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TW202517685A - Anti-transferrin receptor antibodies and uses thereof - Google Patents

Anti-transferrin receptor antibodies and uses thereof Download PDF

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TW202517685A
TW202517685A TW113140918A TW113140918A TW202517685A TW 202517685 A TW202517685 A TW 202517685A TW 113140918 A TW113140918 A TW 113140918A TW 113140918 A TW113140918 A TW 113140918A TW 202517685 A TW202517685 A TW 202517685A
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seq
amino acid
acid sequence
antibody
cdr2
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班傑明 Ａ 史密斯
喬瑟夫 瓦特 艾爾德特
馬克 克里斯多福 朱利安
艾登 肯 寇克索
凱特林 布萊亞納 密克斯
湯瑪斯 Ｏ 卡麥隆
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美商百健Ｍａ公司
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Abstract

The present disclosure provides anti-transferrin receptor antibodies, compositions comprising the same and methods of use for delivery of cargo to brain tissue. This disclosure also provides polynucleotides and vectors encoding the anti-transferrin receptor antibodies and cells comprising the same, methods of making the antibodies, and molecules comprising the antibodies.

Description

抗運鐵蛋白受體抗體及其用途Anti-transferrin receptor antibody and its use

本揭示案係關於抗運鐵蛋白受體抗體、包含該等抗體之組合物及用於將治療性貨物遞送至腦組織之使用方法。本揭示案亦提供編碼抗運鐵蛋白受體抗體之相關多核苷酸及載體以及包含該等多核苷酸及載體之細胞。The present disclosure relates to anti-transferrin receptor antibodies, compositions comprising the antibodies, and methods of use for delivering therapeutic cargo to brain tissue. The present disclosure also provides related polynucleotides and vectors encoding anti-transferrin receptor antibodies and cells comprising the polynucleotides and vectors.

將藥物遞送至中樞神經系統一直係治療神經疾病諸如阿茲海默氏病(Alzheimer's disease)及帕金森氏病(Parkinson's disease)的挑戰。對於到達大腦之藥物，其首先必須穿透血腦障壁，由於血腦障壁之選擇性，此係重大挑戰。血腦障壁充當半透膜，防止大多數分子自血液進入神經系統，且僅允許低分子量(＜400 Da)及親脂性化合物通過。大多數小分子及大分子，諸如單株抗體及反義寡核苷酸，不能穿過此障壁。由於藥物透過血腦障壁之此過程具有挑戰性，一小部分用於神經疾病之治療劑使其進入臨床試驗。Delivering drugs to the central nervous system has been a challenge in treating neurological diseases such as Alzheimer's disease and Parkinson's disease. For drugs to reach the brain, they must first penetrate the blood-brain barrier, which is a significant challenge due to the selectivity of the blood-brain barrier. The blood-brain barrier acts as a semipermeable membrane, preventing most molecules from entering the nervous system from the blood, and only allows low molecular weight (<400 Da) and lipophilic compounds to pass through. Most small molecules and large molecules, such as monoclonal antibodies and antisense oligonucleotides, cannot cross this barrier. Due to the challenging process of drug penetration through the blood-brain barrier, a small number of therapeutics for neurological diseases have made it into clinical trials.

此項技術中需要用於將治療劑遞送至中樞神經系統之改良組合物及方法。What is needed in the art are improved compositions and methods for delivering therapeutic agents to the central nervous system.

本揭示案係關於抗運鐵蛋白受體抗體以及其用於將貨物遞送至腦組織及治療神經病症之使用方法。The present disclosure relates to anti-transferrin receptor antibodies and methods of using the same for delivering cargo to brain tissue and treating neurological disorders.

本文提供結合至人類運鐵蛋白受體之抗體，其包含有包含VH互補決定區(CDR)1、VH CDR2及VH CDR3之重鏈可變區(VH)；及包含VL CDR1、VL CDR2及VL CDR3之輕鏈可變區(VL)，其中：VH CDR1包含胺基酸序列GFTFSSYX 1MN (SEQ ID NO:18)或胺基酸序列SYX 1MN(SEQ ID NO:26)，其中X 1為S或A；VH CDR2包含胺基酸序列SISX 2SSSX 3IYYADSVKG (SEQ ID NO:19)，其中X 2為S或A，且其中X 3為Y或S；且VH CDR3包含胺基酸序列KX 4X 5X 6GDFDY (SEQ ID NO:20)，其中X 4為Y或S，其中X 5為R或S，且其中X 6為A或Y；VL CDR1包含胺基酸序列RASQSVSSX 7X 8LA (SEQ ID NO:21)，其中X 7為S或N，且其中X 8為Y或N；VL CDR2包含胺基酸序列GASX 9RAT (SEQ ID NO:22)，其中X 9為N或S；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)。在一些情況下，VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及/或VL CDR3中之至少一者選自表1中所描繪之突變體CDR，且未選自表1中所描繪之突變體CDR之VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2或VL CDR3中之任一者選自表1中所描繪之親代CDR。在一些情況下，VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2或VL CDR3中之一者選自表1中所描繪之突變體CDR且VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及/或VL CDR3中之五者選自表1中所描繪之親代CDR。在一些情況下，VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及/或VL CDR3中之兩者選自表1中所描繪之突變體CDR且VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及/或VL CDR3中之四者選自表1中所描繪之親代CDR。在一些情況下，VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及/或VL CDR3中之三者選自表1中所描繪之突變體CDR且VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及/或VL CDR3中之三者選自表1中所描繪之親代CDR。在一些情況下，VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及/或VL CDR3中之四者選自表1中所描繪之突變體CDR且VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及/或VL CDR3中之兩者選自表1中所描繪之親代CDR。在一些情況下，VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及/或VL CDR3中之五者選自表1中所描繪之突變體CDR且VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2或VL CDR3中之一者選自表1中所描繪之親代CDR。 Provided herein are antibodies that bind to a human transferrin receptor, comprising a heavy chain variable region (VH) comprising a VH complement determining region (CDR) 1, a VH CDR2, and a VH CDR3; and a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein: VH CDR1 comprises the amino acid sequence GFTFSSYX 1 MN (SEQ ID NO: 18) or the amino acid sequence SYX 1 MN (SEQ ID NO: 26), wherein X 1 is S or A; VH CDR2 comprises the amino acid sequence SISX 2 SSSX 3 IYYADSVKG (SEQ ID NO: 19), wherein X 2 is S or A, and wherein X 3 is Y or S; and VH CDR3 comprises the amino acid sequence KX 4 X 5 X 6 GDFDY (SEQ ID NO: 20), wherein X 4 is S or A; wherein X4 is Y or S, wherein X5 is R or S, and wherein X6 is A or Y; VL CDR1 comprises the amino acid sequence RASQSVSSX 7 X 8 LA (SEQ ID NO: 21), wherein X7 is S or N, and wherein X8 is Y or N; VL CDR2 comprises the amino acid sequence GASX 9 RAT (SEQ ID NO: 22), wherein X9 is N or S; and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO: 8). In some cases, at least one of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 is selected from the mutant CDRs described in Table 1, and any of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, or VL CDR3 that is not selected from the mutant CDRs described in Table 1 is selected from the parent CDRs described in Table 1. In some cases, one of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, or VL CDR3 is selected from the mutant CDRs described in Table 1 and five of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 are selected from the parent CDRs described in Table 1. In some cases, two of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3 are selected from the mutant CDRs described in Table 1 and four of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3 are selected from the parent CDRs described in Table 1. In some cases, three of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3 are selected from the mutant CDRs described in Table 1 and three of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3 are selected from the parent CDRs described in Table 1. In some cases, four of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 are selected from the mutant CDRs described in Table 1 and two of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 are selected from the parent CDRs described in Table 1. In some cases, five of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 are selected from the mutant CDRs described in Table 1 and one of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, or VL CDR3 is selected from the parent CDRs described in Table 1.

在一些情況下，VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)；或 VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)。 In some cases, VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); or VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8).

在一些情況下，VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)，其中VL包含Y57S保守取代； VH CDR1包含胺基酸序列GFTFSSYAMN (SEQ ID NO:9)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)，其中VL包含Y57S保守取代； VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSNYLA (SEQ ID NO:15)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISSSSSSIYYADSVKG (SEQ ID NO:11)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KSRAGDFDY (SEQ ID NO:12)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYSAGDFDY (SEQ ID NO:13)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRYGDFDY (SEQ ID NO:14)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISASSSSIYYADSVKG (SEQ ID NO:25)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；VH CDR3包含胺基酸序列KYSAGDFDY (SEQ ID NO:13)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSNYLA (SEQ ID NO:15)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSNLA (SEQ ID NO:16)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASSRAT (SEQ ID NO:17)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)，其中VL包含Y57S保守取代； VH CDR1包含胺基酸序列SYAMN (SEQ ID NO:24)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)，其中VL包含Y57S保守取代； VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSNYLA (SEQ ID NO:15)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISSSSSSIYYADSVKG (SEQ ID NO:11)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KSRAGDFDY (SEQ ID NO:12)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYSAGDFDY (SEQ ID NO:13)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRYGDFDY (SEQ ID NO:14)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISASSSSIYYADSVKG (SEQ ID NO:25)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；VH CDR3包含胺基酸序列KYSAGDFDY (SEQ ID NO:13)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSNYLA (SEQ ID NO:15)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSNLA (SEQ ID NO:16)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)；或 VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASSRAT (SEQ ID NO:17)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)。 In some cases, VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8), wherein VL comprises a Y57S conservative substitution; VH CDR1 comprises the amino acid sequence GFTFSSYAMN (SEQ ID NO:9); VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8), wherein VL comprises the Y57S conservative substitution; VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSNYLA (SEQ ID NO:15); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISSSSSSIYYADSVKG (SEQ ID NO:11); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KSRAGDFDY (SEQ ID NO:12); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYSAGDFDY (SEQ ID NO:13); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRYGDFDY (SEQ ID NO:14); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISASSSSIYYADSVKG (SEQ ID NO:25); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); VH CDR3 comprises the amino acid sequence KYSAGDFDY (SEQ ID NO:13); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSNYLA (SEQ ID NO:15); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSNLA (SEQ ID NO:16); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASSRAT (SEQ ID NO:17); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8), wherein VL comprises the Y57S conservative substitution; VH CDR1 comprises the amino acid sequence SYAMN (SEQ ID NO:24); VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8), wherein VL comprises the Y57S conservative substitution; VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSNYLA (SEQ ID NO:15); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISSSSSSIYYADSVKG (SEQ ID NO:10); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSNYLA (SEQ ID NO:15); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); ID NO:11); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KSRAGDFDY (SEQ ID NO:12); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYSAGDFDY (SEQ ID NO:13); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRYGDFDY (SEQ ID NO:14); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISASSSSIYYADSVKG (SEQ ID NO:25); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); VH CDR3 comprises the amino acid sequence KYSAGDFDY (SEQ ID NO:13); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSNYLA (SEQ ID NO:15); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); NO:8); VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSNLA (SEQ ID NO:16); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); or VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASSRAT (SEQ ID NO:17); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8).

在一些情況下，(i) VH與SEQ ID NO:100-108中之任一者至少80%、85%、90%、95%、99%或100%一致；且(ii) VL與SEQ ID NO:200-204中之任一者至少80%、85%、90%、95%、99%或100%一致。在一些情況下，VH包含SEQ ID NO:100-108中任一者之胺基酸序列且VL包含SEQ ID NO:200-204中任一者之胺基酸序列。In some cases, (i) VH is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 100-108; and (ii) VL is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 200-204. In some cases, VH comprises the amino acid sequence of any one of SEQ ID NOs: 100-108 and VL comprises the amino acid sequence of any one of SEQ ID NOs: 200-204.

在一些情況下，抗體為(a)單價的且對hTfR1具有＞ 10 nM之單價親和力(K D)，或為二價的且對hTfR1具有＞100 nM之單價親和力(K D)，及/或(b)具有＞= 0.01/s之解離速率(k d)。 In some cases, the antibody is (a) monovalent and has a monovalent affinity ( KD ) >10 nM for hTfR1, or is bivalent and has a monovalent affinity ( KD ) >100 nM for hTfR1, and/or (b) has an off-rate ( kd ) >= 0.01/s.

在一些情況下，VH包含SEQ ID NO:100之胺基酸序列且VL包含SEQ ID NO:200之胺基酸序列。In some instances, VH comprises the amino acid sequence of SEQ ID NO: 100 and VL comprises the amino acid sequence of SEQ ID NO: 200.

在一些情況下，VH包含SEQ ID NO: 101之胺基酸序列且VL包含SEQ ID NO: 200之胺基酸序列；VH包含SEQ ID NO: 102之胺基酸序列且VL包含SEQ ID NO: 200之胺基酸序列；VH包含SEQ ID NO: 102之胺基酸序列且VL包含SEQ ID NO: 201之胺基酸序列；VH包含SEQ ID NO: 102之胺基酸序列且VL包含SEQ ID NO: 203之胺基酸序列；VH包含SEQ ID NO: 103之胺基酸序列且VL包含SEQ ID NO: 200之胺基酸序列；VH包含SEQ ID NO: 104之胺基酸序列且VL包含SEQ ID NO: 200之胺基酸序列；VH包含SEQ ID NO: 105之胺基酸序列且VL包含SEQ ID NO: 200之胺基酸序列；VH包含SEQ ID NO: 106之胺基酸序列且VL包含SEQ ID NO: 200之胺基酸序列；VH包含SEQ ID NO: 107之胺基酸序列且VL包含SEQ ID NO: 200之胺基酸序列；VH包含SEQ ID NO: 108之胺基酸序列且VL包含SEQ ID NO: 200之胺基酸序列；VH包含SEQ ID NO: 100之胺基酸序列且VL包含SEQ ID NO: 201之胺基酸序列；VH包含SEQ ID NO: 100之胺基酸序列且VL包含SEQ ID NO: 202之胺基酸序列；VH包含SEQ ID NO: 100之胺基酸序列且VL包含SEQ ID NO: 203之胺基酸序列；或VH包含SEQ ID NO: 100之胺基酸序列且VL包含SEQ ID NO: 204之胺基酸序列。In some cases, VH comprises the amino acid sequence of SEQ ID NO: 101 and VL comprises the amino acid sequence of SEQ ID NO: 200; VH comprises the amino acid sequence of SEQ ID NO: 102 and VL comprises the amino acid sequence of SEQ ID NO: 200; VH comprises the amino acid sequence of SEQ ID NO: 102 and VL comprises the amino acid sequence of SEQ ID NO: 201; VH comprises the amino acid sequence of SEQ ID NO: 102 and VL comprises the amino acid sequence of SEQ ID NO: 203; VH comprises the amino acid sequence of SEQ ID NO: 103 and VL comprises the amino acid sequence of SEQ ID NO: 200; VH comprises the amino acid sequence of SEQ ID NO: 104 and VL comprises the amino acid sequence of SEQ ID NO: 200; VH comprises the amino acid sequence of SEQ ID NO: 105 and VL comprises the amino acid sequence of SEQ ID NO: 200; VH comprises the amino acid sequence of SEQ ID NO: 106 and VL comprises the amino acid sequence of SEQ ID NO: 200; VH comprises the amino acid sequence of SEQ ID NO: 107 and VL comprises the amino acid sequence of SEQ ID NO: 200; VH comprises the amino acid sequence of SEQ ID NO: 108 and VL comprises the amino acid sequence of SEQ ID NO: 200; VH comprises the amino acid sequence of SEQ ID NO: 100 and VL comprises the amino acid sequence of SEQ ID NO: 201; VH comprises the amino acid sequence of SEQ ID NO: 100 and VL comprises the amino acid sequence of SEQ ID NO: 202; VH comprises the amino acid sequence of SEQ ID NO: 100 and VL comprises the amino acid sequence of SEQ ID NO: 203; or VH comprises the amino acid sequence of SEQ ID NO: 100 and VL comprises the amino acid sequence of SEQ ID NO: 204 amino acid sequence.

在一些情況下，抗體為多特異性抗體、雙特異性抗體、單鏈抗體、Fab片段、F(ab') 2片段、Fab'片段、Fsc片段、Fv片段、scFv、sc(Fv) 2或雙價抗體。 In some cases, the antibody is a multispecific antibody, a bispecific antibody, a single chain antibody, a Fab fragment, a F(ab') 2 fragment, a Fab' fragment, a Fsc fragment, a Fv fragment, scFv, sc(Fv) 2 , or a bivalent antibody.

在一些情況下，抗體包含恆定重鏈(CH)域及恆定輕鏈(CL)域。In some cases, the antibody comprises a constant heavy chain (CH) domain and a constant light chain (CL) domain.

在一些情況下，抗體包含重鏈及輕鏈，其中重鏈包含SEQ ID NO:400中所列出之胺基酸序列或由其組成，且輕鏈包含SEQ ID NO:401中所列出之胺基酸序列或由其組成。In some cases, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:400, and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:401.

在一些情況下，抗體包含Fab片段。在一些情況下，抗體包含Fab片段，其中Fab片段包含SEQ ID NO:401及402中所列出之胺基酸序列。In some cases, the antibody comprises a Fab fragment. In some cases, the antibody comprises a Fab fragment, wherein the Fab fragment comprises the amino acid sequence set forth in SEQ ID NO: 401 and 402.

在一些情況下，抗體包含Fab-Fc。在一些情況下，抗體包含Fab-Fc，其中Fab-Fc包含SEQ ID NO:401、404及406中所列出之胺基酸序列。在一些情況下，抗體包含Fab-Fc，其中Fab-Fc包含SEQ ID NO: 400及401中所列出之胺基酸序列。In some cases, the antibody comprises a Fab-Fc. In some cases, the antibody comprises a Fab-Fc, wherein the Fab-Fc comprises the amino acid sequence listed in SEQ ID NOs: 401, 404, and 406. In some cases, the antibody comprises a Fab-Fc, wherein the Fab-Fc comprises the amino acid sequence listed in SEQ ID NOs: 400 and 401.

在一些情況下，抗體包含Fc-Fab。在一些情況下，抗體包含Fc-Fab，其中Fc-Fab包含SEQ ID NO:401、405及406中所列出之胺基酸序列。在一些情況下，抗體包含Fc-Fab，其中Fc-Fab包含SEQ ID NO:401及403中所列出之胺基酸序列。In some cases, the antibody comprises an Fc-Fab. In some cases, the antibody comprises an Fc-Fab, wherein the Fc-Fab comprises the amino acid sequence listed in SEQ ID NOs: 401, 405, and 406. In some cases, the antibody comprises an Fc-Fab, wherein the Fc-Fab comprises the amino acid sequence listed in SEQ ID NOs: 401 and 403.

在一些情況下，抗體包含：(a)包含400中所列出之胺基酸序列之重鏈，及包含401中所列出之胺基酸序列之輕鏈；(b) SEQ ID NO: 401及402中所列出之胺基酸序列；(c) SEQ ID NO: 401、404及406中所列出之胺基酸序列；(d) SEQ ID NO:400及401中所列出之胺基酸序列；(e) SEQ ID NO: 401、405及406中所列出之胺基酸序列；或(f) SEQ ID NO: 401及403中所列出之胺基酸序列。In some cases, the antibody comprises: (a) a heavy chain comprising the amino acid sequence listed in 400, and a light chain comprising the amino acid sequence listed in 401; (b) the amino acid sequence listed in SEQ ID NOs: 401 and 402; (c) the amino acid sequence listed in SEQ ID NOs: 401, 404, and 406; (d) the amino acid sequence listed in SEQ ID NOs: 400 and 401; (e) the amino acid sequence listed in SEQ ID NOs: 401, 405, and 406; or (f) the amino acid sequence listed in SEQ ID NOs: 401 and 403.

本文亦提供編碼前述抗體中之任一者的一種核酸或多種核酸。Also provided herein is a nucleic acid or nucleic acids encoding any of the aforementioned antibodies.

本文亦提供一種表現載體或多種表現載體，其包含可操作地連接至啟動子之前述一種核酸或多種核酸。Also provided herein is an expression vector or a plurality of expression vectors comprising the aforementioned nucleic acid or nucleic acids operably linked to a promoter.

本文亦提供經分離細胞，其包含前述一種核酸或多種核酸或前述一種表現載體或一種表現載體。Also provided herein are isolated cells comprising the aforementioned nucleic acid or nucleic acids or the aforementioned expression vector or expression vector.

本文亦提供經分離細胞，其包含：包含可操作地連接至啟動子之第一核酸之第一表現載體，該第一核酸編碼包含前述抗體中任一者之VH之第一多肽；及包含可操作地連接至啟動子之第二核酸之第二表現載體，該第二核酸編碼包含前述抗體中任一者之VL之第二多肽。Also provided herein is an isolated cell comprising: a first expression vector comprising a first nucleic acid operably linked to a promoter, the first nucleic acid encoding a first polypeptide comprising the VH of any one of the aforementioned antibodies; and a second expression vector comprising a second nucleic acid operably linked to the promoter, the second nucleic acid encoding a second polypeptide comprising the VL of any one of the aforementioned antibodies.

本文亦提供製備前述抗體中之任一者的方法，其包括培養前述細胞及分離抗體。Also provided herein is a method of preparing any of the aforementioned antibodies, comprising culturing the aforementioned cells and isolating the antibody.

本文亦提供醫藥組合物，其包含前述抗體中之任一者及醫藥學上可接受之載劑。Also provided herein is a pharmaceutical composition comprising any of the aforementioned antibodies and a pharmaceutically acceptable carrier.

本文亦提供結合物，其包含前述抗體中之任一者及劑。在一些情況下，該劑為抗體、蛋白質或肽。在一些情況下，該劑為抗β澱粉樣蛋白抗體。在一些情況下，抗β澱粉樣蛋白抗體為阿杜卡單抗(aducanumab)、巴匹珠單抗(bapineuzumab)、甘特魯單抗(gantenerumab)、索拉珠單抗(solanezumab)、多奈單抗(donanemab)或雷卡奈單抗(lecanemab)。在一些情況下，該劑為抗τ抗體、抗α突觸核蛋白抗體、抗TDP-43抗體、抗LINGO-1抗體、抗LINGO-2抗體、抗LINGO-3抗體、抗LINGO-4抗體、抗TREM2抗體或抗C9orf72二肽重複聚GA抗體。在一些情況下，該劑為蛋白質。在一些情況下，蛋白質為顆粒蛋白前體(progranulin)。在一些情況下，該劑為酶。在一些情況下，酶為葡糖腦苷脂酶。在一些情況下，結合物為包含抗體及劑之重組融合蛋白。在一些情況下，該劑為核酸。在一些情況下，核酸為mRNA、siRNA、反義寡核苷酸、微小RNA (miRNA)、嚮導RNA (gRNA)或胺基磷酸酯嗎啉基寡聚物(PMO)。在一些情況下，核酸經由連接子連接至抗體。在一些情況下，該劑為奈米粒子、脂質體或病毒載體。Also provided herein are conjugates comprising any of the aforementioned antibodies and an agent. In some cases, the agent is an antibody, a protein, or a peptide. In some cases, the agent is an anti-β-amyloid antibody. In some cases, the anti-β-amyloid antibody is aducanumab, bapineuzumab, gantenerumab, solanezumab, donanemab, or lecanemab. In some cases, the agent is an anti-tau antibody, an anti-alpha synaptophysin antibody, an anti-TDP-43 antibody, an anti-LINGO-1 antibody, an anti-LINGO-2 antibody, an anti-LINGO-3 antibody, an anti-LINGO-4 antibody, an anti-TREM2 antibody, or an anti-C9orf72 dipeptide repeat poly-GA antibody. In some cases, the agent is a protein. In some cases, the protein is progranulin. In some cases, the agent is an enzyme. In some cases, the enzyme is glucocerebrosidase. In some cases, the conjugate is a recombinant fusion protein comprising an antibody and an agent. In some cases, the agent is a nucleic acid. In some cases, the nucleic acid is mRNA, siRNA, antisense oligonucleotide, microRNA (miRNA), guide RNA (gRNA), or phosphoamidate morpholino oligomer (PMO). In some cases, the nucleic acid is linked to the antibody via a linker. In some cases, the agent is a nanoparticle, a liposome, or a viral vector.

本文亦提供經由胞吞轉送作用跨血腦障壁轉運劑之方法，該方法包含向人類個體投與任一前述結合物。Also provided herein are methods of transporting an agent across the blood-brain barrier via endocytosis, the method comprising administering any of the aforementioned conjugates to a human subject.

本文亦提供活體內遞送劑之方法，該方法包含向人類個體投與任一前述結合物。在一些情況下，人類個體患有神經病症且該方法將劑遞送至腦組織。在一些情況下，神經病症為阿茲海默氏病、帕金森氏病、額顳葉失智症、ALS、杭丁頓氏病(Huntington’s disease)、多發性硬化症、脊髓性肌萎縮、肌肉營養不良、脊髓損傷、中風、眼科疾患、急性或慢性視神經炎、精神異常(psychiatric disorder)、圖雷特氏病(Tourette's disease)腦損傷、腦腫瘤或癲癇。Also provided herein are methods of delivering an agent in vivo, comprising administering any of the aforementioned conjugates to a human subject. In some cases, the human subject suffers from a neurological disorder and the method delivers the agent to brain tissue. In some cases, the neurological disorder is Alzheimer's disease, Parkinson's disease, frontotemporal dementia, ALS, Huntington's disease, multiple sclerosis, spinal muscular atrophy, muscular dystrophy, spinal cord injury, stroke, ophthalmic disease, acute or chronic optic neuritis, psychiatric disorder, Tourette's disease, brain injury, brain tumor, or epilepsy.

本文亦提供治療有需要之人類個體之阿茲海默氏病之方法，其包含向該個體投與治療有效量之任一前述結合物。Also provided herein are methods of treating Alzheimer's disease in a human subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the foregoing conjugates.

本發明之其他特徵及優點自以下詳細描述及自請求項中顯而易見。Other features and advantages of the present invention will be apparent from the following detailed description and from the claims.

相關申請案之交互參照 本申請案主張2023年10月26日申請之美國臨時申請案第63/545,848號及2024年2月2日申請之美國臨時申請案第63/549,055號之權益，該等美國臨時申請案之全部內容特此以引用方式併入本文中。 Cross-reference to related applications This application claims the benefit of U.S. Provisional Application No. 63/545,848 filed on October 26, 2023 and U.S. Provisional Application No. 63/549,055 filed on February 2, 2024, the entire contents of which are hereby incorporated by reference.

本揭示案提供特異性結合運鐵蛋白受體1 (TfR1)之抗體。亦提供包含該等抗體之相關多肽、多核苷酸、載體、細胞、組合物及結合物、製備該等抗體之方法以及遞送該等組合物及結合物之方法。本揭示案亦提供使用抗TfR抗體之方法。 定義 The present disclosure provides antibodies that specifically bind to transferrin receptor 1 (TfR1). Also provided are related polypeptides, polynucleotides, vectors, cells, compositions and conjugates comprising the antibodies, methods for preparing the antibodies, and methods for delivering the compositions and conjugates. The present disclosure also provides methods of using the anti-TfR antibodies. Definition

除非本文另有定義，否則本說明書中所用之技術及科學術語具有熟習此項技術者通常理解之含義。出於解釋本說明書之目的，將適用以下術語描述，且在適當時，以單數形式使用之術語亦將包括複數，反之亦然。若所闡述之術語之任何描述與以引用方式併入本文中之任何文件衝突，則應以下文所闡述之術語之描述為準。Unless otherwise defined herein, the technical and scientific terms used in this specification have the meanings commonly understood by those skilled in the art. For the purpose of interpreting this specification, the following term descriptions will apply, and where appropriate, terms used in the singular will also include the plural, and vice versa. If any description of the term is inconsistent with any document incorporated herein by reference, the description of the term described below shall prevail.

如本文所用，術語「抗體」係指經由至少一個抗原結合位點識別並結合靶標之免疫球蛋白分子、或包含免疫球蛋白分子之片段之分子。「抗體」在本文中以最廣泛意義使用且涵蓋各種抗體結構，包括「抗體片段」及「抗原結合片段」。因此，術語「抗體」包括但不限於重組抗體、單株抗體、嵌合抗體、人類化抗體、人類抗體、雙特異性抗體、多特異性抗體、雙抗體、三抗體、四抗體、單鏈Fv (scFv)抗體、及抗體片段，只要其表現出所需抗原結合活性即可。As used herein, the term "antibody" refers to an immunoglobulin molecule, or a molecule comprising a fragment of an immunoglobulin molecule, that recognizes and binds to a target via at least one antigen binding site. "Antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including "antibody fragments" and "antigen binding fragments." Therefore, the term "antibody" includes but is not limited to recombinant antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, multispecific antibodies, diabodies, triabodies, tetrabodies, single-chain Fv (scFv) antibodies, and antibody fragments, as long as they exhibit the desired antigen binding activity.

術語「完整抗體」或「全長抗體」係指具有實質上類似於天然抗體結構之結構的抗體。此包括例如如下抗體，其包含各自包含可變區及輕鏈恆定區(CL)之兩個輕鏈以及各自包含可變區及至少重鏈恆定區CH1、CH2及CH3及CH1區與CH2區之間的鉸鏈區之兩個重鏈。The term "intact antibody" or "full-length antibody" refers to an antibody having a structure substantially similar to that of a natural antibody. This includes, for example, an antibody comprising two light chains each comprising a variable region and a light chain constant region (CL) and two heavy chains each comprising a variable region and at least heavy chain constant regions CH1, CH2 and CH3 and a hinge region between the CH1 region and the CH2 region.

如本文所用，術語「抗原結合片段」係指除完整抗體以外的包含抗體之一部分及抗原結合位點之分子。抗體片段之實例包括但不限於Fab、Fab'、F(ab') 2、Fv、單鏈抗體分子(例如scFv、sc(Fv) 2,)、二硫鍵連接之scFv (dsscFv)、雙抗體、三抗體、四抗體、微型抗體、雙可變域抗體(DVD)、單可變域抗體(例如駱駝科抗體)及由抗體片段形成之多特異性抗體。 As used herein, the term "antigen-binding fragment" refers to a molecule that includes a portion of an antibody and an antigen-binding site other than an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , Fv, single-chain antibody molecules (e.g., scFv, sc(Fv) 2 ,), disulfide-linked scFv (dsscFv), diabodies, triabodies, tetrabodies, minibodies, dual variable domain antibodies (DVD), single variable domain antibodies (e.g., camelid antibodies), and multispecific antibodies formed from antibody fragments.

如本文所用，術語「單株抗體」係指參與單一抗原決定位或抗原決定基之高度特異性識別及結合的實質上均質之抗體群體。術語「單株抗體」涵蓋完整及全長單株抗體以及抗體片段(例如Fab、Fab'、F(ab') 2、Fv)、單鏈抗體(例如scFv)、包含抗體片段之融合蛋白及包含至少一個抗原結合位點之任何其他經修飾之免疫球蛋白分子。此外，「單株抗體」係指藉由多種技術製備之此類抗體，該等技術包括但不限於融合瘤產生、噬菌體文庫展示、重組表現及基因轉殖動物。 As used herein, the term "monoclonal antibody" refers to a substantially homogeneous group of antibodies that participate in the highly specific recognition and binding of a single antigenic determinant or antigenic determinant. The term "monoclonal antibody" encompasses intact and full-length monoclonal antibodies as well as antibody fragments (e.g., Fab, Fab', F(ab') 2 , Fv), single-chain antibodies (e.g., scFv), fusion proteins comprising antibody fragments, and any other modified immunoglobulin molecules comprising at least one antigen binding site. In addition, "monoclonal antibody" refers to such antibodies prepared by a variety of techniques, including but not limited to hybridoma production, phage library display, recombinant expression, and genetically modified animals.

術語「嵌合抗體」係指重鏈及/或輕鏈之一部分來源於第一來源或物種，而重鏈及/或輕鏈之其餘部分來源於不同來源或物種之抗體。The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and/or light chain is derived from a first source or species, and the remainder of the heavy chain and/or light chain is derived from a different source or species.

如本文所用，術語「人類化抗體」係指包含人類重鏈可變區及輕鏈可變區之抗體，其中天然CDR胺基酸殘基經來自非人類抗體(例如，小鼠、大鼠、兔或非人類靈長類動物)之相應CDR之殘基置換，其中非人類抗體具有所需特異性、親和力及/或活性。在一些實施例中，人類重鏈或輕鏈可變區之一或多個構架區胺基酸殘基經來自非人類抗體之相應殘基置換。此外，人類化抗體可包含在人類抗體或非人類抗體中未發現之胺基酸殘基。在一些實施例中，進行此等修飾以進一步細化及/或最佳化抗體特徵。在一些實施例中，人類化抗體包含免疫球蛋白恆定區(例如CH1、鉸鏈、CH2、CH3、Fc)之至少一部分，通常為人類免疫球蛋白之恆定區。As used herein, the term "humanized antibody" refers to an antibody comprising a human heavy chain variable region and a light chain variable region, wherein the native CDR amino acid residues are replaced with residues from the corresponding CDR of a non-human antibody (e.g., mouse, rat, rabbit, or non-human primate), wherein the non-human antibody has the desired specificity, affinity, and/or activity. In some embodiments, one or more framework region amino acid residues of the human heavy chain or light chain variable region are replaced with corresponding residues from a non-human antibody. In addition, a humanized antibody may contain amino acid residues not found in a human antibody or a non-human antibody. In some embodiments, such modifications are performed to further refine and/or optimize antibody characteristics. In some embodiments, the humanized antibody comprises at least a portion of an immunoglobulin constant region (e.g., CH1, hinge, CH2, CH3, Fc), typically a constant region of a human immunoglobulin.

如本文所用，術語「人類抗體」係指具有對應於由人類產生之抗體之胺基酸序列的抗體，及/或已使用熟習此項技術者已知用於製備人類抗體之技術來製備之抗體。此等技術包括但不限於噬菌體展示文庫、酵母展示文庫、基因轉殖動物、重組蛋白產生及B細胞融合瘤技術。As used herein, the term "human antibody" refers to an antibody having an amino acid sequence corresponding to an antibody produced by humans and/or an antibody that has been prepared using techniques known to those skilled in the art for preparing human antibodies. Such techniques include, but are not limited to, phage display libraries, yeast display libraries, gene-transfected animals, recombinant protein production, and B-cell fusion tumor technology.

術語「抗原決定基」及「抗原決定位」在本文中可互換使用，且係指抗原或靶標中能夠由特定抗體識別及結合之部分。當抗原或靶標為多肽時，抗原決定基可由藉由蛋白質之三級摺疊並置之鄰接胺基酸及非鄰接胺基酸形成。由鄰接胺基酸形成之抗原決定基(亦稱為線性抗原決定基)通常在蛋白質變性後保留，而藉由三級摺疊形成之抗原決定基(亦稱為構形抗原決定基)通常在蛋白質變性後丟失。抗原決定基通常包括呈獨特空間構形之至少3個且更通常至少5個、6個、7個或8-10個胺基酸。抗原決定基可使用可在網際網路上獲得的大量軟體生物資訊工具中之任一者來預測。X射線晶體學或電子顯微術(例如低溫電子顯微術)可用於藉由分析抗原/抗體複合物之胺基酸殘基相互作用來表徵靶蛋白上之抗原決定基。The terms "antigenic determinant" and "antigenic determinant site" are used interchangeably herein and refer to the portion of an antigen or target that is capable of being recognized and bound by a specific antibody. When the antigen or target is a polypeptide, the antigenic determinant may be formed by adjacent amino acids and non-adjacent amino acids juxtaposed by tertiary folding of the protein. Antigenic determinants formed by adjacent amino acids (also called linear antigenic determinants) are generally retained after protein denaturation, while antigenic determinants formed by tertiary folding (also called conformational antigenic determinants) are generally lost after protein denaturation. An antigenic determinant generally includes at least 3, and more generally at least 5, 6, 7, or 8-10 amino acids in a unique spatial conformation. Epitopes can be predicted using any of a large number of software bioinformatics tools available on the Internet. X-ray crystallography or electron microscopy (e.g., cryogenic electron microscopy) can be used to characterize epitopes on the target protein by analyzing the interactions of the amino acid residues of the antigen/antibody complex.

如本文所用，術語「特異性結合」或「結合」係指抗體與特定抗原、抗原決定基、蛋白質或靶分子之相互作用比與替代物質之相互作用更頻繁、更快速、持續時間更長、親和力更大或上述之某種組合。特異性結合抗原之抗體可例如藉由免疫檢定、ELISA、表面電漿子共振(SPR)或熟習此項技術者已知之其他技術來鑑別。在一些實施例中，特異性結合抗原(例如人類TfR1)之抗體可結合相關抗原(例如石蟹猴TfR1)。特異性結合抗原之抗體結合靶抗原之親和力可以高於靶抗原對不同抗原之親和力。不同抗原可為相關抗原。在一些實施例中，特異性結合抗原之抗體結合靶抗原之親和力可以比其對不同抗原之親和力大至少20倍、大至少30倍、大至少40倍、大至少50倍、大至少60倍、大至少70倍、大至少80倍、大至少90倍或大至少100倍。在一些實施例中，特異性結合特定抗原之抗體以如此低之親和力結合不同抗原，使得使用本文所述或此項技術中另外已知之檢定法無法偵測到結合。在一些實施例中，在如本文所述或如熟習此項技術者已知之Biacore系統中使用SPR技術量測親和力。As used herein, the term "specific binding" or "binding" refers to an interaction of an antibody with a specific antigen, antigenic determinant, protein or target molecule that is more frequent, faster, longer lasting, with greater affinity, or a combination of the foregoing than with a surrogate substance. Antibodies that specifically bind to an antigen can be identified, for example, by immunoassay, ELISA, surface plasmon resonance (SPR), or other techniques known to those skilled in the art. In some embodiments, an antibody that specifically binds to an antigen (e.g., human TfR1) can bind to a related antigen (e.g., cynomolgus monkey TfR1). The affinity of an antibody that specifically binds to an antigen to bind to a target antigen can be higher than the affinity of the target antigen to a different antigen. The different antigens can be related antigens. In some embodiments, an antibody that specifically binds an antigen may bind to a target antigen with an affinity that is at least 20 times greater, at least 30 times greater, at least 40 times greater, at least 50 times greater, at least 60 times greater, at least 70 times greater, at least 80 times greater, at least 90 times greater, or at least 100 times greater than its affinity for a different antigen. In some embodiments, an antibody that specifically binds a particular antigen binds to the different antigen with such low affinity that binding is undetectable using an assay described herein or otherwise known in the art. In some embodiments, affinity is measured using SPR technology in a Biacore system as described herein or as known to those skilled in the art.

術語「多肽」及「肽」及「蛋白質」在本文中可互換使用且係指任何長度之胺基酸之聚合物。聚合物可為直鏈或支鏈的，其可包含經修飾之胺基酸，且其可經非胺基酸中斷。該等術語亦涵蓋已經天然修飾或藉由干預來修飾之胺基酸聚合物；例如，二硫鍵形成、糖基化、脂化、乙醯化、磷酸化或任何其他操縱或修飾。該定義亦包括例如含有胺基酸之一或多種類似物之多肽，該等類似物包括但不限於非天然胺基酸以及此項技術中已知之其他修飾。應理解，由於本揭示案之多肽可基於抗體，故術語「多肽」涵蓋呈單鏈之多肽及具有兩個或更多個相關鏈之多肽。The terms "polypeptide" and "peptide" and "protein" are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. The definition also includes, for example, polypeptides containing one or more analogs of an amino acid, including but not limited to non-natural amino acids and other modifications known in the art. It should be understood that because the polypeptides of the present disclosure may be based on antibodies, the term "polypeptide" encompasses polypeptides that are single chains and polypeptides that have two or more related chains.

術語「多核苷酸」及「核酸」及「核酸分子」在本文中可互換使用且係指任何長度之核苷酸之聚合物，且包括DNA及RNA。核苷酸可為去氧核糖核苷酸、核糖核苷酸、經修飾之核苷酸或鹼基及/或其類似物，或可藉由DNA或RNA聚合酶併入聚合物中之任何受質。The terms "polynucleotide" and "nucleic acid" and "nucleic acid molecule" are used interchangeably herein and refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.

在兩個或更多個核酸或多肽之上下文中，術語「一致」或「一致性百分比」係指在不考慮將任何保守胺基酸取代作為序列一致性之一部分的情況下，當比較及比對(若需要，引入空位)以獲得最大對應性時，兩個或更多個序列或子序列相同或具有指定百分比之相同的核苷酸或胺基酸殘基。一致性百分比可使用序列比較軟體或演算法或藉由目視檢查來量測。可用於獲得胺基酸或核苷酸序列之比對之各種演算法及軟體為此項技術中所熟知的。此等包括但不限於BLAST、ALIGN、Megalign、BestFit、GCG Wisconsin Package及其變體。在一些實施例中，本揭示案之兩個核酸或多肽實質上一致，意謂當比較及比對以獲得最大對應性時，其具有至少70%、至少75%、至少80%、至少85%、至少90%，且在一些實施例中至少95%、96%、97%、98%、99%核苷酸或胺基酸殘基一致性，如使用序列比較演算法或藉由目視檢查所量測。在一些實施例中，一致性存在於至少約10個、至少約20個、至少約20-40個、至少約40-60個核苷酸或胺基酸殘基、至少約60-80個核苷酸或胺基酸殘基或其間之任何整數值之長度的序列區域上。在一些實施例中，一致性存在於長於60-80個核苷酸或胺基酸殘基之區域內，諸如至少約80-100個核苷酸或胺基酸殘基，且在一些實施例中，序列在所比較之該等序列例如(i)核苷酸序列之編碼區或(ii)胺基酸序列之全長上為實質上一致的。In the context of two or more nucleic acids or polypeptides, the term "identical" or "percent identity" means that two or more sequences or subsequences are identical or have a specified percentage of identical nucleotides or amino acid residues when compared and aligned (if necessary, introducing gaps) for maximum correspondence, without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignments of amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, two nucleic acids or polypeptides of the present disclosure are substantially identical, meaning that when compared and aligned for maximum correspondence, they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, the identity exists over a sequence region of at least about 10, at least about 20, at least about 20-40, at least about 40-60 nucleotides or amino acid residues, at least about 60-80 nucleotides or amino acid residues, or any integer value therebetween. In some embodiments, the identity exists over a region longer than 60-80 nucleotides or amino acid residues, such as at least about 80-100 nucleotides or amino acid residues, and in some embodiments, the sequences are substantially identical over the entire length of the compared sequences, e.g., (i) the coding region of a nucleotide sequence or (ii) an amino acid sequence.

如本文所用，片語「保守胺基酸取代」係指一個胺基酸殘基經具有類似側鏈之另一胺基酸殘基置換之取代。具有相似側鏈之胺基酸殘基家族已在此項技術中大體上定義，包括鹼性側鏈(例如離胺酸、精胺酸、組胺酸)、酸性側鏈(例如天冬胺酸、麩胺酸)、不帶電荷之極性側鏈(例如，甘胺酸、天冬醯胺、麩醯胺、絲胺酸、蘇胺酸、酪胺酸、半胱胺酸)、非極性側鏈(例如，丙胺酸、纈胺酸、白胺酸、異白胺酸、脯胺酸、苯丙胺酸、甲硫胺酸、色胺酸)、β-支鏈側鏈(例如，蘇胺酸、纈胺酸、異白胺酸)及芳族側鏈(例如酪胺酸、苯丙胺酸、色胺酸、組胺酸)。舉例而言，苯丙胺酸取代酪胺酸被視為保守取代。通常，多肽及/或抗體序列中之保守取代不會消除多肽或抗體與靶結合位點之結合。鑑別不消除結合之核苷酸及胺基酸保守取代之方法為此項技術中所熟知的。As used herein, the phrase "conservative amino acid substitution" refers to a substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyramine), and amino acid residues having similar side chains. The invention relates to a conservative substitution of a polypeptide or antibody sequence. The conservative substitution of a polypeptide or antibody sequence is generally considered to be a conservative substitution ...

