLVTVSS SEQ ID NO: 3 AEX28899 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRNYYDSGGYGHWGQGTLVTVSSVariable heavy chain reference sequence SEQ ID NO: 1 m13D3-VH EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYIST GGDSANYADNVKGRFTISRDNAKNTLYLQMNSL MSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSA SEQ ID NO: 2 IGHV3-48*03 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYISS SGSTIYYADSVKGRFTISRDNAKNSLYLQMNSL RAEDTAVYYCARYFDYWGQGTLVTVSS SEQ ID NO: 3 AEX28899 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYISS SGSTIYYADSVKGRFTISRDNAKNSLYLQMNSL RAEDTAVYYCARRNYYDSGGY GHWGQGTLVTVSS
人源化13D3 VH設計序列 SEQ ID NO: 4 h13D3VHv1 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMSWVRQAPGKGLEWVAYISTGGDSANYADNVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 5 h13D3VHv2 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQAPGKGLEWVAYISTGGDSANYADNVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 6 h13D3VHv3 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQAPGKGLEWVAYISTGGDSANYADNVKGRFTISRDNAKNSLYAQMNSLRAEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 7 h13D3VHv4 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQAPGKGLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYAQMNSLRAEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 8 h13D3VHv5 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQAPGKGLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTAYAQMNSLRAEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 9 h13D3VHv6 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMSWVRQAPGKGLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTAYAQMNSLRAEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 10 h13D3VHv7 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMGWVRQAPGKGLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTAYAQMNSLRAEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 11 h13D3VHv8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMGWVRQAPGKGLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYGQMNSLRAEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 12 h13D3VHv9 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMGWVRQAPGKGLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTGYLQMNSLRAEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 13 h13D3VHv10 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMGWVRQAPGKGLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSGRAEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 14 h13D3VHv11 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMGWVRQAPGKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTGYLQMNSLMAEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 15 h13D3VHv12 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMGWVRQAPGERLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTGYLQMNSLRAEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 16 h13D3VHv13 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMGWVRQAPGEDLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTGYLQMNSLRAEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 17 h13D3VHv1b ELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRFTITRDNSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 18 h13D3VHv2b ELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRFTITKDTSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 19 h13D3VHv3b ELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRGTITKDTSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 20 h13D3VHv1d EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMSWVRQAPGKGLEWVSYISTGGDSANYADNVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 21 h13D3VHv2d EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMSWVRQAPGKGLEWVSYISTGGDSANYADNVKGRFTISRDNSKNTAYLQMNSLRAEDTAVYYCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 22 h13D3VHv3d EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMSWVRQAPGKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 23 h13D3VHv4d EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMSWVRQAPGKRLEWVSYISTGGDSANYADNVKGRFTISRDNSKNTAYLQMNSLRAEDTAVYYCARQTYYSYGGFPYWGQGTLVTVSSHumanized 13D3 VH design sequence SEQ ID NO: 4 h13D3VHv1 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMSWVRQAPGKGLEWVAYIST GGDSANYADNVKGRFTISRDNAKNSLYLQMNSL RAEDTAVYFCARQTYYSYGGF PYWGQGTLVTVSS SEQ ID NO: 5 h13D3VHv2 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQAPGKGLEWVAYIST GGDSANYADNVKGRFTISSRDNAKNSLYLQMNSL RAEDTAVYFCARQTYYSYGGF PYWGQGTLVTVSS SEQ ID NO: 6 h13D3VHv3EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQAPGKGLEWVAYIST GGDSANYADNVKGRFTISRDNAKNTLYAQMNSL RAEDTAVYFCARQTYYSYGGF PYWGQGTLVTVSS SEQ ID NO: 8 h13D3VHv5 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQAPGKGLEWVAYIST GGDSANYADNVKGRFTISRDNAKNTAYAQMNSL RAEDTAVYFCARQTYYSYGGF PYWGQGTLVTVSS SEQ ID NO: 9 h13D3VHv6 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMSWVRQAPGKGLEWVAYIST GGDSANYADNVKGRFTISRDNAKNTAYAQMNSL RAEDTAVYFCARQTYYSYGGF PYWGQGTLVTVSS SEQ ID NO: 10 h13D3VHv7 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMGWVRQAPGKGLEWVAYIST GGDSANYADNVKGRFTISRDNAKNTAYAQMNSL RAEDTAVYFCARQTYYSYGGF PYWGQGTLVTVSS SEQ ID NO: 11 h13D3VHv8EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMGWVRQAPGKGLEWVAYIST GGDSANYADNVKGRFTISRDNAKNTGYLQMNSL RAEDTAVYFCARQTYYSYGGF PYWGQGTLVTVSS SEQ ID NO: 13 h13D3VHv10 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMGWVRQAPGKGLEWVAYIST GGDSANYADNVKGRFTISRDNAKNTLYLQMNSG RAEDTAVYFCARQTYYSYGGF PYWGQGTLVTVSS SEQ ID NO: 14 h13D3VHv11 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMGWVRQAPGKRLEWVAYIST GGDSANYADNVKGRFTISRDNAKNTGYLQMNSL MAEDTAVYFCARQTYYSYGGF PYWGQGTLVTVSS SEQ ID NO: 15 h13D3VHv12 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMGWVRQAPGERLEWVAYIST GGDSANYADNVKGRFTISRDNAKNTGYLQMNSL RAEDTAVYFCARQTYYSYGGF PYWGQGTLVTVSS SEQ ID NO: 16 h13D3VHv13 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMGWVRQAPGEDLEWVAYIST GGDSANYADNVKGRFTISRDNAKNTGYLQMNSL RAEDTAVYFCARQTYYSYGGF PYWGQGTLVTVSS SEQ ID NO: 17 h13D3VHv1b ELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRFTITRDNSSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 18 h13D3VHv2b ELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRFTITKDTSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 19 h13D3VHv3b ELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRGTITKDTSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 20 h13D3VHv1d EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMSWVRQAPGKGLEWVSYIST GGDSANYADNVKGRFTISRDNSKNSLYLQMNSL RAEDTAVYYCARQTYYSYGGF PYWGQGTLVTVSS SEQ ID NO: 21 h13D3VHv2d EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMSWVRQAPGKGLEWVSYIST GGDSANYADNVKGRFTISRDNSKNTAYLQMNSL RAEDTAVYYCARQTYYSYGGF PYWGQGTLVTVSS SEQ ID NO: 22 h13D3VHv3d EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMSWVRQAPGKRLEWVAYIST GGDSANYADNVKGRFTISSRDNAKNSLYLQMNSL RAEDTAVYYCARQTYYSYGGF PYWGQGTLVTVSS SEQ ID NO: 23 h13D3VHv4d EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMSWVRQAPGKRLEWVSYIST GGDSANYADNVKGRFTISRDNSKNTAYLQMNSL RAEDTAVYYCARQTYYSYGGF PYWGQGTLVTVSS
加下劃線之殘基在下文所定義之位置處。 分別為位置52A*# 位置82A*# 位置82B*# 位置82C*# 位置100A* 位置100B* 位置100C*。 *適用於SEQ ID NO: 1、3-16及20-23 #適用於SEQ ID NO: 2Underlined residues are at the positions defined below.Position 52A*#Position 82A*#Position 82B*#Position 82C*#Position 100A*Position 100B*Position 100C*.*Applies to SEQ ID NOs: 1, 3-16, and 20-23#Applies to SEQ ID NO: 2
可變輕鏈參考序列 SEQ ID NO: 24 m13D3-VL DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELK SEQ ID NO: 25 IGKV2-30*02 DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPYTFGQGTKLEIK SEQ ID NO: 26 ABC66863 DIVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHRPLTFGGGTKVEIKVariable light chain reference sequence SEQ ID NO: 24 m13D3-VL DVVMTQSPLSLPVSLGDQASISCRSSQSLVHS NGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELK SEQ ID NO: 25 IGKV2-30*02SLVHS DGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHRPLTFGGGTKVEIK
人源化13D3 VL設計序列 SEQ ID NO: 27 h13D3VLv1 DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 28 h13D3VLv2 DVVMTQSPLSLPVTLGQQASISCRSSQSLVHSNGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 29 h13D3VLv3 DVVMTQSPSSLPVTLGQQASISCRSSQSLVHSNGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGSGTDFTLKISRVESEDVGVYFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 30 h13D3VLv4 DVVMTQSPSSLPVTLGQQASISCRSSQSLVHSNGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGSGTDFTLKISRVESEDVGVYFCSQSGHVPLTFGGGTKVEIK SEQ ID NO: 31 h13D3VLv5 DVVMTQSPSSLPVTLGQPASISCRSSQSLVHSNGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGSGTDFTLKISRVESEDVGVYFCSQSGHVPLTFGGGTKVEIK SEQ ID NO: 32 h13D3VLv6 DVVMTQSPSSLPVTLGQPASISCRSSQSLVHSNGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGSGTDFTLKISRVESEDVGVYFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 33 h13D3VLv7 DVVMTQSPSSLPVTLGQPASISCRSSQSLVHSNGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGSGTDFTLKISRVESEDVGVYFCSQSLHIPLTFGGGTKVEIK SEQ ID NO: 34 h13D3VLv8 DVVMTQSPSSLPVTLGQPASISCRSSQSLVHSNGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGSGTDFTLKISRVESEDVGVYFCSQSLHAPLTFGGGTKVEIK SEQ ID NO: 35 h13D3VLv9 DVQMTQSPSSLPVTLGQPASISCRSSQSLVHSNGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGSGTDFTLKISRVESEDVGVYFCSQSGHVPLTFGDGTKVEIK SEQ ID NO: 36 h13D3VLv10 DVQMTQSPSSLPVTLGQPASISCRSSQSLVHSNGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGSGTDFTLKISRVESEDVGVYFCSQSGHVPLTFGRGTKVEIK SEQ ID NO: 37 h13D3VLv1b EVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTDFTLTISSLEPEDFAVYFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 38 h13D3VLv2b EVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTEFTLTISSLEPEDFAVYFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 39 h13D3VLv3b EVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTEVTLTISSLEPEDFAVYFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 40 h13D3VLv4b DVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTDFTLTISSLEPEDFAVKFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 41 h13D3VLv5b DVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTEFTLTISSLEPEDFAVKFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 42 h13D3VLv6b DVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTEVTLTISSLEPEDFAVKFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 43 h13D3VLv7b DVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTEFTLTISSLEPEDFAVYFCDQSLHVPLTFGGGTKVEIK SEQ ID NO: 44 h13D3VLv8b DVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTEVTLTISSLEPEDFAVYFCDQSLHVPLTFGGGTKVEIK SEQ ID NO: 45 h13D3VLv9b DVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTEFTLTISSLEPEDFAVYFCSQQLHVPLTFGGGTKVEIK SEQ ID NO: 46 h13D3VLv10b DVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTEVTLTISSLEPEDFAVYFCSQQLHVPLTFGGGTKVEIK SEQ ID NO: 47 h13D3VLv1d DVQMTQSPLSLPVTLGQPASISCRSSQSLVHSNGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSLHVPLTFGDGTKVEIK SEQ ID NO: 48 h13D3VLv2d DVQMTQSPLSLPVTLGQPASISCRSSQSLVHSNGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSAHVPLTFGDGTKVEIKHumanized 13D3 VL design sequence SEQ ID NO: 27 h13D3VLv1 DVVMTQSPLSLPVTLGQPASISCRSSQSLVHS NGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGTDFTLKISRVEAEDVGVYFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 28 h13D3VLv2DVVMTQSPLSLPPTLGQQASISCRSSQSLVHSSLVHSSLVHS NGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGSGTDFTLKISRVESEDVGVYFCSQSGHVPLTFGGGTKVEIK SEQ ID NO: 32h13D3VLv6 DVVMTQSPSSLPVTLGQPASISCRSSQSLVHSSLVHSSLVHSSLVHS NGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGSGTDFTLKISRVESEDVGVYFCSQSGHVPLTFGRGTKVEIK SEQ ID NO: 37h13D3VLv1b EVVMTQSPATLSLSPGERATLSCRSSQSLVHSSLVHSSLVHSSLVHS NGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTEFTLTISSLEPEDFAVKFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 42 h13D3VLv6bDVVMTQSPATLSLSPGERATLSCRSSQSLVHSSLVHS NGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTEVTLTISSLEPEDFAVYFCDQSLHVPLTFGGGTKVEIK SEQ IDSEQ ID NO: 46 h13D3VLv10bSLVHSSLVHS NGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSAHVPLTFGDGTKVEIK
加下劃線之殘基在下文所定義之位置處。 分別地，S為位置27A L為位置27B V為位置27C H為位置27D S為位置27E。 適用於SEQ ID NO: 24-48。 SEQ ID NO: 49 HC CDR1 #1 GFTFSNYFMS SEQ ID NO: 50 HC CDR1 #2 GFTFSNYFMG SEQ ID NO: 51 HC CDR2 #1 YISTGGDSANYADNVKG SEQ ID NO: 52 HC CDR3 #1 QTYYSYGGFPY SEQ ID NO: 53 LC CDR1 #1 RSSQSLVHSNGKTYLH SEQ ID NO: 54 LC CDR2 #1 KVSDRYS SEQ ID NO: 55 LC CDR3 #1 SQSLHVPLT SEQ ID NO: 56 LC CDR3 #2 SQSGHVPLT SEQ ID NO: 57 LC CDR3 #3 SQSLHIPLT SEQ ID NO: 58 LC CDR3 #4 SQSLHAPLT SEQ ID NO: 59 LC CDR3 #5 DQSLHVPLT SEQ ID NO: 60 LC CDR3 #6 SQQLHVPLT SEQ ID NO: 61 LC CDR3 #7 SQSAHVPLTUnderlined residues are at positions defined below.Respectively, S is position 27AL is position 27BV is position 27CH is position 27DS is position 27E.Applicable to SEQ ID NOs: 24-48.SEQ ID NO: 49 HC CDR1 #1 GFTFSNYFMS SEQ ID NO: 50 HC CDR1 #2 GFTFSNYFMG SEQ ID NO: 51 HC CDR2 #1 YISTGGDSANYADNVKG SEQ ID NO: 52 HC CDR3 #1 QTYYSYGGFPY SEQ ID NO: 53 LC CDR1 #1 RSSQSLVHSNGKTYLH SEQ ID NO: 54 LC CDR2 #1 KVSDRYS SEQ ID NO: 55 LC CDR3 #1 SQSLHVPLT SEQ ID NO: 56 LC CDR3 #2 SQSGHVPLT SEQ ID NO: 57 LC CDR3 #3 SQSLHIPLT SEQ ID NO: 58 LC CDR3 #4 SQSLHAPLT SEQ ID NO: 59 LC CDR3 #5 DQSLHVPLT SEQ ID NO: 60 LC CDR3 #6 SQQLHVPLT SEQ ID NO: 61 LC CDR3 #7 SQSAHVPLT
TDP43小鼠抗體可變域序列SEQ ID NO: 62 1B3_JH140 VH訊號肽 MNFGLSLIFLVLVLKGVLC SEQ ID NO: 63 1B3_JH140 可變重域 EVKLVESGGGLVQPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLELVAEISNSGGRTNYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARRRYSDYYYYAMDYWGQGTSVTVSS SEQ ID NO: 64 1B3_JH140 VL訊號肽 MSSAQFLGLLLLCFQGTRC SEQ ID NO: 65 1B3_JH140 可變輕域 DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPRTFGGGTKLEIK SEQ ID NO: 66 1C12_JH104 VH訊號肽 MNFGLSLIFLVLVLKGVLC SEQ ID NO: 67 1C12_JH104 可變重域 EVKLVESGGGLVQPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLELVADISNSGGRTYYPDTVKGRFTISRENAKNSLYLQMSSLKSEDTAMYYCARRRYSDYYYYAMDNWGQGTSVTVSS SEQ ID NO: 68 1C12_JH104 VL訊號肽 MSSAQFLGLLLLCFQGTRC SEQ ID NO: 69 1C12_JH104 可變輕域 DIQMTQTTSSLSASLGDRVTISCRASQDISNYLSWYQQKPDGTVKLLIYYTSRLNSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNALPRTFGGGTKLEIN SEQ ID NO: 70 11A1_LA121 VH訊號肽 MEWPCIFLFLLSVTEGVHS SEQ ID NO: 71 11A1_LA121 可變重域 QVQLQQSGAELVRPGSSVKISCKASGYEFSRYWMNWVKQRPGQGLEWIGQIYHGDGDTNYKGKFKGKAILTADKSSSTAYMQVSSLTSEDSAVYFCVRGGYYGYAMDYWGQGTSVTVSS SEQ ID NO: 72 11A1_LA121 訊號肽 MKLPVRLLVLMFWIPASSS SEQ ID NO: 73 11A1_LA121 可變輕域 DVVMTQTPLSLPVSLGDQASISCRSSQSLLHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTNLEIK SEQ ID NO: 74 11B1_LA121 VH訊號肽 MEWPCIFLFLLSVTEGVHS SEQ ID NO: 75 11B1_LA121 可變重域 QVQLQQSGAELVRPGSSVKISCKASGYEFSRYWMNWVKQRPGQGLEWIGQIYHGDGDTNYKGKFKGKAILTADKSSSTAYMQVSSLTSEDSAVYFCVRGGYYGYAMDYWGQGTSVTVSS SEQ ID NO: 76 11B1_LA121 VL訊號肽 MKLPVRLLVLMFWIPASSS SEQ ID NO: 77 11B1_LA121 可變輕域 DVVMTQTPLSLPVSLGDQASISCRSSQSLLHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTNLEIK SEQ ID NO: 78 16G5_LA121 VH訊號肽 MKCSWVIFFLMAVVTGVNS SEQ ID NO: 79 16G5_LA121 可變重域 EVHLQQSGAELVKPGASVKLSCTGSGFNIIDTYIHWVKQRPEQGLEWIGRIDPANGNTMYASKFQDKATIIADTSSNTVYMRLGSLTSGDTAVYYCSHGDFWWGQGTTLTVSS SEQ ID NO: 80 16G5_LA121 VL訊號肽 MSPAQFLFLLVLWIQETNG SEQ ID NO: 81 16G5_LA121 可變輕域 DVVMTQTPLTLSIPIGQPAYISCKSSQSLLKSNGKTYLNWLFQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCVQGTHLPHTFGGGTRLEIK SEQ ID NO: 82 人類TDP-43蛋白 MSEYIRVTEDENDEPIEIPSEDDGTVLLSTVTAQFPGACGLRYRNPVSQCMRGVRLVEGILHAPDAGWGNLVYVVNYPKDNKRKMDETDASSAVKVKRAVQKTSDLIVLGLPWKTTEQDLKEYFSTFGEVLMVQVKKDLKTGHSKGFGFVRFTEYETQVKVMSQRHMIDGRWCDCKLPNSKQSQDEPLRSRKVFVGRCTEDMTEDELREFFSQYGDVMDVFIPKPFRAFAFVTFADDQIAQSLCGEDLIIKGISVHISNAEPKHNSNRQLERSGRFGGNPGGFGNQGGFGNSRGGGAGLGNNQGSNMGGGMNFGAFSINPAMMAAAQAALQSSWGMMGMLASQQNQSGPSGNNQNQGNMQREPNQAFGSGNNSYSGSNSGAAIGWGSASNAGSGSGFNGGFGSSMDSKSSGWGM SEQ ID NO: 83 TDP-43肽 (pS409/pS410) GSGSGFNGGFGSSMDSKSSGWGM SEQ ID NO: 84 1B3_JH140 可變重鏈CDR1 