如本文所用，術語「載體」意指能夠在宿主細胞中遞送且通常表現一或多種所關注基因或序列之構築體。載體之實例包括但不限於病毒載體、裸DNA或RNA表現載體、質體、黏粒或噬菌體載體、與陽離子縮合劑相關之DNA或RNA表現載體及封裝於脂質體中之DNA或RNA表現載體。As used herein, the term "vector" means a construct capable of delivery and typically expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with a cation condensing agent, and DNA or RNA expression vectors encapsulated in liposomes.

如本文所用，術語「經分離」係指呈自然界中未發現之形式的多肽、可溶性蛋白質、抗體、多核苷酸、載體、細胞或組合物。「經分離」抗體實質上不含來自其所來源之細胞來源之物質。在一些實施例中，經分離之多肽、可溶性蛋白質、抗體、多核苷酸、載體、細胞或組合物為已純化至不再呈其在自然界中發現之形式之程度的彼等。在一些實施例中，經分離之多肽、可溶性蛋白質、抗體、多核苷酸、載體、細胞或組合物為實質上純的。多肽、可溶性蛋白質、抗體、多核苷酸、載體、細胞或組合物可自天然來源(例如組織)或自諸如經工程改造之細胞株之來源分離。As used herein, the term "isolated" refers to a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition that is in a form not found in nature. An "isolated" antibody is substantially free of material from the cell-derived source from which it is derived. In some embodiments, isolated polypeptides, soluble proteins, antibodies, polynucleotides, vectors, cells, or compositions are those that have been purified to the extent that they are no longer in a form found in nature. In some embodiments, isolated polypeptides, soluble proteins, antibodies, polynucleotides, vectors, cells, or compositions are substantially pure. The polypeptide, soluble protein, antibody, polynucleotide, vector, cell or composition can be isolated from a natural source (eg, a tissue) or from a source such as an engineered cell line.

如本文所用，術語「實質上純」係指至少50%純(亦即不含污染物)、至少90%純、至少95%純、至少98%純或至少99%純之材料。As used herein, the term "substantially pure" refers to a material that is at least 50% pure (ie, free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

如本文所用，術語「醫藥學上可接受」係指經監管機構批准或可批准或列於美國藥典、歐洲藥典或其他公認藥典中用於動物(包括人類)之物質。As used herein, the term "pharmaceutically acceptable" refers to a substance that is approved or approvable by a regulatory agency or listed in the U.S. Pharmacopoeia, the European Pharmacopoeia or other generally recognized pharmacopoeia for use in animals (including humans).

如本文所用，術語「醫藥學上可接受之賦形劑、載劑或佐劑」係指可與至少一種本揭示案之抗體一起向個體投與且通常安全，無毒，且對該治療劑之藥理活性無影響的賦形劑、載劑或佐劑。一般而言，熟習此項技術者及U.S. FDA認為醫藥學上可接受之賦形劑、載劑或佐劑係任何調配物之非活性成分。As used herein, the term "pharmaceutically acceptable excipient, carrier or adjuvant" refers to an excipient, carrier or adjuvant that can be administered to a subject together with at least one antibody of the present disclosure and is generally safe, non-toxic, and has no effect on the pharmacological activity of the therapeutic agent. Generally speaking, a pharmaceutically acceptable excipient, carrier or adjuvant is considered by those skilled in the art and the U.S. FDA to be an inactive ingredient of any formulation.

如本文所用，術語「醫藥組合物」係指呈允許抗體之生物活性有效之形式的製劑。醫藥調配物或組合物通常包含額外組分，諸如醫藥學上可接受之賦形劑、載劑、佐劑、緩沖劑等。As used herein, the term "pharmaceutical composition" refers to a preparation in a form that allows the biological activity of the antibody to be effective. Pharmaceutical formulations or compositions usually contain additional components such as pharmaceutically acceptable excipients, carriers, adjuvants, buffers, etc.

如本文所用，術語「結合物」係指其中兩種物質藉由共價鍵連接之組合(例如，本揭示案之抗體連接至治療劑)。在結合物中，兩種物質可直接連接或可經由連接子連接。在本揭示案中，兩種物質中之一者為本揭示案之抗體，且另一種為藥物(例如生理活性物質)。連接子可為可裂解連接子或不可裂解連接子。As used herein, the term "conjugate" refers to a combination in which two substances are linked by a covalent bond (e.g., an antibody of the present disclosure is linked to a therapeutic agent). In a conjugate, the two substances may be directly linked or may be linked via a linker. In the present disclosure, one of the two substances is the antibody of the present disclosure, and the other is a drug (e.g., a physiologically active substance). The linker may be a cleavable linker or a non-cleavable linker.

如本文所用，術語「有效量」或「治療有效量」係指到達所關注組織所需之本揭示案之抗體的量，或係指足以減少及/或改善(i)個體之疾病、病症或疾患，及/或(ii)個體之症狀之嚴重程度及/或持續時間的結合物、融合蛋白或多肽或包含本揭示案之抗體及治療劑之複合物的量。該術語亦涵蓋如下所必需的結合物之量：(i)減少或改善給定疾病、病症或疾患之進程或進展，(ii)減少或改善給定疾病、病症或疾患之複發、發展或發作，及/或(iii)改良或增強另一劑或療法(例如除本文所提供之結合物以外之劑)之預防或治療作用。As used herein, the term "effective amount" or "therapeutically effective amount" refers to the amount of an antibody of the present disclosure required to reach the tissue of interest, or refers to the amount of a conjugate, fusion protein or polypeptide, or a complex comprising an antibody of the present disclosure and a therapeutic agent, sufficient to reduce and/or improve (i) a disease, disorder or condition in an individual, and/or (ii) the severity and/or duration of symptoms in an individual. The term also encompasses the amount of conjugate necessary to: (i) reduce or improve the course or progression of a given disease, disorder or condition, (ii) reduce or improve the recurrence, development or onset of a given disease, disorder or condition, and/or (iii) improve or enhance the preventive or therapeutic effect of another agent or therapy (e.g., an agent other than the conjugate provided herein).

如本文所用，術語「治療作用」係指劑(例如本揭示案之抗體、結合物、融合蛋白或多肽或包含抗體之複合物)減少及/或改善(i)個體之疾病、病症或疾患，及/或(ii)個體之症狀的嚴重程度及/或持續時間之作用及/或能力。該術語亦涵蓋劑(例如結合物)之如下能力：(i)減少或改善給定疾病、病症或疾患之進程或進展，(ii)減少或改善給定疾病、病症或疾患之複發、發展或發作，及/或(iii)改良或增強另一劑或療法(例如除本文所提供之結合物以外之劑)之預防或治療作用。As used herein, the term "therapeutic effect" refers to the effect and/or ability of an agent (e.g., an antibody, conjugate, fusion protein or polypeptide of the present disclosure or a complex comprising an antibody) to reduce and/or improve (i) a disease, disorder or condition in a subject, and/or (ii) the severity and/or duration of symptoms in a subject. The term also encompasses the ability of an agent (e.g., a conjugate) to (i) reduce or improve the course or progression of a given disease, disorder or condition, (ii) reduce or improve the recurrence, development or onset of a given disease, disorder or condition, and/or (iii) improve or enhance the preventive or therapeutic effect of another agent or therapy (e.g., an agent other than a conjugate provided herein).

如本文所用，對「約」或「近似」值或參數之引用包括(且描述)針對該值或參數之實施例。舉例而言，提及「約X」之描述包括對「X」之描述。「約X」意指X之+/-10%。因此，「約10」意指9至11之間的值。 TfR1 及抗TFR1抗體 As used herein, references to "about" or "approximately" a value or parameter include (and describe) embodiments for that value or parameter. For example, a description referring to "about X" includes a description of "X.""AboutX" means +/- 10% of X. Thus, "about 10" means a value between 9 and 11. TfR1 and anti-TFR1 antibodies

運鐵蛋白受體，亦稱為CD71，係在人體之各個位點以不同水準表現之跨膜糖蛋白，其功能係介導細胞自血漿糖蛋白運鐵蛋白攝取鐵。自運鐵蛋白攝取鐵涉及使運鐵蛋白與運鐵蛋白受體結合、藉由受體介導之胞吞轉送作用使運鐵蛋白在胞吞囊泡內內化及藉由內體pH降低自蛋白質釋放鐵。Ponka P, Lok CN.. Int J Biochem Cell Biol. 1999年10月;31(10):1111-37及Xiaopeng Mo, 在Brain Targeted Drug Delivery System, 2019中。載運鐵蛋白(亦即非鐵結合物)在結合至兩個Fe 3+離子時結合至TfR以形成全運鐵蛋白(亦即鐵結合物)。TfR及全運鐵蛋白之複合物藉由受體介導之胞吞轉送作用易位至細胞中。CD71及運鐵蛋白在內體環境中解離，且運鐵蛋白移動至細胞中，而CD71再循環至細胞膜。因此，運鐵蛋白被認為藉由與TfR之適當結合及適當解離而易位至細胞中。運鐵蛋白受體系統已用於將抗癌藥物及蛋白質、治療基因遞送至惡性細胞中，且將其他治療劑穿過血腦障壁遞送至腦。Transferrin receptor, also known as CD71, is a transmembrane glycoprotein expressed at various sites in the human body at varying levels. Its function is to mediate cellular iron uptake from the plasma glycoprotein transferrin. Iron uptake from transferrin involves binding of transferrin to transferrin receptor, internalization of transferrin into endocytic vesicles via receptor-mediated endocytosis, and release of iron from the protein by a decrease in endosomal pH. Ponka P, Lok CN.. Int J Biochem Cell Biol. 1999 Oct;31(10):1111-37 and Xiaopeng Mo, in Brain Targeted Drug Delivery System, 2019. Ferritin (i.e., non-iron binder) binds to TfR when bound to two Fe 3+ ions to form holo-ferritin (i.e., iron binder). The complex of TfR and holo-ferritin is translocated into the cell by receptor-mediated endocytosis. CD71 and ferritin dissociate in the endosomal environment, and ferritin moves into the cell, while CD71 recycles to the cell membrane. Therefore, ferritin is thought to be translocated into the cell by appropriate binding to and appropriate dissociation from TfR. The ferritin receptor system has been used to deliver anticancer drugs and proteins, therapeutic genes into malignant cells, and to deliver other therapeutic agents across the blood-brain barrier to the brain.

在人類及石蟹猴中，已表徵兩種運鐵蛋白受體TfR1及TfR2。TfR1為高親和力遍在表現之受體，而TfR2之表現限於某些細胞類型且不受細胞內鐵濃度影響。TfR2以比TfR1低25-30倍之親和力結合至運鐵蛋白。本揭示案之抗體結合至TfR1。In humans and cynomolgus monkeys, two ferritin receptors, TfR1 and TfR2, have been characterized. TfR1 is a high-affinity ubiquitously expressed receptor, while the expression of TfR2 is restricted to certain cell types and is not affected by intracellular iron concentrations. TfR2 binds to ferritin with an affinity 25-30 times lower than that of TfR1. The antibodies of the present disclosure bind to TfR1.

人類TfR1及石蟹猴TfR1之序列如下： 人類TfR1 (UniProt編號P02786.2；SEQ ID NO:1) MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAVDEEENADNNTKANVTKPKRCSGSICYGTIAVIVFFLIGFMIGYLGYCKGVEPKTECERLAGTESPVREEPGEDFPAARRLYWDDLKRKLSEKLDSTDFTGTIKLLNENSYVPREAGSQKDENLALYVENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKEIKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSGVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQNVKHPVTGQFLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELIERIPELNKVARAAAEVAGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQYRADIKEMGLSLQWLYSARGDFFRATSRLTTDFGNAEKTDRFVMKKLNDRVMRVEYHFLSPYVSPKESPFRHVFWGSGSHTLPALLENLKLRKQNNGAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF 石蟹猴TfR1 (UniProt編號G8F602；SEQ ID NO:2) MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAVDDEENADNNTKANGTKPKRCGGNICYGTIAVIIFFLIGFMIGYLGYCKGVEPKTECERLAGTESPAREEPEEDFPAAPRLYWDDLKRKLSEKLDTTDFTSTIKLLNENLYVPREAGSQKDENLALYIENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGGLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLDSPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVKADLSFFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCKMVTSENKSVKLTVSNVLKETKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSSVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQDVKHPVTGRSLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELVERIPELNKVARAAAEVAGQFVIKLTHDTELNLDYERYNSQLLLFLRDLNQYRADVKEMGLSLQWLYSARGDFFRATSRLTTDFRNAEKRDKFVMKKLNDRVMRVEYYFLSPYVSPKESPFRHVFWGSGSHTLSALLESLKLRRQNNSAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF The sequences of human TfR1 and crab monkey TfR1 are as follows: Human TfR1 (UniProt number P02786.2; SEQ ID NO:1) MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAVDEEENADNNTKANVTKPKRCSGSICYGTIAVIVFFLIGFMIGYLGYCKGVEPKTECERLAGTESPVREEPGEDFPAARRLYWDDLKRKLSEKLDSTDFTGTIKLLNENSYVPREAGSQKDENLALYVENQFREFKLSKVWRDQHFVKI Question LKEIKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSGVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQNVKHPVTGQFLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTM DTYKELIERIPELNKVARAAAEVAGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQYRADIKEMGLSLQWLYSARGDFFRATSRLTTDFGNAEKTDRFVMKKLNDRVMRVEYHFLSPYVSPKESPFRHVFWGSGSHTLPALLENLKLRKQNNGAFNETLFRNQLALATWTIQGAANAALSGDVWDIDNEF Macaca fascicularis TfR1 (UniProt number G8F602; SEQ ID NO:2) MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAVDDEENADNNTKANGTPKRCGGNICYGTIAVIIFFLIGFMIGYLGYCKGVEPKTECERLAGTESPAREEPEEDFPAAPRLYWDDLKRKLSEKLDTTDFTSTIKLLNENLYVPREAGSQKDENLALYIENQFREFKLSKVWRDQHFVKI Question LKETKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSSVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQDVKHPVTGRSLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTM DTYKELVERIPELNKVARAAAEVAGQFVIKLTHDTELNLDYERYNSQLLLFLRDLNQYRADVKEMGLSLQWLYSARGDFFRATSRLTTDFRNAEKRDKFVMKKLNDRVMRVEYYFLSPYVSPKESPFRHVFWGSGSHTLSALLESLKLRRQNNSAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF

本揭示案提供結合TfR1之抗體。The present disclosure provides antibodies that bind to TfR1.

在一些實施例中，抗TfR1抗體為重組抗體。在一些實施例中，抗體為單株抗體。在一些實施例中，抗體為嵌合抗體。在一些實施例中，抗體為人類化抗體。在一些實施例中，抗體為人類抗體。在一些實施例中，抗體為IgA、IgD、IgE、IgG或IgM抗體。在一些實施例中，抗體為IgG抗體。在一些實施例中，抗體為IgG1抗體。在一些實施例中，抗體為IgG2抗體。在一些實施例中，抗體為IgG3抗體。在一些實施例中，抗體為IgG4抗體。在一些情況下，抗體包含人類κ輕鏈恆定區。在其他實施例中，抗體包含人類λ輕鏈恆定區。在一些情況下，抗體為IgG1抗體且包含人類κ輕鏈恆定區。在一些情況下，抗體為IgG1抗體且包含人類λ輕鏈恆定區。在一些實施例中，抗體為包含抗原結合位點之抗體片段。在一些實施例中，抗體為scFv。在一些實施例中，抗體為二硫鍵連接之scFv。在一些實施例中，抗體為雙特異性抗體或多特異性抗體。在一些實施例中，抗體為單價抗體。在一些實施例中，抗體為單特異性抗體。在一些實施例中，抗體為二價抗體。In some embodiments, the anti-TfR1 antibody is a recombinant antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is an IgA, IgD, IgE, IgG or IgM antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG4 antibody. In some cases, the antibody comprises a human kappa light chain constant region. In other embodiments, the antibody comprises a human lambda light chain constant region. In some cases, the antibody is an IgG1 antibody and comprises a human kappa light chain constant region. In some cases, the antibody is an IgG1 antibody and comprises a human lambda light chain constant region. In some embodiments, the antibody is an antibody fragment comprising an antigen binding site. In some embodiments, the antibody is a scFv. In some embodiments, the antibody is a disulfide-linked scFv. In some embodiments, the antibody is a bispecific antibody or a multispecific antibody. In some embodiments, the antibody is a monovalent antibody. In some embodiments, the antibody is a monospecific antibody. In some embodiments, the antibody is a divalent antibody.

在一些情況下，抗體為Fab、Fab'、F(ab) 2、scFv、sc(Fv) 2、雙價抗體或奈米抗體。在一些實施例中，移除抗體或抗原結合片段(例如Fab)中之鏈間二硫鍵。Fab或Fab'含有可變重鏈(VH)及可變輕鏈域(VL)。 In some cases, the antibody is Fab, Fab', F(ab) 2 , scFv, sc(Fv) 2 , bivalent antibody or nanobody. In some embodiments, the interchain disulfide bonds in the antibody or antigen-binding fragment (e.g., Fab) are removed. Fab or Fab' contains a variable heavy chain (VH) and a variable light chain domain (VL).

在一些實施例中，抗體為經分離的。在一些實施例中，抗體為實質上純的。In some embodiments, the antibody is isolated. In some embodiments, the antibody is substantially pure.

在一些實施例中，抗TfR1抗體為人類化抗體。用於產生人類化抗體之各種方法係此項技術中已知的。在一些實施例中，人類化抗體包含一或多個自非人類來源引入其序列中之胺基酸殘基。在一些實施例中，藉由用一或多個非人類CDR序列取代人類抗體之相應CDR序列來進行人類化。In some embodiments, the anti-TfR1 antibody is a humanized antibody. Various methods for producing humanized antibodies are known in the art. In some embodiments, the humanized antibody comprises one or more amino acid residues introduced into its sequence from a non-human source. In some embodiments, humanization is performed by replacing the corresponding CDR sequence of a human antibody with one or more non-human CDR sequences.

可基於多種因素且藉由此項技術中已知之多種方法來選擇使用哪個人類重鏈可變區及/或輕鏈可變區產生人類化抗體。在一些實施例中，使用「最佳擬合」方法，其中針對已知人類可變區序列之整個文庫篩選非人類(例如囓齒動物)抗體之可變區之序列。選擇與非人類(例如囓齒動物)序列之序列最相似之人類序列作為人類化抗體之人類可變區構架。在一些實施例中，選擇源自特定輕鏈或重鏈亞組之所有人類抗體之共有序列的特定可變區構架作為可變區構架。在一些實施例中，可變區構架序列來源於最豐富人類亞類之共有序列。在一些實施例中，使用人類生殖系基因作為可變區構架序列之來源。The selection of which human heavy chain variable region and/or light chain variable region to use to generate a humanized antibody can be based on a variety of factors and by a variety of methods known in the art. In some embodiments, a "best fit" approach is used, in which the sequence of the variable region of a non-human (e.g., rodent) antibody is screened against an entire library of known human variable region sequences. The human sequence that is most similar to the sequence of the non-human (e.g., rodent) sequence is selected as the human variable region framework of the humanized antibody. In some embodiments, a specific variable region framework derived from the consensus sequence of all human antibodies of a specific light chain or heavy chain subgroup is selected as the variable region framework. In some embodiments, the variable region framework sequence is derived from the consensus sequence of the most abundant human subclass. In some embodiments, human germline genes are used as the source of variable region framework sequences.

用於人類化之其他方法包括但不限於(i)稱為「超人類化」之方法，其描述為將CDR直接轉移至人類生殖系構架，(ii)稱為人類字串內容(HSC)之基於「抗體人類性」之度量的方法，(iii)基於人類化變異體之大型文庫(包括噬菌體展示文庫、核醣體展示文庫及酵母展示文庫)之生成的方法，及(iv)基於構架區改組的方法。Other methods for humanization include, but are not limited to, (i) methods referred to as "superhumanization," which describes the direct transfer of CDRs to human germline frameworks, (ii) methods based on a measure of "antibody humanity" referred to as human string content (HSC), (iii) methods based on the generation of large libraries of humanized variants (including phage display libraries, ribosome display libraries, and yeast display libraries), and (iv) methods based on framework region shuffling.

在一些實施例中，抗TfR1抗體為「人類抗體」。人類抗體可使用此項技術中已知之各種技術來製備。在一些實施例中，人類抗體係自活體外免疫之永生化人類B淋巴球產生。在一些實施例中，人類抗體係自分離自經免疫個體之淋巴球產生。在任何情況下，可產生並分離產生針對靶抗原之抗體的細胞。在一些實施例中，人類抗體選自噬菌體文庫，其中該噬菌體文庫表現人類抗體。替代地，噬菌體展示技術可用於在活體外自未免疫供體之免疫球蛋白可變區基因譜系產生人類抗體及抗體片段。用於產生及使用抗體噬菌體文庫之技術為此項技術中所熟知的。一旦鑑別出抗體，則可採用此項技術中已知之親和力成熟策略(包括但不限於鏈改組及定點誘變)來產生更高親和力之人類抗體。在一些實施例中，人類抗體在含有人類免疫球蛋白基因座之基因轉殖小鼠中產生。免疫後，此等小鼠能夠在不產生內源免疫球蛋白之情況下產生人類抗體之全部譜系。In some embodiments, the anti-TfR1 antibody is a "human antibody". Human antibodies can be prepared using various techniques known in the art. In some embodiments, human antibodies are produced from immortalized human B lymphocytes immunized in vitro. In some embodiments, human antibodies are produced from lymphocytes isolated from immunized individuals. In any case, cells that produce antibodies against the target antigen can be produced and isolated. In some embodiments, human antibodies are selected from a phage library, wherein the phage library expresses human antibodies. Alternatively, phage display technology can be used to produce human antibodies and antibody fragments in vitro from the immunoglobulin variable region gene repertoire of unimmunized donors. The techniques for producing and using antibody phage libraries are well known in the art. Once antibodies are identified, affinity maturation strategies known in the art (including but not limited to chain shuffling and site-directed mutagenesis) can be employed to generate higher affinity human antibodies. In some embodiments, human antibodies are generated in transgenic mice containing human immunoglobulin loci. Following immunization, these mice are capable of producing the full repertoire of human antibodies without producing endogenous immunoglobulins.

在一些實施例中，抗TfR1抗體為雙特異性抗體。雙特異性抗體能夠識別及結合至少兩種不同抗原或抗原決定基。不同抗原決定基可在同一分子內(例如TfR1上之兩個抗原決定基)或不同分子上(例如TfR1上之一個抗原決定基及不同靶標上之一個抗原決定基)。在一些實施例中，與個別抗體或與多於一種抗體之組合相比，雙特異性抗體具有增強之效力。在一些實施例中，與個別抗體或與多於一種抗體之組合相比，雙特異性抗體具有減少之毒性。熟習此項技術者已知，任何治療劑可具有獨特的藥物動力學(PK) (例如，循環半衰期)。在一些實施例中，雙特異性抗體具有使兩種活性結合劑之PK同步之能力，其中兩種個別結合劑具有不同PK概況。在一些實施例中，雙特異性抗體能夠將兩種劑之作用集中在個體之共同區域(例如組織)中。在一些實施例中，雙特異性抗體能夠將兩種劑之作用集中至共同靶標(例如特定細胞類型)。在一些實施例中，雙特異性抗體能夠將兩種劑之作用靶向多於一種生物路徑或功能。在一些實施例中，雙特異性抗體能夠靶向兩種不同細胞並使它們靠得更近。In some embodiments, the anti-TfR1 antibody is a bispecific antibody. Bispecific antibodies are capable of recognizing and binding to at least two different antigens or antigenic determinants. The different antigenic determinants can be within the same molecule (e.g., two antigenic determinants on TfR1) or on different molecules (e.g., one antigenic determinant on TfR1 and one antigenic determinant on a different target). In some embodiments, the bispecific antibody has enhanced efficacy compared to an individual antibody or a combination of more than one antibody. In some embodiments, the bispecific antibody has reduced toxicity compared to an individual antibody or a combination of more than one antibody. It is known to those skilled in the art that any therapeutic agent can have unique pharmacokinetics (PK) (e.g., circulation half-life). In some embodiments, bispecific antibodies have the ability to synchronize the PK of two active binding agents, where the two individual binding agents have different PK profiles. In some embodiments, bispecific antibodies can focus the effects of two agents in a common area (e.g., tissue) of an individual. In some embodiments, bispecific antibodies can focus the effects of two agents to a common target (e.g., a specific cell type). In some embodiments, bispecific antibodies can target the effects of two agents to more than one biological pathway or function. In some embodiments, bispecific antibodies can target two different cells and bring them closer together.

在一些實施例中，雙特異性抗體具有降低之毒性及/或副作用。在一些實施例中，與兩種個別抗體之混合物或作為單一劑之抗體相比，雙特異性抗體具有降低之毒性及/或副作用。在一些實施例中，雙特異性抗體具有增加之治療指數。在一些實施例中，與兩種個別抗體之混合物或作為單一劑之抗體相比，雙特異性抗體具有增加之治療指數。In some embodiments, the bispecific antibodies have reduced toxicity and/or side effects. In some embodiments, the bispecific antibodies have reduced toxicity and/or side effects compared to a mixture of two individual antibodies or the antibodies as a single agent. In some embodiments, the bispecific antibodies have an increased therapeutic index. In some embodiments, the bispecific antibodies have an increased therapeutic index compared to a mixture of two individual antibodies or the antibodies as a single agent.

用於製備雙特異性抗體之若干技術為熟習此項技術者已知的。在一些實施例中，雙特異性抗體包含在作為兩個重鏈之間的界面之一部分之胺基酸中具有修飾之重鏈恆定區。進行此等修飾以增強異二聚體形成且通常減少或消除同二聚體形成。在一些實施例中，使用杵入臼(knobs-into-holes，KIH)策略產生雙特異性抗體。參見，例如Ridgway等人 Protein Eng. 1996;9(7):617-21及Klein等人 MAbs. 2012;4(6):653-663。Several techniques for preparing bispecific antibodies are known to those skilled in the art. In some embodiments, the bispecific antibodies comprise a heavy chain constant region having modifications in amino acids that are part of the interface between the two heavy chains. Such modifications are made to enhance heterodimer formation and generally reduce or eliminate homodimer formation. In some embodiments, the bispecific antibodies are generated using a knobs-into-holes (KIH) strategy. See, e.g., Ridgway et al. Protein Eng. 1996;9(7):617-21 and Klein et al. MAbs. 2012;4(6):653-663.

在一些實施例中，雙特異性抗體包含在作為兩個輕鏈之間的界面之一部分之胺基酸中具有修飾之輕鏈恆定區。進行此等修飾以減少或消除輕鏈錯配。參見例如Lewis等人Nat Biotech 2014;32(2):191-98。在一些實施例中，雙特異性抗體包含共價連接VH及VL且移除CH1及CL之scFv。在一些實施例中，雙特異性抗體包含scFab或Fcab (參見例如Wozniak-Knopp等人PEDS 2010;23(4):289-97)、單域抗體(例如具有來自駱駝科物種或鯊魚之VHH)或Duet Mab (參見例如Mazor等人Mabs 2015;7(2):377-89)。In some embodiments, the bispecific antibody comprises a light chain constitutive region with modifications in amino acids that are part of the interface between the two light chains. Such modifications are made to reduce or eliminate light chain mispairing. See, e.g., Lewis et al. Nat Biotech 2014;32(2):191-98. In some embodiments, the bispecific antibody comprises a scFv with VH and VL covalently linked and CH1 and CL removed. In some embodiments, the bispecific antibody comprises a scFab or Fcab (see, e.g., Wozniak-Knopp et al. PEDS 2010; 23(4): 289-97), a single domain antibody (e.g., having a VHH from a camel species or shark), or a Duet Mab (see, e.g., Mazor et al. Mabs 2015; 7(2): 377-89).

雙特異性抗體可為完整抗體或包含抗原結合位點之抗體片段。Bispecific antibodies can be complete antibodies or antibody fragments that contain the antigen-binding site.

本揭示案中考慮具有多於兩種特異性之抗TfR1抗體。在一些實施例中，產生三特異性抗體或四特異性抗體。考慮具有多於兩個化合價之抗TfR1抗體。在一些實施例中，產生三價抗體或四價抗體。Anti-TfR1 antibodies with more than two specificities are contemplated in the present disclosure. In some embodiments, trispecific antibodies or tetraspecific antibodies are generated. Anti-TfR1 antibodies with more than two valencies are contemplated. In some embodiments, trivalent antibodies or tetravalent antibodies are generated.

抗體之CDR由熟習此項技術者使用多種方法/系統來定義。此等系統及/或定義已經開發及細化多年，且包括Kabat、Chothia、IMGT、AbM、Contact及Union。Kabat定義係基於序列變異性且為常用的。Chothia定義係基於結構環區之位置。IMGT系統係基於序列可變性及可變域結構內之位置。AbM定義為Kabat與Chothia之間的折衷。Contact定義係基於對可用抗體晶體結構之分析。示範性系統係Kabat及Chothia之組合。熟習此項技術者可獲得且已知用於分析抗體序列及確定CDR之軟體程式(例如，abYsis)。The CDRs of antibodies are defined by those skilled in the art using a variety of methods/systems. These systems and/or definitions have been developed and refined over the years and include Kabat, Chothia, IMGT, AbM, Contact and Union. The Kabat definition is based on sequence variability and is commonly used. The Chothia definition is based on the position of the structural loop region. The IMGT system is based on sequence variability and the position within the variable domain structure. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on analysis of available antibody crystal structures. An exemplary system is a combination of Kabat and Chothia. Software programs (e.g., abYsis) for analyzing antibody sequences and determining CDRs are available and known to those skilled in the art.

表1中所述之CDR序列包括Union CDR定義(Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S.及Foeller, C. (1991). Sequences of Proteins of Immunological Interest, 第5版National Institutes of Health, Bethesda, MD)及Chothia CDR定義(Chothia, C.及Lesk, A.M. J. Mol. Biol (1987) 196, 901-917) (Chothia, C.等人Nature (1989) 342, 877 -883) (Al-Lazikani, B., Lesk, A.M.及Chothia, C. J. Mol. Biol (1997) 273, 927-948)中所有位置之聯集。CDR之此「聯集」定義亦稱為Bujotzek等人之「Wolfguy」定義(Bujotzek A1, Dunbar J, Lipsmeier F, Schäfer W, Antes I, Deane CM, Georges G. (2015)「Prediction of VH-VL domainorientation for antibody variable domain modeling.」Proteins Apr；83(4):681-95. doi: 10.1002/prot.24756))。在一些實施例中，CDR定義係基於Kabat及Chothia定義之組合(示範性系統)。然而，應理解，提及特定抗體之一或多個VH CDR及/或一或多個VL CDR將涵蓋如熟習此項技術者已知之所有CDR定義。在一種情況下，本文所述之抗TfR1抗體包含基於Wolfguy或Union定義的本文所揭示之抗體X親代抗體之六個CDR。在一種情況下，本文所述之抗TfR1抗體包含基於Chothia定義的本文所揭示之抗體X親代抗體之六個CDR。在一種情況下，本文所述之抗TfR1抗體包含基於Kabat定義的本文所揭示之抗體X親代抗體之六個CDR。在一種情況下，本文所述之抗TfR1抗體包含基於AbM定義的本文所揭示之抗體X親代抗體之六個CDR。在一種情況下，本文所述之抗TfR1抗體包含基於IMGT定義的本文所揭示之抗體X親代抗體之六個CDR。在一種情況下，本文所述之抗TfR1抗體包含基於Contact定義的本文所揭示之抗體X親代抗體之六個CDR。在一種情況下，本文所述之抗TfR1抗體包含基於Wolfguy或Union定義的本文所揭示之任何抗體X突變體抗體之六個CDR。在一種情況下，本文所述之抗TfR1抗體包含基於Chothia定義的本文所揭示之任何抗體X突變體抗體之六個CDR。在一種情況下，本文所述之抗TfR1抗體包含基於Kabat定義的本文所揭示之任何抗體X突變體抗體之六個CDR。在一種情況下，本文所述之抗TfR1抗體包含基於AbM定義的本文所揭示之任何抗體X突變體抗體之六個CDR。在一種情況下，本文所述之抗TfR1抗體包含基於IMGT定義的本文所揭示之任何抗體X突變體抗體之六個CDR。在一種情況下，本文所述之抗TfR1抗體包含基於Contact定義的本文所揭示之任何抗體X突變體抗體之六個CDR。 The CDR sequences described in Table 1 include the union of all positions in the Union CDR definition (Kabat, EA, Wu, TT, Perry, HM, Gottesman, KS, and Foeller, C. (1991). Sequences of Proteins of Immunological Interest, 5th Edition National Institutes of Health, Bethesda, MD) and the Chothia CDR definition (Chothia, C. and Lesk, AM J Mol. Biol (1987) 196, 901-917) (Chothia, C. et al. Nature (1989) 342, 877-883) (Al-Lazikani, B., Lesk, AM, and Chothia, CJ Mol. Biol (1997) 273, 927-948). This "union" definition of CDRs is also known as the "Wolfguy" definition of Bujotzek et al. (Bujotzek A1, Dunbar J, Lipsmeier F, Schäfer W, Antes I, Deane CM, Georges G. (2015) "Prediction of VH-VL domain orientation for antibody variable domain modeling." Proteins Apr; 83(4): 681-95. doi: 10.1002/prot.24756)). In some embodiments, the CDR definition is based on a combination of the Kabat and Chothia definitions (exemplary system). However, it should be understood that reference to one or more VH CDRs and/or one or more VL CDRs of a particular antibody will encompass all CDR definitions known to those skilled in the art. In one instance, the anti-TfR1 antibodies described herein comprise six CDRs of the Antibody X parent antibody disclosed herein based on the Wolfguy or Union definition. In one instance, the anti-TfR1 antibodies described herein comprise six CDRs of the Antibody X parent antibody disclosed herein based on the Chothia definition. In one instance, the anti-TfR1 antibodies described herein comprise six CDRs of the Antibody X parent antibody disclosed herein based on the Kabat definition. In one instance, the anti-TfR1 antibodies described herein comprise six CDRs of the Antibody X parent antibody disclosed herein based on the AbM definition. In one instance, the anti-TfR1 antibodies described herein comprise six CDRs of the Antibody X parent antibody disclosed herein based on the IMGT definition. In one case, the anti-TfR1 antibodies described herein comprise six CDRs of an Antibody X parent antibody disclosed herein based on the Contact definition. In one case, the anti-TfR1 antibodies described herein comprise six CDRs of any Antibody X mutant antibody disclosed herein based on the Wolfguy or Union definition. In one case, the anti-TfR1 antibodies described herein comprise six CDRs of any Antibody X mutant antibody disclosed herein based on the Chothia definition. In one case, the anti-TfR1 antibodies described herein comprise six CDRs of any Antibody X mutant antibody disclosed herein based on the Kabat definition. In one case, the anti-TfR1 antibodies described herein comprise six CDRs of any Antibody X mutant antibody disclosed herein based on the AbM definition. In one aspect, the anti-TfR1 antibodies described herein comprise six CDRs of any Antibody X mutant antibody disclosed herein based on the IMGT definition. In one aspect, the anti-TfR1 antibodies described herein comprise six CDRs of any Antibody X mutant antibody disclosed herein based on the Contact definition.

表1描述參考抗體X抗體之「親代」CDR (根據Union定義)以及本文所述之抗體X突變體中存在的突變體CDR。突變體胺基酸位置用AHo編號來鑑別(Honegger等人, J Mol Biol, 2001, 309(3):657-70)。在不存在突變CDR之情況下，可變區包含相應親代CDR。舉例而言，對於VH-S40A / VL親代，VH包含突變體VH CDR1 (S40A)、親代VH CDR2及親代VH CDR3，且VL包含親代VL CDR1、親代VL CDR2及親代VL CDR3。在一些實施例中，抗TfR1抗體為包含 表1中所述之CDR突變中之至少一者(例如，1-10、1、2、3、4、5、6、7、8、9或10個 表1中所述之CDR突變)，但除此以外含有 表1中所述之親代CDR序列的抗TfR1抗體。在一些實施例中，抗TfR1抗體包含(i) 表1中所述之一個、兩個及/或三個突變體VH CDR，及/或(ii) 表1中所述之一個、兩個及/或三個突變體VL CDR，其中所有剩餘CDR選自 表1中所描繪之親代CDR。 表1：抗體X親代及突變體之Union CDR. 親代CDR VH CDR1 VH CDR2 VH CDR3 VL CDR1 VL CDR2 VL CDR3 GFTFSSYSMN (SEQ ID NO:3) SISSSSSYIYYADSVKG (SEQ ID NO:4) KYRAGDFDY (SEQ ID NO:5) RASQSVSSSYLA (SEQ ID NO:6) GASNRAT (SEQ ID NO:7) QQQSSSPPT (SEQ ID NO:8) 突變體CDR 突變 VH CDR1 VH CDR2 VH CDR3 VL CDR1 VL CDR2 VL CDR3 VH-S40A / VL親代 GFTFSSYAMN (SEQ ID NO:9) VH-S60A / VL親代 SISASSSYIYYADSVKG (SEQ ID NO:10) VH-Y67S / VL親代 SISSSSSSIYYADSVKG (SEQ ID NO:11) VH-Y110S / VL親代 KSRAGDFDY (SEQ ID NO:12) VH-R111S / VL親代 KYSAGDFDY (SEQ ID NO:13) VH-A112Y / VL親代 KYRYGDFDY (SEQ ID NO:14) VH- S60A/Y67S / VL親代 SISASSSSIYYADSVKG (SEQ ID NO:25) VH- S60A/R111S / VL親代 SISASSSYIYYADSVKG (SEQ ID NO:10) KYSAGDFDY (SEQ ID NO:13) VH親代/ VL-S39N RASQSVSSNYLA (SEQ ID NO:15) VH親代/ VL-Y40N RASQSVSSSNLA (SEQ ID NO:16) VH親代/ VL-Y57S GASNRAT (SEQ ID NO:7) VH親代/ VL-N69S GASSRAT (SEQ ID NO:17) VH- S60A / VL-S39N SISASSSYIYYADSVKG (SEQ ID NO:10) RASQSVSSNYLA (SEQ ID NO:15) VH- S60A / VL-Y57S SISASSSYIYYADSVKG (SEQ ID NO:10) GASNRAT (SEQ ID NO:7) Table 1 describes the "parent" CDRs (according to the Union definition) of the reference Antibody X antibody and the mutant CDRs present in the Antibody X mutants described herein. Mutant amino acid positions are identified using AHo numbers (Honegger et al., J Mol Biol, 2001, 309(3):657-70). In the absence of mutant CDRs, the variable regions comprise the corresponding parent CDRs. For example, for the VH-S40A/VL parent, the VH comprises mutant VH CDR1 (S40A), parent VH CDR2, and parent VH CDR3, and the VL comprises parent VL CDR1, parent VL CDR2, and parent VL CDR3. In some embodiments, the anti-TfR1 antibody is an anti-TfR1 antibody comprising at least one of the CDR mutations described in Table 1 (e.g., 1-10, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 CDR mutations described in Table 1), but otherwise contains a parent CDR sequence described in Table 1. In some embodiments, the anti-TfR1 antibody comprises (i) one, two and/or three mutant VH CDRs described in Table 1, and/or (ii) one, two and/or three mutant VL CDRs described in Table 1 , wherein all remaining CDRs are selected from the parent CDRs depicted in Table 1. Table 1: Union CDRs of Antibody X Parent and Mutants. Parent CDR VH CDR1 VH CDR2 VH CDR3 VL CDR1 VL CDR2 VL CDR3 GFTFSSYSMN (SEQ ID NO:3) SISSSSSYIYYADSVKG (SEQ ID NO:4) KYRAGDFDY (SEQ ID NO:5) RASQSVSSSYLA (SEQ ID NO:6) GASNRAT (SEQ ID NO:7) QQQSSSPPT (SEQ ID NO:8) Mutant CDR mutation VH CDR1 VH CDR2 VH CDR3 VL CDR1 VL CDR2 VL CDR3 VH-S40A / VL Parent GFTFSSYAMN (SEQ ID NO:9) VH-S60A / VL Parent SISASSSYIYYADSVKG (SEQ ID NO:10) VH-Y67S / VL parent SISSSSSSIYYADSVKG (SEQ ID NO:11) VH-Y110S / VL parent KSRAGDFDY (SEQ ID NO: 12) VH-R111S / VL parent KYSAGDFDY (SEQ ID NO: 13) VH-A112Y / VL Parent KYRYGDFDY (SEQ ID NO: 14) VH- S60A/Y67S / VL parent SISASSSSIYYADSVKG (SEQ ID NO:25) VH- S60A/R111S / VL parent SISASSSYIYYADSVKG (SEQ ID NO:10) KYSAGDFDY (SEQ ID NO: 13) VH Parent/VL-S39N RASQSVSSNYLA (SEQ ID NO:15) VH Parent/VL-Y40N RASQSVSSSNLA (SEQ ID NO: 16) VH Parent/VL-Y57S GASNRAT (SEQ ID NO:7) VH Parent/VL-N69S GASSRAT (SEQ ID NO:17) VH-S60A / VL-S39N SISASSSYIYYADSVKG (SEQ ID NO:10) RASQSVSSNYLA (SEQ ID NO:15) VH-S60A / VL-Y57S SISASSSYIYYADSVKG (SEQ ID NO:10) GASNRAT (SEQ ID NO:7)

表2描述抗體X抗體之「親代」CDR (根據Kabat定義)以及本文所述之抗體X突變體中存在的突變體CDR。突變體胺基酸位置用AHo編號來鑑別。在不存在突變體CDR之情況下，VH或VL包含相應親代CDR。舉例而言，對於VH-S40A / VL親代，VH包含突變體VH CDR1 (S40A)、親代VH CDR2及親代VH CDR3，且VL包含親代VL CDR1、親代VL CDR2及親代VL CDR3。在一些實施例中，抗TfR1抗體為包含 表2中所述之CDR突變中之至少一者(例如，1-10、1、2、3、4、5、6、7、8、9或10個 表2中所述之CDR突變)，但除此以外含有 表2中所述之親代CDR序列的抗TfR1抗體。在一些實施例中，抗TfR1抗體包含(i) 表2中所述之一個、兩個及/或三個突變體VH CDR，及/或(ii) 表2中所述之一個、兩個及/或三個突變體VL CDR，其中所有剩餘CDR選自 表2中所描繪之親代CDR。 表2：抗體X親代及突變體之Kabat CDR. 親代CDR VH CDR1 VH CDR2 VH CDR3 VL CDR1 VL CDR2 VL CDR3 SYSMN (SEQ ID NO:23) SISSSSSYIYYADSVKG (SEQ ID NO:4) KYRAGDFDY (SEQ ID NO:5) RASQSVSSSYLA (SEQ ID NO:6) GASNRAT (SEQ ID NO:7) QQQSSSPPT (SEQ ID NO:8) 突變體CDR 突變 VH CDR1 VH CDR2 VH CDR3 VL CDR1 VL CDR2 VL CDR3 VH-S40A / VL親代 SYAMN (SEQ ID NO:24) VH-S60A / VL親代 SISASSSYIYYADSVKG (SEQ ID NO:10) VH-Y67S / VL親代 SISSSSSSIYYADSVKG (SEQ ID NO:11) VH-Y110S / VL親代 KSRAGDFDY (SEQ ID NO:12) VH-R111S / VL親代 KYSAGDFDY (SEQ ID NO:13) VH-A112Y / VL親代 KYRYGDFDY (SEQ ID NO:14) VH- S60A/Y67S / VL親代 SISASSSSIYYADSVKG (SEQ ID NO:25) VH- S60A/R111S / VL親代 SISASSSYIYYADSVKG (SEQ ID NO:10) KYSAGDFDY (SEQ ID NO:13) VH親代/ VL-S39N RASQSVSSNYLA (SEQ ID NO:15) VH親代/ VL-Y40N RASQSVSSSNLA (SEQ ID NO:16) VH親代/ VL-Y57S GASNRAT (SEQ ID NO:7) VH親代/ VL-N69S GASSRAT (SEQ ID NO:17) VH- S60A / VL-S39N SISASSSYIYYADSVKG (SEQ ID NO:10) RASQSVSSNYLA (SEQ ID NO:15) VH- S60A / VL-Y57S SISASSSYIYYADSVKG (SEQ ID NO:10) GASNRAT (SEQ ID NO:7) Table 2 describes the "parent" CDRs (according to the Kabat definition) of the Antibody X antibody and the mutant CDRs present in the Antibody X mutants described herein. Mutant amino acid positions are identified by AHo numbering. In the absence of mutant CDRs, the VH or VL comprises the corresponding parent CDRs. For example, for the VH-S40A/VL parent, the VH comprises mutant VH CDR1 (S40A), parent VH CDR2, and parent VH CDR3, and the VL comprises parent VL CDR1, parent VL CDR2, and parent VL CDR3. In some embodiments, the anti-TfR1 antibody is an anti-TfR1 antibody comprising at least one of the CDR mutations described in Table 2 (e.g., 1-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 CDR mutations described in Table 2), but otherwise contains a parent CDR sequence described in Table 2. In some embodiments, the anti-TfR1 antibody comprises (i) one, two, and/or three mutant VH CDRs described in Table 2, and/or (ii) one, two, and/or three mutant VL CDRs described in Table 2 , wherein all remaining CDRs are selected from the parent CDRs depicted in Table 2. Table 2: Kabat CDRs of Antibody X Parent and Mutants. Parent CDR VH CDR1 VH CDR2 VH CDR3 VL CDR1 VL CDR2 VL CDR3 SYSMN (SEQ ID NO:23) SISSSSSYIYYADSVKG (SEQ ID NO:4) KYRAGDFDY (SEQ ID NO:5) RASQSVSSSYLA (SEQ ID NO:6) GASNRAT (SEQ ID NO:7) QQQSSSPPT (SEQ ID NO:8) Mutant CDR mutation VH CDR1 VH CDR2 VH CDR3 VL CDR1 VL CDR2 VL CDR3 VH-S40A / VL Parent SYAMN (SEQ ID NO:24) VH-S60A / VL Parent SISASSSYIYYADSVKG (SEQ ID NO:10) VH-Y67S / VL parent SISSSSSSIYYADSVKG (SEQ ID NO:11) VH-Y110S / VL parent KSRAGDFDY (SEQ ID NO: 12) VH-R111S / VL parent KYSAGDFDY (SEQ ID NO: 13) VH-A112Y / VL Parent KYRYGDFDY (SEQ ID NO: 14) VH- S60A/Y67S / VL parent SISASSSSIYYADSVKG (SEQ ID NO:25) VH- S60A/R111S / VL parent SISASSSYIYYADSVKG (SEQ ID NO:10) KYSAGDFDY (SEQ ID NO: 13) VH Parent/VL-S39N RASQSVSSNYLA (SEQ ID NO:15) VH Parent/VL-Y40N RASQSVSSSNLA (SEQ ID NO: 16) VH Parent/VL-Y57S GASNRAT (SEQ ID NO:7) VH Parent/VL-N69S GASSRAT (SEQ ID NO:17) VH-S60A / VL-S39N SISASSSYIYYADSVKG (SEQ ID NO:10) RASQSVSSNYLA (SEQ ID NO:15) VH-S60A / VL-Y57S SISASSSYIYYADSVKG (SEQ ID NO:10) GASNRAT (SEQ ID NO:7)

在一些實施例中，抗TfR1抗體包含來自本文所述之抗體X親代抗體的VH CDR1、CDR2及CDR3及/或輕鏈可變區CDR1、CDR2及CDR3。在一些實施例中，抗TfR1抗體包含來自本文所述之抗體X親代抗體的VH CDR1、CDR2及CDR3以及VL CDR1、CDR2及CDR3。在一些實施例中，抗TfR1抗體包含本文所述之抗體X突變體抗體之人類化形式或人類化變異體。In some embodiments, the anti-TfR1 antibody comprises VH CDR1, CDR2 and CDR3 and/or light chain variable region CDR1, CDR2 and CDR3 from the Antibody X parent antibody described herein. In some embodiments, the anti-TfR1 antibody comprises VH CDR1, CDR2 and CDR3 and VL CDR1, CDR2 and CDR3 from the Antibody X parent antibody described herein. In some embodiments, the anti-TfR1 antibody comprises a humanized form or humanized variant of the Antibody X mutant antibody described herein.

在一些實施例中，抗TfR1抗體包含有包含VH CDR1、VH CDR2及VH CDR3之VH；及包含VL CDR1、VL CDR2及VL CDR3之VL，其中：(a) VH CDR1包含胺基酸序列GFTFSSYX 1MN (SEQ ID NO:18)或胺基酸序列SYX 1MN(SEQ ID NO:26)，其中X 1為S或A；(b) VH CDR2包含胺基酸序列SISX 2SSSX 3IYYADSVKG (SEQ ID NO:19)，其中X 2為S或A，且其中X 3為Y或S；(c) VH CDR3包含胺基酸序列KX 4X 5X 6GDFDY (SEQ ID NO:20)，其中X 4為Y或S，其中X 5為R或S，且其中X 6為A或Y；(d) VL CDR1包含胺基酸序列RASQSVSSX 7X 8LA (SEQ ID NO:21)，其中X 7為S或N，且其中X 8為Y或N；(e) VL CDR2包含胺基酸序列GASX 9RAT (SEQ ID NO:22)，其中X 9為N或S；且(f) VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)。在一些實施例中，抗體為單價且對hTfR1具有＞ 10 nM之單價親和力(K D)。在一些實施例中，抗體為二價且對hTfR1具有＞10 nM之單價親和力(K D)。在一些實施例中，抗體為二價且對hTfR1具有＞100 nM之單價親和力(K D)。在一些實施例中，抗體為二價且對hTfR1具有＞1000 nM之單價親和力(K D)。在一些實施例中，抗體為二價且對hTfR1具有＞100 nM或＞1000 nM之單價親和力(K D)。在一些實施例中，針對最佳腦暴露靶向抗體之化合價及親和力。 In some embodiments, the anti-TfR1 antibody comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein: (a) VH CDR1 comprises the amino acid sequence GFTFSSYX 1 MN (SEQ ID NO: 18) or the amino acid sequence SYX 1 MN (SEQ ID NO: 26), wherein X 1 is S or A; (b) VH CDR2 comprises the amino acid sequence SISX 2 SSSX 3 IYYADSVKG (SEQ ID NO: 19), wherein X 2 is S or A, and wherein X 3 is Y or S; (c) VH CDR3 comprises the amino acid sequence KX 4 X 5 X 6 GDFDY (SEQ ID NO: 20), wherein X 4 is Y or S, wherein X 5 is R or S, and wherein X 6 is A or Y; (d) VL CDR1 comprises the amino acid sequence RASQSVSSX 7 X 8 LA (SEQ ID NO:21), wherein X 7 is S or N, and wherein X 8 is Y or N; (e) VL CDR2 comprises the amino acid sequence GASX 9 RAT (SEQ ID NO:22), wherein X 9 is N or S; and (f) VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8). In some embodiments, the antibody is monovalent and has a monovalent affinity (K D ) of >10 nM for hTfR1. In some embodiments, the antibody is bivalent and has a monovalent affinity (K D ) of >10 nM for hTfR1. In some embodiments, the antibody is bivalent and has a monovalent affinity (K D ) of >100 nM for hTfR1. In some embodiments, the antibody is bivalent and has a monovalent affinity ( KD ) of >1000 nM for hTfR1. In some embodiments, the antibody is bivalent and has a monovalent affinity ( KD ) of >100 nM or >1000 nM for hTfR1. In some embodiments, the valency and affinity of the antibody are targeted for optimal brain exposure.

在一些實施例中，抗TfR1抗體包含來自本文所述之抗體X突變體抗體的VH CDR1、CDR2及CDR3及/或輕鏈可變區CDR1、CDR2及CDR3。在一些實施例中，抗TfR1抗體包含來自本文所述之抗體X突變體抗體的VH CDR1、CDR2及CDR3以及VL CDR1、CDR2及CDR3。在一些實施例中，抗TfR1抗體包含本文所述之抗體X突變體抗體之人類化形式或人類化變異體。In some embodiments, the anti-TfR1 antibody comprises VH CDR1, CDR2 and CDR3 and/or light chain variable region CDR1, CDR2 and CDR3 from the Antibody X mutant antibody described herein. In some embodiments, the anti-TfR1 antibody comprises VH CDR1, CDR2 and CDR3 and VL CDR1, CDR2 and CDR3 from the Antibody X mutant antibody described herein. In some embodiments, the anti-TfR1 antibody comprises a humanized form or humanized variant of the Antibody X mutant antibody described herein.

在一些實施例中，抗TfR1抗體為本文所述之抗體X親代抗體或抗體X突變體抗體之變異體，其包含一至三十個保守胺基酸取代。在一些實施例中，抗TfR1抗體之變異體包含一至二十五個保守胺基酸取代。在一些實施例中，抗TfR1抗體之變異體包含一至二十個保守胺基酸取代。在一些實施例中，抗TfR1抗體之變異體包含一至十五個保守胺基酸取代。在一些實施例中，抗TfR1抗體之變異體包含一至十個保守胺基酸取代。在一些實施例中，抗TfR1抗體之變異體包含一至五個保守胺基酸取代。在一些實施例中，抗TfR1抗體之變異體包含一至三個保守胺基酸取代。在一些實施例中，保守胺基酸取代在抗體之CDR中。在一些實施例中，保守胺基酸取代在抗體之CDR中(例如，在位置VH-S40、VH-S60、VH-Y67、VH-Y110、VH-R111、VH-A112、VL-S39、VL-Y40及/或VL-N69處，位置用AHo編號鑑別)。在一些實施例中，保守胺基酸取代不在抗體之CDR中。在一些實施例中，保守胺基酸取代在抗體之構架區中(例如，在位置VL-Y57處及/或在位置VL-S83處，位置用AHo編號鑑別)。In some embodiments, the anti-TfR1 antibody is a variant of the Antibody X parent antibody or Antibody X mutant antibody described herein, which comprises one to thirty conservative amino acid substitutions. In some embodiments, the variant of the anti-TfR1 antibody comprises one to twenty five conservative amino acid substitutions. In some embodiments, the variant of the anti-TfR1 antibody comprises one to twenty conservative amino acid substitutions. In some embodiments, the variant of the anti-TfR1 antibody comprises one to fifteen conservative amino acid substitutions. In some embodiments, the variant of the anti-TfR1 antibody comprises one to ten conservative amino acid substitutions. In some embodiments, the variant of the anti-TfR1 antibody comprises one to five conservative amino acid substitutions. In some embodiments, the variant of the anti-TfR1 antibody comprises one to three conservative amino acid substitutions. In some embodiments, conservative amino acid substitutions are in the CDR of an antibody. In some embodiments, conservative amino acid substitutions are in the CDR of an antibody (e.g., at positions VH-S40, VH-S60, VH-Y67, VH-Y110, VH-R111, VH-A112, VL-S39, VL-Y40 and/or VL-N69, positions are identified by AHo numbering). In some embodiments, conservative amino acid substitutions are not in the CDR of an antibody. In some embodiments, conservative amino acid substitutions are in the framework region of an antibody (e.g., at positions VL-Y57 and/or at positions VL-S83, positions are identified by AHo numbering).

表3描述本文所述之抗體X親代及若干抗體X突變體之重鏈可變區序列。 表4描述本文所述之抗體X親代及若干抗體X突變體之輕鏈可變區序列。在兩個表格中，突變體胺基酸位置用AHo編號來鑑別。 表3：抗體X及突變體之重鏈可變區 親代 SEQ ID NO 序列 VH-親代 100 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDYWGQGTLVTVSS 突變體名稱 SEQ ID NO 序列 VH-S40A 101 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDYWGQGTLVTVSS VH-S60A 102 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISASSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDYWGQGTLVTVSS VH-Y67S 103 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSSIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDYWGQGTLVTVSS VH-Y110S 104 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKSRAGDFDYWGQGTLVTVSS VH-R111S 105 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYSAGDFDYWGQGTLVTVSS VH-A112Y 106 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRYGDFDYWGQGTLVTVSS VH-S60A/Y67S 107 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISASSSSIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDYWGQGTLVTVSS VH-S60A/R111S 108 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISASSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYSAGDFDYWGQGTLVTVSS 表4：抗體X及突變體之輕鏈可變區 親代 SEQ ID NO 序列 VL-親代 200 EIVMTQSPGTLSLSPGERATLSC RASQSVSSSYLAWYQQKPGQAPRLLIY GASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQQSSSPPTFGGGTKVEIK 突變體名稱 SEQ ID NO 序列 VL-S39N 201 EIVMTQSPGTLSLSPGERATLSCRASQSVSSNYLAWYQQKPGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQQSSSPPTFGGGTKVEIK VL-Y40N 202 EIVMTQSPGTLSLSPGERATLSCRASQSVSSSNLAWYQQKPGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQQSSSPPTFGGGTKVEIK VL-Y57S 203 EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLISGASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQQSSSPPTFGGGTKVEIK VL-N69S 204 EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQQSSSPPTFGGGTKVEIK Table 3 describes the heavy chain variable region sequences of the antibody X parent and several antibody X mutants described herein. Table 4 describes the light chain variable region sequences of the antibody X parent and several antibody X mutants described herein. In both tables, the mutant amino acid positions are identified by AHo numbers. Table 3: Heavy chain variable regions of antibody X and mutants Parent SEQ ID NO sequence VH-Parenting 100 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDYWGQGTLVTVSS Mutant name SEQ ID NO sequence VH-S40A 101 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDYWGQGTLVTVSS VH-S60A 102 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISASSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDYWGQGTLVTVSS VH-Y67S 103 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSSIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDYWGQGTLVTVSS VH-Y110S 104 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKSRAGDFDYWGQGTLVTVSS VH-R111S 105 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYSAGDFDYWGQGTLVTVSS VH-A112Y 106 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRYGDFDYWGQGTLVTVSS VH-S60A/Y67S 107 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISASSSSIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDYWGQGTLVTVSS VH-S60A/R111S 108 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISASSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYSAGDFDYWGQGTLVTVSS Table 4: Light chain variable regions of antibody X and its mutants Parent SEQ ID NO sequence VL-Parenting 200 EIVMTQSPGTLSLSPGERATLSC RASQSVSSSYLA WYQQKPGQAPRLLIY GASNRAT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQQSSSPPT FGGGTKVEIK Mutant name SEQ ID NO sequence VL-S39N 201 EIVMTQSPGTLSLSPGERATLSCRASQSVSSNYLAWYQQKPGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQQSSSPPTFGGGTKVEIK VL-Y40N 202 EIVMTQSPGTLSLSPGERATLSCRASQSVSSSNLAWYQQKPGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQQSSSPPTFGGGTKVEIK VL-Y57S 203 EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLISGASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQQSSSPPTFGGGTKVEIK VL-N69S 204 EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQQSSSPPTFGGGTKVEIK

在一些實施例中，抗TfR1抗體包含與SEQ ID NO:100包含至少75%、80%、85%、90%、95%、96%、97%、98%、99%或100%一致性之VH。在一些實施例中，抗TfR1抗體包含與SEQ ID NO:200包含至少75%、80%、85%、90%、95%、96%、97%、98%、99%或100%一致性之VL。在一些實施例中，抗TfR1抗體包含與SEQ ID NO:100包含至少75%、80%、85%、90%、95%、96%、97%、98%、99%或100%一致性之VH，及與SEQ ID NO:200包含至少75%、80%、85%、90%、95%、96%、97%、98%、99%或100%一致性之VL。在一些實施例中，由一致性百分比賦予之變異不在抗體X VL之位置Y57及/或S83 (根據SEQ ID NO:200之AHo編號)處。在一些實施例中，VL包含Y57 (或其保守取代，例如Y57S)及/或S83 (或其保守取代) (根據SEQ ID NO:200之AHo編號)。In some embodiments, the anti-TfR1 antibody comprises a VH comprising at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 100. In some embodiments, the anti-TfR1 antibody comprises a VL comprising at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 200. In some embodiments, the anti-TfR1 antibody comprises a VH comprising at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 100, and a VL comprising at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 200. In some embodiments, the variation conferred by the percent identity is not at position Y57 and/or S83 (according to the AHo numbering of SEQ ID NO: 200) of the VL of Antibody X. In some embodiments, the VL comprises Y57 (or a conservative substitution thereof, e.g., Y57S) and/or S83 (or a conservative substitution thereof) (according to the AHo numbering of SEQ ID NO: 200).

在一些實施例中，抗TfR1抗體包含與 表3中所列出之序列包含至少75%、80%、85%、90%、95%、96%、97%、98%、99%或100%一致性的VH及與 表4中所列出之序列包含至少75%、80%、85%、90%、95%、96%、97%、98%、99%或100%一致性的VL，其中VH及VL與親代抗體X VH及VL序列不一致。在一些實施例中，由一致性百分比賦予之變異不在 表3或 表4中所列出之序列之突變位置處。舉例而言，在一些實施例中，抗TfR1抗體包含與SEQ ID NO:101包含至少75%、80%、85%、90%、95%、96%、97%、98%、99%或100%一致性之VH，及與SEQ ID NO:200包含至少75%、80%、85%、90%、95%、96%、97%、98%、99%或100%一致性之VL，其中VH與親代抗體X VH序列不同，且其中VH包含S40A取代(根據SEQ ID NO:100之AHo編號)。在一些實施例中，由一致性百分比賦予之變異不在抗體X VL之位置Y57及/或S83 (根據SEQ ID NO:200之AHo編號)處。在一些實施例中，VL包含Y57 (或其保守取代，例如Y57S)及/或S83 (或其保守取代) (根據SEQ ID NO:200之AHo編號)。 In some embodiments, the anti-TfR1 antibody comprises a VH comprising at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a sequence listed in Table 3 and a VL comprising at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a sequence listed in Table 4 , wherein the VH and VL are not identical to the VH and VL sequences of the parent antibody X. In some embodiments, the variation conferred by the percent identity is not at the mutation position of the sequence listed in Table 3 or Table 4 . For example, in some embodiments, the anti-TfR1 antibody comprises a VH comprising at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 101, and a VL comprising at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 200, wherein the VH differs from the parent antibody X VH sequence, and wherein the VH comprises a S40A substitution (according to the AHo numbering of SEQ ID NO: 100). In some embodiments, the variation conferred by the percent identity is not at position Y57 and/or S83 (according to the AHo numbering of SEQ ID NO: 200) of the Antibody X VL. In some embodiments, VL comprises Y57 (or a conservative substitution thereof, such as Y57S) and/or S83 (or a conservative substitution thereof) (according to the AHo numbering of SEQ ID NO: 200).

在一些實施例中，抗TfR1抗體包含：包含如下胺基酸序列之重鏈可變區，該胺基酸序列具有本文所述之抗體X親代抗體之三個VH CDR且與SEQ ID NO:100具有至少75%、80%、85%、90%、95%、96%、97%、98%、99%或100%一致性；及包含如下胺基酸序列之輕鏈可變區，該胺基酸序列具有本文所述之抗體X親代抗體之三個VL CDR且與SEQ ID NO:200具有至少75%、80%、85%、90%、95%、96%、97%、98%、99%或100%一致性。在一些實施例中，由一致性百分比賦予之變異不在抗體X VL之位置Y57及/或S83 (根據SEQ ID NO:200之AHo編號)處。在一些實施例中，VL包含Y57 (或其保守取代，例如Y57S)及/或S83 (或其保守取代) (根據SEQ ID NO:200之AHo編號)。In some embodiments, the anti-TfR1 antibody comprises: a heavy chain variable region comprising an amino acid sequence having three VH CDRs of the Antibody X parent antibody described herein and having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 100; and a light chain variable region comprising an amino acid sequence having three VL CDRs of the Antibody X parent antibody described herein and having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 200. In some embodiments, the variation conferred by the percent identity is not at position Y57 and/or S83 of the Antibody X VL (according to the AHo numbering of SEQ ID NO: 200). In some embodiments, VL comprises Y57 (or a conservative substitution thereof, such as Y57S) and/or S83 (or a conservative substitution thereof) (according to the AHo numbering of SEQ ID NO: 200).

在一些實施例中，抗TfR1抗體包含：包含如下胺基酸序列之重鏈可變區，該胺基酸序列具有本文所述之任何抗體X突變體抗體之三個VH CDR且與 表3中所列出之VH序列中之任一者具有至少75%、80%、85%、90%、95%、96%、97%、98%、99%或100%一致性；及包含如下胺基酸序列之輕鏈可變區，該胺基酸序列具有本文所述之任何抗體X突變體抗體之三個VL CDR且與 表4中所列出之序列中之任一者具有至少75%、80%、85%、90%、95%、96%、97%、98%、99%或100%一致性。在一些實施例中，由一致性百分比賦予之變異不在抗體X VL之位置Y57及/或S83 (根據SEQ ID NO:200之AHo編號)處。在一些實施例中，VL包含Y57 (或其保守取代，例如Y57S)及/或S83 (或其保守取代) (根據SEQ ID NO:200之AHo編號)。 In some embodiments, the anti-TfR1 antibody comprises: a heavy chain variable region comprising an amino acid sequence having three VH CDRs of any Antibody X mutant antibody described herein and having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of the VH sequences listed in Table 3 ; and a light chain variable region comprising an amino acid sequence having three VL CDRs of any Antibody X mutant antibody described herein and having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of the sequences listed in Table 4. In some embodiments, the variation conferred by the percent identity is not at position Y57 and/or S83 (according to the AHo numbering of SEQ ID NO: 200) of the VL of Antibody X. In some embodiments, the VL comprises Y57 (or a conservative substitution thereof, e.g., Y57S) and/or S83 (or a conservative substitution thereof) (according to the AHo numbering of SEQ ID NO: 200).

在抗TfR1抗體之一些實施例中： VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)，其中VL包含Y57S保守取代； VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)，其中VL包含SEQ ID NO: 203之胺基酸序列； VH CDR1包含胺基酸序列GFTFSSYAMN (SEQ ID NO:9)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)，其中VL包含Y57S保守取代； VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)，其中VL包含SEQ ID NO: 203之胺基酸序列； VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSNYLA (SEQ ID NO:15)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISSSSSSIYYADSVKG (SEQ ID NO:11)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KSRAGDFDY (SEQ ID NO:12)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYSAGDFDY (SEQ ID NO:13)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRYGDFDY (SEQ ID NO:14)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISASSSSIYYADSVKG (SEQ ID NO:25)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；VH CDR3包含胺基酸序列KYSAGDFDY (SEQ ID NO:13)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSNYLA (SEQ ID NO:15)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSNLA (SEQ ID NO:16)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)；或 VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASSRAT (SEQ ID NO:17)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)。在一些實施例中，VL包含Y57 (或其保守取代，例如Y57S)及/或S83 (或其保守取代) (根據SEQ ID NO:200之AHo編號)。 In some embodiments of anti-TfR1 antibodies: VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8), wherein VL comprises the Y57S conservative substitution; VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8), wherein VL comprises SEQ ID NO: 203; VH CDR1 comprises the amino acid sequence GFTFSSYAMN (SEQ ID NO:9); VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8), wherein VL comprises the conservative substitution Y57S; VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); ID NO:3); VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8), wherein VL comprises the amino acid sequence of SEQ ID NO: 203; VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSNYLA (SEQ ID NO:15); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISSSSSSIYYADSVKG (SEQ ID NO:11); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KSRAGDFDY (SEQ ID NO:12); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYSAGDFDY (SEQ ID NO:13); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRYGDFDY (SEQ ID NO:14); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISASSSSIYYADSVKG (SEQ ID NO:25); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); VH CDR3 comprises the amino acid sequence KYSAGDFDY (SEQ ID NO:13); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSNYLA (SEQ ID NO:15); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSNLA (SEQ ID NO:16); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); or VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASSRAT (SEQ ID NO:17); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8). In some embodiments, VL comprises Y57 (or a conservative substitution thereof, such as Y57S) and/or S83 (or a conservative substitution thereof) (according to the AHo numbering of SEQ ID NO:200).

在抗TfR1抗體之一些實施例中： VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)，其中VL包含Y57S保守取代； VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)，其中VL包含SEQ ID NO: 203之胺基酸序列； VH CDR1包含胺基酸序列SYAMN (SEQ ID NO:24)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)，其中VL包含Y57S保守取代； VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)，其中VL包含SEQ ID NO: 203之胺基酸序列； VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSNYLA (SEQ ID NO:15)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISSSSSSIYYADSVKG (SEQ ID NO:11)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KSRAGDFDY (SEQ ID NO:12)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYSAGDFDY (SEQ ID NO:13)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRYGDFDY (SEQ ID NO:14)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISASSSSIYYADSVKG (SEQ ID NO:25)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；VH CDR3包含胺基酸序列KYSAGDFDY (SEQ ID NO:13)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSNYLA (SEQ ID NO:15)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSNLA (SEQ ID NO:16)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)；或 VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASSRAT (SEQ ID NO:17)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)。在一些實施例中，VL包含Y57 (或其保守取代，例如Y57S)及/或S83 (或其保守取代) (根據SEQ ID NO:200之AHo編號)。 In some embodiments of anti-TfR1 antibodies: VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8), wherein VL comprises the Y57S conservative substitution; VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8), wherein VL comprises the amino acid sequence of SEQ ID NO: 203; VH CDR1 comprises the amino acid sequence SYAMN (SEQ ID NO:24); VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8), wherein VL comprises the conservative substitution Y57S; VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8), wherein VL comprises the amino acid sequence of SEQ ID NO: 203; VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSNYLA (SEQ ID NO:15); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISSSSSSIYYADSVKG (SEQ ID NO:11); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KSRAGDFDY (SEQ ID NO: 12); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO: 6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO: 7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO: 8); VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO: 23); VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO: 4); VH CDR3 comprises the amino acid sequence KYSAGDFDY (SEQ ID NO: 13); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO: 6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO: 7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO: 8); VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRYGDFDY (SEQ ID NO:14); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISASSSSIYYADSVKG (SEQ ID NO:25); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); VH CDR3 comprises the amino acid sequence KYSAGDFDY (SEQ ID NO:13); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSNYLA (SEQ ID NO:15); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSNLA (SEQ ID NO:16); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); or VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); VL CDR2 comprises the amino acid sequence GASSRAT (SEQ ID NO:17); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8). In some embodiments, VL comprises Y57 (or a conservative substitution thereof, such as Y57S) and/or S83 (or a conservative substitution thereof) (according to the AHo numbering of SEQ ID NO:200).

在一些實施例中，抗TfR1抗體VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)。在一些實施例中，VL包含Y57 (或其保守取代，例如Y57S)及/或S83 (或其保守取代) (根據SEQ ID NO:200之AHo編號)。In some embodiments, the anti-TfR1 antibody VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO: 3); VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO: 4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO: 5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO: 6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO: 7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO: 8). In some embodiments, VL comprises Y57 (or a conservative substitution thereof, such as Y57S) and/or S83 (or a conservative substitution thereof) (according to the AHo numbering of SEQ ID NO: 200).

在一些實施例中，抗TfR1抗體VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)。在一些實施例中，VL包含Y57 (或其保守取代，例如Y57S)及/或S83 (或其保守取代) (根據SEQ ID NO:200之AHo編號)。In some embodiments, the anti-TfR1 antibody VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO: 23); VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO: 4); VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO: 5); VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO: 6); VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO: 7); and VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO: 8). In some embodiments, VL comprises Y57 (or a conservative substitution thereof, such as Y57S) and/or S83 (or a conservative substitution thereof) (according to the AHo numbering of SEQ ID NO: 200).

在抗TfR1抗體之一些實施例中： VH包含SEQ ID NO:100之胺基酸序列且VL包含SEQ ID NO:200之胺基酸序列； VH包含SEQ ID NO:101之胺基酸序列且VL包含SEQ ID NO:200之胺基酸序列； VH包含SEQ ID NO:102之胺基酸序列且VL包含SEQ ID NO:200之胺基酸序列； VH包含SEQ ID NO:102之胺基酸序列且VL包含SEQ ID NO:201之胺基酸序列； VH包含SEQ ID NO:102之胺基酸序列且VL包含SEQ ID NO:203之胺基酸序列； VH包含SEQ ID NO:103之胺基酸序列且VL包含SEQ ID NO:200之胺基酸序列； VH包含SEQ ID NO:104之胺基酸序列且VL包含SEQ ID NO:200之胺基酸序列； VH包含SEQ ID NO:105之胺基酸序列且VL包含SEQ ID NO:200之胺基酸序列； VH包含SEQ ID NO:106之胺基酸序列且VL包含SEQ ID NO:200之胺基酸序列； VH包含SEQ ID NO:107之胺基酸序列且VL包含SEQ ID NO:200之胺基酸序列； VH包含SEQ ID NO:108之胺基酸序列且VL包含SEQ ID NO:200之胺基酸序列； VH包含SEQ ID NO:100之胺基酸序列且VL包含SEQ ID NO:201之胺基酸序列； VH包含SEQ ID NO:100之胺基酸序列且VL包含SEQ ID NO:202之胺基酸序列； VH包含SEQ ID NO:100之胺基酸序列且VL包含SEQ ID NO:203之胺基酸序列；或 VH包含SEQ ID NO:100之胺基酸序列且VL包含SEQ ID NO:204之胺基酸序列。 In some embodiments of anti-TfR1 antibodies: VH comprises the amino acid sequence of SEQ ID NO: 100 and VL comprises the amino acid sequence of SEQ ID NO: 200; VH comprises the amino acid sequence of SEQ ID NO: 101 and VL comprises the amino acid sequence of SEQ ID NO: 200; VH comprises the amino acid sequence of SEQ ID NO: 102 and VL comprises the amino acid sequence of SEQ ID NO: 200; VH comprises the amino acid sequence of SEQ ID NO: 102 and VL comprises the amino acid sequence of SEQ ID NO: 201; VH comprises the amino acid sequence of SEQ ID NO: 102 and VL comprises the amino acid sequence of SEQ ID NO: 203; VH comprises the amino acid sequence of SEQ ID NO: 103 and VL comprises the amino acid sequence of SEQ ID NO: 200; VH comprises the amino acid sequence of SEQ ID NO: 104 and VL comprises the amino acid sequence of SEQ ID NO:200; VH comprises the amino acid sequence of SEQ ID NO:105 and VL comprises the amino acid sequence of SEQ ID NO:200; VH comprises the amino acid sequence of SEQ ID NO:106 and VL comprises the amino acid sequence of SEQ ID NO:200; VH comprises the amino acid sequence of SEQ ID NO:107 and VL comprises the amino acid sequence of SEQ ID NO:200; VH comprises the amino acid sequence of SEQ ID NO:108 and VL comprises the amino acid sequence of SEQ ID NO:200; VH comprises the amino acid sequence of SEQ ID NO:100 and VL comprises the amino acid sequence of SEQ ID NO:201; VH comprises the amino acid sequence of SEQ ID NO:100 and VL comprises the amino acid sequence of SEQ ID NO:202; VH comprises the amino acid sequence of SEQ ID NO:100 and VL comprises the amino acid sequence of SEQ ID NO:203; or VH comprises the amino acid sequence of SEQ ID NO: 100 and VL comprises the amino acid sequence of SEQ ID NO: 204.

在一些實施例中，抗TfR1抗體包含有包含SEQ ID NO:100之VH及包含SEQ ID NO:200之VL。在一些實施例中，抗TfR1抗體包含由SEQ ID NO:100組成之VH及由SEQ ID NO:200組成之VL。 抗TFR1抗體之恆定區 In some embodiments, the anti-TfR1 antibody comprises a VH comprising SEQ ID NO: 100 and a VL comprising SEQ ID NO: 200. In some embodiments, the anti-TfR1 antibody comprises a VH consisting of SEQ ID NO: 100 and a VL consisting of SEQ ID NO: 200. Constant Region of Anti-TfR1 Antibody

在一些實施例中，本文所述之抗TfR1抗體之可變區與恆定區融合。恆定區具有恆定重鏈(CH)域(例如CH1、鉸鏈、CH2及/或CH3域或其任何組合)及恆定輕鏈(CL)域。在一些實施例中，CH域來自IgG1分子或IgG4分子。在一些實施例中，CH域來自IgG1分子。在一些實施例中，CH域來自IgG2分子、IgG3分子或IgG分子。本文所述之抗TfR1抗體之VH可與如下表 5中所示之以下恆定重鏈(CH)構築體中之任一者融合。本文所述之抗TfR1抗體之VL可與如下 表5中所示之以下恆定輕鏈(CL)構築體中之任一者融合。在一些實施例中，鉸鏈區係此項技術中已知之任何鉸鏈區。在一些實施例中，鉸鏈區係天然存在的，例如來自天然存在之IgG1、IgG2、IgG3或IgG4分子。在其他實施例中，鉸鏈區相對於天然鉸鏈含有修飾。 In some embodiments, the variable region of the anti-TfR1 antibody described herein is fused to a constant region. The constant region has a constant heavy chain (CH) domain (e.g., CH1, hinge, CH2 and/or CH3 domain or any combination thereof) and a constant light chain (CL) domain. In some embodiments, the CH domain is from an IgG1 molecule or an IgG4 molecule. In some embodiments, the CH domain is from an IgG1 molecule. In some embodiments, the CH domain is from an IgG2 molecule, an IgG3 molecule or an IgG molecule. The VH of the anti-TfR1 antibody described herein may be fused with any of the following constant heavy chain (CH) constructs as shown in Table 5 below. The VL of the anti-TfR1 antibody described herein may be fused with any of the following constant light chain (CL) constructs as shown in Table 5 below. In some embodiments, the hinge region is any hinge region known in the art. In some embodiments, the hinge region is naturally occurring, such as from a naturally occurring IgG1, IgG2, IgG3, or IgG4 molecule. In other embodiments, the hinge region contains modifications relative to a natural hinge.

在一些實施例中，本揭示案之抗TfR1抗體為其中至少一或多個恆定區已經修飾或缺失之抗體。在一些實施例中，抗體可包含對重鏈恆定域(CH1、CH2或CH3)及/或輕鏈恆定區(CL)之一或多個修飾。在一些實施例中，經修飾抗體之重鏈恆定區包含至少一個人類恆定區。在一些實施例中，經修飾抗體之重鏈恆定區包含多於一個人類恆定區。在一些實施例中，VH與此項技術中已知之任何CH1構築體融合，且VL與此項技術中已知之任何CL融合。在一些實施例中，構築體之恆定輕鏈(CL)為天然存在之人類κ恆定區。在一些實施例中，對恆定區之修飾包含一或多個區域中一或多個胺基酸之添加、缺失或取代。在一些實施例中，一或多個區域自經修飾抗體之恆定區部分或完全缺失。在一些實施例中，整個CH2域及CH3域已自抗體移除。在一些實施例中，缺失之恆定區經短胺基酸間隔子替代，該短胺基酸間隔子提供一些通常由不存在之恆定區賦予之分子可撓性。在一些實施例中，經修飾抗體包含與抗體之鉸鏈區直接融合之CH1域。在一些實施例中，經修飾抗體包含與Fc底部融合之Fab。In some embodiments, the anti-TfR1 antibody of the present disclosure is an antibody in which at least one or more constant regions have been modified or deleted. In some embodiments, the antibody may include one or more modifications to the heavy chain constant domain (CH1, CH2 or CH3) and/or the light chain constant region (CL). In some embodiments, the heavy chain constant region of the modified antibody includes at least one human constant region. In some embodiments, the heavy chain constant region of the modified antibody includes more than one human constant region. In some embodiments, VH is fused to any CH1 construct known in this technology, and VL is fused to any CL known in this technology. In some embodiments, the constant light chain (CL) of the construct is a naturally occurring human κ constant region. In some embodiments, the modification of the constant region comprises the addition, deletion or substitution of one or more amino acids in one or more regions. In some embodiments, one or more regions are partially or completely missing from the constant region of the modified antibody. In some embodiments, the entire CH2 domain and CH3 domain have been removed from the antibody. In some embodiments, the missing constant region is replaced by a short amino acid spacer, which provides some molecular flexibility usually given by the non-existent constant region. In some embodiments, the modified antibody comprises a CH1 domain directly fused to the hinge region of the antibody. In some embodiments, the modified antibody comprises a Fab fused to the bottom of the Fc.

在一些實施例中，本揭示案之抗TfR1抗體含有連接子(例如，如下 表5中所示之連接子)。在一些實施例中，連接子位於本揭示案之抗TfR1抗體之Fc區與Fab區之間。 表5：恆定區、鉸鏈序列及連接子序列 域 序列 IgG1 或IgG4 CH1 IgG1 CH1 (無鉸鏈) (SEQ ID NO: 300) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV IgG1 CH1 (鉸鏈加下劃線) (SEQ ID NO: 301) ASTKGPSVFPLAPSSKSTSGGTAALG C LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVN HKPSNTKVDKKV EP KS CDKTHT CPP IgG1 CH1 (無鉸鏈) (SEQ ID NO: 302) ASTKGPSVFPLAPSSKSTSGGTAALG C LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVN HKPSNTKVDKRV IgG1 CH1 (鉸鏈加下劃線) (SEQ ID NO: 303) ASTKGPSVFPLAPSSKSTSGGTAALG C LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVN HKPSNTKVDKRV ESKYGPP CP IgG1 CH1 (無鉸鏈) (SEQ ID NO: 304) ASTKGPSVFPLAPSSKSTSGGTAALG C LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVN HKPSNTKVDKKV IgG1 CH1 (鉸鏈加下劃線) (SEQ ID NO: 305) ASTKGPSVFPLAPSSKSTSGGTAALG C LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVN HKPSNTKVDKKV EP KS C IgG4 CH1 (無鉸鏈) (SEQ ID NO: 306) ASTKGPSVFPLAP CSRSTSESTAALG C LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYT CNVD HKPSNTKVDKRV IgG4 CH1 (鉸鏈加下劃線) (SEQ ID NO: 307) ASTKGPSVFPLAP CSRSTSESTAALG C LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYT CNVD HKPSNTKVDKRV ES Fc hIgG1.agly DelPG (SEQ ID NO: 308) KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS Fc hIgG1.agly DelG (SEQ ID NO: 309) KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP Fc hIgG1.agly (SEQ ID NO: 310) KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 鉸鏈Fc hIgG1.agly (SEQ ID NO: 311) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 鉸鏈Fc hIgG1.agly(杵S) (SEQ ID NO: 312) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Fc hIgG1.agly(杵S) (SEQ ID NO: 313) KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 鉸鏈Fc hIgG1.agly (臼S) (SEQ ID NO: 314) KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 鉸鏈區 IgG1鉸鏈A (SEQ ID NO: 315) EPKSCDKTHTCPPCPAPELLGGP IgG1鉸鏈B (SEQ ID NO: 316) EP KS CDKTHT CPP IgG1鉸鏈C (SEQ ID NO: 317) EP KS C IgG4鉸鏈D (SEQ ID NO: 318) ESKYGPPCPPCPAPEFLGGP IgG4鉸鏈E (SEQ ID NO: 319) ESKYGPP CP IgG4鉸鏈F ES IgG1鉸鏈G (SEQ ID NO: 320) EPKS IgG1鉸鏈H (SEQ ID NO: 321) EPKSCD IgG1鉸鏈I (SEQ ID NO: 322) EPKSCDK IgG1鉸鏈J (SEQ ID NO: 323) EPKSCDKT IgG1鉸鏈K (SEQ ID NO: 324) EPKSCDKTH IgG1鉸鏈L (SEQ ID NO: 325) EPKSCDKTHT IgG1鉸鏈M (SEQ ID NO: 326) EPKSCDKTHTC IgG1鉸鏈N (SEQ ID NO: 327) EPKSCDKTHTCP IgG1鉸鏈O (SEQ ID NO: 328) EPKSCDKTHTCPPC IgG1鉸鏈P (SEQ ID NO: 329) EPKSCDKTHTCPPCP IgG1鉸鏈Q (SEQ ID NO: 330) EPKSCDKTHTCPPCPA IgG1鉸鏈R (SEQ ID NO: 331) EPKSCDKTHTCPPCPAP IgG1鉸鏈S (SEQ ID NO: 332) EPKSCDKTHTCPPCPAPE IgG1鉸鏈T (SEQ ID NO: 333) EPKSCDKTHTCPPCPAPEL IgG1鉸鏈U (SEQ ID NO: 334) EPKSCDKTHTCPPCPAPELL IgG1鉸鏈V (SEQ ID NO: 335) EPKSCDKTHTCPPCPAPELLG IgG1鉸鏈W (SEQ ID NO: 336) EPKSCDKTHTCPPCPAPELLGG IgG4鉸鏈X ESK IgG4鉸鏈Y (SEQ ID NO: 337) ESKY IgG4鉸鏈Z (SEQ ID NO: 338) ESKYG IgG4鉸鏈ZA (SEQ ID NO: 339) ESKYGP IgG4鉸鏈ZB (SEQ ID NO: 340) ESKYGPP IgG4鉸鏈ZC (SEQ ID NO: 341) ESKYGPPC IgG4鉸鏈ZD (SEQ ID NO: 342) ESKYGPPCPP IgG4鉸鏈ZE (SEQ ID NO: 343) ESKYGPPCPPC IgG4鉸鏈ZF (SEQ ID NO: 344) ESKYGPPCPPCP IgG4鉸鏈ZG (SEQ ID NO: 345) ESKYGPPCPPCPA IgG4鉸鏈ZH (SEQ ID NO: 346) ESKYGPPCPPCPAP IgG4鉸鏈ZI (SEQ ID NO: 347) ESKYGPPCPPCPAPE IgG4鉸鏈ZJ (SEQ ID NO: 348) ESKYGPPCPPCPAPEF IgG4鉸鏈ZK (SEQ ID NO: 349) ESKYGPPCPPCPAPEFL IgG4鉸鏈ZL (SEQ ID NO: 350) ESKYGPPCPPCPAPEFLG IgG4鉸鏈ZM (SEQ ID NO: 351) ESKYGPPCPPCPAPEFLGG IgG4鉸鏈ZN (SEQ ID NO: 352) ESKYGPPCPPCPAPEFLGGP CL 域 人類κ CL (SEQ ID NO: 353) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 人類κ CL (Δ C214) (SEQ ID NO: 354) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE 連接子 連接子S S 連接子SG 2 SGG 連接子SG 4(SEQ ID NO: 355) SGGGG 連接子SG 4SG 4(SEQ ID NO: 356) SGGGGSGGGG 連接子SG 4SG 4SG 4(SEQ ID NO: 357) SGGGGSGGGGSGGGG 連接子G 3SG 5(SEQ ID NO: 358) GGGSGGGGG 連接子G 4SG 4SG 4S(SEQ ID NO: 359) GGGGSGGGGSGGGGS 連接子TG TG In some embodiments, the anti-TfR1 antibody of the present disclosure contains a linker (e.g., a linker as shown in Table 5 below). In some embodiments, the linker is located between the Fc region and the Fab region of the anti-TfR1 antibody of the present disclosure. Table 5: Constant region, hinge sequence and linker sequence domain sequence IgG1 or IgG4 CH1 IgG1 CH1 (no hinge) (SEQ ID NO: 300) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV IgG1 CH1 (hinge underlined) (SEQ ID NO: 301) ASTKGPSVFPLAPSSKSTSGGTAALG C LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI C NVN HKPSNTKVDK KV EP K S C DKTHT C PP IgG1 CH1 (no hinge) (SEQ ID NO: 302) ASTKGPSVFPLAPSSKSTSGGTAALG C LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI C NVN HKPSNTKVDKR V IgG1 CH1 (hinge underlined) (SEQ ID NO: 303) ASTKGPSVFPLAPSSKSTSGGTAALG C LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI C NVN HKPSNTKVDKR V ESKYGPP C P IgG1 CH1 (no hinge) (SEQ ID NO: 304) ASTKGPSVFPLAPSSKSTSGGTAALG C LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI C NVN HKPSNTKVDK KV IgG1 CH1 (hinge underlined) (SEQ ID NO: 305) ASTKGPSVFPLAPSSKSTSGGTAALG C LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI C NVN HKPSNTKVDK KV EP K S C IgG4 CH1 (no hinge) (SEQ ID NO: 306) ASTKGPSVFPLAP C SRSTSESTAALG C LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYT C NVD HKPSNTKVDK RV IgG4 CH1 (hinge underlined) (SEQ ID NO: 307) ASTKGPSVFPLAP C SRSTSESTAALG C LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYT C NVD HKPSNTKVDK RV ES Fc hIgG1.agly DelPG (SEQ ID NO: 308) KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS Fc hIgG1.aglyDelG (SEQ ID NO: 309) KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP Fc hIgG1.agly (SEQ ID NO: 310) KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Hinge Fc hIgG1.agly (SEQ ID NO: 311) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Hinge Fc hIgG1.agly (knob S) (SEQ ID NO: 312) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Fc hIgG1.agly (knob S) (SEQ ID NO: 313) KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Hinge Fc hIgG1.agly (Hydroxy) (SEQ ID NO: 314) KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Hinge area IgG1 hinge A (SEQ ID NO: 315) EPKSCDKTHTCPPCPAPELLGGP IgG1 hinge B (SEQ ID NO: 316) EP K S C DKTHT C PP IgG1 hinge C (SEQ ID NO: 317) EP K S C IgG4 hinge D (SEQ ID NO: 318) ESKYGPPCPPCPAPEFLGGP IgG4 hinge E (SEQ ID NO: 319) ESKYGPP C P IgG4 hinge F ES IgG1 hinge G (SEQ ID NO: 320) EPKS IgG1 hinge H (SEQ ID NO: 321) EPKSCD IgG1 hinge I (SEQ ID NO: 322) EPKSCDK IgG1 hinge J (SEQ ID NO: 323) EPKSCDKT IgG1 hinge K (SEQ ID NO: 324) EPKSCDKTH IgG1 hinge L (SEQ ID NO: 325) EPKSCDKTHT IgG1 hinge M (SEQ ID NO: 326) EPKSCDKTHTC IgG1 hinge N (SEQ ID NO: 327) EPKSCDKTHTCP IgG1 hinge O (SEQ ID NO: 328) EPKSCDKTHTCPPC IgG1 hinge P (SEQ ID NO: 329) EPKSCDKTHTCPPCP IgG1 hinge Q (SEQ ID NO: 330) EPKSCDKTHTCPPCPA IgG1 hinge R (SEQ ID NO: 331) EPKSCDKTHTCPPCPAP IgG1 hinge S (SEQ ID NO: 332) EPKSCDKTHTCPPCPAPE IgG1 hinge T (SEQ ID NO: 333) EPKSCDKTHTCPPCPAPEL IgG1 hinge U (SEQ ID NO: 334) EPKSCDKTHTCPPCPAPELL IgG1 hinge V (SEQ ID NO: 335) EPKSCDKTHTCPPCPAPELLG IgG1 hinge W (SEQ ID NO: 336) EPKSCDKTHTCPPCPAPELLGG IgG4 Hinge X ESK IgG4 hinge Y (SEQ ID NO: 337) ESKY IgG4 hinge Z (SEQ ID NO: 338) ESKYG IgG4 hinge ZA (SEQ ID NO: 339) ESKYGP IgG4 hinge ZB (SEQ ID NO: 340) ESKYGPP IgG4 hinge ZC (SEQ ID NO: 341) ESKYGPPC IgG4 hinge ZD (SEQ ID NO: 342) ESKYGPPCPP IgG4 hinge ZE (SEQ ID NO: 343) ESKYGPPCPPC IgG4 hinge ZF (SEQ ID NO: 344) ESKYGPPCPPCP IgG4 hinge ZG (SEQ ID NO: 345) ESKYGPPCPPCPA IgG4 hinge ZH (SEQ ID NO: 346) ESKYGPPCPPCPAP IgG4 hinge ZI (SEQ ID NO: 347) ESKYGPPCPPCPAPE IgG4 hinge ZJ (SEQ ID NO: 348) ESKYGPPCPPCPAPEF IgG4 hinge ZK (SEQ ID NO: 349) ESKYGPPCPPCPAPEFL IgG4 hinge ZL (SEQ ID NO: 350) ESKYGPPCPPCPAPEFLG IgG4 hinge ZM (SEQ ID NO: 351) ESKYGPPCPPCPAPEFLGG IgG4 hinge ZN (SEQ ID NO: 352) ESKYGPPCPPCPAPEFLGGP CL domain Human κ CL (SEQ ID NO: 353) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Human κ CL (Δ C214) (SEQ ID NO: 354) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE Connector Connector S S Connector SG 2 SGG Linker SG 4 (SEQ ID NO: 355) SGGGG Linker SG 4 SG 4 (SEQ ID NO: 356) SGGGGSGGGG Linker SG 4 SG 4 SG 4 (SEQ ID NO: 357) SGGGGSGGGGSGGGG Linker G 3 SG 5 (SEQ ID NO: 358) GGGSGGGGG Linker G 4 SG 4 SG 4 S (SEQ ID NO: 359) GGGGSGGGGSGGGGS Connector TG TG