SGFTFSSYTMS SEQ ID NO: 85 1B3_JH140 可變重鏈CDR2 EISNSGGRTNY SEQ ID NO: 86 1B3_JH140 可變重鏈CDR3 RRYSDYYYYAMDY SEQ ID NO: 87 1B3_JH140 可變輕鏈CDR1 RASQDISNYLN SEQ ID NO: 88 1B3_JH140 可變輕鏈CDR2 YTSRLHS SEQ ID NO: 89 1B3_JH140 可變輕鏈CDR3 QQGNTLPRT SEQ ID NO: 90 1C12_JH104 可變重鏈CDR1 SGFTFSSYTMS SEQ ID NO: 91 1C12_JH104 可變重鏈CDR2 DISNSGGRTYYPDTVKG SEQ ID NO: 92 1C12_JH104 可變重鏈CDR3 RRYSDYYYYAMDN SEQ ID NO: 93 1C12_JH104 可變輕鏈CDR1 RASQDISNYLS SEQ ID NO: 94 1C12 JH104 可變輕鏈CDR2 YTSRLNS SEQ ID NO: 95 1C12_JH104 可變輕鏈CDR3 QQGNALPRT SEQ ID NO: 96 11A1_LA121 可變重鏈CDR1 SGYEFSRYWMN SEQ ID NO: 97 11A1_LA121 可變重鏈CDR2 QIYHGDGDTNYKGKFKG SEQ ID NO: 98 11A1_LA121 可變重鏈CDR3 GGYYGYAMDY SEQ ID NO: 99 11A1_LA121 可變輕鏈CDR1 RSSQSLLHSNGNTYLH SEQ ID NO: 100 11A1_LA121 可變輕鏈CDR2 KVSNRFS SEQ ID NO: 101 11A1_LA121 可變輕鏈CDR3 SQSTHVPYT SEQ ID NO: 102 11B1_LA121 可變重鏈CDR1 SGYEFSRYWMN SEQ ID NO: 103 11B1_LA121 可變重鏈CDR2 QIYHGDGDTNYKGKFKG SEQ ID NO: 104 11B1_LA121 可變重鏈CDR3 GGYYGYAMDY SEQ ID NO: 105 11B1_LA121 可變輕鏈CDR1 RSSQSLLHSNGNTYLH SEQ ID NO: 106 11B1_LA121 可變輕鏈CDR2 KVSNRFS SEQ ID NO: 107 11B1_LA121 可變輕鏈CDR3 SQSTHVPYT SEQ ID NO: 108 16G5_LA121 可變重鏈CDR1 SGFNIIDTYIH SEQ ID NO: 109 16G5_LA121 可變重鏈CDR2 RIDPANGNTMYA SEQ ID NO: 110 16G5_LA121 可變重鏈CDR3 GDFW SEQ ID NO: 111 16G5_LA121 可變輕鏈CDR1 KSSQSLLKSNGKTYLN SEQ ID NO: 112 16G5_LA121 可變輕鏈CDR2 LVSKLDS SEQ ID NO: 113 16G5 LA121 可變輕鏈CDR3 VQGTHLPHT SEQ ID NO: 114 m13D3重鏈 - 具有小鼠IgG2a重鏈恆定域；恆定區加下劃線 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKSEQ ID NO: 115 m13D3輕鏈 - 具有小鼠κ輕鏈恆定域；恆定區加下劃線 DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC*SEQ ID NO: 114及115中加下劃線之序列表示恆定域。 SEQ ID NO: 116 13D3_Ab_LC-Ct-CMIP4重鏈 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 117 13D3_Ab_LC-Ct-CMIP4輕鏈 DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSGGGGSIWLTALKFSGKAAAKAEAKQFLSKLSEQ ID NO: 118 13D3_Ab_HC-C末端_TAT重鏈 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGRKKRRQRRRPPQSEQ ID NO: 119 13D3_Ab_HC-C末端_TAT輕鏈 DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 120 13D3_Ab_LC-L17E_M-Lycotoxin重鏈 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 121 13D3_Ab_LC-L17E_M-Lycotoxin輕鏈 DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSGGGGSIWLTALKFLGKHAAKHEAKQQLSKLSEQ ID NO: 122 13D3_Ab_HC-Ct-L17E_M-Lycotoxin重鏈 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGGGGSGGGGSIWLTALKFLGKHAAKHEAKQQLSKLSEQ ID NO: 123 13D3_Ab_HC-Ct-L17E_M-Lycotoxin輕鏈 DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 124 13D3_Ab_HC-AH-PEPTH重鏈 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPGSVKKKKIKAEIKIGAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 125 13D3_Ab_HC-AH-PEPTH輕鏈 DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 126 13D3_Ab_HC-C末端-cycR9重鏈 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGGGCG*RRRRRRRRRG*CSEQ ID NO: 127 13D3_Ab_HC-C末端-cycR9輕鏈 DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 128 13D3_Ab_HC-C末端-cycTAT1修飾之重鏈 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGGGCG*GRKKRRQRRRPQG*CSEQ ID NO: 129 13D3_Ab_HC-C末端-cycTAT1修飾之輕鏈 DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 130 13D3_Ab_HC-C末端-TAT重鏈 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGRKKRRQRRRPPQSEQ ID NO: 131 13D3_Ab_HC-C末端-TAT輕鏈 DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 132 13D3_Ab_LC-C末端-Penetain重鏈 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 133 13D3_Ab_LC-C末端-Penetain輕鏈 DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSGGGGSRQIKIWFQNRRMKWKKG*SEQ ID NO: 134 13D3_Ab_LC-C末端-L17E-M-Lycotoxin重鏈 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 135 13D3_Ab_LC-C末端-L17E-M-Lycotoxin輕鏈 DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSGGGGSIWLTALKFLGKHAAKHEAKQQLSKLSEQ ID NO: 136 13D3_Ab_LC-C末端-Pepth重鏈 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 137 13D3_Ab_LC-C末端-Pepth輕鏈 DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSGGGGSVKKKKIKAEIKIG*SEQ ID NO: 138 13D3_Ab_HC-C末端-TAT3重鏈 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGGGGSGGGGSGRKKRRQRRRPQGRKKRRQRRRPQGRKKRRQRRRPQG*G*SEQ ID NO: 139 13D3_Ab_HC-C末端-TAT3輕鏈 DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 140 13D3_Ab_LC-C末端_TAT3重鏈 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 141 13D3_Ab_LC-C末端_TAT3輕鏈 DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSGGGGSGRKKRRQRRRPQGRKKRRQRRRPQGRKKRRQRRRPQG*G*SEQ ID NO: 142 13D3_Ab_LC-C末端_cycTAT3重鏈 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 143 13D3_Ab_LC-C末端_cycTAT3輕鏈 DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSGGGGSCG*G*GRKKRRQRRRPQGRKKRRQRRRPQGRKKRRQRRRPQG*G*CSEQ ID NO: 144 13D3_Ab_LC-C末端_cycR8x3重鏈 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGGGGSGGGGSCG*G*G*RRRRRRRRG*G*G*RRRRRRRRG*G*G*RRRRRRRRG*G*G*CSEQ ID NO: 145 13D3_Ab_LC-C末端_cycR8x3輕鏈 DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 146 13D3_Ab_HC-C末端_cycTAT3重鏈 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGGGGSGGGGSCG*G*G*GRKKRRQRRRPQGRKKRRQRRRPQGRKKRRQRRRPQG*G*G*CSEQ ID NO: 147 13D3_Ab_HC-C末端_cycTAT3輕鏈 DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 148 13D3_Ab_LC-C末端_R8x3重鏈 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 149 13D3_Ab_LC-C末端_R8x3輕鏈 DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSRRRRRRRRG*G*G*G*S*RRRRRRRRG*G*G*G*S*RRRRRRRRG*G*SEQ ID NO: 150 13D3_Ab_HC-C末端_R6H4重鏈 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGGGGSGGGGSRRRRRRHHHHSEQ ID NO: 151 13D3_Ab_HC-C末端_R6H4輕鏈 DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 152 13D3_Ab_HC-C末端_TAT-H4重鏈 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGGGGSGRKKRRQRRRPHHHHSEQ ID NO: 153 13D3_Ab_HC-C末端_TAT-H4輕鏈 DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSgalDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 154 h13D3H1bL1b_Ab_LC-C末端_CMIP4重鏈 EVELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRFTITRDNSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 155 h13D3H1bL1b_Ab_LC-C末端_CMIP4輕鏈 DVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTDFTLTISSLEPEDFAVYFCSQSLHVPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGSGGGSIWLTALKFSGKAAAKAEAKQFLSKLSEQ ID NO: 156 h13D3H1bL2b_Ab_LC-C末端_CMIP4重鏈 EVELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRFTITRDNSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 157 h13D3H1bL2b_Ab_LC-C末端_CMIP4輕鏈 DVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTEFTLTISSLEPEDFAVYFCSQSLHVPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGSGGGSIWLTALKFSGKAAAKAEAKQFLSKLSEQ ID NO: 158 h13D3H2bL1b_Ab_LC-C末端_CMIP4重鏈 EVELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRFTITKDTSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 159 h13D3H2bL1b_Ab_LC-C末端_CMIP4輕鏈 DVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTDFTLTISSLEPEDFAVYFCSQSLHVPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGSGGGSIWLTALKFSGKAAAKAEAKQFLSKLSEQ ID NO: 160 h13D3H2bL2b_Ab_LC-C末端_CMIP4重鏈 EVELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRFTITKDTSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 161 h13D3H2bL2b_Ab_LC-C末端_CMIP4輕鏈 DVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTEFTLTISSLEPEDFAVYFCSQSLHVPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGSGGGSIWLTALKFSGKAAAKAEAKQFLSKLSEQ ID NO: 162 h13D3H3bL1b_ Ab_LC-C末端_CMIP4重鏈 EVELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRGTITKDTSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 163 h13D3H3bL1b_ Ab_LC-C末端_CMIP4輕鏈 DVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTDFTLTISSLEPEDFAVYFCSQSLHVPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGSGGGSIWLTALKFSGKAAAKAEAKQFLSKLSEQ ID NO: 164 h13D3H3bL2b_ Ab_LC-C末端_CMIP4重鏈 EVELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRGTITKDTSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 165 h13D3H3bL2b_ Ab_LC-C末端_CMIP4輕鏈 DVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTEFTLTISSLEPEDFAVYFCSQSLHVPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGSGGGSIWLTALKFSGKAAAKAEAKQFLSKLSEQ ID NO: 166 m13D3_ Ab_LC-C末端_CMIP1重鏈 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 167 m13D3_ Ab_LC-C末端_CMIP1輕鏈 DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSGGGGSIWLTALKFLGKAAAKAEAKQQLSKLSEQ ID NO: 168 m13D3_ Ab_LC-C末端_CMIP2重鏈 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 169 m13D3_ Ab_LC-C末端_CMIP2輕鏈 DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSGGGGSIWLTALKFSGKAAAKAEAKQQLSKLSEQ ID NO: 170 m13D3_ Ab_LC-C末端_CMIP3重鏈 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 171 m13D3_ Ab_LC-C末端_CMIP3輕鏈 DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSGGGGSIWLTASKFSGKAAAKAEAKQQLSKLSEQ ID NO: 172 m13D3_ Ab_LC-C末端_CMIP4重鏈 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 173 m13D3_ Ab_LC-C末端_CMIP4輕鏈 DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSGGGGSIWLTALKFSGKAAAKAEAKQFLSKLSEQ ID NO: 174 m13D3_ Ab_LC-C末端_CMIP5重鏈 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 175 m13D3_ Ab_LC-C末端_CMIP5輕鏈 DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSGGGGSIWLTALKFSGKAAAKAEAKQWLSKL以下適用於SEQ ID NO: 116-175 連接子序列以粗體表示。 