可與本文所述之抗體可變區組合之其他示範性恆定區(例如鉸鏈區)包括但不限於以下文獻中描述之鉸鏈區：Peters SJ等人J Biol Chem. 2012年7月13日;287(29):24525-33；及Heads JT等人Protein Sci. 2012年9月;21(9):1315-22；該等文獻以引用之方式整體併入本文中。Other exemplary constant regions (e.g., hinge regions) that can be combined with the antibody variable regions described herein include, but are not limited to, hinge regions described in Peters SJ et al. J Biol Chem. 2012 Jul 13;287(29):24525-33; and Heads JT et al. Protein Sci. 2012 Sep;21(9):1315-22; which are incorporated herein by reference in their entirety.

在一些實施例中，抗TfR1抗體包含 表6中所述之重鏈。在一些實施例中，抗TfR1抗體包含 表6中所述之輕鏈。在一些實施例中，抗TfR1抗體包含 表6中所述之重鏈及 表6中所述之輕鏈。 表6：抗體X及突變體之重鏈及輕鏈. 名稱 SEQ ID NO 序列 抗體X HC 400 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 抗體X LC 401 EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQQSSSPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC ANTIBODY X Fab片段HC 402 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH 抗體X二價Fc-Fab HC 403 KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 抗體X單價Fab-Fc HC1 (杵) 404 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 抗體X單價Fc-Fab HC1 (杵) 405 KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Fc (臼) 406 KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 抗BACE1/二價抗體X HC (恆定區) 407 ASTKGPSVFPRAPSSKSTSGGTAALGCLVRDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCQVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYELSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 抗BACE1 /單價抗體X HC1 (杵) (恆定區) 408 ASTKGPSVFPRAPSSKSTSGGTAALGCLVRDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCQVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYELSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 抗BACE1 /單價抗體X HC2 (臼) (恆定區) 409 ASTKGPSVFPRAPSSKSTSGGTAALGCLVRDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 抗BACE1 /抗體X LC1 (恆定區) 410 RTVAAPSVFIFPPSDEELKSGTASVQCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSELTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 抗BACE1/抗體X LC2 411 EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQQSSSPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGRASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSRLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 抗體X S442C HC 412 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPG 單價抗體X S442C HC1 (杵) 413 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 單價抗體X S442C HC2 (臼) 414 KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPG In some embodiments, the anti-TfR1 antibody comprises a heavy chain as described in Table 6. In some embodiments, the anti-TfR1 antibody comprises a light chain as described in Table 6. In some embodiments, the anti-TfR1 antibody comprises a heavy chain as described in Table 6 and a light chain as described in Table 6. Table 6: Heavy and light chains of Antibody X and mutants. Name SEQ ID NO sequence Antibody X HC 400 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Antibody X LC 401 EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQQSSSPPTFGGGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC ANTIBODY X Fab fragment HC 402 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDYWGQGT LVTVSSASTKGPSVFPLPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH Antibody X Bivalent Fc-Fab HC 403 KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGG GSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Antibody X Monovalent Fab-Fc HC1 (Pestle) 404 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Antibody X Monovalent Fc-Fab HC1 (Pestle) 405 KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGG GSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Fc (Mortar) 406 KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Anti-BACE1/Bivalent Antibody X HC (Constant Region) 407 ASTKGPSVFPRAPSSKSTSGGTAALGCLVRDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCARKYRAGDFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCQVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYELSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Anti-BACE1 / Monovalent Antibody X HC1 (Pestle) (Constant Region) 408 ASTKGPSVFPRAPSSKSTSGGTAALGCLVRDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCARKYRAGDFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCQVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYELSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Anti-BACE1 / Monovalent Antibody X HC2 (Mouse) (Constant Region) 409 ASTKGPSVFPRAPSSKSTSGGTAALGCLVRDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Anti-BACE1 / Antibody X LC1 (Constant Region) 410 RTVAAPSVFIFPPSDEELKSGTASVQCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSELTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Anti-BACE1/Antibody X LC2 411 EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQQSSSPPTFGGGTKVEI KRTVAAPSVFIFPPSDEQLKSGRASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSRLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Antibody X S442C HC 412 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDYWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPG Monovalent Antibody X S442C HC1 (Pestle) 413 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDYWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Monovalent Antibody X S442C HC2 (Molar) 414 KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPG

在一些情況下，抗TfR1抗體包含重鏈及輕鏈，其中重鏈包含SEQ ID NO:400中所列出之胺基酸序列或由其組成，且輕鏈包含SEQ ID NO:401中所列出之胺基酸序列或由其組成。In some cases, the anti-TfR1 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence listed in SEQ ID NO:400, and the light chain comprises or consists of the amino acid sequence listed in SEQ ID NO:401.

在一些情況下，抗TfR1抗體包含Fab片段。在一些情況下，抗體包含Fab片段，其中Fab片段包含SEQ ID NO:401及402中所列出之胺基酸序列。In some cases, the anti-TfR1 antibody comprises a Fab fragment. In some cases, the antibody comprises a Fab fragment, wherein the Fab fragment comprises the amino acid sequence set forth in SEQ ID NO: 401 and 402.

在一些情況下，抗TfR1抗體包含Fab-Fc。在一些情況下，抗體包含Fab-Fc，其中Fab-Fc包含SEQ ID NO:401、404及406中所列出之胺基酸序列。在一些情況下，抗體包含Fab-Fc，其中Fab-Fc包含SEQ ID NO: 400及401中所列出之胺基酸序列。In some cases, the anti-TfR1 antibody comprises Fab-Fc. In some cases, the antibody comprises Fab-Fc, wherein the Fab-Fc comprises the amino acid sequence listed in SEQ ID NOs: 401, 404, and 406. In some cases, the antibody comprises Fab-Fc, wherein the Fab-Fc comprises the amino acid sequence listed in SEQ ID NOs: 400 and 401.

在一些情況下，抗TfR1抗體包含Fc-Fab。在一些情況下，抗體包含Fc-Fab，其中Fc-Fab包含SEQ ID NO:401、405及406中所列出之胺基酸序列。在一些情況下，抗體包含Fc-Fab，其中Fc-Fab包含SEQ ID NO:401及403中所列出之胺基酸序列。In some cases, the anti-TfR1 antibody comprises an Fc-Fab. In some cases, the antibody comprises an Fc-Fab, wherein the Fc-Fab comprises the amino acid sequence listed in SEQ ID NOs: 401, 405, and 406. In some cases, the antibody comprises an Fc-Fab, wherein the Fc-Fab comprises the amino acid sequence listed in SEQ ID NOs: 401 and 403.

在一些實施例中，抗TfR1抗體包含重鏈，該重鏈與本文所鑑別之重鏈序列具有至少80%、至少85%、至少90%或至少95%序列一致性，其中抗TfR1抗體具有與抗體X之VH及VL一致的VH及VL。在一些實施例中，抗TfR1抗體包含輕鏈，該輕鏈與本文所鑑別之輕鏈序列具有至少80%、至少85%、至少90%或至少95%序列一致性，其中抗TfR1抗體具有與抗體X之VH及VL一致的VH及VL。在一些實施例中，抗TfR1抗體包含重鏈，該重鏈與本文所鑑別之重鏈序列具有至少80%、至少85%、至少90%或至少95%序列一致性；及輕鏈，該輕鏈與本文所鑑別之輕鏈序列具有至少80%、至少85%、至少90%或至少95%序列一致性，其中抗TfR1抗體具有與抗體X之VH及VL一致的VH及VL。In some embodiments, the anti-TfR1 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a heavy chain sequence identified herein, wherein the anti-TfR1 antibody has a VH and VL that are identical to the VH and VL of antibody X. In some embodiments, the anti-TfR1 antibody comprises a light chain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a light chain sequence identified herein, wherein the anti-TfR1 antibody has a VH and VL that are identical to the VH and VL of antibody X. In some embodiments, the anti-TfR1 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90% or at least 95% sequence identity to a heavy chain sequence identified herein; and a light chain having at least 80%, at least 85%, at least 90% or at least 95% sequence identity to a light chain sequence identified herein, wherein the anti-TfR1 antibody has VH and VL that are consistent with the VH and VL of antibody X.

在一些實施例中，抗TfR1抗體包含重鏈，該重鏈與本文所鑑別之重鏈序列具有至少80%、至少85%、至少90%或至少95%序列一致性，其中抗TfR1抗體不具有與抗體X之VH及VL一致的VH及VL。在一些實施例中，抗TfR1抗體包含輕鏈，該輕鏈與本文所鑑別之輕鏈序列具有至少80%、至少85%、至少90%或至少95%序列一致性，其中抗TfR1抗體不具有與抗體X之VH及VL一致的VH及VL。在一些實施例中，抗TfR1抗體包含重鏈，該重鏈與本文所鑑別之重鏈序列具有至少80%、至少85%、至少90%或至少95%序列一致性；及輕鏈，該輕鏈與本文所鑑別之輕鏈序列具有至少80%、至少85%、至少90%或至少95%序列一致性，其中抗TfR1抗體不具有與抗體X之VH及VL一致的VH及VL。In some embodiments, the anti-TfR1 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a heavy chain sequence identified herein, wherein the anti-TfR1 antibody does not have a VH and VL that are identical to the VH and VL of antibody X. In some embodiments, the anti-TfR1 antibody comprises a light chain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a light chain sequence identified herein, wherein the anti-TfR1 antibody does not have a VH and VL that are identical to the VH and VL of antibody X. In some embodiments, the anti-TfR1 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a heavy chain sequence identified herein; and a light chain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a light chain sequence identified herein, wherein the anti-TfR1 antibody does not have VH and VL that are identical to the VH and VL of antibody X.

在一些實施例中，抗TfR1抗體包含重鏈，該重鏈具有對本文所鑑別之重鏈序列具有一個、兩個或三個或更多個修飾(例如取代、缺失或插入)之胺基酸序列，其中抗TfR1抗體具有與抗體X之VH及VL一致的VH及VL。在一些實施例中，抗TfR1抗體包含輕鏈，該輕鏈具有對本文所鑑別之輕鏈序列具有一個、兩個或三個或更多個修飾(例如取代、缺失或插入)之胺基酸序列，其中抗TfR1抗體具有與抗體X之VH及VL一致的VH及VL。在一些實施例中，抗TfR1抗體包含重鏈，該重鏈具有對本文所鑑別之重鏈序列具有一個、兩個或三個或更多個修飾(例如取代、缺失或插入)之胺基酸序列；及輕鏈，該輕鏈具有對本文所鑑別之輕鏈序列具有一個、兩個或三個或更多個修飾(例如取代、缺失或插入)之胺基酸序列，其中抗TfR1抗體具有與抗體X之VH及VL一致的VH及VL。In some embodiments, the anti-TfR1 antibody comprises a heavy chain having an amino acid sequence with one, two, or three or more modifications (e.g., substitutions, deletions, or insertions) to a heavy chain sequence identified herein, wherein the anti-TfR1 antibody has a VH and VL that are consistent with the VH and VL of antibody X. In some embodiments, the anti-TfR1 antibody comprises a light chain having an amino acid sequence with one, two, or three or more modifications (e.g., substitutions, deletions, or insertions) to a light chain sequence identified herein, wherein the anti-TfR1 antibody has a VH and VL that are consistent with the VH and VL of antibody X. In some embodiments, the anti-TfR1 antibody comprises a heavy chain having an amino acid sequence having one, two, or three or more modifications (e.g., substitutions, deletions, or insertions) to the heavy chain sequence identified herein; and a light chain having an amino acid sequence having one, two, or three or more modifications (e.g., substitutions, deletions, or insertions) to the light chain sequence identified herein, wherein the anti-TfR1 antibody has VH and VL that are consistent with the VH and VL of antibody X.

在一些實施例中，抗TfR1抗體包含重鏈，該重鏈具有對本文所鑑別之重鏈序列具有一個、兩個或三個或更多個修飾(例如取代、缺失或插入)之胺基酸序列，其中抗TfR1抗體不具有與抗體X之VH及VL一致的VH及VL。在一些實施例中，抗TfR1抗體包含輕鏈，該輕鏈具有對本文所鑑別之輕鏈序列具有一個、兩個或三個或更多個修飾(例如取代、缺失或插入)之胺基酸序列，其中抗TfR1抗體不具有與抗體X之VH及VL一致的VH及VL。在一些實施例中，抗TfR1抗體包含重鏈，該重鏈具有對本文所鑑別之重鏈序列具有一個、兩個或三個或更多個修飾(例如取代、缺失或插入)之胺基酸序列；及輕鏈，該輕鏈具有對本文所鑑別之輕鏈序列具有一個、兩個或三個或更多個修飾(例如取代、缺失或插入)之胺基酸序列，其中抗TfR1抗體不具有與抗體X之VH及VL一致的VH及VL。In some embodiments, the anti-TfR1 antibody comprises a heavy chain having an amino acid sequence with one, two, or three or more modifications (e.g., substitutions, deletions, or insertions) to a heavy chain sequence identified herein, wherein the anti-TfR1 antibody does not have a VH and a VL that are identical to the VH and VL of antibody X. In some embodiments, the anti-TfR1 antibody comprises a light chain having an amino acid sequence with one, two, or three or more modifications (e.g., substitutions, deletions, or insertions) to a light chain sequence identified herein, wherein the anti-TfR1 antibody does not have a VH and a VL that are identical to the VH and VL of antibody X. In some embodiments, the anti-TfR1 antibody comprises a heavy chain having an amino acid sequence having one, two, or three or more modifications (e.g., substitutions, deletions, or insertions) to the heavy chain sequence identified herein; and a light chain having an amino acid sequence having one, two, or three or more modifications (e.g., substitutions, deletions, or insertions) to the light chain sequence identified herein, wherein the anti-TfR1 antibody does not have VH and VL that are consistent with the VH and VL of antibody X.

如實例3中所述，抗體X不連續抗原決定基由來自hu TfR1 (SEQ ID NO:1)之頂端域之殘基Q285、T286、K287、P289、E343、D352、C353、P354、S355、K358、D360、S361、R364及蛋白酶樣域之殘基S492、D560、T561、R579形成。在一些實施例中，抗TfR1抗體與抗體X結合至相同抗原決定基(參見實例3)。在一些實施例中，抗TfR1抗體結合至抗體X抗原決定基之一或多個(例如，1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16或17個)胺基酸(例如hu TfR1 (SEQ ID NO:1)之殘基Q285、T286、K287、P289、E343、D352、C353、P354、S355、K358、D360、S361、R364、S492、D560、T561及R579中之一或多者)。在一些實施例中，抗TfR1抗體結合至抗體X抗原決定基之一或多個(例如，1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16或17個)胺基酸(例如hu TfR1 (SEQ ID NO:1)之殘基T286、K287、E343、D352、C353、P354、S355、K358、D360、S361、R364、S492、D560、T561及R579中之一或多者)。在一些實施例中，抗體為單價且對hTfR1具有＞ 10 nM之單價親和力(K D)。在一些實施例中，抗體為二價且具有＞10 nM之單價親和力(K D)。在一些實施例中，抗體為二價且對hTfR1具有＞100 nM之單價親和力(K D)。在一些實施例中，抗體為二價且對hTfR1具有＞1000 nM之單價親和力(K D)。在一些實施例中，抗體為二價且對hTfR1具有＞100 nM或＞1000 nM之單價親和力(K D)。在一些實施例中，針對最佳腦暴露靶向抗體之化合價及親和力。 As described in Example 3, the Antibody X discontinuous epitope is formed by residues Q285, T286, K287, P289, E343, D352, C353, P354, S355, K358, D360, S361, R364 from the apical domain of hu TfR1 (SEQ ID NO: 1) and residues S492, D560, T561, R579 from the protease-like domain. In some embodiments, the anti-TfR1 antibody binds to the same epitope as Antibody X (see Example 3). In some embodiments, the anti-TfRl antibody binds to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) amino acids of the Antibody X epitope (e.g., one or more of residues Q285, T286, K287, P289, E343, D352, C353, P354, S355, K358, D360, S361, R364, S492, D560, T561, and R579 of huTfRl (SEQ ID NO: 1)). In some embodiments, the anti-TfRl antibody binds to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) amino acids of the Antibody X epitope (e.g., one or more of residues T286, K287, E343, D352, C353, P354, S355, K358, D360, S361, R364, S492, D560, T561, and R579 of huTfRl (SEQ ID NO: 1)). In some embodiments, the antibody is monovalent and has a monovalent affinity ( KD ) of >10 nM for hTfRl. In some embodiments, the antibody is bivalent and has a monovalent affinity ( KD ) of >10 nM. In some embodiments, the antibody is bivalent and has a monovalent affinity ( KD ) of >100 nM for hTfR1. In some embodiments, the antibody is bivalent and has a monovalent affinity ( KD ) of >1000 nM for hTfR1. In some embodiments, the antibody is bivalent and has a monovalent affinity ( KD ) of >100 nM or >1000 nM for hTfR1. In some embodiments, the valency and affinity of the antibody are targeted for optimal brain exposure.

本揭示案進一步涵蓋與本文所述之重組抗體、單株抗體、嵌合抗體、人類化抗體及人類抗體或其抗體片段實質上同源之額外變異體及等效物。在一些實施例中，期望調節抗體之結合親和力。在一些實施例中，期望調節抗體之生物性質，包括但不限於特異性、熱穩定性、表現水準、效應子功能、糖基化、免疫原性及/或溶解度。熟習此項技術者將瞭解，胺基酸變化可改變抗體之轉譯後過程，諸如改變糖基化位點之數目或位置或改變膜錨定特性。The present disclosure further encompasses additional variants and equivalents that are substantially homologous to the recombinant antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and human antibodies or antibody fragments thereof described herein. In some embodiments, it is desirable to modulate the binding affinity of the antibody. In some embodiments, it is desirable to modulate the biological properties of the antibody, including but not limited to specificity, thermal stability, expression level, effector function, glycosylation, immunogenicity, and/or solubility. Those skilled in the art will appreciate that amino acid changes can alter the post-translational processes of the antibody, such as changing the number or location of glycosylation sites or altering membrane anchoring properties.

抗體之效應子功能可藉由胺基酸突變及/或域取代(例如，包括但不限於Dumet等人MABS 2019; 11(8):1341-50中所述之彼等，及Wilkinson等人, 2021, PLoS One. 16(12):e0260954中所述之彼等，例如其中表4中之任一胺基酸改變，例如「LALA」雙突變(人類IgG1 L234A/L235A)及「LALAPG」三重突變(人類IgG1 L234A/L235A/P329G))。其他特徵，諸如藥物動力學(例如Dall'acqua等人J of Immunology 2002;169 (9) 5171-80)、糖基化、免疫原性、溶解度及穩定性可藉由突變或取代對Fc之修飾來工程化。另外，新穎抗原特異性可經工程改造至恆定域中以產生新互補位(例如，Wozniak-Knopp等人PEDS 2010;23(4):289-97)。Fab之親和力或親合力可藉由改變抗體之域之間的鍵聯來調節，諸如自抗體頂部移除Fab且藉由零至40個胺基酸之任何長度之連接子將Fab連接至Fc C端，且融合至Fab之VH域或VL域之N端，產生「倒置」抗體，該抗體具有潛在地經調節的與抗原結合之親和力或親合力，及經調節之效應子功能(例如，Weber等人Cell Reports 2018;22:149-62)。The effector function of the antibody can be achieved by amino acid mutation and/or domain substitution (e.g., including but not limited to those described in Dumet et al. MABS 2019; 11(8):1341-50, and those described in Wilkinson et al., 2021, PLoS One. 16(12):e0260954, such as any one of the amino acid changes in Table 4, such as "LALA" double mutation (human IgG1 L234A/L235A) and "LALAPG" triple mutation (human IgG1 L234A/L235A/P329G)). Other characteristics, such as pharmacokinetics (e.g., Dall'acqua et al. J of Immunology 2002;169(9):5171-80), glycosylation, immunogenicity, solubility, and stability can be engineered by modifying the Fc by mutation or substitution. In addition, novel antigenic specificities can be engineered into the homeostatic domain to create new complementary sites (e.g., Wozniak-Knopp et al. PEDS 2010;23(4):289-97). The affinity or avidity of a Fab can be modulated by changing the linkage between the domains of the antibody, such as removing the Fab from the top of the antibody and linking the Fab to the Fc C-terminus via a linker of any length from zero to 40 amino acids and fused to the N-terminus of the VH domain or VL domain of the Fab, creating an "inverted" antibody that has potentially modulated affinity or avidity for binding to an antigen, and modulated effector function (e.g., Weber et al. Cell Reports 2018;22:149-62).

變異可為編碼抗體或多肽之一或多個核苷酸之取代、缺失或插入，其導致胺基酸序列與天然抗體或多肽序列相比發生變化。在一些實施例中，胺基酸取代係將一個胺基酸用具有相似結構及/或化學性質之另一胺基酸置換之結果，諸如將白胺酸用絲胺酸置換，例如保守胺基酸置換。插入或缺失可視情況在約1至5個胺基酸之範圍內。在一些實施例中，取代、缺失或插入包括相對於親代分子，少於25個胺基酸取代、少於20個胺基酸取代、少於15個胺基酸取代、少於10個胺基酸取代、少於5個胺基酸取代、少於4個胺基酸取代、少於3個胺基酸取代或少於2個胺基酸取代。在一些實施例中，可藉由在序列中系統地進行插入、缺失或取代且測試所得變異體蛋白與親代蛋白相比之活性來確定生物學上有用及/或相關之胺基酸序列之變異。The variation can be a substitution, deletion or insertion of one or more nucleotides encoding an antibody or polypeptide, which results in a change in the amino acid sequence compared to a natural antibody or polypeptide sequence. In some embodiments, amino acid substitution is the result of replacing an amino acid with another amino acid having a similar structure and/or chemical property, such as replacing leucine with serine, such as a conservative amino acid substitution. Insertion or deletion may be within the range of about 1 to 5 amino acids, as appropriate. In some embodiments, substitution, deletion or insertion includes less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions or less than 2 amino acid substitutions relative to the parent molecule. In some embodiments, variations in biologically useful and/or relevant amino acid sequences can be determined by systematically making insertions, deletions or substitutions in the sequence and testing the resulting variant proteins for activity compared to the parent protein.

在一些實施例中，變異體可包括在抗體或多肽之胺基端及/或羧基端添加胺基酸殘基。額外胺基酸殘基之長度可在一個殘基至一百個或更多個殘基範圍內。在一些實施例中，變異體包含N端甲硫醯基殘基。在一些實施例中，變異體包含額外多肽/蛋白質(例如Fc區)以產生融合蛋白。在一些實施例中，變異體經工程改造為可偵測的且可包含可偵測標記及/或蛋白質(例如螢光標籤或酶)。In some embodiments, variants may include the addition of amino acid residues at the amino and/or carboxyl termini of antibodies or polypeptides. The length of the additional amino acid residues may range from one residue to one hundred or more residues. In some embodiments, variants include an N-terminal methionyl residue. In some embodiments, variants include additional polypeptides/proteins (e.g., Fc regions) to produce fusion proteins. In some embodiments, variants are engineered to be detectable and may include detectable markers and/or proteins (e.g., fluorescent tags or enzymes).

在一些實施例中，不參與維持抗體之適當構形之半胱胺酸殘基經取代或缺失以調節抗體之特徵，例如以改良氧化穩定性及/或防止異常二硫化物交聯及/或促進劑之连接。相反，在一些實施例中，添加一或多個半胱胺酸殘基以產生二硫鍵以改良穩定性。In some embodiments, cysteine residues that are not involved in maintaining the proper conformation of the antibody are substituted or deleted to modulate the characteristics of the antibody, for example to improve oxidative stability and/or prevent abnormal disulfide cross-linking and/or the attachment of promoters. Conversely, in some embodiments, one or more cysteine residues are added to generate disulfide bonds to improve stability.

在一些實施例中，本揭示案之抗體包含不能在相同重鏈之間形成二硫鍵聯(例如減少同二聚體形成)之變異體鉸鏈區。在一些實施例中，抗體包含具有導致靜電相互作用改變之胺基酸變化之重鏈。在一些實施例中，抗體包含具有導致疏水性/親水性相互作用改變之胺基酸變化之重鏈。In some embodiments, the antibodies of the present disclosure comprise variant hinge regions that are unable to form disulfide bonds between identical heavy chains (e.g., reduce homodimer formation). In some embodiments, the antibodies comprise heavy chains with amino acid changes that result in altered electrostatic interactions. In some embodiments, the antibodies comprise heavy chains with amino acid changes that result in altered hydrophobic/hydrophilic interactions.

在一些實施例中，本揭示案之抗體經「去免疫化」。抗體之去免疫化通常由引入特定胺基酸突變(例如取代、缺失、添加)組成，該等特定胺基酸突變導致經預測之T細胞抗原決定基之移除而不顯著降低抗體之結合親和力或其他期望特徵。In some embodiments, the antibodies of the present disclosure are "deimmunized." Deimmunization of an antibody generally consists of introducing specific amino acid mutations (e.g., substitutions, deletions, additions) that result in the removal of predicted T cell antigenic determinants without significantly reducing the binding affinity or other desired characteristics of the antibody.

在一些實施例中，本文所述之抗TfR1抗體具有一或多個(例如，1、2、3、4、5、6、7、8、9、10個)修飾以使得能夠將抗TfR1抗體連接至如下文所述之劑。In some embodiments, an anti-TfR1 antibody described herein has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) modifications that enable the anti-TfR1 antibody to be linked to an agent as described below.

本文所述之變異體抗體或多肽可使用此項技術中已知之方法產生，該等方法包括但不限於定點誘變、丙胺酸掃描誘變及PCR誘變。Variant antibodies or polypeptides described herein can be generated using methods known in the art, including but not limited to site-directed mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis.

在一些實施例中，本文所述之抗TfR1抗體經化學修飾。在一些實施例中，抗TfR1抗體已藉由糖基化、乙醯化、聚乙二醇化、磷酸化、醯胺化、藉由已知保護/阻斷基團進行之衍生化、蛋白水解裂解及/或與細胞配位體或其他蛋白質連接來進行化學修飾。多種化學修飾中之任一者可藉由已知技術進行。In some embodiments, the anti-TfR1 antibodies described herein are chemically modified. In some embodiments, the anti-TfR1 antibodies have been chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, and/or attachment to cellular ligands or other proteins. Any of a variety of chemical modifications can be performed by known techniques.

一般而言，抗原-抗體相互作用為非共價且可逆的，由氫鍵、疏水相互作用、靜電及凡得瓦力之組合形成。當描述抗原-抗體複合物之強度時，通常使用術語親和力及/或親合力。抗體與其抗原之結合係可逆過程，且結合之親和力通常報告為平衡解離常數(K D)。K D為抗體解離速率(koff，在本文中亦稱為k d) (其自其抗原解離之速度)與抗體締合速率(kon) (其與其抗原結合之速度)的比率。在一些實施例中，藉由量測特定抗體/抗原相互作用之kon及koff速率且接著使用此等值之比率計算K D值來確定K D值。在一些實施例中，使用K D值來評估個別抗體/抗原相互作用之強度並對其進行排序。抗體之K D越低，抗體對其靶標之親和力就越高。在一些實施例中，在Biacore系統中使用SPR技術量測親和力。親合力給出抗體-抗原複合物之總體強度的量度。其取決於三個主要參數：(i)抗體對靶標之親和力，(ii)抗體及抗原兩者之化合價，及(iii)相互作用部分之結構排列。 In general, antigen-antibody interactions are non-covalent and reversible, formed by a combination of hydrogen bonds, hydrophobic interactions, electrostatic and van der Waals forces. When describing the strength of an antigen-antibody complex, the terms affinity and/or avidity are often used. The binding of an antibody to its antigen is a reversible process, and the affinity of the binding is often reported as an equilibrium dissociation constant ( KD ). KD is the ratio of the antibody dissociation rate (koff, also referred to herein as kd ) (the speed at which it dissociates from its antigen) to the antibody association rate (kon) (the speed at which it binds to its antigen). In some embodiments, the KD value is determined by measuring the kon and koff rates of a specific antibody/antigen interaction and then calculating the KD value using the ratio of these equivalent values. In some embodiments, the KD value is used to assess the strength of individual antibody/antigen interactions and to rank them. The lower the KD of an antibody, the higher the affinity of the antibody for its target. In some embodiments, affinity is measured using SPR technology in a Biacore system. Avidity gives a measure of the overall strength of the antibody-antigen complex. It depends on three main parameters: (i) the affinity of the antibody for the target, (ii) the valency of both the antibody and the antigen, and (iii) the structural arrangement of the interacting parts.

在一些實施例中，本文所述之抗TfR1抗體具有＞ 10 nM (例如，＞25 nM、＞50 nM、＞ 100 nM、＞500 nM、＞5000 nM、＞1 uM、＞2 uM、＞10 nM至100 nM、＞10 nM至1000 nM、＞10 nM至2000 nM、＞10nM至5000 nM、＞10 nM至10000 nM、100 nM至1000 nM、100 nM至5000 nM、100 nM至5000 nM、100 nM至500 nM、500 nM至1 uM、50 nM至1 uM、100 nM至1 uM、500 nM至10 uM或1 uM至10 uM)之對hTfR1的單價親和力(K D)及/或＞= 0.01/s之解離速率(k d)。在一些實施例中，抗TfR1抗體為單價或多價(例如二價)的。在一些實施例中，本文所述之抗TfR1抗體為多價(例如二價)抗體且具有＞100 nM之單價親和力。在一些實施例中，本文所述之抗TfR1抗體為多價(例如二價)抗體且具有＞1000 nM之單價親和力。在一些實施例中，本文所述之抗TfR1抗體為多價(例如二價)抗體且具有100 nM至1000 nM之單價親和力。在一些實施例中，本文所述之抗TfR1抗體為多價(例如二價)抗體且具有100 nM至10000 nM之單價親和力。在一些實施例中，本文所述之抗TfR1抗體為多價(例如二價)抗體且具有100 nM至5000 nM之單價親和力。在一些實施例中，本文所述之抗TfR1抗體為單價抗體且對hTfR1具有＞ 10 nM之單價親和力。 製備抗TFR1抗體之方法 In some embodiments, the anti-TfR1 antibodies described herein have a concentration >10 nM (e.g., >25 nM, >50 nM, >100 nM, >500 nM, >5000 nM, >1 uM, >2 uM, >10 nM to 100 nM, >10 nM to 1000 nM, >10 nM to 2000 nM, >10 nM to 5000 nM, >10 nM to 10000 nM, 100 nM to 1000 nM, 100 nM to 5000 nM, 100 nM to 5000 nM, 100 nM to 1000 nM, 500 nM to 1 uM, 50 nM to 1 uM, 100 nM to 1 uM, 500 nM to 10 uM, or 1 uM to 10 uM) of monovalent affinity (K D ) for hTfR1 and/or a dissociation rate (k d ) of >= 0.01/s. In some embodiments, the anti-TfR1 antibody is monovalent or multivalent (e.g., bivalent). In some embodiments, the anti-TfR1 antibody described herein is a multivalent (e.g., bivalent) antibody and has a monovalent affinity of >100 nM. In some embodiments, the anti-TfR1 antibody described herein is a multivalent (e.g., bivalent) antibody and has a monovalent affinity of >1000 nM. In some embodiments, the anti-TfR1 antibody described herein is a multivalent (e.g., bivalent) antibody and has a monovalent affinity of 100 nM to 1000 nM. In some embodiments, the anti-TfR1 antibodies described herein are multivalent (e.g., bivalent) antibodies and have a monovalent affinity of 100 nM to 10000 nM. In some embodiments, the anti-TfR1 antibodies described herein are multivalent (e.g., bivalent) antibodies and have a monovalent affinity of 100 nM to 5000 nM. In some embodiments, the anti-TfR1 antibodies described herein are monovalent antibodies and have a monovalent affinity for hTfR1 of > 10 nM. Methods for preparing anti-TFR1 antibodies

本文所述之抗TfR1抗體可藉由此項技術中已知之任何合適方法產生。此類方法之範圍為自直接蛋白質合成方法至構築編碼多肽序列之DNA序列且在合適宿主中表現彼等序列。在一些實施例中，使用重組技術藉由分離或合成編碼所關注野生型蛋白之DNA序列來構築DNA序列。視情況，可藉由位點特異性誘變來誘變序列以提供其功能變異體。在一些實施例中，使用寡核苷酸合成器藉由化學合成來構築編碼所關注多肽之DNA序列。寡核苷酸可基於所需多肽之胺基酸序列且選擇在將產生所關注重組多肽之宿主細胞中有利之彼等密碼子來設計。可應用標準方法來合成編碼所關注之經分離多肽之多核苷酸序列。舉例而言，完整胺基酸序列可用於構築回譯基因。此外，可合成含有編碼特定經分離多肽之核苷酸序列之DNA寡聚物。舉例而言，可合成編碼所需多肽之部分的若干小寡核苷酸，然後連接。個別寡核苷酸通常含有用於互補組裝之5'或3'懸垂。The anti-TfR1 antibodies described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing DNA sequences encoding polypeptide sequences and expressing those sequences in suitable hosts. In some embodiments, recombinant technology is used to construct DNA sequences by isolating or synthesizing DNA sequences encoding wild-type proteins of interest. Depending on the circumstances, the sequence can be induced by site-specific mutation to provide its functional variants. In some embodiments, an oligonucleotide synthesizer is used to construct a DNA sequence encoding a polypeptide of interest by chemical synthesis. Oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and selecting those codons that are favorable in the host cell that will produce the recombinant polypeptide of interest. Standard methods can be applied to synthesize polynucleotide sequences encoding isolated polypeptides of interest. For example, the entire amino acid sequence can be used to construct a backtranslated gene. In addition, DNA oligomers containing a nucleotide sequence encoding a specific isolated polypeptide can be synthesized. For example, several small oligonucleotides encoding portions of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

一旦組裝(藉由合成、定點誘變或另一方法)，編碼特定所關注多肽之多核苷酸序列可插入表現載體中且可操作地連接至適於在所需宿主中表現蛋白質的表現控制序列。正確組裝可藉由核苷酸定序、限制酶作圖及/或生物活性多肽在合適宿主中之表現來確認。如此項技術中所熟知，為了在宿主中獲得經轉染基因之高表現水準，該基因必須可操作地連接至在所選表現宿主中起作用之轉錄及轉譯表現控制序列。Once assembled (by synthesis, site-directed mutagenesis or another method), the polynucleotide sequence encoding a particular polypeptide of interest can be inserted into an expression vector and operably linked to expression control sequences suitable for expression of the protein in the desired host. Correct assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping and/or expression of the biologically active polypeptide in a suitable host. As is well known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene must be operably linked to transcriptional and translational expression control sequences that are functional in the selected expression host.

在一些實施例中，使用重組表現載體擴增及表現編碼針對人類TfR1之抗體之DNA。舉例而言，重組表現載體可為可複製DNA構築體，其包括編碼抗TfR1抗體之多肽鏈的合成或cDNA衍生性DNA片段，該等片段可操作地連接至源自哺乳動物、微生物、病毒或昆蟲基因之合適轉錄及/或轉譯調控元件。轉錄單元通常包含以下各者之組裝體：(1)在基因表現中具有調控作用之一或多種遺傳元件，例如轉錄啟動子或增強子，(2)轉錄成mRNA且轉譯成蛋白質之結構序列或編碼序列，及(3)適當轉錄及轉譯起始及終止序列。調控元件可包括控制轉錄之操縱子序列。亦可包括通常由複製起點賦予之在宿主中複製之能力，及促進識別轉移體之選擇基因。當DNA區域在功能上彼此相關時，其「可操作地連接」。舉例而言，若信號肽(分泌前導序列)之DNA表現為參與多肽分泌之前驅體，則該DNA可操作地連接至該多肽之DNA；若啟動子控制序列之轉錄，則啟動子可操作地連接至編碼序列；或若核醣體結合位點定位成允許轉譯，則核醣體結合位點可操作地連接至編碼序列。在一些實施例中，意欲用於酵母表現系統中之結構元件包括使得宿主細胞能夠細胞外分泌經轉譯蛋白質之前導序列。在一些實施例中，在重組蛋白在無前導序列或轉運序列之情況下表現之情形下，多肽可包括N端甲硫胺酸殘基。此殘基可視情況隨後自所表現之重組蛋白裂解以提供最終產物。In some embodiments, a recombinant expression vector is used to amplify and express DNA encoding an antibody against human TfR1. For example, a recombinant expression vector can be a replicable DNA construct comprising synthetic or cDNA-derived DNA fragments encoding a polypeptide chain of an anti-TfR1 antibody, which fragments are operably linked to appropriate transcriptional and/or translational regulatory elements derived from mammalian, microbial, viral or insect genes. A transcriptional unit typically comprises an assembly of: (1) one or more genetic elements that have a regulatory role in gene expression, such as a transcriptional promoter or enhancer, (2) a structural sequence or coding sequence that is transcribed into mRNA and translated into protein, and (3) appropriate transcriptional and translational start and stop sequences. The regulatory element may include an operator sequence that controls transcription. It may also include the ability to replicate in the host, usually conferred by an origin of replication, and a selection gene that facilitates recognition of the transposon. DNA regions are "operably linked" when they are functionally related to each other. For example, if the DNA of a signal peptide (secretion leader sequence) is expressed as a promotor involved in the secretion of a polypeptide, the DNA is operably linked to the DNA of the polypeptide; if the promoter controls the transcription of the sequence, the promoter is operably linked to the coding sequence; or if the ribosome binding site is positioned to allow translation, the ribosome binding site is operably linked to the coding sequence. In some embodiments, the structural elements intended for use in a yeast expression system include a leader sequence that enables the host cell to secrete the translated protein extracellularly. In some embodiments, where the recombinant protein is expressed without a leader or transit sequence, the polypeptide may include an N-terminal methionine residue. This residue may be subsequently cleaved from the expressed recombinant protein to provide the final product, as appropriate.

表現控制序列及表現載體之選擇通常取決於宿主之選擇。可採用多種表現宿主/載體組合。可用於真核宿主之表現載體包括例如包含來自SV40、牛乳頭狀瘤病毒、腺病毒及細胞巨大病毒之表現控制序列之載體。可用於細菌宿主之表現載體包括已知細菌質體，諸如來自大腸桿菌( E. coli)之質體，包括pCR1、pBR322、pMB9及其衍生物，以及更廣泛宿主範圍之質體，諸如M13及其他絲狀單股DNA噬菌體。 The choice of expression control sequences and expression vectors generally depends on the choice of host. A variety of expression host/vector combinations can be employed. Expression vectors useful for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Expression vectors useful for bacterial hosts include known bacterial plasmids, such as those from E. coli , including pCR1, pBR322, pMB9, and derivatives thereof, as well as plasmids with a broader host range, such as M13 and other filamentous single-stranded DNA bacteriophages.

在一些實施例中，本揭示案之抗TfR1抗體自一或多種載體表現。在一些實施例中，重鏈多肽由一種載體表現且輕鏈多肽由第二載體表現。在一些實施例中，重鏈多肽及輕鏈多肽由一種載體表現。因此，本揭示案提供編碼本文所述之抗TfR1抗體之載體。在一個實施例中，載體編碼本文所述之抗TfR1抗體之重鏈多肽。在一個實施例中，載體編碼本文所述之抗TfR1抗體之輕鏈多肽。在一個實施例中，載體編碼本文所述之抗TfR1抗體之重鏈多肽及輕鏈多肽。In some embodiments, the anti-TfR1 antibodies of the present disclosure are expressed from one or more vectors. In some embodiments, the heavy chain polypeptide is expressed by one vector and the light chain polypeptide is expressed by a second vector. In some embodiments, the heavy chain polypeptide and the light chain polypeptide are expressed by one vector. Therefore, the present disclosure provides vectors encoding the anti-TfR1 antibodies described herein. In one embodiment, the vector encodes the heavy chain polypeptide of the anti-TfR1 antibody described herein. In one embodiment, the vector encodes the light chain polypeptide of the anti-TfR1 antibody described herein. In one embodiment, the vector encodes the heavy chain polypeptide and the light chain polypeptide of the anti-TfR1 antibody described herein.

用於表現抗TfR1抗體或TfR1蛋白或其片段以用作抗原或免疫原之合適宿主細胞包括在適當啟動子控制下之原核生物、酵母細胞、昆蟲細胞或高等真核細胞。原核生物包括革蘭氏陰性或革蘭氏陽性生物體，例如大腸桿菌或芽孢桿菌屬( Bacillus)。高等真核細胞包括如本文所述之哺乳動物來源之已建立細胞株。亦可採用無細胞轉譯系統。用於細菌、真菌、酵母及哺乳動物細胞宿主之適當選殖及表現載體，以及蛋白質產生方法(包括抗體產生)係此項技術中熟知的。 Suitable host cells for expressing anti-TfR1 antibodies or TfR1 proteins or fragments thereof for use as antigens or immunogens include prokaryotes, yeast cells, insect cells or higher eukaryotic cells under the control of an appropriate promoter. Prokaryotes include Gram-negative or Gram-positive organisms, such as Escherichia coli or Bacillus . Higher eukaryotic cells include established cell lines of mammalian origin as described herein. Cell-free translation systems may also be used. Appropriate selection and expression vectors for bacterial, fungal, yeast and mammalian cell hosts, and protein production methods (including antibody production) are well known in the art.

各種哺乳動物培養系統可用於表現重組多肽。重組蛋白在哺乳動物細胞中之表現可能係合乎需要的，因為此等蛋白質通常經正確摺疊、適當修飾且具有生物功能。合適哺乳動物宿主細胞株之實例包括但不限於COS-7 (猴腎源性)、L-929 (鼠類纖維母細胞源性)、C127 (鼠類乳腺腫瘤源性)、3T3 (鼠類纖維母細胞源性)、CHO (中國倉鼠卵巢源性)、HeLa (人類子宮頸癌源性)、BHK (倉鼠腎纖維母細胞源性)、HEK-293 (人類胚胎腎源性)細胞株及其變異體。哺乳動物表現載體可包含非轉錄元件，諸如複製起點、連接至待表現基因之合適啟動子及增強子、及其他5'或3'側接非轉錄序列、及5'或3'非轉譯序列，諸如必需的核醣體結合位點、多聚腺苷酸化位點、剪接供體及受體位點以及轉錄終止序列。A variety of mammalian culture systems can be used to express recombinant polypeptides. Expression of recombinant proteins in mammalian cells may be desirable because such proteins are generally correctly folded, appropriately modified, and biologically functional. Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (monkey kidney-derived), L-929 (murine fibroblast-derived), C127 (murine breast tumor-derived), 3T3 (murine fibroblast-derived), CHO (Chinese hamster ovary-derived), HeLa (human cervical carcinoma-derived), BHK (hamster kidney fibroblast-derived), HEK-293 (human embryonic kidney-derived) cell lines and variants thereof. Mammalian expression vectors may comprise non-transcribed elements such as an origin of replication, an appropriate promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences, and 5' or 3' non-translated sequences such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcriptional termination sequences.

重組蛋白在昆蟲細胞培養系統(例如桿狀病毒)中之表現亦提供產生正確摺疊且具有生物功能之蛋白質的穩健方法。用於在昆蟲細胞中產生異源蛋白質之桿狀病毒系統為熟習此項技術者所熟知的。Expression of recombinant proteins in insect cell culture systems (e.g., baculovirus) also provides a robust method for producing properly folded and biologically functional proteins. Baculovirus systems for producing heterologous proteins in insect cells are well known to those skilled in the art.

因此，本揭示案提供包含本文所述之抗TfR1抗體之細胞。本揭示案亦提供細胞，其包含編碼本文所述之抗TfR1抗體之一或多個多核苷酸或編碼本文所述之抗TfR1抗體之一或多個載體。在一個實施例中，細胞包含編碼本文所述之抗TfR1抗體之多核苷酸。在一個實施例中，細胞包含編碼本文所述之抗TfR1抗體之重鏈的第一多核苷酸及編碼本文所述之抗TfR1抗體之輕鏈的第二多核苷酸。在一個實施例中，細胞包含編碼本文所述之抗TfR1抗體之重鏈及輕鏈的多核苷酸。在一個實施例中，細胞包含編碼本文所述之抗TfR1抗體之載體。在一個實施例中，細胞包含編碼本文所述之抗TfR1抗體之重鏈的第一載體及編碼本文所述之抗TfR1抗體之輕鏈的第二載體。在一個實施例中，細胞包含編碼本文所述之抗TfR1抗體之重鏈及輕鏈的載體。在一些實施例中，細胞產生本文所述之抗TfR1抗體。在一些實施例中，細胞產生抗體。在一些實施例中，細胞產生結合人類TfR1之抗體。在一些實施例中，細胞產生結合石蟹猴TfR1之抗體。在一些實施例中，細胞產生結合人類TfR1及石蟹猴TfR1之抗體。在一些實施例中，細胞為原核細胞(例如大腸桿菌)。在一些實施例中，細胞為真核細胞。在一些實施例中，細胞為哺乳動物細胞。在一些實施例中，細胞為融合瘤細胞。Therefore, the present disclosure provides cells comprising anti-TfR1 antibodies described herein. The present disclosure also provides cells comprising one or more polynucleotides encoding anti-TfR1 antibodies described herein or one or more vectors encoding anti-TfR1 antibodies described herein. In one embodiment, the cell comprises a polynucleotide encoding an anti-TfR1 antibody described herein. In one embodiment, the cell comprises a first polynucleotide encoding the heavy chain of an anti-TfR1 antibody described herein and a second polynucleotide encoding the light chain of an anti-TfR1 antibody described herein. In one embodiment, the cell comprises a polynucleotide encoding the heavy chain and light chain of an anti-TfR1 antibody described herein. In one embodiment, the cell comprises a vector encoding an anti-TfR1 antibody described herein. In one embodiment, the cell comprises a first vector encoding the heavy chain of an anti-TfR1 antibody described herein and a second vector encoding the light chain of an anti-TfR1 antibody described herein. In one embodiment, the cell comprises a vector encoding the heavy chain and light chain of an anti-TfR1 antibody described herein. In some embodiments, the cell produces an anti-TfR1 antibody described herein. In some embodiments, the cell produces antibodies. In some embodiments, the cell produces antibodies that bind to human TfR1. In some embodiments, the cell produces antibodies that bind to cynomolgus monkey TfR1. In some embodiments, the cell produces antibodies that bind to human TfR1 and cynomolgus monkey TfR1. In some embodiments, the cell is a prokaryotic cell (e.g., Escherichia coli). In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a fusion tumor cell.

宿主細胞產生之蛋白質可根據任何合適方法純化。標準方法包括層析(例如離子交換層析、親和層析及大小管柱層析)、離心、差異溶解度或藉由用於蛋白質純化之任何其他標準技術。親和標籤，諸如六組胺酸(SEQ ID NO:360)、麥芽糖結合域、流感外殼序列及麩胱甘肽-S-轉移酶可連接至蛋白質以允許藉由通過適當親和管柱來容易地純化。用於純化免疫球蛋白之親和層析包括但不限於蛋白A、蛋白G及蛋白L層析。可使用熟習此項技術者已知之技術對經分離之蛋白質進行物理表徵，該等技術包括但不限於蛋白水解、尺寸排阻層析(SEC)、質譜(MS)、核磁共振(NMR)、等電聚焦(IEF)、高效液相層析(HPLC)及x射線晶體學。經分離蛋白質之純度可使用熟習此項技術者已知之技術來確定，該等技術包括但不限於SDS-PAGE、SEC、毛細管凝膠電泳、IEF及毛細管等電聚焦(cIEF)。The protein produced by the host cells can be purified according to any suitable method. Standard methods include chromatography (e.g., ion exchange chromatography, affinity chromatography, and size column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. Affinity tags, such as hexahistidine (SEQ ID NO: 360), maltose binding domain, influenza capsid sequence, and glutathione-S-transferase can be attached to the protein to allow for easy purification by passage through an appropriate affinity column. Affinity chromatography used to purify immunoglobulins includes, but is not limited to, protein A, protein G, and protein L chromatography. The isolated proteins can be physically characterized using techniques known to those skilled in the art, including but not limited to proteolysis, size exclusion chromatography (SEC), mass spectrometry (MS), nuclear magnetic resonance (NMR), isoelectric focusing (IEF), high performance liquid chromatography (HPLC), and x-ray crystallography. The purity of the isolated proteins can be determined using techniques known to those skilled in the art, including but not limited to SDS-PAGE, SEC, capillary gel electrophoresis, IEF, and capillary isoelectric focusing (cIEF).

在一些實施例中，首先使用市售蛋白質濃縮過濾器(例如Amicon®或Millipore Pellicon®超濾單元)濃縮來自將重組蛋白分泌至培養基中之表現系統的上清液。在濃縮步驟之後，可將濃縮物施加至適合純化基質。在一些實施例中，採用陰離子交換樹脂，例如具有二乙基胺基乙基(DEAE)側基之基質或受質。基質可為丙烯醯胺、瓊脂糖、葡聚醣、纖維素或蛋白質純化中常用之其他類型。在一些實施例中，採用陽離子交換步驟。合適陽離子交換劑包括包含磺丙基或羧甲基之各種不溶性基質。在一些實施例中，採用羥磷灰石介質，包括但不限於陶瓷羥磷灰石(CHT)。在一些實施例中，採用一或多個反相HPLC步驟來進一步純化重組蛋白，該一或多個反相HPLC步驟採用疏水性RP-HPLC介質，例如具有側甲基或其他脂族基團之矽膠。在一些實施例中，使用疏水相互作用層析(HIC)基於重組蛋白之疏水性來分離重組蛋白。由於使用在與一些其他技術相比更少之變性條件下操作之條件及基質，HIC係用於純化蛋白質同時維持生物活性之有用分離技術。可採用各種組合的一些或所有前述純化步驟來提供均質重組蛋白。In some embodiments, the supernatant from a expression system that secretes the recombinant protein into a culture medium is first concentrated using a commercially available protein concentrator filter (e.g., Amicon® or Millipore Pellicon® ultrafiltration unit). After the concentration step, the concentrate can be applied to a suitable purification matrix. In some embodiments, an anion exchange resin is used, such as a matrix or substrate having diethylaminoethyl (DEAE) side groups. The matrix can be acrylamide, agarose, dextran, cellulose, or other types commonly used in protein purification. In some embodiments, a cation exchange step is used. Suitable cation exchangers include various insoluble matrices containing sulfopropyl or carboxymethyl groups. In some embodiments, a hydroxyapatite medium is used, including but not limited to ceramic hydroxyapatite (CHT). In some embodiments, the recombinant protein is further purified using one or more reverse phase HPLC steps, and the one or more reverse phase HPLC steps use hydrophobic RP-HPLC media, such as silica gel with pendant methyl or other aliphatic groups. In some embodiments, hydrophobic interaction chromatography (HIC) is used to separate the recombinant protein based on the hydrophobicity of the recombinant protein. HIC is a useful separation technique for purifying proteins while maintaining biological activity due to the use of conditions and matrices that operate under less denaturing conditions than some other techniques. Various combinations of some or all of the foregoing purification steps may be employed to provide homogeneous recombinant protein.

在一些實施例中，本揭示案之抗體為Fab，其可藉由以下方式產生：首先製備全單株Ab，然後藉由化學或酶促裂解(例如，胃蛋白酶、木瓜蛋白酶或無花果蛋白酶消化)消化單株抗體以產生F(ab') 2片段，隨後還原彼等片段以產生Fab片段。此類技術為此項技術中已知的。參見例如Victor C-G等人, Biosensors and Bioelectronics, 2016 (85):32-45。 In some embodiments, the antibody of the present disclosure is Fab, which can be produced by first preparing a whole monoclonal Ab, then digesting the monoclonal antibody by chemical or enzymatic cleavage (e.g., pepsin, papain or ficin digestion) to produce F(ab') 2 fragments, and then reducing those fragments to produce Fab fragments. Such techniques are known in the art. See, for example, Victor CG et al., Biosensors and Bioelectronics, 2016 (85): 32-45.

替代地，本揭示案之抗體藉由重組合成F(ab') 2抗體片段、隨後化學還原此等片段以產生Fab單元來製得。 多核苷酸 Alternatively, the antibodies of the present disclosure are prepared by recombinant synthesis of F(ab') 2 antibody fragments followed by chemical reduction of these fragments to generate Fab units.

在一些實施例中，本揭示案涵蓋多核苷酸，其包含編碼本文所述之多肽(例如，抗TfR1抗體)之多核苷酸。術語「編碼多肽之多核苷酸」涵蓋僅包括多肽之編碼序列之多核苷酸以及包括額外編碼序列及/或非編碼序列之多核苷酸。本揭示案之多核苷酸可呈RNA形式或呈DNA形式。DNA包括cDNA、基因體DNA及合成DNA；且可為雙股或單股，且若為單股，則可為編碼股或非編碼(反義)股。在一些實施例中，多核苷酸包含編碼本文所述之抗TfR1抗體之重鏈的多核苷酸(例如核苷酸序列)。在一些實施例中，多核苷酸包含編碼本文所述之抗TfR1抗體之輕鏈的多核苷酸(例如，核苷酸序列)。在一些實施例中，多核苷酸包含編碼本文所述之抗TfR1抗體之重鏈的多核苷酸(例如核苷酸序列)及編碼抗TfR1抗體之輕鏈的多核苷酸(例如核苷酸序列)。In some embodiments, the present disclosure encompasses polynucleotides comprising polynucleotides encoding a polypeptide described herein (e.g., an anti-TfR1 antibody). The term "polynucleotide encoding a polypeptide" encompasses polynucleotides that include only the coding sequence of the polypeptide as well as polynucleotides that include additional coding sequences and/or non-coding sequences. The polynucleotides of the present disclosure may be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and may be double-stranded or single-stranded, and if single-stranded, may be a coding strand or a non-coding (antisense) strand. In some embodiments, the polynucleotide comprises a polynucleotide (e.g., a nucleotide sequence) encoding the heavy chain of an anti-TfR1 antibody described herein. In some embodiments, the polynucleotide comprises a polynucleotide (e.g., a nucleotide sequence) encoding the light chain of an anti-TfR1 antibody described herein. In some embodiments, the polynucleotide comprises a polynucleotide (eg, a nucleotide sequence) encoding the heavy chain of an anti-TfR1 antibody described herein and a polynucleotide (eg, a nucleotide sequence) encoding the light chain of an anti-TfR1 antibody.

在一些實施例中，多核苷酸包含編碼包含 表3中所描繪之VH胺基酸序列之多肽的多核苷酸(例如核苷酸序列)。在一些實施例中，多核苷酸包含編碼包含 表4中所描繪之VL胺基酸序列之多肽的多核苷酸(例如核苷酸序列)。在一些實施例中，多核苷酸包含編碼包含 表3中所描繪之VH胺基酸序列之多肽及包含 表4中所描繪之VL胺基酸序列之多肽的多核苷酸(例如核苷酸序列)。在一些實施例中，多核苷酸包含編碼包含 表6中所描繪之重鏈胺基酸序列之多肽的多核苷酸(例如核苷酸序列)。在一些實施例中，多核苷酸包含編碼包含 表6中所描繪之輕鏈胺基酸序列之多肽的多核苷酸(例如核苷酸序列)。在一些實施例中，多核苷酸包含編碼包含 表6中所描繪之重鏈胺基酸序列之多肽及包含 表6中所描繪之輕鏈胺基酸序列之多肽的多核苷酸(例如核苷酸序列)。 In some embodiments, the polynucleotide comprises a polynucleotide (e.g., a nucleotide sequence) encoding a polypeptide comprising a VH amino acid sequence described in Table 3. In some embodiments, the polynucleotide comprises a polynucleotide (e.g., a nucleotide sequence) encoding a polypeptide comprising a VL amino acid sequence described in Table 4. In some embodiments, the polynucleotide comprises a polynucleotide (e.g., a nucleotide sequence) encoding a polypeptide comprising a VH amino acid sequence described in Table 3 and a polypeptide comprising a VL amino acid sequence described in Table 4. In some embodiments, the polynucleotide comprises a polynucleotide (e.g., a nucleotide sequence) encoding a polypeptide comprising a heavy chain amino acid sequence described in Table 6. In some embodiments, the polynucleotide comprises a polynucleotide (e.g., a nucleotide sequence) encoding a polypeptide comprising a light chain amino acid sequence described in Table 6. In some embodiments, the polynucleotide comprises a polynucleotide (e.g., a nucleotide sequence) encoding a polypeptide comprising a heavy chain amino acid sequence described in Table 6 and a polypeptide comprising a light chain amino acid sequence described in Table 6 .

多核苷酸變異體可含有編碼區、非編碼區或兩者中之改變。在一些實施例中，多核苷酸變異體含有產生沉默取代、添加或缺失之改變，但不改變經編碼多肽之性質或活性。在一些實施例中，多核苷酸變異體包含不導致多肽之胺基酸序列變化(歸因於遺傳密碼之簡併性)之沉默取代。在一些實施例中，多核苷酸變異體包含一或多個突變密碼子，該等突變密碼子包含對密碼子之一或多個(例如1、2或3個)取代，該等取代改變由彼密碼子編碼之胺基酸。用於將一或多個取代引入密碼子中之方法為此項技術中已知，諸如PCR誘變及定點誘變。可出於多種原因產生多核苷酸變異體，例如以最佳化特定宿主之密碼子表現(例如，將人類mRNA中之密碼子改變為細菌宿主(諸如大腸桿菌)所偏好之彼等密碼子)。在一些實施例中，多核苷酸變異體在序列之非編碼區或編碼區中包含至少一個沉默突變。Polynucleotide variants may contain changes in coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants contain changes that produce silent substitutions, additions, or deletions, but do not change the properties or activity of the encoded polypeptide. In some embodiments, polynucleotide variants include silent substitutions that do not cause changes in the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code). In some embodiments, polynucleotide variants include one or more mutant codons, which include one or more (e.g., 1, 2, or 3) substitutions to the codons, which change the amino acid encoded by the codons. Methods for introducing one or more substitutions into codons are known in the art, such as PCR mutagenesis and site-directed mutagenesis. Polynucleotide variants can be generated for a variety of reasons, such as to optimize codon expression for a particular host (e.g., changing codons in human mRNA to those preferred by a bacterial host such as E. coli). In some embodiments, the polynucleotide variant comprises at least one silent mutation in a non-coding or coding region of the sequence.

在一些實施例中，產生多核苷酸變異體以調節或改變所編碼多肽之表現(或表現水準)。在一些實施例中，產生多核苷酸變異體以增加所編碼多肽之表現。在一些實施例中，產生多核苷酸變異體以降低所編碼多肽之表現。在一些實施例中，與親代多核苷酸序列相比，多核苷酸變異體具有增加之所編碼多肽之表現。在一些實施例中，與親代多核苷酸序列相比，多核苷酸變異體具有降低之所編碼多肽之表現。In some embodiments, polynucleotide variants are generated to modulate or alter the expression (or expression level) of an encoded polypeptide. In some embodiments, polynucleotide variants are generated to increase the expression of an encoded polypeptide. In some embodiments, polynucleotide variants are generated to reduce the expression of an encoded polypeptide. In some embodiments, the polynucleotide variants have increased expression of an encoded polypeptide compared to the parental polynucleotide sequence. In some embodiments, the polynucleotide variants have reduced expression of an encoded polypeptide compared to the parental polynucleotide sequence.

在一些實施例中，多核苷酸包含多肽(例如，抗體)之編碼序列，該編碼序列在同一閱讀框中與有助於多肽自宿主細胞表現及分泌之多核苷酸(例如，用作控制多肽轉運之分泌序列之前導序列)融合。多肽可具有由宿主細胞裂解以形成多肽之「成熟」形式的前導序列。In some embodiments, the polynucleotide comprises a coding sequence for a polypeptide (e.g., an antibody) fused in the same reading frame to a polynucleotide that facilitates expression and secretion of the polypeptide from a host cell (e.g., a leader sequence used as a secretory sequence to control polypeptide trafficking). A polypeptide may have a leader sequence that is cleaved by a host cell to form a "mature" form of the polypeptide.

在一些實施例中，多核苷酸包含在同一閱讀框中與標記或標籤序列融合之多肽(例如抗體)之編碼序列。舉例而言，在一些實施例中，標記序列為六組胺酸(SEQ ID NO:360)標籤(HIS標籤)，其允許有效純化與該標記融合之多肽。在一些實施例中，當使用哺乳動物宿主(例如COS-7細胞)時，標記序列為源自流感血球凝集素蛋白之血球凝集素(HA)標籤。在一些實施例中，標記序列為FLAG™標籤。在一些實施例中，標記與其他標記或標籤結合使用。In some embodiments, the polynucleotide comprises a coding sequence for a polypeptide (e.g., an antibody) fused in the same reading frame to a marker or tag sequence. For example, in some embodiments, the marker sequence is a hexa-histidine (SEQ ID NO: 360) tag (HIS tag), which allows efficient purification of the polypeptide fused to the marker. In some embodiments, when a mammalian host (e.g., COS-7 cells) is used, the marker sequence is a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein. In some embodiments, the marker sequence is a FLAG™ tag. In some embodiments, the marker is used in combination with other markers or tags.

在一些實施例中，多核苷酸經分離。在一些實施例中，多核苷酸係實質上純的。 載體及細胞 In some embodiments, the polynucleotide is isolated. In some embodiments, the polynucleotide is substantially pure. Vectors and Cells

亦提供包含本文所述之各個及每一個多核苷酸之載體及細胞。在一些實施例中，表現載體包含編碼本文所述之抗TfR1抗體之多核苷酸分子。在一些實施例中，表現載體包含編碼作為本文所述之抗TfR1抗體之一部分的多肽的多核苷酸分子。在一些實施例中，表現載體包含編碼本文所述之抗TfR1抗體之重鏈多肽的多核苷酸分子。在一些實施例中，表現載體包含編碼本文所述之抗TfR1抗體之輕鏈多肽的多核苷酸分子。在一些實施例中，表現載體包含編碼本文所述之抗TfR1抗體之重鏈多肽及輕鏈多肽的多核苷酸分子。在一些實施例中，宿主細胞包含有包含編碼本文所述之抗TfR1抗體之多核苷酸分子的表現載體。在一些實施例中，宿主細胞包含有包含編碼作為本文所述之抗TfR1抗體之一部分的多肽的多核苷酸分子的表現載體。在一些實施例中，宿主細胞包含編碼本文所述之抗TfR1抗體之多核苷酸分子。在一些實施例中，宿主細胞包含有包含編碼本文所述之抗TfR1抗體之重鏈多肽的多核苷酸分子的表現載體。在一些實施例中，宿主細胞包含有包含編碼本文所述之抗TfR1抗體之輕鏈多肽的多核苷酸分子的表現載體。在一些實施例中，宿主細胞包含表現載體，該表現載體包含編碼本文所述之抗TfR1抗體之重鏈多肽及第二多核苷酸輕鏈多肽的第一多核苷酸。在一些實施例中，宿主細胞包含：(ii)第一表現載體，其包含編碼本文所述之抗TfR1抗體之重鏈多肽之多核苷酸分子，及(ii)第二表現載體，其包含編碼該抗TfR1抗體之輕鏈多肽之多核苷酸分子。 抗TFR1抗體之物理/化學性質之分析 Vectors and cells comprising each and every polynucleotide described herein are also provided. In some embodiments, the expression vector comprises a polynucleotide molecule encoding an anti-TfR1 antibody described herein. In some embodiments, the expression vector comprises a polynucleotide molecule encoding a polypeptide that is part of an anti-TfR1 antibody described herein. In some embodiments, the expression vector comprises a polynucleotide molecule encoding a heavy chain polypeptide of an anti-TfR1 antibody described herein. In some embodiments, the expression vector comprises a polynucleotide molecule encoding a light chain polypeptide of an anti-TfR1 antibody described herein. In some embodiments, the expression vector comprises a polynucleotide molecule encoding a heavy chain polypeptide and a light chain polypeptide of an anti-TfR1 antibody described herein. In some embodiments, a host cell comprises an expression vector comprising a polynucleotide molecule encoding an anti-TfR1 antibody described herein. In some embodiments, the host cell comprises an expression vector comprising a polynucleotide molecule encoding a polypeptide that is part of an anti-TfR1 antibody described herein. In some embodiments, the host cell comprises a polynucleotide molecule encoding an anti-TfR1 antibody described herein. In some embodiments, the host cell comprises an expression vector comprising a polynucleotide molecule encoding a heavy chain polypeptide of an anti-TfR1 antibody described herein. In some embodiments, the host cell comprises an expression vector comprising a polynucleotide molecule encoding a light chain polypeptide of an anti-TfR1 antibody described herein. In some embodiments, the host cell comprises an expression vector comprising a first polynucleotide encoding a heavy chain polypeptide of an anti-TfR1 antibody described herein and a second polynucleotide light chain polypeptide. In some embodiments, the host cell comprises: (ii) a first expression vector comprising a polynucleotide molecule encoding a heavy chain polypeptide of an anti-TfR1 antibody described herein, and (ii) a second expression vector comprising a polynucleotide molecule encoding a light chain polypeptide of the anti-TfR1 antibody. Analysis of physical/chemical properties of anti-TfR1 antibodies

可藉由此項技術中已知之各種方法來分析本揭示案之抗TfR1抗體之物理/化學性質及/或生物活性。在一些實施例中，測試抗TfR1抗體結合TfR1 (例如人類TfR1及/或石蟹猴TfR1)之能力。結合檢定包括但不限於SPR (例如Biacore)、ELISA及流式細胞術。在一些實施例中，測試抗TfR1抗體抑制、減少或阻斷運鐵蛋白與其TfR1受體結合之能力。在一些實施例中，測試抗TfR1抗體抑制、降低或阻斷TfR1活性之能力。在一些實施例中，測試抗TfR1抗體與TFR1一起內化及誘導TfR1內化增加之能力。另外，可評估抗體之溶解度、穩定性、熱穩定性、黏度、表現水準、表現品質及/或純化效率。The physical/chemical properties and/or biological activity of the anti-TfR1 antibodies of the present disclosure can be analyzed by various methods known in the art. In some embodiments, the ability of anti-TfR1 antibodies to bind to TfR1 (e.g., human TfR1 and/or cynomolgus monkey TfR1) is tested. Binding assays include but are not limited to SPR (e.g., Biacore), ELISA, and flow cytometry. In some embodiments, the ability of anti-TfR1 antibodies to inhibit, reduce, or block the binding of ferritin to its TfR1 receptor is tested. In some embodiments, the ability of anti-TfR1 antibodies to inhibit, reduce, or block the activity of TfR1 is tested. In some embodiments, the ability of anti-TfR1 antibodies to internalize together with TFR1 and induce increased internalization of TfR1 is tested. Additionally, antibodies may be evaluated for solubility, stability, thermostability, viscosity, expression level, expression quality, and/or purification efficiency.

在一些實施例中，提供用於鑑別影響TfR1活性之抗TfR1抗體之檢定。在一些實施例中，使用SPR、ELISA或FACS檢定評估抗TfR1抗體阻斷TfR1與Tf結合之能力。在一些實施例中，使用細胞毒性檢定評估抗TfR1抗體影響自然殺手(NK)細胞活性之能力。在一些實施例中，使用增殖檢定評估抗TfR1抗體影響T細胞活性之能力。In some embodiments, an assay is provided for identifying anti-TfR1 antibodies that affect TfR1 activity. In some embodiments, the ability of anti-TfR1 antibodies to block TfR1 binding to Tf is assessed using SPR, ELISA, or FACS assays. In some embodiments, the ability of anti-TfR1 antibodies to affect natural killer (NK) cell activity is assessed using cytotoxicity assays. In some embodiments, the ability of anti-TfR1 antibodies to affect T cell activity is assessed using proliferation assays.

在一些實施例中，本文所述之抗TfR1抗體為人類TfR1拮抗劑。在一些情況下，術語「抑制」、「誘導」、「減少」、「增加」、「增強」係相對於在包含抗TfR1抗體之結合物之治療不存在下的水準/活性。在一些情況下，術語「抑制」、「誘導」、「減少」、「增加」、「增強」係相對於在包含抗TfR1抗體之結合物之治療之前的水準/活性。 抗TFR1抗體結合物及複合物 In some embodiments, the anti-TfR1 antibodies described herein are human TfR1 antagonists. In some cases, the terms "inhibit", "induce", "reduce", "increase", "enhance" are relative to the level/activity in the absence of treatment with a conjugate comprising an anti-TfR1 antibody. In some cases, the terms "inhibit", "induce", "reduce", "increase", "enhance" are relative to the level/activity before treatment with a conjugate comprising an anti-TfR1 antibody. Anti-TFR1 Antibody Conjugates and Complexes

本揭示案亦提供結合物，其包含結合至第二分子之本文所述之抗TfR1抗體。在一些實施例中，第二分子包含本文所述之任何劑，例如治療劑。The present disclosure also provides conjugates comprising an anti-TfR1 antibody described herein conjugated to a second molecule. In some embodiments, the second molecule comprises any agent described herein, such as a therapeutic agent.

包含本文所述之抗TfR1抗體之結合物可使用此項技術中已知之任何合適方法來製備。在一些實施例中，結合物之組分藉由共價相互作用連接。在一些實施例中，使用多種雙功能蛋白質偶合劑製備結合物，該等偶合劑諸如N-琥珀醯亞胺基-3-(2-吡啶二硫醇)丙酸酯(SPDP)、亞胺基硫雜環戊烷(IT)、亞胺基酯之雙功能衍生物(諸如己二亞胺酸二甲酯HCl)、活性酯(諸如辛二酸二琥珀醯亞胺酯)、醛(諸如戊二醛)、雙疊氮基化合物(諸如雙(對疊氮基苯甲醯基)己二胺)、雙-重氮鎓衍生物(諸如雙-(對-重氮鎓苯甲醯基)-乙二胺)、二異氰酸酯(諸如甲苯2,6-二異氰酸酯)及雙活性氟化合物(諸如1,5-二氟-2,4-二硝基苯)。Conjugates comprising the anti-TfR1 antibodies described herein can be prepared using any suitable method known in the art. In some embodiments, the components of the conjugate are linked by covalent interactions. In some embodiments, conjugates are prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imino esters (such as dimethyl adipimidate HCl), active esters (such as disuccinyl suberate), and the like. imine esters), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis-(p-azidobenzyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

在一些實施例中，將游離半胱胺酸引入本文所述之抗TfR1抗體之恆定區中以促進與劑之結合。合適游離半胱胺酸取代為此項技術中已知的，諸如例如Zhou等人, Pharmaceuticals. 2021, 4(7):672 (參見例如表1)中所述之彼等，該文獻之全文以引用方式併入本文中。在一些實施例中，本文所述之抗TfR1抗體包含S442C取代(EU編號；Stimmel等人, JBC. 275(39):30445-30450)，該取代促進劑(例如ASO)之基於順丁烯二醯亞胺之結合。In some embodiments, free cysteine is introduced into the constant region of the anti-TfR1 antibody described herein to promote binding to the agent. Suitable free cysteine substitutions are known in the art, such as those described in, for example, Zhou et al., Pharmaceuticals. 2021, 4(7):672 (see, for example, Table 1), the entire text of which is incorporated herein by reference. In some embodiments, the anti-TfR1 antibody described herein comprises an S442C substitution (EU numbering; Stimmel et al., JBC. 275(39):30445-30450), which promotes cis-butylene diimide-based binding of an agent (e.g., ASO).

在一些實施例中，結合物之組分藉由Ohri等人, Bioconjugate Chem. 2018, 29, 473-485；Yamazoe等人, Bioconjugate Chemistry 2020 31 (4), 1199-1208；Shen等人, Nat Biotechnol. 2012年1月22日;30(2):184-9；Dimasi等人, Mol. Pharmaceutics 2017, 14, 1501-1516, Stimmel等人, JBC. 275(39):30445-30450及Sussman等人, Protein Engineering, Design & Selection, 2017, 31(2):47-54中所述之方法連接，該等文獻中之每一者之全文皆以引用方式併入本文中。In some embodiments, the components of the conjugate are linked by the methods described in Ohri et al., Bioconjugate Chem. 2018, 29, 473-485; Yamazoe et al., Bioconjugate Chemistry 2020 31 (4), 1199-1208; Shen et al., Nat Biotechnol. 2012 Jan 22;30(2):184-9; Dimasi et al., Mol. Pharmaceutics 2017, 14, 1501-1516, Stimmel et al., JBC. 275(39):30445-30450 and Sussman et al., Protein Engineering, Design & Selection, 2017, 31(2):47-54, each of which is incorporated herein by reference in its entirety.

在一些實施例中，本文所述之抗TfR1抗體結合至允許該劑用於診斷及/或偵測之可偵測物質或分子。可偵測物質可包括但不限於酶，諸如辣根過氧化物酶、鹼性磷酸酶、β-半乳糖苷酶及乙醯膽鹼酯酶；輔基，諸如生物素及黃素；螢光材料，諸如繖形酮、螢光素、異硫氰酸螢光素(FITC)、玫紅(rhodamine)、異硫氰酸四甲基玫紅(TRITC)、二氯三嗪基胺螢光素、丹磺醯氯、花青(Cy3)及藻紅蛋白；生物發光材料，諸如螢光素酶；放射性材料，諸如 212Bi、 14C、 57Co、 51Cr、 67Cu、 18F、 68Ga、 67Ga、 153Gd、 159Gd、 68Ge、 3H、 166Ho、 131I、 125I、 123I、 121I、 115In、 113In、 112In、 111In、 140La、 177Lu、 54Mn、 99Mo、 32P、 103Pd、 149Pm、 142Pr、 186Re、 188Re、 105Rh、 97Ru、 35S、 47Sc、 75Se、 153Sm、 113Sn、 117Sn、 85Sr、 99mTc、 201Ti、 133Xe、 90Y、 69Yb、 175Yb、 65Zn；正電子發射金屬；及磁性金屬離子。 In some embodiments, the anti-TfR1 antibodies described herein bind to a detectable substance or molecule that allows the agent to be used for diagnosis and/or detection. Detectable substances may include, but are not limited to, enzymes such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholine esterase; cofactors such as biotin and flavin; fluorescent materials such as umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, tetramethyl rhodamine isothiocyanate (TRITC), dichlorotriazinylamine fluorescein, dansyl chloride, cyanine (Cy3), and phycoerythrin; bioluminescent materials such as luciferase; radioactive materials such as 212 Bi, 14 C, 57 Co, 51 Cr, 67 Cu, 18 F, 68 Ga, 67 Ga, 153 Gd, 159 Gd, 68 Ge, 3 H, 166 Ho, 131 I, 125 I, 123 I, 121 I, 115 In, 113 In, 112 In, 111 In, 140 La, 177 Lu, 54 Mn, 99 Mo, 32 P, 103 Pd, 149 Pm, 142 Pr, 186 Re, 188 Re, 105 Rh, 97 Ru, 35 S, 47 Sc, 75 Se, 153 Sm, 113 Sn, 117 Sn, 85 Sr, 99m Tc, 201 Ti, 133 Xe, 90 Y, 69 Yb, 175 Yb, 65 Zn; positron emitting metals; and magnetic metal ions.

本文所述之抗TfR1抗體亦可結合(例如，經由連接子)至第二抗體(例如，抗β澱粉樣蛋白抗體，例如阿杜卡單抗、巴匹珠單抗、甘特魯單抗、索拉珠單抗、多奈單抗或雷卡奈單抗；抗BACE1抗體、抗τ抗體、抗α突觸核蛋白抗體、抗TDP-43抗體、抗LINGO-1抗體、抗LINGO-2抗體、抗LINGO-3抗體、抗LINGO-4抗體、抗TREM2抗體、抗C9orf72二肽重複聚GA抗體、抗CD20抗體、抗CD40抗體、抗CD40L抗體、抗VLA4抗體或抗MerTK抗體)以形成抗體異源結合物(heteroconjugate)。The anti-TfR1 antibodies described herein can also be conjugated (e.g., via a linker) to a second antibody (e.g., an anti-β-amyloid antibody, such as aducanumab, bapineumab, gantelumab, sorazumab, donetumab, or lecanezumab; an anti-BACE1 antibody, an anti-tau antibody, an anti-α-synaptophysin antibody, an anti-TDP-43 antibody, an anti-LINGO-1 antibody, an anti-LINGO-2 antibody, an anti-LINGO-3 antibody, an anti-LINGO-4 antibody, an anti-TREM2 antibody, an anti-C9orf72 dipeptide repeat poly-GA antibody, an anti-CD20 antibody, an anti-CD40 antibody, an anti-CD40L antibody, an anti-VLA4 antibody, or an anti-MerTK antibody) to form an antibody heteroconjugate.

在一些實施例中，本揭示案之抗TfR1抗體可結合至劑，例如分子或藥物，諸如抗體、蛋白質(例如，顆粒蛋白前體)、肽、酶(例如，葡糖腦苷脂酶)、核酸，例如反義寡核苷酸、短干擾RNA (siRNA)、RNA諸如信使RNA (mRNA)、微小RNA (miRNA)、嚮導RNA (gRNA)、胺基磷酸酯嗎啉基寡聚物或適配體等。在一些實施例中，抗TfR1抗體結合至粒子，例如脂質粒子或奈米粒子，其可含有治療劑，諸如本文所述之治療劑。在一些實施例中，抗TfR1抗體結合至病毒粒子，例如包含治療性核酸及/或蛋白質之病毒粒子，例如用於基因療法之病毒粒子(例如腺相關病毒或慢病毒)。抗TfR1抗體可藉由連接子連接至劑，例如藥物。在一些實施例中，抗TfR1抗體結合至小分子，諸如細胞毒性劑(例如美坦辛(maitansine))。在一些實施例中，抗TfR1抗體結合至消炎劑(例如糖皮質激素)。在一些實施例中，抗TfR1抗體結合至半衰期延長部分(例如聚乙二醇)。製備抗體-核酸結合物(諸如本揭示案中所考慮之結合物)之方法為此項技術中所熟知。參見例如美國專利申請公開案第US20190240346號及美國專利第US10881743號及第US10550188號，及國際專利申請公開案第WO1991004753號，其揭示內容之全文皆以引用方式併入本文中。In some embodiments, the anti-TfR1 antibodies of the present disclosure can be conjugated to an agent, such as a molecule or drug, such as an antibody, a protein (e.g., a progranulin), a peptide, an enzyme (e.g., a glucocerebrosidase), a nucleic acid, such as an antisense oligonucleotide, a short interfering RNA (siRNA), an RNA such as a messenger RNA (mRNA), a microRNA (miRNA), a guide RNA (gRNA), an aminophosphocyanine oligomer or an aptamer, etc. In some embodiments, the anti-TfR1 antibody is conjugated to a particle, such as a lipid particle or a nanoparticle, which may contain a therapeutic agent, such as a therapeutic agent described herein. In some embodiments, the anti-TfR1 antibody is conjugated to a viral particle, such as a viral particle containing a therapeutic nucleic acid and/or protein, such as a viral particle used for gene therapy (e.g., an adeno-associated virus or a lentivirus). The anti-TfR1 antibody can be linked to an agent, such as a drug, via a linker. In some embodiments, the anti-TfR1 antibody is conjugated to a small molecule, such as a cytotoxic agent (e.g., maitansine). In some embodiments, the anti-TfR1 antibody is conjugated to an anti-inflammatory agent (e.g., a glucocorticoid). In some embodiments, the anti-TfR1 antibody is conjugated to a half-life extending moiety (e.g., polyethylene glycol). Methods for preparing antibody-nucleic acid conjugates (such as the conjugates contemplated in the present disclosure) are well known in the art. See, for example, U.S. Patent Application Publication No. US20190240346, U.S. Patent Nos. US10881743 and US10550188, and International Patent Application Publication No. WO1991004753, the entire disclosures of which are incorporated herein by reference.