間隔區或胺基酸在胺基酸殘基(例如，G*)之後用星號(*)表示。 細胞內化模組序列用雙下劃線表示。 SEQ ID NO: 176 CIM #1 CMIP1 IWLTALKFLGKAAAKAEAKQQLSKL SEQ ID NO: 177 CIM #2 CMIP2 IWLTALKFSGKAAAKAEAKQQLSKL SEQ ID NO: 178 CIM #3 CMIP3 IWLTASKFSGKAAAKAEAKQQLSKL SEQ ID NO: 166 CIM #4 CMIP4 IWLTALKFSGKAAAKAEAKQFLSKL SEQ ID NO: 180 CIM #5 CMIP5 IWLTALKFSGKAAAKAEAKQWLSKL SEQ ID NO: 181 CIM #6 TAT GRKKRRQRRRPPQ SEQ ID NO: 182 CIM #7野生型M-lycotoxin IWLTALKFLGKHAAKHEAKQQLSKL SEQ ID NO: 183 CIM #8 L17E_M-lycotoxin IWLTALKFLGKHAAKHEAKQQLSKL SEQ ID NO: 184 CIM #9 PEPTH VKKKKIKAEIKIG SEQ ID NO: 185 CIM #10 cycR9 CG*RRRRRRRRRG*C SEQ ID NO: 186 CIM #11 cycTAT1 CG*GRKKRRQRRRPQG*C SEQ ID NO: 187 CIM #12 Penetain RQIKIWFQNRRMKWKKG* SEQ ID NO: 188 CIM #13 Tat3 GRKKRRQRRRPQGRKKRRQRRRPQGRKKRRQRRRPQG*G* SEQ ID NO: 189 CIM #14 cycTat3 CG*G*GRKKRRQRRRPQGRKKRRQRRRPQGRKKRRQRRRPQG*G*C SEQ ID NO: 190 CIM #15 R8x3 RRRRRRRRG*G*G*G*S*RRRRRRRRG*G*G*G*S*RRRRRRRRG*G* SEQ ID NO: 191 CIM #16 cycR8x3 CG*G*G*RRRRRRRRG*G*G*RRRRRRRRG*G*G*RRRRRRRRG*G*G*C SEQ ID NO: 192 CIM #17 R6H4 RRRRRRHHHH SEQ ID NO: 193 CIM #18 TatH4 GRKKRRQRRRPHHHH 以下適用於SEQ ID NO: 176-193 間隔區或胺基酸在胺基酸殘基(例如，G*)之後用星號(*)表示。 SEQ ID NO: 194連接子序列#1 GGGGSGGGGS SEQ ID NO: 195連接子序列#2 GGGGS SEQ ID NO: 196連接子序列#3 GGGGS SEQ ID NO: 197連接子序列#4 GS SEQ ID NO: 198連接子序列#5 GGG SEQ ID NO: 199：連接子序列#6 GGGSGGGS SEQ ID NO: 200間隔區#1 G SEQ ID NO: 201間隔區#2 GG SEQ ID NO: 202間隔區#3 GGG SEQ ID NO: 203間隔區#4 GGGGSTDP43mouse antibody variable domain sequence SEQ ID NO: 62 1B3_JH140 VH signal peptide MNFGLSLIFLVLVLKGVLC SEQ ID NO: 63 1B3_JH140 variable heavy domain EVKLVESGGGLVQPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLELVAEISNSGGRTNYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARRRYSDYYYYAMDYWGQGTSVTVSS SEQ ID NO: 64 1B3_JH140 VL signal peptide MSSAQFLGLLLLCFQGTRC SEQ ID NO: 65 1B3_JH140 1C12_JH104 VH signal peptide MNFGLSLIFLVLVLKGVLC SEQ ID NO: 67 1C12_JH104 variable heavy domain EVKLVESGGGLVQPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLELVADISNSGGRTYYPDTVKGRFTISRENAKNSLYLQMSSLKSEDTAMYYCARRRYSDYYYYAMDNWGQGTSVTVSS SEQ ID NO: 68 1C12_JH104 VL signal peptide MSSAQFLGLLLLCFQGTRC SEQ ID NO: 69 1C12_JH104 variable light domain DIQMTQTTSSLSASLGDRVTISCRASQDISNYLSWYQQKPDGTVKLLIYYTSRLNSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNALPRTFGGGTKLEIN SEQ ID NO: 70 11A1_LA121 VH signal peptide MEWPCIFLFLLSVTEGVHS SEQ ID NO: 71 11A1_LA121 variable heavy domain QVQLQQSGAELVRPGSSVKISCKASGYEFSRYWMNWVKQRPGQGLEWIGQIYHGDGDTNYKGKFKGKAILTADKSSSTAYMQVSSLTSEDSAVYFCVRGGYYGYAMDYWGQGTSVTVSS SEQ ID NO: 72 11A1_LA121 signal peptide MKLPVRLLVLMFWIPASSS SEQ ID NO: 73 11A1_LA121 Variable light domain DVVMTQTPLSLPVSLGDQASISCRSSQSLLHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTNLEIK SEQ ID NO: 74 11B1_LA121 VH signal peptide MEWPCIFLFLLSVTEGVHS SEQ ID NO: 75 11B1_LA121 Variable heavy domain QVQLQQSGAELVRPGSSVKISCKASGYEFSRYWMNWVKQRPGQGLEWIGQIYHGDGDTNYKGKFKGKAILTADKSSSTAYMQVSSLTSEDSAVYFCVRGGYYGYAMDYWGQGTSVTVSS SEQ ID NO: 76 11B1_LA121 VL signal peptide MKLPVRLLVLMFWIPASSS SEQ ID NO: 77 11B1_LA121 variable light domain DVVMTQTPLSLPVSLGDQASISCRSSQSLLHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTNLEIK SEQ ID NO: 78 16G5_LA121 VH signal peptide MKCSWVIFFLMAVVTGVNS SEQ ID NO: 79 16G5_LA121 Variable heavy domain EVHLQQSGAELVKPGASVKLSCTGSGFNIIDTYIHWVKQRPEQGLEWIGRIDPANGNTMYASKFQDKATIIADTSSNTVYMRLGSLTSGDTAVYYCSHGDFWWGQGTTLTVSS SEQ ID NO: 80 16G5_LA121 VL signal peptide MSPAQFLFLLVLWIQETNG SEQ ID NO: 81 16G5_LA121 Variable light domain DVVMTQTPLTLSIPIGQPAYISCKSSQSLLKSNGKTYLNWLFQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCVQGTHLPHTFGGGTRLEIK SEQ ID NO: 82 Human TDP-43 proteinMSEYIRVTEDENDEPIEIPSEDDGTVLLSTVTAQFPGACGLRYRNPVSQCMRGVRLVEGILHAPDAGWGNLVYVVNYPKDNKRKMDETDASSAVKV KRAVQKTSDLIVLGLPWKTTEQDLKEYFSTFGEVLMVQVKKDLKTGHSKGFGFVRFTEYETQVKVMSQRHMIDGRWCDCKLPNSKQSQDEPLRSRKVFVGRCTEDM TEDELREFFSQYGDVMDVFIPKPFRAFAFVTFADDQIAQSLCGEDLIIKGISVHISNAEPKHNSNRQLERSGRFGGNPGGFGNQGGFGNSRGGGAGLGNNQGSNMG GGMNFGAFSINPAMMAAAQAALQSSWGMMGMLASQQNQSGPSGNNQNQGNMQREPNQAFGSGNNSYSGSNSGAAIGWGSASNAGSGSGFNGGFGSSMDSKSSGWGM SEQ ID NO: 83 TDP-43 peptide (pS409/pS410) GSGSGFNGGFGSSMDSKSSGWGM SEQ ID NO: 84 1B3_JH140 variable heavy chain CDR1 SGFTFSSYTMS SEQ ID NO: 85 1B3_JH140 variable heavy chain CDR2 EISNSGGRTNY SEQ ID NO: 86 1B3_JH140 variable heavy chain CDR3 RRYSDYYYYAMDY SEQ ID NO: 87 1B3_JH140 variable light chain CDR1 RASQDISNYLN SEQ ID NO: 88 1B3_JH140 variable light chain CDR2 YTSRLHS SEQ ID NO: 89 1B3_JH140 variable light chain CDR3 1C12_JH104 variable heavy chain CDR1 SGFTFSSYTMS SEQ ID NO: 91 1C12_JH104 variable heavy chain CDR2 DISNSGGRTYYPDTVKG SEQ ID NO: 92 1C12_JH104 variable heavy chain CDR3 RRYSDYYYYAMDN SEQ ID NO: 93 1C12_JH104 variable light chain CDR1 RASQDISNYLS SEQ ID NO: 94 1C12 JH104 variable light chain CDR2 YTSRLNS SEQ ID NO: 95 1C12_JH104 variable light chain CDR3 QQGNALPRT SEQ ID NO: 96 11A1_LA121 11A1_LA121 Variable heavy chain CDR1 SGYEFSRYWMN SEQ ID NO: 97 11A1_LA121 Variable heavy chain CDR2 QIYHGDGDTNYKGKFKG SEQ ID NO: 98 11A1_LA121 Variable heavy chain CDR3 GGYYGYAMDY SEQ ID NO: 99 11A1_LA121 Variable light chain CDR1 RSSQSLLHSNGNTYLH SEQ ID NO: 100 11A1_LA121 Variable light chain CDR2 KVSNRFS SEQ ID NO: 101 11A1_LA121 Variable light chain CDR3 SQSTHVPYT SEQ ID NO: 102 11B1_LA121 Variable heavy chain CDR1 SGYEFSRYWMN SEQ ID NO: NO: 103 11B1_LA121 variable heavy chain CDR2 QIYHGDGDTNYKGKFKG SEQ ID NO: 104 11B1_LA121 variable heavy chain CDR3 GGYYGYAMDY SEQ ID NO: 105 11B1_LA121 variable light chain CDR1 RSSQSLLHSNGNTYLH SEQ ID NO: 106 11B1_LA121 variable light chain CDR2 KVSNRFS SEQ ID NO: 107 11B1_LA121 variable light chain CDR3 SQSTHVPYT SEQ ID NO: 108 16G5_LA121 variable heavy chain CDR1 SGFNIIDTYIH SEQ ID NO: 109 16G5_LA121 Variable heavy chain CDR2 RIDPANGNTMYA SEQ ID NO: 110 16G5_LA121 Variable heavy chain CDR3 GDFW SEQ ID NO: 111 16G5_LA121 Variable light chain CDR1 KSSQSLLKSNGKTYLN SEQ ID NO: 112 16G5_LA121 Variable light chain CDR2 LVSKLDS SEQ ID NO: 113 16G5 LA121 Variable light chain CDR3 VQGTHLPHT SEQ ID NO: 114 m13D3 heavy chain Contains mouse IgG2a heavy chain constitutive domain; constitutive region underlined EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNV EVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 115 m13D3 light chain- Possesses a mouse kappa light chain homeostasis domain; homeostasis region is underlined: DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC *The underlined sequences in SEQ ID NOs: 114 and 115 represent the homeostasis domain. SEQ ID NO: 116 13D3_Ab_LC-Ct-CMIP4 heavy chainEVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFC ARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVD KKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLP APIERTISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 