在一些實施例中，結合物為融合蛋白。包含本文所述之抗TfR1抗體之融合蛋白可使用此項技術中已知之任何合適方法來製得。此類融合蛋白可包括本揭示案之抗TfR1抗體(包括雙特異性、多特異性或多價抗TfR1抗體)與治療性多肽或抗體(例如抗β澱粉樣蛋白抗體，例如阿杜卡單抗、巴匹珠單抗、甘特魯單抗、索拉珠單抗、多奈單抗或雷卡奈單抗；抗BACE1抗體、抗τ抗體、抗α突觸核蛋白抗體、抗TDP-43抗體、抗LINGO-1抗體、抗LINGO-2抗體、抗LINGO-3抗體、抗LINGO-4抗體、抗TREM2抗體、抗C9orf72二肽重複聚GA抗體、抗CD20抗體、抗CD40抗體、抗CD40L抗體、抗VLA4抗體或抗MerTK抗體)。在一種情況下，本文所述之抗TfR1抗體經由融合蛋白結合至抗β澱粉樣蛋白抗體(例如阿杜卡單抗)。在一種情況下，本文所述之抗TfR1抗體經由融合蛋白結合至利妥昔單抗。在一種情況下，本文所述之抗TfR1抗體經由融合蛋白結合至酶(例如艾杜醣醛酸2-硫酸酯酶、葡糖腦苷脂酶、α-L-艾杜醣醛酸苷酶或磺胺醯胺酶)。在一種情況下，抗生物素蛋白可添加至重鏈之C端以產生融合蛋白，如Candelaria PV等人 Front Immunol. 2021;12:607692中所描述。融合蛋白可進一步結合或複合至第二分子或藥物，諸如生物素化藥物，如Daniels TR等人Biochim Biophys Acta. 2012;1820(3):291-317中所描述。In some embodiments, the conjugate is a fusion protein. Fusion proteins comprising the anti-TfR1 antibodies described herein can be prepared using any suitable method known in the art. Such fusion proteins may include an anti-TfR1 antibody (including a bispecific, multispecific or multivalent anti-TfR1 antibody) of the present disclosure and a therapeutic polypeptide or antibody (e.g., an anti-β-amyloid antibody, such as aducanumab, bapineumab, gantelumab, sorazumab, donetumab or lecanezumab; an anti-BACE1 antibody, an anti-tau antibody, an anti-α-synaptophysin antibody, an anti-TDP-43 antibody, an anti-LINGO-1 antibody, an anti-LINGO-2 antibody, an anti-LINGO-3 antibody, an anti-LINGO-4 antibody, an anti-TREM2 antibody, an anti-C9orf72 dipeptide repeat poly-GA antibody, an anti-CD20 antibody, an anti-CD40 antibody, an anti-CD40L antibody, an anti-VLA4 antibody or an anti-MerTK antibody). In one case, the anti-TfR1 antibody described herein is bound to an anti-β-amyloid antibody (e.g., aducanumab) via a fusion protein. In one case, the anti-TfR1 antibody described herein is bound to rituximab via a fusion protein. In one case, the anti-TfR1 antibody described herein is bound to an enzyme (e.g., iduronate 2-sulfatase, glucocerebrosidase, α-L-iduronasidase, or sulfadiazine) via a fusion protein. In one case, avidin can be added to the C-terminus of the heavy chain to produce a fusion protein as described in Candelaria PV et al. Front Immunol. 2021;12:607692. The fusion protein can be further conjugated or complexed to a second molecule or drug, such as a biotinylated drug, as described in Daniels TR et al. Biochim Biophys Acta. 2012;1820(3):291-317.

在一些實施例中，包含本文所述之抗TfR1抗體之複合物可使用此項技術中已知之任何合適方法來製備。在一些實施例中，複合物之組分藉由非共價相互作用連接。此類化合物包含與另一劑(例如治療劑)複合或與具有治療性多肽或蛋白質之脂質或奈米粒子複合之抗TfR1抗體。 抗TfR1抗體之組織靶向及用途 In some embodiments, complexes comprising the anti-TfR1 antibodies described herein can be prepared using any suitable method known in the art. In some embodiments, the components of the complex are linked by non-covalent interactions. Such compounds include anti-TfR1 antibodies complexed with another agent (e.g., a therapeutic agent) or with lipids or nanoparticles having a therapeutic polypeptide or protein. Tissue Targeting and Uses of Anti-TfR1 Antibodies

在一些實施例中，所述抗TfR1抗體可用於靶向腦組織且跨血腦障壁轉運劑以治療神經病症。示範性神經病症包括阿茲海默氏病、帕金森氏病、額顳葉失智症、ALS、杭丁頓氏病、多發性硬化症、脊髓性肌萎縮、肌肉營養不良、脊髓損傷、中風、眼科疾患、急性或慢性視神經炎、精神異常、圖雷特氏病腦損傷、腦腫瘤及癲癇。In some embodiments, the anti-TfR1 antibody can be used to target brain tissue and transport agents across the blood-brain barrier to treat neurological disorders. Exemplary neurological disorders include Alzheimer's disease, Parkinson's disease, frontotemporal dementia, ALS, Huntington's disease, multiple sclerosis, spinal muscular atrophy, muscular dystrophy, spinal cord injury, stroke, ophthalmic disease, acute or chronic optic neuritis, mental disorders, Tourette's disease brain damage, brain tumors, and epilepsy.

用於治療阿茲海默氏病之示範性治療劑包括辛酸甘油三酯、抗τ抗體、抗β澱粉樣蛋白抗體、抗DKK1抗體、APOE拮抗劑抗體、多奈哌齊(donepezil)、奎寧定(quinidine)、血清素6受體拮抗劑、β-分泌酶抑制劑、RAGE拮抗劑、BACE抑制劑、澱粉樣β蛋白抑制劑、磷酸二酯酶9A抑制劑、雙諾西絲胺酸(bisnorcymserine)、苔蘚蟲素-1、α-7增效劑、嘌呤受體P2Y6促效劑、τ蛋白聚集/TDP-43聚集抑制劑、N3pG-Aß mAb、mGlu2促效劑、喹唑啉酮、粒線體蛋白刺激劑、澱粉樣前體蛋白分泌酶抑制劑、5HT6拮抗劑、R-苯絲胺酸(R-phenserine)、澱粉樣β/τ蛋白抑制劑、MAO-B抑制劑、Lp-PLA2抑制劑、5-HT6受體拮抗劑、BET蛋白抑制劑、抗初原纖維AB mAb、諾美噻唑(nomethiazole)、組胺H3受體拮抗劑、PPAR-δ/γ促效劑、阿貝紫杉烷(abeotaxane)及p38促分裂原活化蛋白激酶抑制劑。Exemplary therapeutic agents for treating Alzheimer's disease include caprylic triglyceride, anti-tau antibodies, anti-beta amyloid antibodies, anti-DKK1 antibodies, APOE antagonist antibodies, donepezil, quinidine, serotonin 6 receptor antagonists, beta-secretase inhibitors, RAGE antagonists, BACE inhibitors, beta amyloid inhibitors, phosphodiesterase 9A inhibitors, bisnorcymserine, lichen planus-1, alpha-7 potentiators, purine receptor P2Y6 agonists, tau aggregation/TDP-43 aggregation inhibitors, N3pG-Aß mAb, mGlu2 agonist, quinazolinone, mitochondrial protein stimulator, amyloid precursor protein secretase inhibitor, 5HT6 antagonist, R-phenserine, amyloid β/tau protein inhibitor, MAO-B inhibitor, Lp-PLA2 inhibitor, 5-HT6 receptor antagonist, BET protein inhibitor, anti-protofibrillary AB mAb, nomethiazole, histamine H3 receptor antagonist, PPAR-δ/γ agonist, abeotaxane and p38 mitogen-activated protein kinase inhibitor.

用於治療ALS之示範性治療劑包括抗SOD1抗體、抗DR6抗體、抗DPR抗體、右旋普拉克索(dexpramipexole)、阿利莫洛爾(arimoclomal)、GM6、異布司特(ibudilast)、巨噬細胞調節劑、NOGO-A抑制劑及肌鈣蛋白複合物刺激劑。Exemplary therapeutic agents for treating ALS include anti-SOD1 antibodies, anti-DR6 antibodies, anti-DPR antibodies, dexpramipexole, arimoclomal, GM6, ibudilast, macrophage regulators, NOGO-A inhibitors, and sarcoma complex stimulators.

用於治療腦損傷之示範性治療劑包括阿樸嗎啡(apomorphine)、細胞介素抑制劑/神經肽受體調節劑及黃體酮受體促效劑。Exemplary therapeutic agents used to treat brain injury include apomorphine, interleukin inhibitors/neuropeptide receptor modulators, and progesterone receptor agonists.

用於治療腦腫瘤之示範性治療劑包括IDH1抑制劑、多柔比星(doxorubicin)、太平洋紫杉醇(paclitaxel)、抗EGFRvIII抗體-藥物結合物、貝伐單抗(bevacizumab)、FGF-R激酶抑制劑、PI3K抑制劑、卡博替尼(cabozantinib)、碘I 131德羅妥昔單抗(derlotuximab)生物素、PDGFR抑制劑、乳清酸羧胺三唑、本可麥定(penclomidine)之非神經毒性衍生物、戈伐替尼(golvatinib)、地塞比諾(dexanabinol)、TGF-β1激酶抑制劑、阿法替尼(afatinib)、IDO抑制劑、卡巴他賽(cabazitaxel)、Src激酶/前微管蛋白抑制劑、SMO蛋白抑制劑、內皮素A/B受體拮抗劑、蛋白酶體抑制劑、T型鈣通道拮抗劑、毒胡蘿蔔素(thapsigargin)類似物、伊立替康(irinotecan)、納武單抗(nivolumab)、CSF-1R抑制劑、派拉瑞普(pelareorep)、EGFR拮抗劑、輸出蛋白-1蛋白抑制劑/核蛋白抑制劑、BIRC5蛋白抑制劑、依伏磷醯胺(evofosfamide)、阿貝紫杉烷、ENG蛋白抑制劑、反式藏紅花酸鈉(trans-sodium crocetinate)、N7-烷基化劑、靶向抗血管生成劑及維利帕尼(veliparib)。Exemplary therapeutic agents for the treatment of brain tumors include IDH1 inhibitors, doxorubicin, paclitaxel, anti-EGFRvIII antibody-drug conjugates, bevacizumab, FGF-R kinase inhibitors, PI3K inhibitors, cabozantinib, iodine I 131 derlotuximab biotin, PDGFR inhibitor, orotate carboxyamide triazole, non-neurotoxic derivatives of penclomidine, golvatinib, dexanabinol, TGF-β1 kinase inhibitor, afatinib, IDO inhibitor, cabazitaxel, Src kinase/protubulin inhibitor, SMO protein inhibitor, endothelin A/B receptor antagonist , proteasome inhibitors, T-type calcium channel antagonists, thapsigargin analogs, irinotecan, nivolumab, CSF-1R inhibitors, pelareorep, EGFR antagonists, exportin-1 protein inhibitors/nucleoprotein inhibitors, BIRC5 protein inhibitors, evofosfamide, abetaxanes, ENG protein inhibitors, trans-sodium crocetinate, N7-alkylating agents, targeted antiangiogenic agents, and veliparib.

用於治療癲癇之示範性治療劑包括依維莫司(everolimus)、乙酸艾司利卡西平(eslicarbazepine acetate)、阿普唑侖(alprazolam)、布瓦西坦(brivaracetam)、卡巴馬平(carbamazepine)、大麻二酚、4-胺基丁酸轉胺酶抑制劑、吡崙帕奈(perampanel)、GABA-A受體促效劑、合成石杉鹼、普瑞巴林(pregabalin)、氯巴占(clobazam)、二氮平(diazepam)、GABA A突觸及突觸外受體調節劑、托比拉邁(topiramate)IV、拉考沙胺(lacosamide)及血清素受體促效劑。 Exemplary therapeutic agents used to treat epilepsy include everolimus, eslicarbazepine acetate, alprazolam, brivaracetam, carbamazepine, cannabidiol, 4-aminobutyrate aminotransferase inhibitors, perampanel, GABA-A receptor agonists, synthetic huperzine, pregabalin, clobazam, diazepam, GABA A synaptic and extrasynaptic receptor modulators, topiramate IV, lacosamide, and serotonin receptor agonists.

用於治療遺傳病症(例如，弗里德里希氏共濟失調(Friedrich's ataxia)、晚期嬰兒神經元蠟樣質、脊髓及延髓性肌萎縮、共濟失調毛細血管擴張症、泛酸激酶相關之神經退化、脊髓性肌萎縮、家族性澱粉樣多發性神經病變、雷特症候群(Rett syndrome)、萊氏症候群(Leigh syndrome)、威爾遜氏病(Wilson’s disease))之示範性治療劑包括NF/E2相關因子2刺激劑、干擾素γ-1b、rhTPP1酶替代療法、凡替醌(vatiquinone)、去鐵酮(deferiprone)、諾西那生(nusinersen)、奧瑪韋隆(omaveloxolone)、ISIS-TTRRX、血清素1A受體促效劑、細胞介素抑制劑/神經肽受體調節劑、靶向TTR之siRNA抑制劑、磷酸泛酸酯置換、DcpS抑制劑、半胱胺重酒石酸鹽、吲哚丙酸、轉甲狀腺素蛋白解離抑制劑及雙膽鹼四硫代鉬酸鹽。For the treatment of genetic diseases (e.g., Friedrich's ataxia, late infantile neuromyelitis, spinal and bulbar muscular atrophy, ataxia telangiectasia, pantothenate kinase-related neurodegeneration, spinal muscular atrophy, familial amyloid polyneuropathy, Rett syndrome, Leigh syndrome, Wilson's Exemplary therapeutic agents for treating TTR-related disease include NF/E2-related factor 2 stimulators, interferon gamma-1b, rhTPP1 enzyme replacement therapy, vatiquinone, deferiprone, nusinersen, omaveloxolone, ISIS-TTRRX, serotonin 1A receptor agonists, interleukin inhibitors/neuropeptide receptor modulators, siRNA inhibitors targeting TTR, phosphopanthocyanate replacement, DcpS inhibitors, cysteamine bitartrate, indolepropionic acid, transthyretin dissociation inhibitors, and dicholine tetrathiomolybdate.

用於治療頭痛之示範性治療劑包括抗CGRP mAb、CGRP受體拮抗劑mAb、舒馬曲坦(sumatriptan)、右美沙芬(dextromethorphan)/奎寧定、A型奧那肉毒桿菌毒素(onabotulinumtoxinA)、血清素-1F受體促效劑、nNOS抑制劑/5HT、二氫麥角胺、環苯扎林(cyclobenzaprine)及阿司匹靈(aspirin)/舒馬曲坦組合。Exemplary therapeutic agents for the treatment of headache include anti-CGRP mAb, CGRP receptor antagonist mAb, sumatriptan, dextromethorphan/quinidine, onabotulinumtoxinA, serotonin-1F receptor agonists, nNOS inhibitors/5HT, dihydroergotamine, cyclobenzaprine, and aspirin/sumatriptan combinations.

用於治療杭丁頓氏病之示範性治療劑包括拉喹莫德(laquinimod)、PDE10抑制劑、普多比啶(pridopidine)、半胱胺重酒石酸鹽及aVMAT2抑制劑。Exemplary therapeutic agents used to treat Huntington's disease include laquinimod, PDE10 inhibitors, pridopidine, cysteamine bitartrate, and aVMAT2 inhibitors.

用於治療多發性硬化症之示範性治療劑包括那他珠單抗(natalizumab)、反丁烯二酸單甲酯前藥、抗LINGO-1抗體、Nck蛋白調節劑、S1PR-1/5受體促效劑、芬戈莫德(fingolimod)、抗CD52 mAb、艾地苯醌(idebenone)、PPAR-γ促效劑/調節劑、拉喹莫德、酪胺酸激酶抑制劑、抗CD19 mAb、異布司特、胍那芐(guanabenz)、抗CD20 mAb (例如，利妥昔單抗、歐瑞珠單抗(ocrelizumab)、奧法木單抗(ofatumumab)或烏妥昔單抗(ublituximab))、干擾素β-1b、IL-7受體抑制劑、S1P1受體促效劑、髓磷脂蛋白刺激劑、琥珀酸雌三醇、伊米塞爾-T (imilecleucel-T)、抗VLA 2 mAb、BAFF-R調節劑、CD100抗原抑制劑、抗DR6抗體及NF-κB抑制劑。Exemplary therapeutic agents for treating multiple sclerosis include natalizumab, fumarate monomethyl ester prodrug, anti-LINGO-1 antibody, Nck protein modulator, S1PR-1/5 receptor agonist, fingolimod, anti-CD52 mAb, idebenone, PPAR-γ agonist/modulator, laquinimod, tyrosine kinase inhibitor, anti-CD19 mAb, isobupifact, guanabenz, anti-CD20 mAb (e.g., rituximab, ocrelizumab, ofatumumab, or ublituximab), interferon beta-1b, IL-7 receptor inhibitors, S1P1 receptor agonists, myelin protein stimulators, estriol succinate, imilecleucel-T, anti-VLA 2 mAb, BAFF-R modulators, CD100 antigen inhibitors, anti-DR6 antibodies, and NF-κB inhibitors.

用於治療肌肉營養不良之示範性治療劑包括肌肉生長抑制素(myostatin)抑制劑、屈沙培森(drisapersen)、依特普森(eteplirsen)、鹵夫酮(halofuginone)、艾地苯醌、ISIS-DMPKR X、類固醇受體促效劑、GAPDH抑制劑、遺傳轉錄抑制劑、他達拉非(tadalafil)、阿他盧侖(ataluren)及糖皮質激素受體促效劑。 Exemplary therapeutic agents used to treat muscular dystrophy include myostatin inhibitors, drisapersen, eteplirsen, halofuginone, idebenone, ISIS-DMPKR X , steroid receptor agonists, GAPDH inhibitors, genetic transcription inhibitors, tadalafil, ataluren, and glucocorticoid receptor agonists.

用於治療疼痛之示範性治療劑包括神經胚素、P2X3嘌呤受體拮抗劑、SNARE蛋白拮抗劑、羥考酮(oxycodone)-納曲酮(naltrexone)核心(抗濫用)、阿米替林(amitriptyline)/氯胺酮(ketamine)、林他托莫德(rintatolimod)、大麻素受體CB2促效劑、非紅血球生成肽、PPAR-γ促效劑、糖原磷酸化酶抑制劑、NMDA受體拮抗劑、唑來膦酸(zoledronic acid)、早期生長反應蛋白1抑制劑、(組胺-3受體拮抗劑、丁基原啡因(buprenorphine)、細胞介素抑制劑、西博帕多(cebranopadol)、塞來昔布(celecoxib)、花生四烯酸類似物、合成辣椒素、Nav1.7鈉通道抑制劑、類鴉片κ受體促效劑、度洛西汀(duloxetine)、神經生長因子刺激劑、右美托咪定(dexmedetomidine)、電壓閘控鈉通道抑制劑、布比卡因(bupivacaine)、血管收縮素2型受體拮抗劑、神經生長因子抑制劑、p38抑制劑、拉帕斯內(rapastinel)、左啡諾(levorphanol)、CGRP mAb、普瑞巴林、mGlu2/3受體促效劑、CACNA2D1蛋白調節劑、骨再吸收因子抑制劑、神經胚素、μ-阿片類鎮痛劑、nNOS抑制劑、O-去甲基曲馬多(O-desmethyltramadol)、棕櫚醯乙醇醯胺、GABA A促效劑、TRPV-1受體促效劑、納比西莫斯(nabiximols)、環加氧酶2抑制劑、神經生長因子調節劑、環苯扎林、氟比洛芬(flurbiprofen)、脂肪酸醯胺水解酶抑制劑及伊布洛芬(ibuprofen)/磷脂醯膽鹼。Exemplary therapeutic agents for the treatment of pain include neuroblastoma, P2X3 purine receptor antagonists, SNARE protein antagonists, oxycodone-naltrexone core (anti-abuse), amitriptyline/ketamine, rintatolimod, cannabinoid receptor CB2 agonists, non-erythropoietic peptides, PPAR-γ agonists, glycogen phosphorylase inhibitors, NMDA receptor antagonists, zoledronic acid, acid), early growth response protein 1 inhibitors, (histamine-3 receptor antagonists, buprenorphine, interleukin inhibitors, cebanopadol, celecoxib, arachidonic acid analogs, synthetic capsaicin, Nav1.7 sodium channel inhibitors, opioid kappa receptor agonists, duloxetine, neural growth factor stimulators, dexmedetomidine, voltage-gated sodium channel inhibitors, bupivacaine, vasopressin type 2 receptor antagonists, neural growth factor inhibitors, p38 inhibitors, rapastinel, levorphanol, CGRP mAbs, pregabalin, mGlu2/3 receptor agonists, CACNA2D1 protein modulators, bone resorption factor inhibitors, neuroblastin, μ-opioid analgesics, nNOS inhibitors, O-desmethyltramadol, palmitoylethanolamide, GABA A agonists, TRPV-1 receptor agonists, nabiximols, cyclooxygenase 2 inhibitors, neural growth factor regulators, cyclobenzaprine, flurbiprofen, fatty acid amide hydrolase inhibitors, and ibuprofen/phosphatidylcholine.

用於治療帕金森氏病之示範性治療劑包括金剛烷胺、阿樸嗎啡、α7尼古丁乙醯膽鹼受體部分促效劑、抗α-突觸核蛋白抗體、α-突觸核蛋白抑制劑、左旋多巴、D1增效劑、二聚尿劑、血清素1A/1B部分促效劑、非帕美唑(fipamezole)、GM6、類視色素X受體促效劑、伊曲茶鹼(istradefylline)、羅替戈汀(rotigotine)、普拉克索(pramipexole)/雷沙吉蘭(rasagiline)、R-苯絲胺酸、血清素2A/6受體拮抗劑、腺苷A2A受體拮抗劑、沙芬醯胺(safinamide)及多巴胺受體促效劑。Exemplary therapeutic agents for treating Parkinson's disease include ramantadine, apomorphine, α7 nicotinic acetylcholine receptor partial agonist, anti-α-synuclein antibody, α-synuclein inhibitor, levodopa, D1 potentiator, dimerurea, serotonin 1A/1B partial agonist, fipamezole, GM6, retinoid X receptor agonist, istradefylline, rotigotine, pramipexole/rasagiline, R-phenylsine, serotonin 2A/6 receptor antagonists, adenosine A2A receptor antagonists, safinamide, and dopamine receptor agonists.

用於治療痙攣之示範性治療劑包括巴氯芬(baclofen)、A型奧那肉毒桿菌毒素、A型阿波肉毒桿菌毒素(abobotulinumtoxinA)、阿巴氯芬(arbaclofen)、納比西莫斯及A型因可肉毒桿菌毒素(incobotulinumtoxinA)。Exemplary therapeutic agents used to treat spasticity include baclofen, onabotulinumtoxinA, abobotulinumtoxinA, arbaclofen, nabiximols, and incobotulinumtoxinA.

用於治療脊髓損傷之示範性治療劑包括抗Lingo-1抗體、抗NgR1抗體、神經胚素、神經系統調節劑、Rho GTP結合蛋白抑制劑及纖維母細胞生長因子受體。Exemplary therapeutic agents for treating spinal cord injury include anti-Lingo-1 antibodies, anti-NgR1 antibodies, neuroblastogens, nervous system modulators, Rho GTP-binding protein inhibitors, and fibroblast growth factor receptors.

用於治療中風之示範性治療劑包括那他珠單抗、人類野生型活化蛋白C之重組突變形式、替格瑞洛(ticagrelor)、達法吡啶、阿司匹靈、尼莫地平微粒、GM6、PARP抑制劑、PDZ域抑制劑、β澱粉樣蛋白抑制劑、達比加群(dabigatran)、及亞硝酸鈉。Exemplary therapeutic agents for treating stroke include natalizumab, recombinant mutant forms of human wild-type activated protein C, ticagrelor, dafacillin, aspirin, nimodipine microparticles, GM6, PARP inhibitors, PDZ domain inhibitors, beta-amyloid inhibitors, dabigatran, and sodium nitrite.

用於治療圖雷特氏症候群之示範性治療劑包括組胺-3受體拮抗劑、4-胺基丁酸轉胺酶抑制劑、A型阿波肉毒桿菌毒素、依考匹泮(ecopipam)、VMAT2抑制劑、阿坎酸(acamprosate)及氨己烯酸。Exemplary therapeutic agents used to treat Tourette syndrome include histamine-3 receptor antagonists, 4-aminobutyrate transaminase inhibitors, botulinum toxin type A, ecopipam, VMAT2 inhibitors, acamprosate, and vigabatrin.

用於治療其他神經病症之其他示範性治療劑包括肌肉生長抑制素抑制劑、NF/E2相關因子2刺激劑、抗τ抗體、髓過氧化物酶抑制劑、粒線體通透性轉變孔抑制劑、貝利單抗(belimumab)、II-B型活化素受體調節劑mAb、C1酯酶抑制劑、羧基麥芽糖鐵、胺苯吡啶、芬戈莫德、單胺氧化酶B抑制劑、神經遞質調節劑、多巴胺受體促效劑、抗CD19 mAb、VMAT2抑制劑、CD20 mAb、胸腺素β-4、抗IL-6受體mAb、依庫珠單抗(eculizumab)、AMPA受體調節劑、類固醇羥化酶抑制劑、磷酸吡哆醛(pyridoxal phosphate)、阿貝紫杉烷、阿醋脲酸(aceneuramic acid)及羥丁酸鈉。Other exemplary therapeutic agents for treating other neurological disorders include myostatin inhibitors, NF/E2-related factor 2 stimulators, anti-tau antibodies, myeloperoxidase inhibitors, mitochondrial permeability transition pore inhibitors, belimumab, type II-B activin receptor modulator mAb, C1 esterase inhibitors, carboxymaltose ferric, amifenpyridine, fingolimod, monoamine oxidase B inhibitors, neurotransmitter modulators, dopamine receptor agonists, anti-CD19 mAb, VMAT2 inhibitors, CD20 mAb, thymosin beta-4, anti-IL-6 receptor mAb, eculizumab, AMPA receptor modulators, steroid hydroxylation inhibitors, pyridoxal phosphate, phosphate), abetaxane, aceneuramic acid, and sodium hydroxybutyrate.

在一些實施例中，結合物為包含本文所述之抗TfR1抗體及完整抗體或抗體片段(治療劑)之融合多肽。在某些實施例中，完整抗體或抗體片段為抗β澱粉樣蛋白抗體(例如，阿杜卡單抗、巴匹珠單抗、甘特魯單抗、索拉珠單抗、多奈單抗或雷卡奈單抗)、抗BACE1抗體、抗τ抗體、抗α突觸核蛋白抗體、抗TDP-43抗體、抗LINGO-1抗體、抗LINGO-2抗體、抗LINGO-3抗體、抗LINGO-4抗體、抗TREM2抗體、抗C9orf72二肽重複聚GA抗體(亦即，如自染色體9開放閱讀框72 (C9orf72)基因轉譯的能夠結合具有至少6個重複(GA) 6之聚甘胺酸-丙胺酸(GA)之二肽重複序列(DPR)的抗體)、抗CD20抗體、抗CD40抗體、抗CD40L抗體、抗VLA4抗體、抗MerTK抗體、抗TWEAK抗體或抗TWEAK-R抗體。 醫藥組合物 In some embodiments, the conjugate is a fusion polypeptide comprising an anti-TfR1 antibody described herein and a complete antibody or antibody fragment (therapeutic agent). In certain embodiments, the complete antibody or antibody fragment is an anti-β-amyloid antibody (e.g., aducanumab, bapilizumab, gantelumab, sorazumab, donetumab or lecaneluzumab), an anti-BACE1 antibody, an anti-tau antibody, an anti-α-synaptophysin antibody, an anti-TDP-43 antibody, an anti-LINGO-1 antibody, an anti-LINGO-2 antibody, an anti-LINGO-3 antibody, an anti-LINGO-4 antibody, an anti-TREM2 antibody, an anti-C9orf72 dipeptide repeat poly-GA antibody (i.e., an antibody capable of binding to a polypeptide having at least 6 repeats (GA) as translated from the chromosome 9 open reading frame 72 (C9orf72) gene) 6 ), anti-CD20 antibody, anti-CD40 antibody, anti-CD40L antibody, anti-VLA4 antibody, anti-MerTK antibody, anti-TWEAK antibody or anti-TWEAK-R antibody. Pharmaceutical composition

本揭示案提供包含本文所述之抗TfR1抗體之組合物。本揭示案亦提供醫藥組合物，其包含本文所述之抗TfR1抗體及醫藥學上可接受之媒劑。The present disclosure provides compositions comprising the anti-TfR1 antibodies described herein. The present disclosure also provides pharmaceutical compositions comprising the anti-TfR1 antibodies described herein and a pharmaceutically acceptable vehicle.

藉由將本揭示案之抗TfR1抗體與醫藥學上可接受之媒劑(例如，載劑或賦形劑)組合來製備調配物以用於儲存及/或使用。熟習此項技術者通常認為醫藥學上可接受之載劑、賦形劑及/或穩定劑為調配物或醫藥組合物之非活性成分。The formulation is prepared by combining the anti-TfR1 antibody of the present disclosure with a pharmaceutically acceptable vehicle (e.g., carrier or excipient) for storage and/or use. Those skilled in the art generally consider pharmaceutically acceptable carriers, excipients and/or stabilizers to be inactive ingredients of formulations or pharmaceutical compositions.

合適醫藥學上可接受之媒劑包括但不限於無毒緩衝劑，例如磷酸鹽、檸檬酸鹽及其他有機酸；鹽，諸如氯化鈉；抗氧化劑，包括抗壞血酸及甲硫胺酸；防腐劑，例如十八烷基二甲基芐基氯化銨、氯化六甲季銨、苯扎氯銨、氯化本索寧、酚、丁醇或苯甲醇、對羥基苯甲酸烷基酯例如對羥基苯甲酸甲酯或對羥基苯甲酸丙酯、兒茶酚、間苯二酚、環己醇、3-戊醇、及間甲酚；低分子量多肽(小於約10個殘基)；蛋白質，例如血清白蛋白、明膠或免疫球蛋白；親水性聚合物，例如聚乙烯吡咯啶酮；胺基酸，諸如甘胺酸、麩醯胺酸、天冬胺酸、組胺酸、精胺酸或離胺酸；碳水化合物，諸如單糖、雙糖、葡萄糖、甘露糖或糊精；螯合劑，例如EDTA；糖類，例如蔗糖、甘露醇、海藻糖或山梨醇；成鹽相對離子，例如鈉；金屬錯合物，例如Zn-蛋白質錯合物；及非離子表面活性劑，例如TWEEN或聚乙二醇(PEG)。( Remington: The Science and Practice of Pharmacy, 第22版, 2012, Pharmaceutical Press, London.)。在一些實施例中，調配物係呈水溶液形式。在一些實施例中，調配物經凍乾或呈替代性乾燥形式。 Suitable pharmaceutically acceptable vehicles include, but are not limited to, nontoxic buffers such as phosphates, citrates and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzylammonium chloride, hexamethylammonium chloride, benzalkonium chloride, benzathonine chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl parabens, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight polypeptides (less than about 10 residues); proteins; Proteins, such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, aspartic acid, histidine, arginine or lysine; carbohydrates, such as monosaccharides, disaccharides, glucose, mannose or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming relative ions, such as sodium; metal complexes, such as Zn-protein complexes; and non-ionic surfactants, such as TWEEN or polyethylene glycol (PEG). ( Remington: The Science and Practice of Pharmacy, 22nd edition , 2012, Pharmaceutical Press, London.). In some embodiments, the formulation is in the form of an aqueous solution. In some embodiments, the formulation is lyophilized or in an alternative dried form.

治療調配物可呈單位劑型。此類調配物包括錠劑、丸劑、膠囊、散劑、顆粒、於水或非水性介質中之溶液或懸浮液、或栓劑。在固體組合物(諸如錠劑)中，將主要活性成分與醫藥載劑混合。習知壓錠成分包括玉米澱粉、乳糖、蔗糖、山梨醇、滑石、硬脂酸、硬脂酸鎂、磷酸二鈣或樹膠及稀釋劑(例如水)。此等可用於形成含有本揭示案之化合物或其無毒醫藥學上可接受之鹽之均質混合物的固體預調配組合物。然後將固體預調配組合物細分成上述類型之單位劑型。調配物或組合物之錠劑、丸劑等可經包衣或以其他方式配混以提供具有延長作用之優點的劑型。舉例而言，錠劑或丸劑可包含由外部組分覆蓋之內部組合物。此外，兩種組分可由腸溶層隔開，該腸溶層用於抵抗崩解且允許內部組分完整地通過胃或延遲釋放。多種材料可用於此類腸溶層或包衣，此類材料包括多種聚合酸及聚合酸與諸如蟲膠、鯨蠟醇及乙酸纖維素之材料之混合物。The therapeutic formulation may be in unit dosage form. Such formulations include tablets, pills, capsules, powders, granules, solutions or suspensions in water or non-aqueous media, or suppositories. In solid compositions such as tablets, the main active ingredient is mixed with a pharmaceutical carrier. Conventional compressed tablet ingredients include corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, calcium phosphate or resin and a diluent such as water. These can be used to form a solid preformulated composition containing a homogeneous mixture of the compounds of the present disclosure or their non-toxic pharmaceutically acceptable salts. The solid preformulated composition is then subdivided into unit dosage forms of the above type. Tablets, pills, etc. of the formulation or composition may be coated or otherwise compounded to provide a dosage form with the advantage of prolonged action. For example, a tablet or pill may comprise an inner composition covered by an outer component. In addition, the two components may be separated by an enteric layer that resists disintegration and allows the inner component to pass through the stomach intact or to be released later. A variety of materials may be used for such enteric layers or coatings, including a variety of polymeric acids and mixtures of polymeric acids with materials such as wormwood, cetyl alcohol, and cellulose acetate.

本揭示案之結合劑可以任何適合形式調配以用於遞送至靶細胞/組織。在一些實施例中，抗TfR1抗體可經調配為脂質體、微粒、微膠囊、白蛋白微球、微乳液、奈米顆粒、奈米膠囊或巨乳液。在一些實施例中，醫藥調配物包括與脂質體複合之本揭示案之抗TfR1抗體。產生脂質體之方法為熟習此項技術者已知。舉例而言，一些脂質體可藉由用包含磷脂醯膽鹼、膽固醇及PEG衍生性磷脂醯乙醇胺(PEG-PE)之脂質組合物進行逆相蒸發來產生。The binding agents of the present disclosure may be formulated in any suitable form for delivery to target cells/tissues. In some embodiments, the anti-TfR1 antibody may be formulated as liposomes, microparticles, microcapsules, albumin microspheres, microemulsions, nanoparticles, nanocapsules, or macroemulsions. In some embodiments, the pharmaceutical formulation includes the anti-TfR1 antibody of the present disclosure complexed with liposomes. Methods for producing liposomes are known to those skilled in the art. For example, some liposomes may be produced by reverse phase evaporation of a lipid composition comprising phospholipid acylcholine, cholesterol, and PEG-derivatized phospholipid acylethanolamine (PEG-PE).

在一些實施例中，抗TfR1抗體經調配為持續釋放製劑。持續釋放製劑之合適實例包括含有劑之固體疏水性聚合物之半滲透性基質，其中該等基質呈成形製品(例如，膜或微膠囊)之形式。持續釋放基質包括但不限於聚酯、水凝膠諸如聚(甲基丙烯酸2-羥乙酯)或聚(乙烯醇)、聚丙交酯、L-麩胺酸與7乙基-L-麩胺酸之共聚物、不可降解乙烯-乙酸乙烯酯、可降解乳酸-乙醇酸共聚物，諸如LUPRON DEPOT™ (由乳酸-乙醇酸共聚物及乙酸亮丙瑞林組成之可注射微球)、乙酸異丁酸蔗糖酯及聚-D-(−)-3-羥基丁酸。In some embodiments, the anti-TfR1 antibody is formulated as a sustained release formulation. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing agents, wherein the matrices are in the form of shaped articles (e.g., films or microcapsules). Sustained release matrices include, but are not limited to, polyesters, hydrogels such as poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol), polylactides, copolymers of L-glutamine and 7-ethyl-L-glutamine, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(−)-3-hydroxybutyric acid.

本揭示案之醫藥組合物或調配物可以多種方式投與以用於局部或全身治療。在一些實施例中，投與係藉由表皮或經皮貼片、軟膏、洗劑、乳霜、凝膠、滴劑、栓劑、噴霧劑、液體及散劑進行局部投與。在一些實施例中，藉由吸入或吹入粉末或氣溶膠(包括藉由霧化器、氣管內及鼻內)進行肺部投與。在一些實施例中，投與為經口投與。在一些實施例中，投與為非經腸投與，包括靜脈內、動脈內、腫瘤內、皮下、腹膜內、肌內(例如注射或輸注)或顱內(例如鞘內或心室內)。在一些實施例中，藉由靜脈內注射或靜脈內輸注投與。在一些實施例中，藉由肌肉內注射投與。在一些實施例中，投與為鞘內(例如，經由直接注射至鞘內空間中或註射至用於鞘內遞送之端口中之鞘內投與)。在一些實施例中，皮下投與。The pharmaceutical compositions or formulations of the present disclosure can be administered in a variety of ways for local or systemic treatment. In some embodiments, administration is topical by epidermal or transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. In some embodiments, pulmonary administration is performed by inhalation or insufflation of powders or aerosols (including by nebulizer, intratracheal and intranasal). In some embodiments, administration is oral administration. In some embodiments, administration is parenteral administration, including intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion) or intracranial (e.g., intrathecal or intraventricular). In some embodiments, administration is by intravenous injection or intravenous infusion. In some embodiments, administration is by intramuscular injection. In some embodiments, administration is intrathecal (e.g., intrathecal administration via direct injection into the intrathecal space or injection into a port for intrathecal delivery). In some embodiments, administration is subcutaneous.

各種遞送系統為已知的且可用於投與本文所述之抗TfR1抗體。在一些實施例中，本文所述之抗TfR1抗體或組合物係在受控釋放或持續釋放系統中遞送。在一些實施例中，使用泵來達成控制或持續釋放。在一些實施例中，使用聚合材料達成本文抗TfR1抗體之控制或持續釋放。持續釋放調配物中所用之聚合物之實例包括但不限於聚甲基丙烯酸2-羥基乙酯、聚甲基丙烯酸甲酯、聚丙烯酸、聚乙烯-共-乙酸乙烯酯、聚甲基丙烯酸、聚乙交酯(PLG)、聚酸酐、聚N-乙烯基吡咯啶酮、聚乙烯醇(PVA)、聚丙烯醯胺、聚乙二醇(PEG)、聚丙交酯(PLA)、聚丙交酯-共-乙交酯(PLGA)及聚原酸酯。持續釋放調配物中所用之任何聚合物應為惰性的、不含可浸出雜質、儲存時穩定、無菌且可生物降解的。Various delivery systems are known and can be used to administer the anti-TfR1 antibodies described herein. In some embodiments, the anti-TfR1 antibodies or compositions described herein are delivered in a controlled release or sustained release system. In some embodiments, a pump is used to achieve controlled or sustained release. In some embodiments, a polymeric material is used to achieve controlled or sustained release of the anti-TfR1 antibodies herein. Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), polyacrylic acid, polyethylene-co-vinyl acetate, polymethacrylic acid, polyglycolide (PLG), polyanhydrides, poly-N-vinyl pyrrolidone, polyvinyl alcohol (PVA), polyacrylamide, polyethylene glycol (PEG), polylactide (PLA), polylactide-co-glycolide (PLGA) and polyorthoesters. Any polymer used in a sustained-release formulation should be inert, free of leachable impurities, stable in storage, sterile, and biodegradable.