117 13D3_Ab_LC-Ct-CMIP4 light chainDVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHV PLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSGGGGSIWLTALKFSGKAAAKAEAKQFLSKL SEQ ID NO: 118 13D3_Ab_HC-C-terminal_TAT heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCA RQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDK KIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLP APIERTISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGRKKRRQRRRPPQ SEQ ID NO: 119 13D3_Ab_HC-C-terminal_TAT light chainDVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHV PLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 120 13D3_Ab_LC-L17E_M-Lycotoxin heavy chainEVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSED TAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASS TKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKD LPAPIERTISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 121 13D3_Ab_LC-L17E_M-Lycotoxin light chainDVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQ SLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSGGGGSIWLTALKFLGKHAAKHEAKQQLSKL SEQ ID NO: 122 13D3_Ab_HC-Ct-L17E_M-Lycotoxin heavy chainEVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMS EDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPAS STKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNK DLPAPIERTISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGGGGSGGGGSIWLTALKFLGKHAAKHEAKQQLSKL SEQ ID NO: 123 13D3_Ab_HC-Ct-L17E_M-Lycotoxin light chainDVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFC SQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 124 13D3_Ab_HC-AH-PEPTH heavy chainEVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGG FPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPGSVKKKKIKAEIKIG APNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISK PKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 125 13D3_Ab_HC-AH-PEPTH light chainDVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHV PLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 126 13D3_Ab_HC-C-terminal-cycR9 heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFC ARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVD KKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLP APIERTISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGGGCG*RRRRRRRRRG*C SEQ ID NO: 127 13D3_Ab_HC-C-terminal-cycR9 light chainDVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDTLKISRVETEDLGVYFCSQSLH VPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 128 13D3_Ab_HC-C terminal-cycTAT1 modified heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTA MYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST KVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKD LPAPIERTISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGGGCG*GRKKRRQRRRPQG*C SEQ ID NO: 129 13D3_Ab_HC-C-terminal-cycTAT1 modified light chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQS LHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 130 13D3_Ab_HC-C-terminal-TAT heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCA RQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDK KIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLP APIERTISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGRKKRRQRRRPPQ SEQ ID NO: 131 13D3_Ab_HC-C-terminal-TAT light chainDVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHV PLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 132 13D3_Ab_LC-C-terminal-Penetain heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAM YFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTK VDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDL PAPIERTISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 133 13D3_Ab_LC-C-terminal-Penetain light chainDVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQS LHVPLTFGAGTKLELKADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSGGGGSRQIKIWFQNRRMKWKKG* SEQ ID NO: 134 13D3_Ab_LC-C-terminal-L17E-M-Lycotoxin heavy chainEVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLM SEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPA SSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNK DLPAPIERTISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 135 13D3_Ab_LC-C-terminal-L17E-M-Lycotoxin light chainDVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYF CSQSLHVPLTFGAGTKLELKADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSGGGGSIWLTALKFLGKHAAKHEAKQQLSKL SEQ ID NO: 136 13D3_Ab_LC-C terminus-Pepth heavy chainEVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFC ARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVD KKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLP APIERTISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 137 13D3_Ab_LC-C terminus-Pepth light chainDVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDTLKISRVETEDLGVYFCSQSLH VPLTFGAGTKLELKADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSGGGGSVKKKKIKAEIKIG* SEQ ID NO: 138 13D3_Ab_HC-C-terminal-TAT3 heavy chainEVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFC ARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVD KKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLP APIERTISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGGGGSGGGGSGRKKRRQRRRPQGRKKRRQRRRPQGRKKRRQRRRPQG*G* SEQ ID NO: 139 13D3_Ab_HC-C-terminal-TAT3 light chainDVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDTLKISRVETEDLGVYFCSQSLHV PLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 140 13D3_Ab_LC-C-terminal_TAT3 heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFC ARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVD KKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLP APIERTISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 141 13D3_Ab_LC-C-terminal_TAT3 light chainDVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDTLKISRVETEDLGVYFCSQSLHV PLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSGGGGSGRKKRRQRRRPQGRKKRRQRRRPQGRKKRRQRRRPQG*G* SEQ ID NO: 142 13D3_Ab_LC-C-terminal_cycTAT3 heavy chainEVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMY FCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKV DKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDL PAPIERTISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 143 13D3_Ab_LC-C-terminal_cycTAT3 light chainDVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDTLKISRVETEDLGVYFCSQSL HVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSGGGGSCG*G*GRKKRRQRRRPQGRKKRRQRRRPQGRKKRRQRRRPQG*G*C SEQ ID NO: 144 13D3_Ab_LC-C-terminal_cycR8x3 heavy chainEVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMY FCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKV DKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDL PAPIERTISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGGGGSGGGGSCG*G*G*RRRRRRRRRG*G*G*RRRRRRRRRG*G*G*RRRRRRRRRG*G*G*C SEQ ID NO: 145 13D3_Ab_LC-C-terminal_cycR8x3 light chainDVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDTLKISRVETEDLGVYFCSQSL HVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 146 13D3_Ab_HC-C-terminal_cycTAT3 heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMY FCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKV DKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDL PAPIERTISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGGGGSGGGGSCG*G*G*GRKKRRQRRRPQGRKKRRQRRRPQGRKKRRQRRRPQG*G*G*C SEQ ID NO: 147 13D3_Ab_HC-C-terminal_cycTAT3 light chainDVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDTLKISRVETEDLGVYFCSQSL HVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 148 13D3_Ab_LC-C-terminal_R8x3 heavy chainEVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFC ARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVD KKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLP APIERTISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 149 13D3_Ab_LC-C-terminal_R8x3 light chainDVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDTLKISRVETEDLGVYFCSQSLHV PLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSRRRRRRRRG*G*G*G*S*RRRRRRRRG*G*G*G*S*RRRRRRRRRG*G* SEQ ID NO: 150 13D3_Ab_HC-C-terminal_R6H4 heavy chainEVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFC ARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVD KKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLP APIERTISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGGGGSGGGGSRRRRRRHHHHSEQ ID NO: 151 13D3_Ab_HC-C-terminal_R6H4 light chainDVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDTLKISRVETEDLGVYFCSQSLHV PLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 152 13D3_Ab_HC-C-terminal_TAT-H4 heavy chainEVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYF CARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKV DKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDL PAPIERTISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGGGGSGRKKRRQRRRPHHHH SEQ ID NO: 153 13D3_Ab_HC-C-terminal_TAT-H4 light chainDVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSgalDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 154 h13D3H1bL1b_Ab_LC-C-terminal_CMIP4 heavy chain EVELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRFTITRDNSSTLYMELSSLRSED TAVYFCARQTYYSYGGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 155 h13D3H1bL1b_Ab_LC-C-terminal_CMIP4 light chainDVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTDFTLTISSLEPEDFAVYFCSQ SLHVPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGSGGGSIWLTALKFSGKAAAKAEAKQFLSKL SEQ ID NO: 156 h13D3H1bL2b_Ab_LC-C-terminal_CMIP4 heavy chain EVELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRFTITRDNSSTLYMELSSLRSED TAVYFCARQTYYSYGGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 157 h13D3H1bL2b_Ab_LC-C-terminal_CMIP4 light chainDVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTEFTLTISSLEPEDFAVYFCSQ SLHVPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGSGGGSIWLTALKFSGKAAAKAEAKQFLSKL SEQ ID NO: 158 h13D3H2bL1b_Ab_LC-C-terminal_CMIP4 heavy chain EVELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRFTITKDTSTSTLYMELSSLRSED TAVYFCARQTYYSYGGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 159 h13D3H2bL1b_Ab_LC-C-terminal_CMIP4 light chainDVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTDFTLTISSLEPEDFAVYFCSQ SLHVPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGSGGGSIWLTALKFSGKAAAKAEAKQFLSKL SEQ ID NO: 160 h13D3H2bL2b_Ab_LC-C-terminal_CMIP4 heavy chain EVELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRFTITKDTSTSTLYMELSSLRSED TAVYFCARQTYYSYGGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 161 h13D3H2bL2b_Ab_LC-C-terminal_CMIP4 light chainDVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTEFTLTISSLEPEDFAVYFCSQ SLHVPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGSGGGSIWLTALKFSGKAAAKAEAKQFLSKL SEQ ID NO: 162 h13D3H3bL1b_ Ab_LC-C-terminal_CMIP4 heavy chain EVELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRGTITKDTSTSTLYMELSSLRSEDTAVYFCARQ TYYSYGGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 163 h13D3H3bL1b_ Ab_LC-C-terminal_CMIP4 light chainDVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTDFTLTISSLEPEDFAVYFCSQSLHVPL TFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGSGGGSIWLTALKFSGKAAAKAEAKQFLSKL SEQ ID NO: 164 h13D3H3bL2b_ Ab_LC-C-terminal_CMIP4 heavy chain EVELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRGTITKDTSTSTLYMELSSLRSEDTAVYFCARQ TYYSYGGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 165 h13D3H3bL2b_ Ab_LC-C-terminal_CMIP4 light chainDVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTEFTLTISSLEPEDFAVYFCSQSLHVPL TFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGSGGGSIWLTALKFSGKAAAKAEAKQFLSKL SEQ ID NO: 166 m13D3_ Ab_LC-C-terminal_CMIP1 heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAV LQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLT CMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 167 m13D3_ Ab_LC-C-terminal_CMIP1 light chainDVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPL TFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSGGGGSIWLTALKFLGKAAAKAEAKQQLSKL SEQ ID NO: 168 m13D3_ Ab_LC-C-terminal_CMIP2 heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQ TYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKK IEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPA PIERTISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 169 m13D3_ Ab_LC-C-terminal_CMIP2 light chainDVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPL TFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSGGGGSIWLTALKFSGKAAAKAEAKQQLSKL SEQ ID NO: 170 m13D3_ Ab_LC-C-terminal_CMIP3 heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQ TYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKK IEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPA PIERTISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 171 m13D3_ Ab_LC-C-terminal_CMIP3 light chainDVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPL TFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSGGGGSIWLTASKFSGKAAAKAEAKQQLSKL SEQ ID NO: 172 m13D3_ Ab_LC-C-terminal_CMIP4 heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQ TYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKK IEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPA PIERTISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 173 m13D3_ Ab_LC-C-terminal_CMIP4 light chainDVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPL TFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSGGGGSIWLTALKFSGKAAAKAEAKQFLSKL SEQ ID NO: 174 m13D3_ Ab_LC-C-terminal_CMIP5 heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQ TYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKK IEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPA PIERTISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 175 m13D3_Ab_LC-Cterminus_CMIP5 light chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSGGGGSIWLTALKFSGKAAAKAEAKQWLSKL The following applies to SEQ ID NOs: 116-175. Linker sequences are in bold. Spacers or amino acids following an amino acid residue (e.g., G*) are indicated by an asterisk (*). The internalization module sequence is double-underlined. 176 CIM CMIP4 IWLTALKFSGKAAAKAEAKQFLSKL SEQ ID NO: 180 CIM #5 CMIP5 IWLTALKFSGKAAAKAEAKQWLSKL SEQ ID NO: 181 CIM #6 TAT GRKKRRQRRRPPQ SEQ ID NO: 182 CIM #7Wild type M-lycotoxin IWLTALKFLGKHAAKHEAKQQLSKL SEQ ID NO: 183 CIM #8 L17E_M-lycotoxin IWLTALKFLGKHAAKHEAKQQLSKL SEQ ID NO: 184 CIM #9 PEPTH VKKKKIKAEIKIG SEQ ID NO: 185 CIM #10 cycR9 CG*RRRRRRRRRG*C SEQ ID NO: 186 CIM #11 cycTAT1 CG*GRKKRRQRRRPQG*C SEQ ID NO: 187 CIM #12 Penetain RQIKIWFQNRRMKWKKG* SEQ ID NO: 188 CIM #13 Tat3 GRKKRRQRRRPQGRKKRRQRRRPQGRKKRRQRRRPQG*G* SEQ ID NO: 189 CIM #14 cycTat3 CG*G*GRKKRRQRRRPQGRKKRRQRRRPQGRKKRRQRRRPQG*G*C SEQ ID NO: 190 CIM #15 R8x3 RRRRRRRRG*G*G*G*S*RRRRRRRRRG*G*G*G*S*RRRRRRRRRG*G* SEQ ID NO: 191 CIM #16 cycR8x3 CG*G*G*RRRRRRRRRG*G*G*RRRRRRRRG*G*G*RRRRRRRRRG*G*G*C SEQ ID NO: 192 CIM #17 R6H4 RRRRRRHHHH SEQ ID NO: 193 CIM #18 TatH4 GRKKRRQRRRPHHHHH The following applies to SEQ ID NO: 176-193 SEQ ID NO: 194 Linker sequence #1 GGGGSGGGGS SEQ ID NO: 195 Linker sequence #2 GGGGS SEQ ID NO: 196 Linker sequence #3 GGGGS SEQ ID NO: 197 Linker sequence #4 GS SEQ ID NO: 198 Linker sequence #5 GGG SEQ ID NO: 199 Linker sequence #6 GGGSGGGS SEQ ID NO: 200 Spacer #1 G SEQ ID NO: 201 Spacer #2 GG SEQ ID NO: 202 Spacer #3 GGG SEQ ID NO: 203 Spacer #4 GGGGS
以下圖式圖解說明本揭示內容之特徵及優點之某些實施例。此等實施例不欲以任何方式限制所附申請專利範圍之範疇。圖式中相同之參考符號指示相同之元件。The following drawings illustrate certain embodiments of the features and advantages of the present disclosure. These embodiments are not intended to limit the scope of the appended patent applications in any way. Like reference symbols in the drawings indicate like elements.