額外遞送系統可用於投與本文所述之抗TfR1抗體，包括但不限於可注射藥物遞送裝置及滲透泵。可注射藥物遞送裝置包括例如手持裝置(例如，自動注射器)或可穿戴裝置。不同類型之滲透泵系統可包括單區室系統、雙區室系統及多區室系統。 示範性實施例 Additional delivery systems can be used to administer the anti-TfR1 antibodies described herein, including but not limited to injectable drug delivery devices and osmotic pumps. Injectable drug delivery devices include, for example, handheld devices (e.g., autoinjectors) or wearable devices. Different types of osmotic pump systems can include single compartment systems, dual compartment systems, and multi-compartment systems. Exemplary Embodiments

如下文所述之示范性實施例亦在本揭示案之範疇內： 實施例1. 一種結合至人類運鐵蛋白受體之抗體，其包含有包含VH互補決定區(CDR)1、VH CDR2及VH CDR3之重鏈可變區(VH)及包含VL CDR1、VL CDR2及VL CDR3之輕鏈可變區(VL)，其中： 該VH CDR1包含胺基酸序列GFTFSSYX 1MN (SEQ ID NO:18)或胺基酸序列SYX 1MN (SEQ ID NO:26)，其中X 1為S或A； 該VH CDR2包含胺基酸序列SISX 2SSSX 3IYYADSVKG (SEQ ID NO:19)，其中X 2為S或A，且其中X 3為Y或S；且 該VH CDR3包含胺基酸序列KX 4X 5X 6GDFDY (SEQ ID NO:20)，其中X 4為Y或S，其中X 5為R或S，且其中X 6為A或Y； 該VL CDR1包含胺基酸序列RASQSVSSX 7X 8LA (SEQ ID NO:21)，其中X 7為S或N，且其中X 8為Y或N； 該VL CDR2包含胺基酸序列GASX 9RAT (SEQ ID NO:22)，其中X 9為N或S；且 該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)。 實施例2. 如實施例1之抗體，其中VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及/或VL CDR3中之至少一者選自表1中所描繪之突變體CDR，且未選自表1中所描繪之突變體CDR之VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2或VL CDR3中之任一者選自表1中所描繪之親代CDR。 實施例3. 如實施例1之抗體，其中VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2或VL CDR3中之一者選自表1中所描繪之突變體CDR且VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及/或VL CDR3中之五者選自表1中所描繪之親代CDR。 實施例4. 如實施例1之抗體，其中VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及/或VL CDR3中之兩者選自表1中所描繪之突變體CDR且VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及/或VL CDR3中之四者選自表1中所描繪之親代CDR。 實施例5. 如實施例1之抗體，其中VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及/或VL CDR3中之三者選自表1中所描繪之突變體CDR且VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及/或VL CDR3中之三者選自表1中所描繪之親代CDR。 實施例6. 如實施例1之抗體，其中VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及/或VL CDR3中之四者選自表1中所描繪之突變體CDR且VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及/或VL CDR3中之兩者選自表1中所描繪之親代CDR。 實施例7. 如實施例1之抗體，其中VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及/或VL CDR3中之五者選自表1中所描繪之突變體CDR且VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2或VL CDR3中之一者選自表1中所描繪之親代CDR。 實施例8. 如實施例1之抗體，其中： 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)；或 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)。 實施例9. 如實施例1之抗體，其中： 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)，其中該VL包含Y57S保守取代； 該VH CDR1包含胺基酸序列GFTFSSYAMN (SEQ ID NO:9)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)，其中該VL包含Y57S保守取代； 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSNYLA (SEQ ID NO:15)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISSSSSSIYYADSVKG (SEQ ID NO:11)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KSRAGDFDY (SEQ ID NO:12)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYSAGDFDY (SEQ ID NO:13)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRYGDFDY (SEQ ID NO:14)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISASSSSIYYADSVKG (SEQ ID NO:25)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；該VH CDR3包含胺基酸序列KYSAGDFDY (SEQ ID NO:13)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSNYLA (SEQ ID NO:15)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSNLA (SEQ ID NO:16)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASSRAT (SEQ ID NO:17)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)，其中該VL包含Y57S保守取代； 該VH CDR1包含胺基酸序列SYAMN (SEQ ID NO:24)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)，其中該VL包含Y57S保守取代； 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSNYLA (SEQ ID NO:15)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISSSSSSIYYADSVKG (SEQ ID NO:11)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KSRAGDFDY (SEQ ID NO:12)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYSAGDFDY (SEQ ID NO:13)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRYGDFDY (SEQ ID NO:14)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISASSSSIYYADSVKG (SEQ ID NO:25)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；該VH CDR3包含胺基酸序列KYSAGDFDY (SEQ ID NO:13)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSNYLA (SEQ ID NO:15)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSNLA (SEQ ID NO:16)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)；或 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASSRAT (SEQ ID NO:17)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)。 實施例10. 如前述實施例中任一項之抗體，其中： (i) 該VH與SEQ ID NO:100-108中之任一者至少80%、85%、90%、95%、99%或100%一致；且 (ii) 該VL與SEQ ID NO:200-204中之任一者至少80%、85%、90%、95%、99%或100%一致。 實施例11. 如實施例1之抗體，其中該VH包含SEQ ID NO:100-108中任一者之胺基酸序列且該VL包含SEQ ID NO:200-204中任一者之胺基酸序列。 實施例12. 如前述實施例中任一項之抗體，其中該抗體為(a)單價的且對hTfR1具有＞ 10 nM之單價親和力(K D)，或為二價的且對hTfR1具有＞100 nM之單價親和力(K D)，及/或(b)具有＞= 0.01/s之解離速率(k d)。 實施例13. 如實施例1之抗體，其中： 該VH包含該SEQ ID NO:100之胺基酸序列且該VL包含該SEQ ID NO:200之胺基酸序列。 實施例14. 如實施例1之抗體，其中： 該VH包含該SEQ ID NO:101之胺基酸序列且該VL包含該SEQ ID NO:200之胺基酸序列； 該VH包含該SEQ ID NO:102之胺基酸序列且該VL包含該SEQ ID NO:200之胺基酸序列； 該VH包含該SEQ ID NO:102之胺基酸序列且該VL包含該SEQ ID NO:201之胺基酸序列； 該VH包含該SEQ ID NO:102之胺基酸序列且該VL包含該SEQ ID NO:203之胺基酸序列； 該VH包含該SEQ ID NO:103之胺基酸序列且該VL包含該SEQ ID NO:200之胺基酸序列； 該VH包含該SEQ ID NO:104之胺基酸序列且該VL包含該SEQ ID NO:200之胺基酸序列； 該VH包含該SEQ ID NO:105之胺基酸序列且該VL包含該SEQ ID NO:200之胺基酸序列； 該VH包含該SEQ ID NO:106之胺基酸序列且該VL包含該SEQ ID NO:200之胺基酸序列； 該VH包含該SEQ ID NO:107之胺基酸序列且該VL包含該SEQ ID NO:200之胺基酸序列； 該VH包含該SEQ ID NO:108之胺基酸序列且該VL包含該SEQ ID NO:200之胺基酸序列； 該VH包含該SEQ ID NO:100之胺基酸序列且該VL包含該SEQ ID NO:201之胺基酸序列； 該VH包含該SEQ ID NO:100之胺基酸序列且該VL包含該SEQ ID NO:202之胺基酸序列； 該VH包含該SEQ ID NO:100之胺基酸序列且該VL包含該SEQ ID NO:203之胺基酸序列；或 該VH包含該SEQ ID NO:100之胺基酸序列且該VL包含該SEQ ID NO:204之胺基酸序列。 實施例15. 如實施例1至14中任一項之抗體，其為多特異性抗體、雙特異性抗體、單鏈抗體、Fab片段、F(ab') 2片段、Fab'片段、Fsc片段、Fv片段、scFv、sc(Fv) 2、或雙價抗體。 實施例16. 如實施例1至14中任一項之抗體，其包含恆定重鏈(CH)域及恆定輕鏈(CL)域。 實施例17. 如實施例1之抗體，其中該抗體包含： (a) 包含400中所列出之胺基酸序列之重鏈，及包含401中所列出之胺基酸序列之輕鏈； (b) SEQ ID NO: 401及402中所列出之胺基酸序列； (c) SEQ ID NO: 401、404及406中所列出之胺基酸序列； (d) SEQ ID NO:400及401中所列出之胺基酸序列； (e) SEQ ID NO: 401、405及406中所列出之胺基酸序列；或 (f) SEQ ID NO: 401及403中所列出之胺基酸序列。 實施例18. 一種核酸或多種核酸，其編碼如實施例1至17中任一項之抗體。 實施例19. 一種表現載體或多種表現載體，其包含可操作地連接至啟動子之如實施例18之該核酸或該等核酸。 實施例20. 一種經分離細胞，其包含如實施例18之該核酸或該等核酸或如實施例19之該表現載體或該等表現載體。 實施例21. 一種經分離細胞，其包含：包含可操作地連接至啟動子之第一核酸之第一表現載體，該第一核酸編碼包含如實施例1至17中任一項之抗體之VH之第一多肽；及包含可操作地連接至啟動子之第二核酸之第二表現載體，該第二核酸編碼包含如實施例1至17中任一項之抗體之VL之第二多肽。 實施例22. 一種製備如實施例1至17中任一項之抗體的方法，其包含培養如實施例20或21之細胞且分離該抗體。 實施例23. 一種醫藥組合物，其包含如實施例1至17中任一項之抗體及醫藥學上可接受之載劑。 實施例24. 一種結合物，其包含如實施例1至17中任一項之抗體及劑。 實施例25. 如實施例24之結合物，其中該劑為抗體、蛋白質或肽。 實施例26. 如實施例24之結合物，其中該劑為抗β澱粉樣蛋白抗體。 實施例27. 如實施例26之結合物，其中抗β澱粉樣蛋白抗體為阿杜卡單抗、巴匹珠單抗、甘特魯單抗、索拉珠單抗、多奈單抗或雷卡奈單抗。 實施例28. 如實施例24之結合物，其中該劑為抗τ抗體、抗α突觸核蛋白抗體、抗TDP-43抗體、抗LINGO-1抗體、抗LINGO-2抗體、抗LINGO-3抗體、抗LINGO-4抗體、抗TREM2抗體或抗C9orf72二肽重複聚GA抗體。 實施例29. 如實施例24之結合物，其中該劑為蛋白質。 實施例30. 如實施例29之結合物，其中該蛋白質為顆粒蛋白前體(progranulin)。 實施例31. 如實施例24之結合物，其中該劑為酶。 實施例32. 如實施例31之結合物，其中該酶為葡糖腦苷脂酶。 實施例33. 如實施例24至32中任一項之結合物，其中該結合物為包含該抗體及該劑之重組融合蛋白。 實施例34. 如實施例24之結合物，其中該劑為核酸。 實施例35. 如實施例34之結合物，其中該核酸為mRNA、siRNA、反義寡核苷酸、微小RNA (miRNA)、嚮導RNA (gRNA)或胺基磷酸酯嗎啉基寡聚物(PMO)。 實施例36. 如實施例34或35之結合物，其中該核酸經由連接子連接至該抗體。 實施例37. 如實施例24之結合物，其中該劑為奈米粒子、脂質體或病毒載體。 實施例38. 一種經由胞吞轉送作用跨血腦障壁轉運劑之方法，該方法包含向人類個體投與如實施例24至37中任一項之結合物。 實施例39. 一種活體內遞送劑之方法，該方法包含向人類個體投與如實施例24至37中任一項之結合物。 實施例40. 如實施例39之方法，其中該人類個體患有神經病症且該方法將該劑遞送至腦組織。 實施例41. 如實施例39之方法，其中該神經病症為阿茲海默氏病、帕金森氏病、額顳葉失智症、ALS、杭丁頓氏病、多發性硬化症、脊髓性肌萎縮、肌肉營養不良、脊髓損傷、中風、眼科疾患、急性或慢性視神經炎、精神異常、圖雷特氏病腦損傷、腦腫瘤或癲癇。 實施例42. 一種治療有需要之人類個體之阿茲海默氏病之方法，其包含向該人類個體投與治療有效量之如實施例26或27之結合物。 Exemplary embodiments as described below are also within the scope of the present disclosure: Embodiment 1. An antibody that binds to a human transferrin receptor, comprising a heavy chain variable region (VH) comprising a VH complementation determining region (CDR) 1, a VH CDR2, and a VH CDR3, and a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein: the VH CDR1 comprises the amino acid sequence GFTFSSYX 1 MN (SEQ ID NO: 18) or the amino acid sequence SYX 1 MN (SEQ ID NO: 26), wherein X 1 is S or A; the VH CDR2 comprises the amino acid sequence SISX 2 SSSX 3 IYYADSVKG (SEQ ID NO: 19), wherein X 2 is S or A, and wherein X 3 is Y or S; and the VH The CDR3 comprises the amino acid sequence KX4X5X6GDFDY (SEQ ID NO: 20 ), wherein X4 is Y or S, wherein X5 is R or S, and wherein X6 is A or Y; the VL CDR1 comprises the amino acid sequence RASQSVSSX7X8LA (SEQ ID NO: 21 ), wherein X7 is S or N, and wherein X8 is Y or N; the VL CDR2 comprises the amino acid sequence GASX9RAT (SEQ ID NO:22), wherein X9 is N or S; and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8). Embodiment 2. The antibody of Embodiment 1, wherein at least one of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3 is selected from the mutant CDRs described in Table 1, and any of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 or VL CDR3 not selected from the mutant CDRs described in Table 1 is selected from the parent CDRs described in Table 1. Embodiment 3. The antibody of Embodiment 1, wherein one of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 or VL CDR3 is selected from the mutant CDRs described in Table 1 and five of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3 are selected from the parent CDRs described in Table 1. Embodiment 4. The antibody of Embodiment 1, wherein two of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3 are selected from the mutant CDRs described in Table 1 and four of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3 are selected from the parent CDRs described in Table 1. Embodiment 5. The antibody of Embodiment 1, wherein three of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3 are selected from the mutant CDRs described in Table 1 and three of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3 are selected from the parent CDRs described in Table 1. Embodiment 6. The antibody of Embodiment 1, wherein four of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3 are selected from the mutant CDRs described in Table 1 and two of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3 are selected from the parent CDRs described in Table 1. Embodiment 7. The antibody of Embodiment 1, wherein five of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3 are selected from the mutant CDRs described in Table 1 and one of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 or VL CDR3 is selected from the parent CDRs described in Table 1. Embodiment 8. The antibody of embodiment 1, wherein: the VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); or the VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); the VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8). Embodiment 9. The antibody of embodiment 1, wherein: the VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8), wherein the VL comprises a Y57S conservative substitution; the VH CDR1 comprises the amino acid sequence GFTFSSYAMN (SEQ ID NO:9); the VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); the VH the VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); NO:8); the VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8), wherein the VL comprises a Y57S conservative substitution; the VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8), wherein the VL comprises a Y57S conservative substitution; NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSNYLA (SEQ ID NO:15); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence SISSSSSSIYYADSVKG (SEQ ID NO:11); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH The VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KSRAGDFDY (SEQ ID NO:12); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KYSAGDFDY (SEQ ID NO:13); the VL The CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KYRYGDFDY (SEQ ID NO:14); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KYRYGDFDY (SEQ ID NO:14); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence SISASSSSIYYADSVKG (SEQ ID NO:25); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); the VH CDR3 comprises the amino acid sequence KYSAGDFDY (SEQ ID NO:13); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL The VH CDR1 comprises the amino acid sequence of GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence of SISSSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence of KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence of RASQSVSSNYLA (SEQ ID NO:15); the VL CDR2 comprises the amino acid sequence of GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence of QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence of GFTFSSYSMN (SEQ ID NO:3); the VH The VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSNLA (SEQ ID NO:16); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL the VH CDR1 comprises the amino acid sequence SYAMN (SEQ ID NO:24); the VH CDR2 comprises the amino acid sequence SYAMN (SEQ ID NO:25); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8), wherein the VL comprises a Y57S conservative substitution; the VH CDR1 comprises the amino acid sequence SYAMN (SEQ ID NO:24); the VH the VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); the VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); the VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8), wherein the VL comprises a Y57S conservative substitution; the VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); the VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); NO:10); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSNYLA (SEQ ID NO:15); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); the VH CDR2 comprises the amino acid sequence SISSSSSSIYYADSVKG (SEQ ID NO:11); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL The VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); the VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KSRAGDFDY (SEQ ID NO:12); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); the VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KYSAGDFDY (SEQ ID NO:12); ID NO:13); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); the VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KYRYGDFDY (SEQ ID NO:14); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH the VH CDR2 comprises the amino acid sequence SISASSSSIYYADSVKG (SEQ ID NO:25); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); the VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); the VH CDR3 comprises the amino acid sequence KYSAGDFDY (SEQ ID NO:13); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); the VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSNYLA (SEQ ID NO:15); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); the VH the VH CDR2 comprises the amino acid sequence of SISSSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence of KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence of RASQSVSSSNLA (SEQ ID NO:16); the VL CDR2 comprises the amino acid sequence of GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence of QQQSSSPPT (SEQ ID NO:8); or the VH CDR1 comprises the amino acid sequence of SYSMN (SEQ ID NO:23); the VH CDR2 comprises the amino acid sequence of SISSSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence of KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence of RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence of GASSRAT (SEQ ID NO: 17); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO: 8). Embodiment 10. The antibody of any of the preceding embodiments, wherein: (i) the VH is at least 80%, 85%, 90%, 95%, 99% or 100% identical to any of SEQ ID NOs: 100-108; and (ii) the VL is at least 80%, 85%, 90%, 95%, 99% or 100% identical to any of SEQ ID NOs: 200-204. Embodiment 11. The antibody of embodiment 1, wherein the VH comprises the amino acid sequence of any of SEQ ID NOs: 100-108 and the VL comprises the amino acid sequence of any of SEQ ID NOs: 200-204. Embodiment 12. The antibody of any of the preceding embodiments, wherein the antibody is (a) monovalent and has a monovalent affinity ( KD ) of >10 nM for hTfR1, or is bivalent and has a monovalent affinity ( KD ) of >100 nM for hTfR1, and/or (b) has a dissociation rate ( kd ) of >= 0.01/s. Embodiment 13. The antibody of embodiment 1, wherein: the VH comprises the amino acid sequence of SEQ ID NO: 100 and the VL comprises the amino acid sequence of SEQ ID NO: 200. Embodiment 14. The antibody of Embodiment 1, wherein: the VH comprises the amino acid sequence of SEQ ID NO: 101 and the VL comprises the amino acid sequence of SEQ ID NO: 200; the VH comprises the amino acid sequence of SEQ ID NO: 102 and the VL comprises the amino acid sequence of SEQ ID NO: 200; the VH comprises the amino acid sequence of SEQ ID NO: 102 and the VL comprises the amino acid sequence of SEQ ID NO: 201; the VH comprises the amino acid sequence of SEQ ID NO: 102 and the VL comprises the amino acid sequence of SEQ ID NO: 203; the VH comprises the amino acid sequence of SEQ ID NO: 103 and the VL comprises the amino acid sequence of SEQ ID NO: 200; the VH comprises the amino acid sequence of SEQ ID NO: 104 and the VL comprises the amino acid sequence of SEQ ID NO: 200; The VH comprises the amino acid sequence of SEQ ID NO:105 and the VL comprises the amino acid sequence of SEQ ID NO:200; The VH comprises the amino acid sequence of SEQ ID NO:106 and the VL comprises the amino acid sequence of SEQ ID NO:200; The VH comprises the amino acid sequence of SEQ ID NO:107 and the VL comprises the amino acid sequence of SEQ ID NO:200; The VH comprises the amino acid sequence of SEQ ID NO:108 and the VL comprises the amino acid sequence of SEQ ID NO:200; The VH comprises the amino acid sequence of SEQ ID NO:100 and the VL comprises the amino acid sequence of SEQ ID NO:201; The VH comprises the amino acid sequence of SEQ ID NO:100 and the VL comprises the amino acid sequence of SEQ ID NO:202; The VH comprises the amino acid sequence of SEQ ID NO:107 and the VL comprises the amino acid sequence of SEQ ID NO:200; NO:100 and the VL comprises the amino acid sequence of SEQ ID NO:203; or the VH comprises the amino acid sequence of SEQ ID NO:100 and the VL comprises the amino acid sequence of SEQ ID NO:204. Embodiment 15. The antibody of any one of Embodiments 1 to 14, which is a multispecific antibody, a bispecific antibody, a single chain antibody, a Fab fragment, a F(ab') 2 fragment, a Fab' fragment, a Fsc fragment, a Fv fragment, scFv, sc(Fv) 2 , or a bivalent antibody. Embodiment 16. The antibody of any one of Embodiments 1 to 14, which comprises a constant heavy chain (CH) domain and a constant light chain (CL) domain. Embodiment 17. The antibody of embodiment 1, wherein the antibody comprises: (a) a heavy chain comprising the amino acid sequence listed in 400, and a light chain comprising the amino acid sequence listed in 401; (b) the amino acid sequence listed in SEQ ID NOs: 401 and 402; (c) the amino acid sequence listed in SEQ ID NOs: 401, 404, and 406; (d) the amino acid sequence listed in SEQ ID NOs: 400 and 401; (e) the amino acid sequence listed in SEQ ID NOs: 401, 405, and 406; or (f) the amino acid sequence listed in SEQ ID NOs: 401 and 403. Embodiment 18. A nucleic acid or a plurality of nucleic acids encoding the antibody of any one of embodiments 1 to 17. Example 19. An expression vector or multiple expression vectors, comprising the nucleic acid or nucleic acids of Example 18 operably linked to a promoter. Example 20. An isolated cell, comprising the nucleic acid or nucleic acids of Example 18 or the expression vector or expression vectors of Example 19. Example 21. An isolated cell, comprising: a first expression vector comprising a first nucleic acid operably linked to a promoter, the first nucleic acid encoding a first polypeptide comprising the VH of an antibody as described in any one of Examples 1 to 17; and a second expression vector comprising a second nucleic acid operably linked to a promoter, the second nucleic acid encoding a second polypeptide comprising the VL of an antibody as described in any one of Examples 1 to 17. Example 22. A method for preparing an antibody as described in any one of Examples 1 to 17, comprising culturing the cells as described in Example 20 or 21 and isolating the antibody. Example 23. A pharmaceutical composition comprising an antibody as described in any one of Examples 1 to 17 and a pharmaceutically acceptable carrier. Example 24. A conjugate comprising an antibody as described in any one of Examples 1 to 17 and an agent. Example 25. The conjugate as described in Example 24, wherein the agent is an antibody, a protein or a peptide. Example 26. The conjugate as described in Example 24, wherein the agent is an anti-β-amyloid antibody. Example 27. The conjugate of Example 26, wherein the anti-β-amyloid antibody is aducanumab, bapineumab, gantelumab, sorazumab, donetumab or lecanezumab. Example 28. The conjugate of Example 24, wherein the agent is an anti-tau antibody, an anti-α-synaptophysin antibody, an anti-TDP-43 antibody, an anti-LINGO-1 antibody, an anti-LINGO-2 antibody, an anti-LINGO-3 antibody, an anti-LINGO-4 antibody, an anti-TREM2 antibody or an anti-C9orf72 dipeptide repeat poly-GA antibody. Example 29. The conjugate of Example 24, wherein the agent is a protein. Example 30. The conjugate of Example 29, wherein the protein is progranulin. Embodiment 31. The conjugate of embodiment 24, wherein the agent is an enzyme. Embodiment 32. The conjugate of embodiment 31, wherein the enzyme is glucocerebrosidase. Embodiment 33. The conjugate of any one of embodiments 24 to 32, wherein the conjugate is a recombinant fusion protein comprising the antibody and the agent. Embodiment 34. The conjugate of embodiment 24, wherein the agent is a nucleic acid. Embodiment 35. The conjugate of embodiment 34, wherein the nucleic acid is mRNA, siRNA, antisense oligonucleotide, micro RNA (miRNA), guide RNA (gRNA) or phosphamidonomorpholino oligomer (PMO). Embodiment 36. The conjugate of embodiment 34 or 35, wherein the nucleic acid is linked to the antibody via a linker. Embodiment 37. The conjugate of embodiment 24, wherein the agent is a nanoparticle, a liposome or a viral vector. Embodiment 38. A method of transporting an agent across the blood-brain barrier via endocytosis, the method comprising administering the conjugate of any one of embodiments 24 to 37 to a human subject. Embodiment 39. A method of delivering an agent in vivo, the method comprising administering the conjugate of any one of embodiments 24 to 37 to a human subject. Embodiment 40. The method of embodiment 39, wherein the human subject suffers from a neurological disorder and the method delivers the agent to brain tissue. Embodiment 41. The method of embodiment 39, wherein the neurological disorder is Alzheimer's disease, Parkinson's disease, frontotemporal dementia, ALS, Huntington's disease, multiple sclerosis, spinal muscular atrophy, muscular dystrophy, spinal cord injury, stroke, ophthalmic disease, acute or chronic optic neuritis, mental disorder, Tourette's disease brain injury, brain tumor or epilepsy. Embodiment 42. A method for treating Alzheimer's disease in a human subject in need thereof, comprising administering to the human subject a therapeutically effective amount of a conjugate of embodiment 26 or 27.

提供以下實例以更好地說明所主張之發明且不應解釋為限制本發明之範疇。就提及特定材料而言，其僅用於說明目的且不意欲限制本發明。熟習此項技術者可在不行使發明能力且不背離本發明之範疇的情況下開發等效手段或反應物。 實例 實例1：抗體產生 The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. To the extent that specific materials are mentioned, they are for illustrative purposes only and are not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without exercising inventive power and without departing from the scope of the invention. Examples Example 1: Antibody Production

根據美國專利公開案20100056386及20090181855中所揭示之方法篩選阿迪單抗(Adimab)表現文庫。在對人類TfR1胞外域(ECD)進行迭代輪次之選擇性壓力後，之後對石蟹猴TfR1 ECD進行選擇性壓力，之後對用重組人類全運鐵蛋白(Sigma; T0665)預阻斷之人類TfR1 ECD進行選定壓力。隨後使用此項技術中已知之技術對集落進行定序以鑑別獨特純系。在此活動之後，自酵母中表現出768種抗體，且在蛋白A樹脂上自酵母純化。使用生物層干涉術(BLI)測試酵母表現抗體與人類及石蟹猴TfR ECD重組蛋白兩者之結合。在用重組人類全運鐵蛋白預阻斷人類TfR ECD後，類似地針對人類TfR進行抗體結合。根據標準程序在由ForteBio製造之Octet RED384及Octet HTX儀器上進行BLI。接著測試來自BLI分類之酵母表現抗體與過表現全長人類TfR (huTfR-CHO)或石蟹猴TfR (cyTfR-CHO)之BAF3細胞的結合。將細胞與單一200 nM濃度之抗體在冰上孵育2小時，用等滲緩衝液洗滌兩次，接著與經螢光標記之抗IgG二級抗體(Jackson; 109-116-097)孵育，固定於1% PFA中且在流式細胞儀上分析。The Adimab expression library was screened according to the methods disclosed in U.S. Patent Publications 20100056386 and 20090181855. After iterative rounds of selective pressure on the human TfR1 extracellular domain (ECD), selective pressure was then applied to the cynomolgus monkey TfR1 ECD, followed by selective pressure on the human TfR1 ECD pre-blocked with recombinant human holotransferrin (Sigma; T0665). Colonies were then sequenced using techniques known in the art to identify unique clones. Following this campaign, 768 antibodies were expressed from yeast and purified from yeast on protein A resin. Yeast expressed antibodies were tested for binding to both human and cynomolgus monkey TfR ECD recombinant proteins using biolayer interferometry (BLI). Antibody binding was similarly performed against human TfR after pre-blocking human TfR ECD with recombinant human holotransferrin. BLI was performed on Octet RED384 and Octet HTX instruments manufactured by ForteBio according to standard procedures. Yeast expressed antibodies from BLI sorting were then tested for binding to BAF3 cells overexpressing full-length human TfR (huTfR-CHO) or cynomolgus monkey TfR (cyTfR-CHO). Cells were incubated with a single antibody at 200 nM concentration for 2 h on ice, washed twice with isotonic buffer, then incubated with a fluorescently labeled anti-IgG secondary antibody (Jackson; 109-116-097), fixed in 1% PFA and analyzed on a flow cytometer.

藉由在無血清培養基中瞬時轉染懸浮CHO-S細胞將抗體表現為非糖基化(aglycosylated) hIgG1。藉由離心及過濾收集經調節之上清液。藉由將上清液加載至蛋白A瓊脂糖凝膠FF (Protein A Sepharose FF)上，用25 mM磷酸鈉、100 mM NaCl，pH 2.8溶析，接著用1:60 (v:v) 500 mM磷酸鈉pH 8.6中和來純化蛋白質。藉由使中和之溶離液通過在PBS pH 7.0中平衡之TMAE陰離子交換樹脂來進行進一步純化。評估聚集體含量，且若總蛋白質＞5%，則藉由尺寸排阻層析(PBS中之superdex 200)進一步純化抗體。The antibody was expressed as aglycosylated hIgG1 by transient transfection of suspension CHO-S cells in serum-free medium. The conditioned supernatant was collected by centrifugation and filtration. The protein was purified by loading the supernatant onto Protein A Sepharose FF, eluting with 25 mM sodium phosphate, 100 mM NaCl, pH 2.8, followed by neutralization with 1:60 (v:v) 500 mM sodium phosphate, pH 8.6. Further purification was performed by passing the neutralized eluate through TMAE anion exchange resin equilibrated in PBS pH 7.0. Aggregate content was assessed and if total protein was >5%, antibodies were further purified by size exclusion chromatography (superdex 200 in PBS).

評估抗體之TfR結合、Tf阻斷及胞吞轉送性質。對於細胞結合研究，二價抗體結合至表現全長人類TfR1 (huTfR-CHO)或石蟹猴TfR1 (cyTfR-CHO)之CHO細胞，無內源性倉鼠TfR1 (TfR KO CHO背景)。將細胞與濃度高達1000 nM之抗體(4倍7 pt稀釋系列)在冰上孵育1-2小時，接著用等滲緩衝液洗滌三次，與結合人類IgG之螢光二級試劑(PE結合)孵育，再次洗滌，固定於1%多聚甲醛中且在流式細胞儀上分析。計算PE螢光團之平均螢光強度(MFI)且代表抗體與細胞之結合。藉由1000 nM抗體與TfR KO CHO細胞之結合來評估非特異性結合。藉由比較在不存在及存在1000 nM人類全運鐵蛋白(Sigma T0665)之情況下抗體與huTfR-CHO之滴定結合來評估運鐵蛋白競爭結合。抗體X、抗體1及抗體2之結果提供於 表7中。 Antibodies were evaluated for TfR binding, Tf blocking, and endocytic trafficking properties. For cell binding studies, bivalent antibodies were bound to CHO cells expressing full-length human TfR1 (huTfR-CHO) or cynomolgus monkey TfR1 (cyTfR-CHO) without endogenous hamster TfR1 (TfR KO CHO background). Cells were incubated with antibodies up to 1000 nM (4-fold 7 pt dilution series) on ice for 1-2 hours, then washed three times with isotonic buffer, incubated with a fluorescent secondary reagent conjugated to human IgG (PE conjugated), washed again, fixed in 1% paraformaldehyde, and analyzed on a flow cytometer. The mean fluorescence intensity (MFI) of the PE fluorophore was calculated and represents the binding of the antibody to the cells. Nonspecific binding was assessed by binding of 1000 nM antibody to TfR KO CHO cells. Competitive binding to transferrin was assessed by comparing titrated binding of the antibody to huTfR-CHO in the absence and presence of 1000 nM human holo-transferrin (Sigma T0665). The results for Antibody X, Antibody 1, and Antibody 2 are provided in Table 7 .

對於胞吞轉送作用檢定，將Caco2細胞以25,000個/孔接種於Corning 0.4微米transwell插入物中且在完全培養基(DMEM、10% FBS、1 mM丙酮酸鈉、2 mM麩醯胺酸、1% NEAA及1% Pen/Strep)中培養21天。跨上皮電阻(TEER)值在1000-2000 Ohm.cm 2範圍內。將抗體以100 nM添加至頂部孔中，且在兩天後，自底部孔收集樣品，接著在中尺度發現檢定系統中使用通常可用之抗人類IgG試劑分析hIgG濃度，自同一測試物品之滴定標準曲線外推出結果。結果報告為測試抗TfR抗體之底部孔濃度相較於針對單獨transwell中非靶向hIgG對照抗體所量測之底部孔濃度的比率。抗體X、抗體1及抗體2之結果提供於 表7中。 表7. 抗TfR huTfR-CHO EC 50(nM) cyTfR-CHO EC 50(nM) TfR KO CHO 1 uM 之MFI Tf 阻斷 Caco2 胞吞轉送作用 ( 倍數/對照) 抗體X 1.8 1.5 7 編號 7 抗體1 0.3 約0.6 47 是 6 抗體2 12 5.4 3 是 5 For the endocytosis assay, Caco2 cells were seeded at 25,000/well in Corning 0.4 micron transwell inserts and cultured for 21 days in complete medium (DMEM, 10% FBS, 1 mM sodium pyruvate, 2 mM glutamine, 1% NEAA, and 1% Pen/Strep). Transepithelial electrical resistance (TEER) values ranged from 1000-2000 Ohm.cm 2. Antibodies were added to the top wells at 100 nM, and after two days, samples were collected from the bottom wells and analyzed for hIgG concentrations in a mesoscale discovery assay system using a commonly available anti-human IgG reagent, with results extrapolated from a titrated standard curve of the same test article. Results are reported as the ratio of the bottom well concentration of the tested anti-TfR antibody compared to the bottom well concentration measured for a non-targeting hIgG control antibody in a single transwell. Results for Antibody X, Antibody 1, and Antibody 2 are provided in Table 7. Table 7. Anti-TfR huTfR-CHO EC 50 (nM) cyTfR-CHO EC 50 (nM) TfR KO CHO 1 uM of MFI Tf blocking Caco2 endocytosis ( fold/control) Antibody X 1.8 1.5 7 No. 7 Antibody 1 0.3 About 0.6 47 yes 6 Antibody 2 12 5.4 3 yes 5

出於與人類及石蟹猴TfR1之良好匹配之結合、低非特異性細胞結合、不與運鐵蛋白競爭及在Caco2細胞中之積極胞吞轉送作用之組合而選擇抗體X ( 表7)。 Antibody X was selected for its combination of well-matched binding to human and cynomolgus monkey TfR1, low nonspecific cell binding, no competition with ferritin, and active endocytic transport in Caco2 cells ( Table 7 ).

抗體X之可變區之胺基酸序列提供於下文。 抗體X VH： EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDYWGQGTLVTVSS (SEQ ID NO:100) 抗體X VL： EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQQSSSPPTFGGGTKVEIK (SEQ ID NO:200) 實例2：抗體X之表徵 The amino acid sequence of the variable region of Antibody X is provided below. Antibody X VH: EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARKYRAGDFDYWGQGTLVTVSS (SEQ ID NO: 100) Antibody X VL: EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQQSSSPPTFGGGTKVEIK (SEQ ID NO: 200) Example 2: Characterization of Antibody X

藉由表面電漿子共振(SPR)評估抗體X與運鐵蛋白受體結合之親和力及動力學。在包被有抗His捕獲試劑(Cytiva)之SPR晶片(Biacore 8K+中之CM5晶片)上以30-50 pg/mm 2捕獲帶組胺酸標籤之人類或食蟹猴運鐵蛋白受體-1胞外域(分別為huTfR1 ECD或cyTfR1 ECD)。以4至4000 nM範圍內之濃度注射包含抗體X之VH及VL之單價抗體或Fab片段(用10 ug/mg抗體之木瓜蛋白酶(Roche 108014)消化，在37℃下孵育4小時)。使用Biacore Insight評估軟體以1:1結合模型量測並分析結合感測圖( 圖1A及圖1B)。觀察到抗體X之快速解離動力學(k d為約0.02 /s)及適中之單價親和力(K D為約50 nM)，如血腦障壁處之功能結合、轉運及釋放所期望。 The affinity and kinetics of binding of Antibody X to the ferritin receptor were assessed by surface plasmon resonance (SPR). Histidine-tagged human or cynomolgus monkey ferritin receptor-1 extracellular domain (huTfR1 ECD or cyTfR1 ECD, respectively) was captured at 30-50 pg/ mm2 on an SPR chip (CM5 chip in Biacore 8K+) coated with anti-His capture reagent (Cytiva). Monovalent antibodies or Fab fragments containing the VH and VL of Antibody X (digested with papain (Roche 108014) at 10 ug/mg antibody, incubated at 37°C for 4 hours) were injected at concentrations ranging from 4 to 4000 nM. The binding sensorgrams were measured and analyzed using Biacore Insight evaluation software in a 1:1 binding model ( Figure 1A and Figure 1B ). Rapid dissociation kinetics ( kd of approximately 0.02/s) and moderate monovalent affinity ( Kd of approximately 50 nM) were observed for Antibody X, as expected for functional binding, trafficking, and release at the blood-brain barrier.

如上文所述進行單價hIgG型式之結合性質之進一步研究，其中使用杵入臼突變將抗體X Fab與人類Fc片段之N末端或C末端融合，以驅動與不含Fab之Fc鏈之異二聚化(分別為Fab -Fc或Fc-Fab)。在單價抗體型式之間未觀察到對huTfR1 ECD之親和力的顯著差異，但Fc融合物與cyTfR1 ECD之結合比Fab片段稍弱( 表8)。 Further investigation of the binding properties of monovalent hIgG formats was performed as described above, in which the Antibody X Fab was fused to the N-terminus or C-terminus of a human Fc fragment using knob-in-hole mutations to drive heterodimerization with a Fab-free Fc chain (Fab-Fc or Fc-Fab, respectively). No significant differences in affinity for huTfR1 ECD were observed between the monovalent antibody formats, but the Fc fusions bound slightly weaker to cyTfR1 ECD than the Fab fragments ( Table 8 ).

藉由流式細胞術研究不同型式變異體(包括單價及二價型式)之結合細胞表面人類TfR1方面之差異。將內源倉鼠TfR敲除且人類TfR過表現之CHO細胞與含有來自抗體X之Fab區之抗體(0.001-1000 nM) (包括Fab-Fc及Fc-Fab二價融合物，及兩種單價型式Fab-Fc及Fc-Fab)在冰上孵育60-90分鐘，洗滌三次，接著與經螢光標記之抗hIgG Fc (Jackson 109-116-098)孵育60分鐘，固定於1% PFA中，且在流式細胞儀中分析。單價抗體型式之結合弱於二價型式( 圖1C、表8)。在流式細胞術中觀察到Fab-Fc型式與Fc-Fab型式之間的差異，該等差異藉由SPR確定不明顯( 圖1C、表8)。 The differences in binding of the different variants (including monovalent and bivalent forms) to cell surface human TfR1 were investigated by flow cytometry. CHO cells with endogenous hamster TfR knockout and human TfR overexpression were incubated with antibodies (0.001-1000 nM) containing the Fab region from Antibody X (including Fab-Fc and Fc-Fab bivalent fusions, and two monovalent forms Fab-Fc and Fc-Fab) on ice for 60-90 minutes, washed three times, then incubated with fluorescently labeled anti-hIgG Fc (Jackson 109-116-098) for 60 minutes, fixed in 1% PFA, and analyzed in a flow cytometer. The monovalent antibody forms bound less strongly than the bivalent forms ( Figure 1C, Table 8 ). Differences between Fab-Fc and Fc-Fab formats were observed in flow cytometry, but were not significant as determined by SPR ( Fig. 1C, Table 8 ).

為評估與運鐵蛋白之競爭性結合，將藉由SPR所確定的單獨抗體與TfR1 ECD之結合與在有1000 nM全人類運鐵蛋白(T0665, Sigma)时之抗體進行比較，該全人類運鐵蛋白在所捕獲受體上經預飽和，與該抗體混合且在解離階段(在Biacore控制軟體中之ABA模式下)期間加以包括。在存在運鐵蛋白之情況下未觀察到結合親和力減弱，而是偵測到稍強之結合( 表8)。 To assess competitive binding to transferrin, binding of individual antibodies to TfR1 ECD determined by SPR was compared to the antibody in the presence of 1000 nM whole human transferrin (T0665, Sigma) presaturated on captured receptors, mixed with the antibody and included during the dissociation phase (ABA mode in the Biacore control software). No reduction in binding affinity was observed in the presence of transferrin, but rather slightly stronger binding was detected ( Table 8 ).

藉由流式細胞術進行細胞表面運鐵蛋白競爭實驗。在不存在及存在全人類運鐵蛋白(1000 nM)之情況下評估抗體X二價Fab-Fc (0.001-1000 nM)與huTfR CHO之結合。與藉由SPR所觀察到的類似，在運鐵蛋白存在下抗體結合沒有損失，而是在存在運鐵蛋白之情況下抗體染色水準略有升高( 圖2A)。 Cell surface transferrin competition experiments were performed by flow cytometry. Binding of Antibody X bivalent Fab-Fc (0.001-1000 nM) to huTfR CHO was assessed in the absence and presence of fully human transferrin (1000 nM). Similar to what was observed by SPR, there was no loss of antibody binding in the presence of transferrin, but rather a slight increase in antibody staining levels in the presence of transferrin ( Figure 2A ).

藉由抗體X二價Fab-Fc进行運鐵蛋白替換之特徵在於，將huTfR CHO與次飽和水準之经AlexaFluor647標記之人類運鐵蛋白(3 nM, Thermo T23366)以及抗TfR或未標記之全人類運鐵蛋白一起孵育。與陽性阻斷性抗TfR (抗體1)或全人類運鐵蛋白相比，對抗體X二價Fab-Fc觀察到最小運鐵蛋白替換( 圖2B)。 表8. 抗體X 蛋白質描述 抗體分子包含以下多肽鏈序列： huTfR ECD k a(M -1s -1) huTfR ECD k d(s -1) huTfR ECD K D(nM) huTfR CHO EC 50(nM) Fab片段 SEQ ID NO:401 SEQ ID NO:402 5.0×10 5 2.1×10 -2 43 單價Fab-Fc SEQ ID NO:401 SEQ ID NO:404 SEQ ID NO:406 3.8×10 5 2.1×10 -2 55 47 單價Fab-Fc + holo huTf (1 uM) SEQ ID NO:401 SEQ ID NO:404 SEQ ID NO:406 4.4×10 5 2.2×10 -2 49 單價Fc-Fab SEQ ID NO:401 SEQ ID NO:405 SEQ ID NO:406 3.2×10 5 1.7×10 -2 53 6.9 單價Fc-Fab + holo huTf (1 uM) SEQ ID NO:401 SEQ ID NO:405 SEQ ID NO:406 3.5×10 5 1.7×10 -2 48 二價Fab-Fc SEQ ID NO:400 SEQ ID NO:401 0.20 二價Fc-Fab SEQ ID NO:401 SEQ ID NO:403 0.45 抗體X 蛋白質描述 抗體分子包含以下多肽鏈序列： cyTfR ECD k a(M -1s -1) cyTfR ECD k d(s -1) cyTfR ECD K D(nM) cyTfR CHO EC 50(nM) Fab片段 SEQ ID NO:401 SEQ ID NO:402 5.6×10 5 3.7×10 -2 67 單價Fab-Fc SEQ ID NO:401 SEQ ID NO:404 SEQ ID NO:406 2.6×10 5 3.2×10 -2 125 84 單價Fab-Fc + holo huTf (1 uM) SEQ ID NO:401 SEQ ID NO:404 SEQ ID NO:406 2.8×10 5 3.2×10 -2 115 單價Fc-Fab SEQ ID NO:401 SEQ ID NO:405 SEQ ID NO:406 2.4×10 5 3.0×10 -2 128 21 單價Fc-Fab + holo huTf (1 uM) SEQ ID NO:401 SEQ ID NO:405 SEQ ID NO:406 2.8×10 5 3.1×10 -2 111 二價Fab-Fc SEQ ID NO:400 SEQ ID NO:401 0.13 二價Fc-Fab SEQ ID NO:401 SEQ ID NO:403 0.59 實例3：抗體X互補位映射 Transferrin replacement by Antibody X Bivalent Fab-Fc was characterized by incubating huTfR CHO with subsaturated levels of AlexaFluor647 labeled human transferrin (3 nM, Thermo T23366) and anti-TfR or unlabeled whole human transferrin. Minimal transferrin replacement was observed for Antibody X Bivalent Fab-Fc compared to positive blocking anti-TfR (Antibody 1) or whole human transferrin ( Figure 2B ). Table 8. Antibody X protein description The antibody molecule contains the following polypeptide chain sequence: huTfR ECD k a (M -1 s -1 ) huTfR ECD k d (s -1 ) huTfR ECD K D (nM) huTfR CHO EC 50 (nM) Fab fragment SEQ ID NO:401 SEQ ID NO:402 5.0×10 5 2.1×10 -2 43 Unit price Fab-Fc SEQ ID NO:401 SEQ ID NO:404 SEQ ID NO:406 3.8×10 5 2.1×10 -2 55 47 Monovalent Fab-Fc + holo huTf (1 uM) SEQ ID NO:401 SEQ ID NO:404 SEQ ID NO:406 4.4×10 5 2.2×10 -2 49 Unit price Fc-Fab SEQ ID NO:401 SEQ ID NO:405 SEQ ID NO:406 3.2×10 5 1.7×10 -2 53 6.9 Unit price Fc-Fab + holo huTf (1 uM) SEQ ID NO:401 SEQ ID NO:405 SEQ ID NO:406 3.5×10 5 1.7×10 -2 48 Bivalent Fab-Fc SEQ ID NO:400 SEQ ID NO:401 0.20 Bivalent Fc-Fab SEQ ID NO:401 SEQ ID NO:403 0.45 Antibody X protein description The antibody molecule contains the following polypeptide chain sequence: cyTfR ECD k a (M -1 s -1 ) cyTfR ECD k d (s -1 ) cyTfR ECD KD (nM) cyTfR CHO EC 50 (nM) Fab fragment SEQ ID NO:401 SEQ ID NO:402 5.6×10 5 3.7×10 -2 67 Unit price Fab-Fc SEQ ID NO:401 SEQ ID NO:404 SEQ ID NO:406 2.6×10 5 3.2×10 -2 125 84 Monovalent Fab-Fc + holo huTf (1 uM) SEQ ID NO:401 SEQ ID NO:404 SEQ ID NO:406 2.8×10 5 3.2×10 -2 115 Unit price Fc-Fab SEQ ID NO:401 SEQ ID NO:405 SEQ ID NO:406 2.4×10 5 3.0×10 -2 128 twenty one Unit price Fc-Fab + holo huTf (1 uM) SEQ ID NO:401 SEQ ID NO:405 SEQ ID NO:406 2.8×10 5 3.1×10 -2 111 Bivalent Fab-Fc SEQ ID NO:400 SEQ ID NO:401 0.13 Bivalent Fc-Fab SEQ ID NO:401 SEQ ID NO:403 0.59 Example 3: Antibody X complementary site mapping

為鑑別抗體X之抗原識別之分子基礎，以3.6 Å之解析度產生抗體X Fab片段、huTfR1 ECD及全人類運鐵蛋白(Sigma T0665)之三元復合物之低溫EM結構。藉由將抗體X Fab之同源模型以及TfR1及Tf之可用晶體結構擬合至低溫EM密度圖中來獲得複合物之原子模型( 圖3A)。最終模型揭示，抗體X結合至跨越TfR1之頂端及蛋白酶樣域之構形抗原決定基。有17個來自TfR1之殘基及19個來自抗體X Fab之殘基在結合搭配物之4 Å內。不連續抗原決定基由來自hu TfR1之頂端域之殘基Q285、T286、K287、P289、E343、D352、C353、P354、S355、K358、D360、S361、R364及蛋白酶樣域之殘基S492、D560、T561、R579形成。抗體X之抗原結合位點(基於低溫EM模型中之原子距離)由所有六個CDR (VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及VL CDR3)形成，且由重鏈之S32、S33、S40、S60、S61、S66、Y67、K109、Y110、R111及A112 (SEQ ID NO:100之AHo編號)，以及輕鏈之S33、S39、Y40、N69、Q109及S110 (SEQ ID NO:200之AHo編號)，加上來自輕鏈之Y57及S83之額外構架接觸( 表9)構成。 To identify the molecular basis of the antigenic recognition of Antibody X, a low-temperature EM structure of the ternary complex of Antibody X Fab fragment, huTfR1 ECD, and holohuman transferrin (Sigma T0665) was generated at 3.6 Å resolution. An atomic model of the complex was obtained by fitting the homology model of Antibody X Fab and the available crystal structures of TfR1 and Tf into the low-temperature EM density map ( Figure 3A ). The final model revealed that Antibody X binds to a conformational epitope spanning the top and protease-like domains of TfR1. There are 17 residues from TfR1 and 19 residues from Antibody X Fab within 4 Å of the binding partner. The discontinuous epitope is formed by residues Q285, T286, K287, P289, E343, D352, C353, P354, S355, K358, D360, S361, R364 from the apical domain of hu TfR1 and residues S492, D560, T561, R579 from the protease-like domain. The antigen binding site of Antibody X (based on atomic distances in the low temperature EM model) is formed by all six CDRs (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3) and is composed of S32, S33, S40, S60, S61, S66, Y67, K109, Y110, R111 and A112 of the heavy chain (AHo numbering of SEQ ID NO: 100), and S33, S39, Y40, N69, Q109 and S110 of the light chain (AHo numbering of SEQ ID NO: 200), plus additional framework contacts from Y57 and S83 of the light chain ( Table 9 ).