圖1顯示鼠類抗體13D3重鏈可變域之經註明之版本。Figure 1 shows an annotated version of the heavy chain variable domain of murine antibody 13D3.
圖2顯示鼠類抗體13D3輕鏈可變域之經註明之版本。Figure 2 shows an annotated version of the light chain variable domain of murine antibody 13D3.
圖3為顯示嵌合13D3抗體及其人源化形式與磷酸化人類TDP-43肽之結合資料的圖。FIG3 is a graph showing binding data of the chimeric 13D3 antibody and its humanized form to phosphorylated human TDP-43 peptide.
圖4為顯示嵌合13D3抗體及其人源化形式與磷酸化人類TDP-43肽之結合資料的圖。FIG4 is a graph showing binding data of the chimeric 13D3 antibody and its humanized form to phosphorylated human TDP-43 peptide.
圖5為顯示13D3抗體之人源化形式hu13D2Hd5Ld2與磷酸化人類TDP-43肽之結合資料的圖。FIG5 is a graph showing binding data of the humanized form of the 13D3 antibody, hu13D2Hd5Ld2, to phosphorylated human TDP-43 peptide.
圖6A至圖6C顯示用13D3抗體染色之額顳葉失智症(「FTD」)腦組織(圖6A及圖6B)及健康腦組織(圖6C)的免疫組織化學圖像。圖6B為圖6A之插圖，顯示13D3抗體與磷酸化人類TDP-43 FTD相關之神經元細胞質聚集體之共定位。Figures 6A to 6C show immunohistochemical images of frontotemporal dementia (FTD) brain tissue (Figures 6A and 6B) and healthy brain tissue (Figure 6C) stained with the 13D3 antibody. Figure 6B is an inset to Figure 6A, showing colocalization of the 13D3 antibody with FTD-associated neuronal cytoplasmic aggregates of phosphorylated human TDP-43.
圖7A至圖7C為來自用13D3抗體染色之TDP-43蛋白病的rNLS8 dox可抑制模型之腦組織的免疫組織化學圖像。資料顯示，13D3抗體結合TDP-43蛋白病之rNLS8 dox可抑制模型中的細胞質聚集體。Figures 7A-7C are immunohistochemical images of brain tissue from an rNLS8-dox-inhibitory model of TDP-43 proteinopathy stained with the 13D3 antibody. The data demonstrate that the 13D3 antibody binds to cytoplasmic aggregates in the rNLS8-dox-inhibitory model of TDP-43 proteinopathy.
圖8A至圖8C顯示用GFP-2a-TDP43或僅用GFP轉染之HEK細胞的結果。圖8A顯示經轉染細胞中pTDP-43、GFP及細胞核之染色。圖8B為顯示細胞計數之圖。圖8C顯示HEK細胞之免疫組織化學圖像，該等圖像顯示抗體13D3、13C13及2D4偵測HEK細胞中過表現之錯誤定位的人類TDP-43之能力。Figures 8A to 8C show the results of HEK cells transfected with GFP-2a-TDP43 or GFP alone. Figure 8A shows staining of pTDP-43, GFP, and nuclei in transfected cells. Figure 8B is a graph showing cell counts. Figure 8C shows immunohistochemistry images of HEK cells, demonstrating the ability of antibodies 13D3, 13C13, and 2D4 to detect overexpressed, mislocalized human TDP-43 in HEK cells.
圖9A至圖9B為顯示與鼠類13D3 (m13D3) CPA一起培育後CPA陽性細胞之百分比(圖9A)及與m13D3 CPA一起培育後各細胞之CPA陽性斑點數目(圖9B)的圖。Figures 9A and 9B are graphs showing the percentage of CPA-positive cells after incubation with mouse 13D3 (m13D3) CPA (Figure 9A) and the number of CPA-positive spots per cell after incubation with m13D3 CPA (Figure 9B).
圖10A至圖10E為顯示用不同m13D3 CPA轉染之HEK細胞之結果的圖，該等結果包括各孔面積之病灶數目(圖10A)、平均聚焦強度(圖10B)、各孔細胞計數(圖10C)、藉由細胞計數正規化之TDP-43病灶數目(圖10D)及平均病灶面積(圖10E)。Figures 10A to 10E are graphs showing the results for HEK cells transfected with different m13D3 CPAs, including the number of foci per well area (Figure 10A), mean focus intensity (Figure 10B), cell counts per well (Figure 10C), the number of TDP-43 foci normalized by cell counts (Figure 10D), and mean foci area (Figure 10E).
圖11A至圖11D為顯示用不同m13D3 CPA轉染之HEK細胞之結果的圖，該等結果包括各孔面積之病灶數目(圖11A)、細胞計數(圖11B)、病灶計數(圖11C)及平均病灶大小(圖11D)。Figures 11A to 11D are graphs showing the results of HEK cells transfected with different m13D3 CPAs, including the number of foci per well area (Figure 11A), cell counts (Figure 11B), foci counts (Figure 11C), and average foci size (Figure 11D).
圖12A至圖12D為顯示用不同m13D3 CPA轉染之HEK細胞之結果的圖，該等結果包括各孔面積之總病灶面積(圖12A)、細胞計數(圖12B)、pTDP-43病灶計數(圖12C)及平均病灶大小(圖12D)。Figures 12A to 12D are graphs showing the results of HEK cells transfected with different m13D3 CPAs, including total lesion area per well (Figure 12A), cell counts (Figure 12B), pTDP-43 lesion counts (Figure 12C), and average lesion size (Figure 12D).
圖13為顯示未轉染HEK細胞(左)或經GFP-2a-TDP43轉染之細胞(右)之細胞死亡百分比的圖。FIG13 is a graph showing the percentage of cell death in untransfected HEK cells (left) or cells transfected with GFP-2a-TDP43 (right).
圖14A至圖14E為顯示HEK細胞中TDP-43之磷酸化細胞質聚集體之內化及共定位的圖。具體而言，該等圖顯示各孔面積之pTDP-43病灶之總和(圖14A)、平均聚焦強度(圖14B)、細胞計數(圖14C)、p-TDP-43病灶數目(圖14D)及平均病灶面積(圖14E)。Figures 14A to 14E are graphs showing the internalization and colocalization of phosphorylated cytoplasmic aggregates of TDP-43 in HEK cells. Specifically, these graphs show the sum of pTDP-43 foci per well area (Figure 14A), mean focus intensity (Figure 14B), cell count (Figure 14C), number of p-TDP-43 foci (Figure 14D), and mean foci area (Figure 14E).
圖15A至圖15D為顯示HEK細胞與m13D3 m-Lycotoxin [L17E] CPA或m13D3 CMIP4 CPA一起培育之結果的圖，該等結果包括總病灶面積(圖15A)、細胞計數(圖15B)、p-TDP-43病灶數目(圖15C)及平均病灶面積(圖15D)。Figures 15A to 15D are graphs showing the results of incubating HEK cells with m13D3 m-Lycotoxin [L17E] CPA or m13D3 CMIP4 CPA, including total lesion area (Figure 15A), cell counts (Figure 15B), number of p-TDP-43 foci (Figure 15C), and mean lesion area (Figure 15D).
圖16A至圖16E為顯示在乙酸鹽緩衝液或PBS中用m13D3 CMIP4 CPA或未標記抗體轉染之HEK細胞之結果的圖，該等結果包括各孔面積之病灶(圖16A)、平均聚焦強度(圖16B)、細胞計數(圖16C)、p-TDP-43病灶數目(圖16D)及平均病灶面積(圖16E)。Figures 16A to 16E are graphs showing the results for HEK cells transfected with m13D3 CMIP4 CPA or unlabeled antibody in acetate buffer or PBS, including lesion area per well (Figure 16A), mean focus intensity (Figure 16B), cell count (Figure 16C), number of p-TDP-43 foci (Figure 16D), and mean lesion area (Figure 16E).
圖17A至圖17B顯示用GFP-2A-TDP43及嵌合13D3 CPA轉染之HEK細胞之結果，包括共定位成像(圖17A)，及顯示與GFP-2A-TDP43及嵌合13D3 CPA共定位之p-TDP-43之百分比的圖(圖17B)。Figures 17A and 17B show the results of HEK cells transfected with GFP-2A-TDP43 and chimeric 13D3 CPA, including colocalization imaging (Figure 17A) and a graph showing the percentage of p-TDP-43 colocalized with GFP-2A-TDP43 and chimeric 13D3 CPA (Figure 17B).
圖18A至圖18D為顯示抗TDP-43 CPA之內化資料的圖，包括與抗TDP-43 CPA共定位之pTDP-43之百分比(圖18A)、各細胞之13D3抗體斑點之平均數目(圖18B)、平均斑點大小(圖18C)及平均斑點大小(針對斑點強度校正) (圖18D)。Figures 18A to 18D are graphs showing anti-TDP-43 CPA internalization data, including the percentage of pTDP-43 colocalized with anti-TDP-43 CPA (Figure 18A), the average number of 13D3 antibody spots per cell (Figure 18B), the average spot size (Figure 18C), and the average spot size (corrected for spot intensity) (Figure 18D).
圖19A至圖19D為顯示pTDP-43之磷酸化細胞質聚集體之內化及共定位的圖，包括與人源化13D3抗體、嵌合13D3抗體及對照共定位之pTDP-43的百分比(圖19A)、細胞計數(圖19B)、各孔斑點面積(圖19C)及各細胞之CPA斑點(圖19D)。Figures 19A to 19D are graphs showing the internalization and colocalization of phosphorylated cytoplasmic aggregates of pTDP-43, including the percentage of pTDP-43 colocalized with the humanized 13D3 antibody, chimeric 13D3 antibody, and control (Figure 19A), cell counts (Figure 19B), spot area per well (Figure 19C), and CPA spots per cell (Figure 19D).
圖20A至圖20C為顯示神經膠質母細胞瘤細胞中TPD-43之磷酸化細胞質聚集體之內化及共定位的圖，包括總病灶面積(圖20A)、平均病灶大小(圖20B)及總病灶計數(圖20C)。20A to 20C are graphs showing the internalization and colocalization of phosphorylated cytoplasmic aggregates of TPD-43 in glioblastoma cells, including total lesion area ( FIG. 20A ), average lesion size ( FIG. 20B ), and total lesion count ( FIG. 20C ).
圖21A至圖21B顯示TDP-43之磷酸化細胞質聚集體在原代大鼠皮質神經元細胞中之內化及共定位。圖21A顯示用各種CPA處理之原代大鼠皮質神經元細胞的圖像。圖21B為顯示各孔之總病灶面積的圖。Figures 21A and 21B show the internalization and colocalization of phosphorylated TDP-43 cytoplasmic aggregates in primary rat cortical neurons. Figure 21A shows images of primary rat cortical neurons treated with various CPAs. Figure 21B is a graph showing the total lesion area in each well.
圖22A至圖22F為顯示經轉染之原代大鼠皮質神經元細胞之結果的圖，該等結果包括各細胞之13D3斑點之總數(圖22A)、各孔之13D3斑點之總面積(圖22B)、平均斑點大小(圖22C)、斑點積分強度(圖22D)、與早期胞內體抗原1 (EEA1)共定位之13D3之百分比(圖22E)及細胞計數(圖22F)。Figures 22A to 22F are graphs showing the results for transfected primary rat cortical neurons, including the total number of 13D3 puncta per cell (Figure 22A), the total area of 13D3 puncta per well (Figure 22B), the average puncta size (Figure 22C), the integrated puncta intensity (Figure 22D), the percentage of 13D3 colocalized with early endosomal antigen 1 (EEA1) (Figure 22E), and the cell count (Figure 22F).
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5208036A (en) | 1985-01-07 | 1993-05-04 | Syntex (U.S.A.) Inc. | N-(ω, (ω-1)-dialkyloxy)- and N-(ω, (ω-1)-dialkenyloxy)-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor |
| EP0832981A1 (en) | 1987-02-17 | 1998-04-01 | Pharming B.V. | DNA sequences to target proteins to the mammary gland for efficient secretion |
| EP0307434B2 (en) | 1987-03-18 | 1998-07-29 | Scotgen Biopharmaceuticals, Inc. | Altered antibodies |
| US5633076A (en) | 1989-12-01 | 1997-05-27 | Pharming Bv | Method of producing a transgenic bovine or transgenic bovine embryo |
| US5279833A (en) | 1990-04-04 | 1994-01-18 | Yale University | Liposomal transfection of nucleic acids into animal cells |
| US5264618A (en) | 1990-04-19 | 1993-11-23 | Vical, Inc. | Cationic lipids for intracellular delivery of biologically active molecules |
| US5283185A (en) | 1991-08-28 | 1994-02-01 | University Of Tennessee Research Corporation | Method for delivering nucleic acids into cells |
| AU6014094A (en) | 1992-12-02 | 1994-06-22 | Baylor College Of Medicine | Episomal vectors for gene therapy |
| US5827690A (en) | 1993-12-20 | 1998-10-27 | Genzyme Transgenics Corporatiion | Transgenic production of antibodies in milk |
| US5505947A (en) | 1994-05-27 | 1996-04-09 | The University Of North Carolina At Chapel Hill | Attenuating mutations in Venezuelan Equine Encephalitis virus |
| US5786464C1 (en) | 1994-09-19 | 2012-04-24 | Gen Hospital Corp | Overexpression of mammalian and viral proteins |
| US7153510B1 (en) | 1995-05-04 | 2006-12-26 | Yale University | Recombinant vesiculoviruses and their uses |
| DE19539493A1 (en) | 1995-10-24 | 1997-04-30 | Thomae Gmbh Dr K | Strong homologous promoter from hamster |
| US5834597A (en) | 1996-05-20 | 1998-11-10 | Protein Design Labs, Inc. | Mutated nonactivating IgG2 domains and anti CD3 antibodies incorporating the same |
| US6114148C1 (en) | 1996-09-20 | 2012-05-01 | Gen Hospital Corp | High level expression of proteins |
| US5888809A (en) | 1997-05-01 | 1999-03-30 | Icos Corporation | Hamster EF-1α transcriptional regulatory DNA |
| MY138084A (en) | 1999-02-05 | 2009-04-30 | Samsung Electronics Co Ltd | Method for image texture describing |
| PE20020574A1 (en) | 2000-12-06 | 2002-07-02 | Wyeth Corp | HUMANIZED ANTIBODIES THAT RECOGNIZE THE AMYLOID PEPTIDE BETA |
| ES2727425T3 (en) | 2000-12-12 | 2019-10-16 | Medimmune Llc | Molecules with prolonged half-lives, compositions and uses thereof |
| DE60220719T2 (en) | 2001-12-28 | 2008-03-06 | Amgen Fremont Inc. | ANTIBODIES TO THE MUC18 ANTIGEN |
| EP1567652B1 (en) | 2002-11-29 | 2007-08-22 | BOEHRINGER INGELHEIM PHARMA GMBH & CO. KG | Novel neomycin phosphotransferase genes and method for the selection of high-producing recombinant cells |
| DE10338531A1 (en) | 2003-08-19 | 2005-04-07 | Boehringer Ingelheim Pharma Gmbh & Co. Kg | Method for recloning production cells |
| PL1820022T3 (en) | 2004-11-10 | 2009-11-30 | Boehringer Ingelheim Pharma | Application of flow cytometric analysis in optimization of strategies for Chinese hamster ovary cell banks |
| US20080124760A1 (en) | 2006-07-26 | 2008-05-29 | Barbara Enenkel | Regulatory Nucleic Acid Elements |
| US8455622B2 (en) | 2006-12-01 | 2013-06-04 | Seattle Genetics, Inc. | Variant target binding agents and uses thereof |
| PL2126093T3 (en) | 2007-03-02 | 2013-03-29 | Boehringer Ingelheim Pharma | Improvement of protein production |
| EP2031064A1 (en) | 2007-08-29 | 2009-03-04 | Boehringer Ingelheim Pharma GmbH & Co. KG | Method for increasing protein titres |
| US20220313618A1 (en)* | 2019-12-20 | 2022-10-06 | Case Western Reserve University | Plant viral nucleic acid delivery particles and uses thereof |
| KR20230025659A (en)* | 2020-04-28 | 2023-02-22 | 솔라 바이오사이언시즈 엘엘씨 | Compositions and methods for the treatment of TDP-43 proteinopathy |
| US20230174630A1 (en)* | 2020-04-29 | 2023-06-08 | The University Of British Columbia | Single chain antibodies and intrabodies to misfolded tdp-43 and methods of use |
| EP4305058A2 (en)* | 2021-03-10 | 2024-01-17 | Mabylon AG | Antibodies against tdp-43 and methods of using the same |
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|---|---|
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| Publication | Publication Date | Title |
|---|---|---|
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