抗體X識別人類及石蟹猴中之TfR1。此等結構研究提供對抗體X之結合性質的詳細見解。TfR1之頂端及蛋白酶樣域中之結合抗原決定基在人類與石蟹猴之間高度保守，僅具有單一保守R364K突變(hu＞石蟹猴TfR1) ( 圖3B)，與來自此等物種之抗體X結合之交叉反應性一致。 表9：接觸TfR1之抗TfR抗體X互補位殘基. TfR1之4.0 Å內之接觸殘基為粗體小寫。 CDR 接觸殘基(AHo) (＜4Å) CDR 序列 VH CDR1 S32、S33、S40 GFTF ssY sMN (SEQ ID NO:3) VH CDR2 S60、S61、S66、Y67 SIS ssS syIYYADSVKG (SEQ ID NO:4) VH CDR3 K109、Y110、R111、A112 kyraGDFDY (SEQ ID NO:5) VL CDR1 S33、S39、Y40 RASQSVS ssyLA (SEQ ID NO:6) VL CDR2 N69 GAS nRAT (SEQ ID NO:7) VL CDR3 Q109、S110 QQ qsSSPPT (SEQ ID NO:8) LC構架 Y57、S83 實例4：抗體X之細胞運輸 Antibody X recognizes TfR1 in humans and cynomolgus monkeys. These structural studies provide detailed insights into the binding properties of Antibody X. The binding epitopes in the top and protease-like domains of TfR1 are highly conserved between humans and cynomolgus monkeys, with only a single conserved R364K mutation (hu>cynomolgus monkey TfR1) ( Figure 3B ), consistent with the cross-reactivity of Antibody X binding from these species. Table 9: Anti-TfR Antibody X complementary residues that contact TfR1. Contact residues within 4.0 Å of TfR1 are in bold lowercase. CDR Contact residue (AHo) (＜4Å) CDR sequences VH CDR1 S32, S33, S40 GFTF ss Y s MN (SEQ ID NO:3) VH CDR2 S60, S61, S66, Y67 SIS ss S sy IYYADSVKG (SEQ ID NO:4) VH CDR3 K109, Y110, R111, A112 kyra GDFDY (SEQ ID NO:5) VL CDR1 S33, S39, Y40 RASQSVS ssy LA (SEQ ID NO:6) VL CDR2 N69 GAS n RAT (SEQ ID NO:7) VL CDR3 Q109, S110 QQ qs SSPPT (SEQ ID NO:8) LC Structure Y57, S83 Example 4: Cellular transport of antibody X

為研究抗體X之細胞運輸，表徵模型血腦障壁(BBB)細胞中與轉移受體之pH依賴性結合及胞吞轉送作用。To study the cellular trafficking of Antibody X, we characterized its pH-dependent binding to transfer receptors and endocytic transport in model blood-brain barrier (BBB) cells.

由於pH沿著胞內體、溶酶體及跨細胞運輸路徑自細胞表面下降，因此抗TfR抗體之pH依賴性結合可影響分選成不同次細胞區室且可影響胞吞轉送作用之效率。藉由表面電漿子共振(如上文所述)在pH 7.4及pH 5.5下量測2 - 2000 nM之單價Fab-Fc型式之抗體X與帶His標籤之hTfR1 ECD的結合。儘管SPR晶片上之hTfR1 ECD水準相似，但在pH 5.5下量測到與pH 7.4相比低得多之結合反應( 圖4A)，表明抗體X之結合之強pH敏感性。 Since pH decreases from the cell surface along the endosomes, lysosomes, and transcellular transport pathways, pH-dependent binding of anti-TfR antibodies can affect sorting into different subcellular compartments and can affect the efficiency of endocytic transport. Binding of 2-2000 nM monovalent Fab-Fc format of Antibody X to His-tagged hTfR1 ECD was measured by surface plasmon resonance (as described above) at pH 7.4 and pH 5.5. Despite similar levels of hTfR1 ECD on the SPR chip, much lower binding responses were measured at pH 5.5 compared to pH 7.4 ( Figure 4A ), indicating strong pH sensitivity of Antibody X binding.

為了解抗體型式/化合價之變化是否可改良細胞轉運，如上文所述，在transwell中培養之Caco2細胞中測試多種抗體型式之抗體X之胞吞轉送作用。在Caco2 transwell頂部孔中以100 nM孵育兩天後，在底部孔中偵測到水準比非靶向hIgG對照抗體高7.6倍的呈二價Fab-Fc型式之抗體X，而呈二價Fc-Fc、單價Fab-Fc及單價Fc-Fab型式之抗體X各自顯示水準比對照hIgG高15-20倍的顯著更高之胞吞轉運作用( 圖4B)。此等數據證實，所測試之抗體X型式中之每一者皆對於活體外細胞內化及胞吞作用具有活性，且抗體X之型式可改變胞吞作用程度，使得呈各種型式之抗體X可用於各種應用，此取決於例如胞吞轉送作用之期望程度及所連接之貨物。 實例5：抗體X之腦分佈 To understand whether changes in antibody format/valency could improve cellular trafficking, various antibody formats of Antibody X were tested for endocytic trafficking in Caco2 cells cultured in transwells as described above. After two days of incubation at 100 nM in the top wells of Caco2 transwells, Antibody X in bivalent Fab-Fc format was detected in the bottom wells at 7.6-fold higher levels than the non-targeting hIgG control antibody, while Antibody X in bivalent Fc-Fc, monovalent Fab-Fc, and monovalent Fc-Fab formats each showed significantly higher endocytic trafficking at 15-20-fold higher levels than the control hIgG ( FIG. 4B ). These data demonstrate that each of the forms of Antibody X tested is active for in vitro cellular internalization and endocytosis, and that the form of Antibody X can alter the extent of endocytosis, allowing Antibody X in various forms to be useful in a variety of applications, depending on, for example, the desired extent of endocytic transport and the attached cargo. Example 5: Brain Distribution of Antibody X

為測試抗體X是否可在活體內穿透血腦障壁，向人類TfR敲入小鼠(hTfR KI，含有抗體X抗原決定基)靜脈內註射20 mg/kg之抗體X二價Fab-Fc或非靶向對照hIgG，兩者皆含有突變N297Q以經由非糖基化降低Fc效應子功能。經由面頰採血收集活體(In life)血清樣品。在4、24、72或168小時後，用氯胺酮/甲苯噻嗪(xylazine) (100/10 mg/kg ip)麻醉小鼠，經由心臟穿刺收集血液樣品，且經由左心室以2 ml/分鐘向小鼠灌注冰冷PBS/肝素(1 ug/mL)持續10分鐘以清除血液之脈管系統。接著取出腦並半切開，一個半球在液氮中急速冷凍，且另一半球在10%中性緩衝福馬林中固定24小時。在收集后24小時內分析血液樣品的包括網狀紅血球在內之全血球計數(IDEXX)。另外，藉由使血液在室溫下凝結15-30分鐘、以2000 g離心10分鐘、冷凍上清液來產生血清用於進一步分析。To test whether Antibody X can cross the blood-brain barrier in vivo, human TfR knock-in mice (hTfR KI, containing Antibody X epitopes) were intravenously infused with 20 mg/kg of Antibody X bivalent Fab-Fc or non-targeting control hIgG, both containing the mutation N297Q to reduce Fc effector function by aglycosylation. In life serum samples were collected by buccal bleed. After 4, 24, 72, or 168 hours, mice were anesthetized with ketamine/xylazine (100/10 mg/kg ip), blood samples were collected by cardiac puncture, and mice were perfused with ice-cold PBS/heparin (1 ug/mL) via the left ventricle at 2 ml/min for 10 minutes to clear the vasculature of blood. The brain was then removed and hemisectomized, one hemisphere was snap frozen in liquid nitrogen, and the other hemisphere was fixed in 10% neutral buffered formalin for 24 hours. Blood samples were analyzed for complete blood counts (IDEXX) including reticulocytes within 24 hours of collection. In addition, serum was generated by allowing blood to clot at room temperature for 15-30 minutes, centrifuging at 2000 g for 10 minutes, and freezing the supernatant for further analysis.

將冷凍腦半球在具有氧化鋯珠粒(ZROB05及ZROB10)之溶解緩衝液(50 mM Tris pH 7.5、150 mM NaCl、0.25%去氧膽酸鈉、1 mM EDTA、1% NP40、完全蛋白酶抑制劑)中在組織均質器(NextAdvance Bullet Blender)攪拌10分鐘，接著在4℃下孵育，旋轉1小時。接著藉由以12,000 g離心20分鐘來清除溶解產物之碎片。Frozen brain hemispheres were blended in lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.25% sodium deoxycholate, 1 mM EDTA, 1% NP40, complete protease inhibitor) with zirconia beads (ZROB05 and ZROB10) for 10 min in a tissue homogenizer (NextAdvance Bullet Blender), followed by incubation at 4°C with rotation for 1 h. Lysate debris was then cleared by centrifugation at 12,000 g for 20 min.

藉由MSD免疫檢定分析血清及腦溶解產物中之人類IgG水準。將樣品在包被有抗hIgG Fc捕獲試劑(Jackson ImmunoResearch，目錄號709-006-098)之MSD板(MSD，目錄號L15XB-3/L11XB-3)上孵育，用帶磺基標籤之抗hIgG (MSD，目錄號R32AJ-1)偵測，且藉由在針對每一測試抗體產生之標準曲線上內插來定量。Human IgG levels in serum and brain lysates were analyzed by MSD immunoassay. Samples were incubated on MSD plates (MSD, Catalog No. L15XB-3/L11XB-3) coated with anti-hIgG Fc capture reagent (Jackson ImmunoResearch, Catalog No. 709-006-098), detected with sulfo-tagged anti-hIgG (MSD, Catalog No. R32AJ-1), and quantified by interpolation on a standard curve generated for each test antibody.

將福馬林固定之腦半球轉移至磷酸鹽緩衝鹽水中，石蠟包埋，分離成六個5 mm冠狀區段，切成3-5 mm厚，蛋白酶K處理用於抗原修復，且針對人類IgG染色(Southern Biotech 6145-01, 2 ug/ml，接著Leica Refine HRP聚合物及DAB色原體)。Formalin-fixed brain hemispheres were transferred to phosphate-buffered saline, paraffin-embedded, separated into six 5-mm coronal sections, cut into 3-5 mm thickness, treated with proteinase K for antigen retrieval, and stained for human IgG (Southern Biotech 6145-01, 2 ug/ml, followed by Leica Refine HRP polymer and DAB chromogen).

抗體X在血清中顯示出比對照IgG快得多之清除率，在靜脈內投與後3天下降＞10倍且到7天下降＞1000倍( 圖5A)，如運鐵蛋白受體介導之處置所預期。抗體X之網狀紅血球計數在第1天瞬時減少至總血球之約1%，但到7天恢復至約3%，相比之下，對照hIgG為約3%。在靜脈內投與後最長達3天，抗體X之腦暴露比對照hIgG大約10倍，但在7天時降至小於對照hIgG ( 圖5B)。hIgG染色之免疫組織化學結果顯示，相對於非靶向對照hIgG，抗體X跨多個腦區(尤其大腦皮質中)具有增強之生物分佈( 圖5C；來自第3天之影像)。在較高放大率下，在血管以及整個實質及大腦皮質中之錐體神經元上可見hIgG染色( 圖5D)。總之，抗體X顯示出極大增強之對CNS之生物分佈。 Antibody X showed much faster clearance in serum than control IgG, decreasing >10-fold by 3 days after intravenous administration and >1000-fold by 7 days ( FIG. 5A ), as expected for transferrin receptor-mediated disposal. Reticulocyte counts for Antibody X decreased transiently to approximately 1% of total blood cells on day 1, but recovered to approximately 3% by day 7, compared to approximately 3% for control hIgG. Brain exposure of Antibody X was approximately 10-fold greater than control hIgG for up to 3 days after intravenous administration, but decreased to less than control hIgG by day 7 ( FIG. 5B ). Immunohistochemistry results of hIgG staining showed that Antibody X had enhanced biodistribution across multiple brain regions, particularly in the cerebral cortex, relative to the non-targeted control hIgG ( FIG. 5C ; images from day 3). At higher magnification, hIgG staining was seen on blood vessels and pyramidal neurons throughout the parenchyma and cerebral cortex ( Fig. 5D ). In summary, Antibody X showed greatly enhanced biodistribution to the CNS.

為驗證非人類靈長類動物中抗體X之腦攝取活性之可轉譯性，向石蟹猴靜脈內投與20 mg/kg劑量之二價Fab-Fc抗體X或非靶向對照hIgG。在48小時後，遵循與上述小鼠研究類似之方法，在氯胺酮/甲苯噻嗪麻醉下灌注動物，且自來自各半球之額葉皮質、海馬迴及小腦收集腦樣品。將來自一個半球之腦生檢切片進行急速冷凍、均質化，且藉由MSD免疫檢定來分析總hIgG。來自第二半球之腦生檢切片經福馬林固定處理以用於hIgG免疫組織化學。To validate the translatability of the brain uptake activity of Antibody X in non-human primates, cynomolgus monkeys were administered intravenously with a dose of 20 mg/kg of bivalent Fab-Fc Antibody X or a non-targeting control hIgG. After 48 hours, animals were perfused under ketamine/xylazine anesthesia and brain samples were collected from the frontal cortex, hippocampus, and cerebellum from each hemisphere, following a similar approach to the mouse studies described above. Brain biopsies from one hemisphere were snap-frozen, homogenized, and analyzed for total hIgG by MSD immunoassay. Brain biopsies from the second hemisphere were formalin-fixed for hIgG immunohistochemistry.

在所有取樣區域中，在腦生檢切片中皆偵測到抗體X之水準比對照hIgG高20-25倍( 圖6A)。如在hTfR KI小鼠中所觀察到，抗體X之腦攝取升高對應於IHC對血管、實質及神經元hIgG染色之增加( 圖6B)。此外，在大腦皮質之錐體神經元內之囊泡中偵測到抗體X ( 圖6C)，此支持需要神經元溶酶體遞送之治療應用。 實例6：抗體X之劑量依賴性腦暴露 Antibody X was detected in brain biopsies at levels 20-25-fold higher than control hIgG in all sampled regions ( Fig. 6A ). As observed in hTfR KI mice, the increased brain uptake of Antibody X corresponded to increased IHC staining of vascular, parenchymal, and neuronal hIgG ( Fig. 6B ). In addition, Antibody X was detected in vesicles within pyramidal neurons in the cerebral cortex ( Fig. 6C ), supporting therapeutic applications requiring neuronal lysosomal delivery. Example 6: Dose-dependent brain exposure of Antibody X

預期BBB處之經受體介導之轉運為可飽和過程，其中遞送能力取決於受體在腦內皮細胞中之表現。為更好地理解TfR1介導之對抗體X之腦攝取之限制，在hTfR KI小鼠中進行劑量依賴性生物分佈研究。由於與二價型式相比，單價型式之抗體X可能因TfR1相互作用之1:1化學計量而具有更高之腦攝取，因此將其作為單價Fab-Fc融合物(由非糖基化之重鏈、輕鏈及Fc片段組成之三鏈分子，利用杵入臼突變進行Fc異二聚化)以3、10、30及90 mg/kg莫耳IgG當量(即2、6.7、20及60 mg/kg Fab-Fc)靜脈內給予，且與等效劑量之非靶向二價對照抗體進行比較。一天後，自每隻小鼠收集血液及心臟灌注之腦樣品，且藉由MSD免疫檢定來定量血清及腦溶解產物中之hIgG水準。Receptor-mediated transport at the BBB is expected to be a saturable process in which delivery capacity depends on receptor expression in brain endothelial cells. To better understand the TfR1-mediated limitation of antibody X's brain uptake, a dose-dependent biodistribution study was performed in hTfR KI mice. Since the monovalent version of Antibody X may have higher brain uptake compared to the bivalent version due to the 1:1 stoichiometry of the TfR1 interaction, it was given intravenously as a monovalent Fab-Fc fusion (a three-chain molecule composed of non-glycosylated heavy chain, light chain and Fc fragment, with Fc heterodimerization using knob-in-hole mutations) at 3, 10, 30 and 90 mg/kg molar IgG equivalents (i.e., 2, 6.7, 20 and 60 mg/kg Fab-Fc) and compared to an equivalent dose of a non-targeting bivalent control antibody. One day later, blood and cardiac perfused brain samples were collected from each mouse, and hIgG levels in serum and brain lysates were quantified by MSD immunoassay.

血清暴露與增加之劑量成比例地增加( 圖17A)，但單價抗體X之血清暴露低於二價hIgG對照，其係數在所有劑量水準下皆一致(0.3-0.45倍/對照)。此表明TfR1介導之清除效應在此1天時間點不顯著。然而，在所有劑量水準下，單價抗體X之腦暴露皆高於hIgG對照( 圖17B)。單價抗體X亦顯示腦攝取之非線性劑量依賴性，自10 mg/kg莫耳IgG等效劑量在腦中達到約15 nM，但對於90 mg/kg莫耳IgG等效劑量增加至僅25-30 nM。隨著劑量增加至高於10 mg/kg，腦暴露之此適度增加可能歸因於被動生物分佈，乃因對照IgG在此劑量範圍內顯示腦攝取之類似增加。因此，表示為相較於對照之倍數的腦暴露之改良在所測試之最低劑量下最高，但由於TfR1介導之轉運飽和及被動腦攝取增加，因此對於較高劑量而言下降( 圖17C)。 實例7：抗體X-抗BACE1 Serum exposure increased proportionally with increasing dose ( Figure 17A ), but serum exposure of monovalent Antibody X was lower than that of the bivalent hIgG control by a factor that was consistent at all dose levels (0.3-0.45-fold/control). This suggests that TfR1-mediated clearance was not significant at this 1-day time point. However, brain exposure of monovalent Antibody X was higher than that of the hIgG control at all dose levels ( Figure 17B ). Monovalent Antibody X also showed nonlinear dose dependence of brain uptake, reaching approximately 15 nM in the brain from a 10 mg/kg molar IgG equivalent dose, but increasing to only 25-30 nM for a 90 mg/kg molar IgG equivalent dose. This modest increase in brain exposure with increasing doses above 10 mg/kg is likely attributable to passive biodistribution, as the control IgG showed similar increases in brain uptake over this dose range. Thus, the improvement in brain exposure expressed as a fold relative to control was greatest at the lowest dose tested, but declined for higher doses due to increased saturation of TfR1-mediated transport and passive brain uptake ( Figure 17C ). Example 7: Antibody X - Anti-BACE1

為了證實抗TfR介導之抗體以足夠濃度遞送至CNS以達成腦特異性藥理學活性，測試了組合抗體X及抗β分泌酶1 (BACE1)抗體之雙特異性抗體，其對重組人及小鼠BACE1 ECD具有K D= 1.3 nM之親和力。設計此雙特異性抗體之兩個形式，兩者皆對於BACE1為二價，但(i)一者對於hTfR為二價(自N末端至C末端具有抗BACE1 VH序列後跟SEQ ID NO:407之鏈，自N末端至C末端具有抗BACE1 VL序列後跟SEQ ID NO:410之鏈，以及具有序列SEQ ID NO:411之鏈)，且(ii)另一者對於hTfR為單價(自N末端至C末端具有抗BACE1 VH序列後跟SEQ ID NO:408之鏈，自N末端至C末端具有抗BACE1 VH序列後跟SEQ ID NO:409之鏈、自N末端至C末端具有抗BACE1 VL序列後跟SEQ ID NO:410之鏈，以及具有序列SEQ ID NO:411之鏈) ( 圖7)。抗BACE1 Fab (CH1：L128R、K147R；CL：Q124E、V133Q T178E)及抗TfR Fab (CH1：L145Q、K147E、S181E；CL：T129R、T178R、T180Q)中之突變用於促進適當輕鏈配對。Fc區中之突變用於降低效應子功能(L234A、L235A、P329G)及促進單價抗TfR雙特異性抗體(杵入臼)之異質Fc形成。將抗體在CHO中瞬時表現且藉由標準親和層析(MabSelect SuRe，在pH 2.8磷酸鹽中溶析)、之後藉由尺寸排阻層析(Superdex 200)純化。為評估此等抗體之活體內PKPD行為，向hTfR KI小鼠靜脈內投與50 mg/kg莫耳IgG當量劑量之對照hIgG、抗BACE1、抗BACE1/二價抗體X或抗BACE1/單價抗體X(藉由尾靜脈注射，每組四隻動物)。在1、3或7天後，用氯胺酮/甲苯噻嗪腹膜內麻醉動物，藉由心臟穿刺收集血液樣品，用冰冷PBS/肝素灌注脈管系統，且收穫腦，如上文所述。將冷凍之腦半球粉碎且藉由MSD免疫檢定(V-PLEX Aβ肽小組1 4G8套組)單獨分析總hIgG及澱粉樣蛋白β1-40 (Ab40)。 To demonstrate that anti-TfR-mediated antibodies are delivered to the CNS at sufficient concentrations to achieve brain-specific pharmacological activity, a bispecific antibody combining Antibody X and an anti-β-secretase 1 (BACE1) antibody was tested, which has an affinity of K D = 1.3 nM for recombinant human and mouse BACE1 ECD. Two forms of this bispecific antibody were designed, both of which were bivalent for BACE1, but (i) one was bivalent for hTfR (having a chain of anti-BACE1 VH sequence followed by SEQ ID NO:407 from N-terminus to C-terminus, a chain of anti-BACE1 VL sequence followed by SEQ ID NO:410 from N-terminus to C-terminus, and a chain with sequence SEQ ID NO:411), and (ii) the other was monovalent for hTfR (having a chain of anti-BACE1 VH sequence followed by SEQ ID NO:408 from N-terminus to C-terminus, a chain of anti-BACE1 VH sequence followed by SEQ ID NO:409 from N-terminus to C-terminus, a chain of anti-BACE1 VL sequence followed by SEQ ID NO:410 from N-terminus to C-terminus, and a chain with sequence SEQ ID NO:411) ( FIG. 7 ). Mutations in anti-BACE1 Fab (CH1: L128R, K147R; CL: Q124E, V133Q T178E) and anti-TfR Fab (CH1: L145Q, K147E, S181E; CL: T129R, T178R, T180Q) were used to promote proper light chain pairing. Mutations in the Fc region were used to reduce effector function (L234A, L235A, P329G) and promote heterogeneous Fc formation of monovalent anti-TfR bispecific antibodies (knobs-in-holes). Antibodies were transiently expressed in CHO and purified by standard affinity analysis (MabSelect SuRe, eluted in pH 2.8 phosphate) followed by size exclusion analysis (Superdex 200). To evaluate the in vivo PKPD behavior of these antibodies, hTfR KI mice were intravenously administered 50 mg/kg molar IgG equivalent doses of control hIgG, anti-BACE1, anti-BACE1/bivalent Antibody X, or anti-BACE1/monovalent Antibody X (by tail vein injection, four animals per group). After 1, 3, or 7 days, animals were anesthetized intraperitoneally with ketamine/xylazine, blood samples were collected by cardiac puncture, the vasculature was perfused with ice-cold PBS/heparin, and brains were harvested as described above. Frozen brain hemispheres were crushed and analyzed separately for total hIgG and amyloid beta 1-40 (Ab40) by MSD immunoassay (V-PLEX Aβ peptide panel 1 4G8 kit).

兩種抗BACE1/TfR雙特異性抗體自血漿中清除之速度遠快於抗BACE1或對照hIgG，且對TfR為單價之雙特異性抗體之血漿濃度低於對TfR為二價之雙特異性抗體的血漿濃度( 圖8A)。然而，雙特異性抗體之腦水準高於單特異性抗體或對照抗體，其中偵測到的單價TfR結合物之水準高於二價TfR結合物( 圖8B)。兩種雙特異性抗體相較於對照皆持續腦暴露至7天。兩種抗BACE1/TfR雙特異性抗體皆顯著降低腦溶解產物中之Ab40水準(約50%，如對BACE1抑制所預期)，且此藥效學效應亦持續至靜脈內投與後7天( 圖8C)。 實例8：抗體X寡核苷酸 Both anti-BACE1/TfR bispecific antibodies were cleared from plasma much faster than anti-BACE1 or control hIgG, and the plasma concentration of the monovalent bispecific antibody to TfR was lower than that of the bivalent bispecific antibody to TfR ( Figure 8A ). However, the brain levels of the bispecific antibodies were higher than those of the monospecific antibodies or the control antibodies, with higher levels of monovalent TfR binders detected than bivalent TfR binders ( Figure 8B ). Both bispecific antibodies sustained brain exposure to 7 days compared to the control. Both anti-BACE1/TfR bispecific antibodies significantly reduced Ab40 levels in brain lysates (approximately 50%, as expected for BACE1 inhibition), and this pharmacodynamic effect also persisted up to 7 days after intravenous administration ( Figure 8C ). Example 8: Antibody X Oligonucleotide

為證實抗體X可自靜脈內投與增強寡核苷酸之CNS活性，產生抗體X之二價形式(包含SEQ ID NO:401及SEQ ID NO:412)及抗體X之單價形式(包含SEQ ID NO:401、SEQ ID NO:413及SEQ ID NO:414)以及對照hIgG，其具有用於結合之半胱胺酸修飾(S442C)。合成靶向轉移相關之肺腺癌轉錄物1 (Malat-1)之3–10–3 cEt間隙聚體反義寡核苷酸(ASO) (其在每週三劑量後用於減少小鼠肝臟中之Malat-1 mRNA之效力為ED50 = 0.3 umol/kg/週) (WuXi)，且藉由與順丁烯二醯亞胺-PEG 2-琥珀醯亞胺基酯(SM(PEG)2，Thermo 22103)反應進行官能化以用於結合。將抗體還原(1 mM TCEP，60-90分鐘，室溫)，加載至Mabselect SuRe管柱上，再氧化(2 mM DHAA，2小時，室溫)，洗滌，溶析(25 mM NaPi pH 2.8，0.1 M NaCl)且中和至pH 5.0，接著以約1:1莫耳當量與順丁烯二醯亞胺-ASO反應。藉由陰離子交換層析(TMAE管柱，用10 mM NaPi pH7.0、0.6M NaCl溶析)分離具有1:1抗體(Ab)/ASO比率之結合物，藉由SDS-PAGE及UV分光光度法確認。將最終匯集物透析至PBS中，且藉由BCA檢定(Thermo A53226)確定蛋白質濃度。 To demonstrate that Antibody X can enhance the CNS activity of oligonucleotides by intravenous administration, a bivalent form of Antibody X (comprising SEQ ID NO:401 and SEQ ID NO:412) and a monovalent form of Antibody X (comprising SEQ ID NO:401, SEQ ID NO:413 and SEQ ID NO:414) were generated, as well as a control hIgG with a cysteine modification (S442C) for binding. A 3–10–3 cEt gapmer antisense oligonucleotide (ASO) targeting metastasis-associated lung adenocarcinoma transcript 1 (Malat-1) with an ED50 of 0.3 umol/kg/week for reducing Malat-1 mRNA in mouse liver after three weekly doses (WuXi) was synthesized and functionalized for conjugation by reaction with cis(PEG) 2- succinimidyl ester (SM(PEG)2, Thermo 22103). The antibody was reduced (1 mM TCEP, 60-90 min, room temperature), loaded onto a Mabselect SuRe column, reoxidized (2 mM DHAA, 2 h, room temperature), washed, eluted (25 mM NaPi pH 2.8, 0.1 M NaCl) and neutralized to pH 5.0, then reacted with cis-butylenediamide-ASO at approximately 1:1 molar equivalents. The conjugate with a 1:1 antibody (Ab)/ASO ratio was isolated by anion exchange chromatography (TMAE column, eluted with 10 mM NaPi pH 7.0, 0.6 M NaCl) and confirmed by SDS-PAGE and UV spectrophotometry. The final pool was dialyzed into PBS and the protein concentration was determined by BCA assay (Thermo A53226).

為確認Ab:ASO結合物之腦生物分佈性質，藉由尾靜脈注射游離ASO、對照hIgG-ASO、二價抗體X-ASO或單價抗體X-ASO，以20 mg/kg IgG:0.75 mg/kg ASO莫耳當量向hTfR KI小鼠給藥。在24小時後，利用心臟灌注處死小鼠，且收集腦及血清樣品(如上文所述)。藉由hIgG MSD免疫檢定來定量血清及腦溶解產物中之總抗體濃度。為定量腦中之游離ASO濃度，藉由使用包被有與Malat1 ASO互補之生物素化DNA探針之磁性鏈黴抗生物素蛋白珠進行雜交來提取腦均質物。將組織均質物中之ASO捕獲於珠粒上，洗滌，且接著利用熱變性將其釋放至新盤中。藉由LC-MS/MS使用C18反相管柱及多反應監測對提取之ASO進行定量。To confirm the brain biodistribution properties of Ab:ASO conjugates, hTfR KI mice were dosed with free ASO, control hIgG-ASO, bivalent Antibody X-ASO, or monovalent Antibody X-ASO by tail vein injection at 20 mg/kg IgG:0.75 mg/kg ASO molar equivalents. After 24 hours, mice were sacrificed by cardiac perfusion, and brain and serum samples were collected (as described above). Total antibody concentrations in serum and brain lysates were quantified by hIgG MSD immunoassay. To quantify free ASO concentrations in the brain, brain homogenates were extracted by hybridization using magnetic streptavidin beads coated with biotinylated DNA probes complementary to Malat1 ASO. ASOs in tissue homogenates were captured on beads, washed, and then released into a new plate using heat denaturation. The extracted ASOs were quantified by LC-MS/MS using a C18 reverse phase column and multiple reaction monitoring.

在給予二價或單價抗體X-ASO結合物之小鼠中，在血清中偵測到抗體之水準比給予對照hIgG-ASO結合物之小鼠低約4倍( 圖9A)。然而，在腦溶解產物中，與對照hIgG相比，二價抗體X高約5倍，且單價抗體X高約9倍( 圖9B)。類似地，對於給予任一抗體X-ASO之動物，皆偵測到腦溶解產物中之游離ASO，該游離ASO之水準對於單價抗體而言高於二價抗體，但低於給予單獨ASO或對照hIgG-ASO之動物腦中之定量極限( 圖9C)。 In mice given either bivalent or monovalent antibody X-ASO conjugates, antibody was detected in serum at levels approximately 4-fold lower than in mice given control hIgG-ASO conjugates ( FIG. 9A ). However, in brain lysates, bivalent antibody X was approximately 5-fold higher and monovalent antibody X was approximately 9-fold higher compared to control hIgG ( FIG. 9B ). Similarly, free ASO was detected in brain lysates for animals given either antibody X-ASO, with levels higher for the monovalent than for the bivalent antibody but below the limit of quantification in the brains of animals given either ASO alone or control hIgG-ASO ( FIG. 9C ).

為在Malat1 mRNA之敲低中誘發藥效學反應，在hTfR KI小鼠中以40 mg/kg IgG:1.5 mg/kg ASO莫耳當量每週四次靜脈內給予結合物，進行多劑量研究。為防止小鼠中之抗藥物抗體反應，在測試物品之第一劑量及第三劑量之前四天，以20 mg/kg腹膜內投與抗小鼠CD4 GK1.5 (BioXcell BP003-1)。在末次劑量後三天處死小鼠，收穫腦半球、脊髓、脛骨前肌及肝臟，急速冷凍且儲存用於分析。如上文所述，藉由LC-MS/MS分析來自各動物之腦半球之游離ASO累積。將組織在TRIzol試劑(Qiagen)中均質化。接著使用PureLink RNA微型套組(Invitrogen)組合用DNase I (Qiagen)進行管柱內DNA消化來分離總RNA。使用SuperScript VILO主混合物(Master Mix) (Invitrogen)進行逆轉錄以產生cDNA。根據製造商之說明書，使用QX200 AutoDG微滴數位PCR系統(Bio-Rad)來偵測及定量 Malat1及持家基因之轉錄物。QX200自動化微滴發生器(Bio-Rad)提供微滴產生，且在T100熱循環儀(Bio-Rad)上進行PCR。在QX200微滴讀取器(Bio-Rad)上讀取板且使用QuantaSoft分析軟體(Bio-Rad)進行分析以定量每孔轉錄物之拷貝。將 Malat1之表現水準相對於 Actb之表現水準作正規化，且將其進一步相對於媒劑治療之動物中之水準作正規化。 To induce a pharmacodynamic response in the knockdown of Malat1 mRNA, a multiple dose study was performed in hTfR KI mice with the conjugate administered intravenously four times a week at 40 mg/kg IgG:1.5 mg/kg ASO molar equivalents. To prevent anti-drug antibody reactions in mice, anti-mouse CD4 GK1.5 (BioXcell BP003-1) was administered intraperitoneally at 20 mg/kg four days before the first and third doses of the test article. Mice were sacrificed three days after the last dose, and the brain hemispheres, spinal cord, tibialis anterior muscle, and liver were harvested, snap frozen, and stored for analysis. Free ASO accumulation from the brain hemispheres of each animal was analyzed by LC-MS/MS as described above. Tissues were homogenized in TRIzol reagent (Qiagen). Total RNA was then isolated using the PureLink RNA Mini Kit (Invitrogen) in combination with on-column DNA digestion with DNase I (Qiagen). Reverse transcription was performed using SuperScript VILO Master Mix (Invitrogen) to generate cDNA. Transcripts of Malat1 and housekeeping genes were detected and quantified using the QX200 AutoDG Droplet Digital PCR System (Bio-Rad) according to the manufacturer's instructions. Droplet generation was provided by the QX200 Automated Droplet Generator (Bio-Rad), and PCR was performed on a T100 Thermal Cycler (Bio-Rad). Plates were read on a QX200 microtiter reader (Bio-Rad) and analyzed using QuantaSoft analysis software (Bio-Rad) to quantify the copies of transcripts per well. Expression levels of Malat1 were normalized to those of Actb and further normalized to levels in vehicle-treated animals.

與來自單劑量研究之結果類似，抗體X將ASO以遠高於來自單獨給予ASO或對照hIgG-ASO結合物之水準遞送至腦( 圖9D)。投與二價抗體X-ASO結合物使ASO在腦溶解產物中累積至約9 nM，且單價抗體X-ASO結合物使其累積至約18 nM。相應地，雖然相對於給予DPBS媒劑之小鼠，自給予單獨ASO或對照hIgG-ASO結合物未觀察到腦中Malat1 mRNA之降低，但二價及單價抗體X-ASO結合物使Malat1 mRNA分別降低約40%及約50% ( 圖9E)。在脊髓中觀察到類似趨勢，其中投與二價及單價抗體X-ASO結合物使Malat1敲低分別為約50%及約60%。亦在周邊組織(諸如肌肉(脛骨前肌))中觀察到Malat1敲低，其中單獨給予ASO具有有限作用，且在肝臟中單獨給予ASO導致比Ab-ASO結合物更大之敲低。抗體X以足以誘導靶mRNA敲低之水準有效地將經結合之ASO自周邊投與遞送至CNS，同時亦遞送至其他治療相關組織，而不增加至肝臟之遞送(其中可能出現毒性)。 Similar to the results from the single-dose studies, Antibody X delivered ASO to the brain at levels much higher than from administration of ASO alone or control hIgG-ASO conjugate ( FIG. 9D ). Administration of the bivalent Antibody X-ASO conjugate resulted in accumulation of ASO in brain lysates to approximately 9 nM, and the monovalent Antibody X-ASO conjugate resulted in accumulation to approximately 18 nM. Correspondingly, while no reduction in Malat1 mRNA in the brain was observed from administration of ASO alone or control hIgG-ASO conjugate relative to mice given DPBS vehicle, the bivalent and monovalent Antibody X-ASO conjugates resulted in reductions in Malat1 mRNA by approximately 40% and approximately 50%, respectively ( FIG. 9E ). Similar trends were observed in the spinal cord, where administration of the bivalent and monovalent Antibody X-ASO conjugates resulted in Malat1 knockdown of approximately 50% and approximately 60%, respectively. Malat1 knockdown was also observed in peripheral tissues such as muscle (tibialis anterior), where ASO administration alone had limited effect, and in the liver where ASO administration alone resulted in greater knockdown than the Ab-ASO conjugate. Antibody X effectively delivers the conjugated ASO from peripheral administration to the CNS at levels sufficient to induce knockdown of target mRNA, as well as to other therapeutically relevant tissues, without increasing delivery to the liver, where toxicity may occur.

為評估抗體X是否亦可自靜脈內投與將有效濃度之短干擾核糖核酸(siRNA)遞送至CNS，使抗體X之單價形式(包含SEQ ID NO:401、SEQ ID NO:413及SEQ ID NO:414)以及對照hIgG (其具有用於結合之半胱胺酸修飾(S442C))結合至靶向次黃嘌呤-鳥嘌呤磷酸核糖基轉移酶(HPRT)之siRNA。此siRNA具有混合硫代磷酸酯-磷酸酯主鏈、混合2 '-O-甲基及2 '-氟修飾、有義股3 '端上之(CH 2) 6NH 2及反義5 '端上之乙烯基膦酸酯。使有義股與 N-琥珀醯亞胺基3-順丁烯二醯亞胺基丙酸酯異雙官能交聯劑(BMPS, TCI S0427)反應，與反義股退火，且結合至對照hIgG (S442C)或單價抗體X (S442C)，如上文所述。藉由陰離子交換層析分離抗體-siRNA 1:1結合物。 To assess whether Antibody X can also deliver effective concentrations of short interfering RNA (siRNA) to the CNS via intravenous administration, monovalent forms of Antibody X (comprising SEQ ID NO: 401, SEQ ID NO: 413, and SEQ ID NO: 414) and control hIgG (which has a cysteine modification (S442C) for binding) were conjugated to siRNA targeting hypoxanthine-guanine phosphoribosyltransferase (HPRT). This siRNA has a mixed phosphorothioate-phosphate backbone, mixed 2' - O-methyl and 2' -fluoro modifications, ( CH2 ) 6NH2 on the 3 ' end of the sense strand, and vinylphosphonate on the 5 ' end of the antisense strand. The sense strand was reacted with N- succinimidyl 3-butylene diimidopropionate heterobifunctional crosslinker (BMPS, TCI S0427), annealed with the antisense strand, and conjugated to control hIgG (S442C) or monovalent Antibody X (S442C) as described above. Antibody-siRNA 1:1 conjugates were isolated by anion exchange chromatography.

在HEK293T細胞中針對HPRT mRNA敲低，且在hTfR-MDCKII transwell中針對胞吞轉送作用測試抗體-siRNA結合物之細胞活性。將各種濃度之結合物或單獨siRNA (最高達6000 nM siRNA)與HEK293T (5000個細胞/孔)在DMEM + 10% FBS中孵育72 h，接著在冷PBS中洗滌，溶解且相對於未經處理之細胞或用siRNA及轉染試劑(Lipofectamine RNAiMax, Invitrogen)處理之細胞，藉由qPCR (TaqMan 4399002, Invitrogen)分析藉由ACTB (4333762T, Invitrogen) mRNA正規化之HPRT (Hs01003270_g1, Initrogen)。未結合之siRNA處理導致HPRT mRNA之敲低，但效力較低(IC50為約1300 nM； 圖16A)。雖然與對照hIgG結合不影響此細胞檢定中之siRNA活性(IC50為約1200 nM)，但與單價抗體X結合使HEK293T中HPRT敲低之效力增加超過40倍(IC50為約30 nM)。此證實用單價抗體X靶向人類運鐵蛋白受體足以增強siRNA在活體外向細胞中之功能遞送。 The cellular activity of antibody-siRNA conjugates was tested for HPRT mRNA knockdown in HEK293T cells and for endocytic transport in hTfR-MDCKII transwells. Various concentrations of conjugate or siRNA alone (up to 6000 nM siRNA) were incubated with HEK293T (5000 cells/well) in DMEM + 10% FBS for 72 h, then washed in cold PBS, lysed and analyzed by qPCR (TaqMan 4399002, Invitrogen) for HPRT (Hs01003270_g1, Initrogen) normalized by ACTB (4333762T, Invitrogen) mRNA relative to untreated cells or cells treated with siRNA and transfection reagent (Lipofectamine RNAiMax, Invitrogen). Unconjugated siRNA treatment resulted in knockdown of HPRT mRNA, but with lower potency (IC50 of approximately 1300 nM; Figure 16A ). While binding to control hIgG did not affect siRNA activity in this cell assay (IC50 of approximately 1200 nM), binding to monovalent Antibody X increased the potency of HPRT knockdown in HEK293T by more than 40-fold (IC50 of approximately 30 nM). This demonstrates that targeting the human ferritin receptor with monovalent Antibody X is sufficient to enhance the functional delivery of siRNA into cells in vivo.

為評估siRNA結合對運鐵蛋白受體介導之抗體X跨細胞運輸之影響，在hTfR細胞胞吞作用中將抗體-siRNA結合物與未結合抗體進行比較。用編碼人類TfR1基因之慢病毒粒子穩定轉導馬丁達比犬腎(Madin-Darby canine) II細胞(MDCK II, ECACC 00062107)，且在流式細胞術中使用抗TfR抗體驗證人類TfR1之表現。將MDCKII/hTfR細胞平鋪至Corning 0.4微米孔transwell插入物上，在完全培養基中每個插入物25,000個細胞，培養五天，且在頂部孔中用100 nM之抗體或抗體-siRNA結合物處理。在兩天處理後，自頂部室及底部室收集樣品且在中尺度發現檢定系統中使用通常可用之抗人類IgG試劑來分析測試物品之濃度。在transwell底部室中累積極少對照hIgG (＜2 nM)，但在高約100倍濃度下偵測到單價抗體X，表明發生主動TfR1介導之細胞轉運( 圖16B)。siRNA結合之單價抗體X亦顯示出主動轉運，但其水準為未結合之單價抗體X之大約一半。此指示siRNA結合在限制抗體X之細胞轉運方面具有適度效應。 To assess the effect of siRNA binding on the transferrin receptor-mediated transcellular transport of Antibody X, antibody-siRNA conjugates were compared with unconjugated antibody in hTfR endocytosis. Madin-Darby canine kidney II cells (MDCK II, ECACC 00062107) were stably transduced with lentiviral particles encoding the human TfR1 gene, and the expression of human TfR1 was verified in flow cytometry using anti-TfR antibodies. MDCKII/hTfR cells were plated onto Corning 0.4 μm pore transwell inserts at 25,000 cells per insert in complete medium, cultured for five days, and treated with 100 nM of antibody or antibody-siRNA conjugate in the top well. After two days of treatment, samples were collected from the top and bottom chambers and analyzed for test article concentrations using a commonly available anti-human IgG reagent in a mesoscale discovery assay system. Very little control hIgG (<2 nM) accumulated in the bottom chamber of the transwell, but monovalent antibody X was detected at approximately 100-fold higher concentrations, indicating that active TfR1-mediated cell trafficking occurred ( FIG. 16B ). siRNA-bound monovalent Antibody X also showed active transport, but at a level approximately half that of unbound monovalent Antibody X. This indicates that siRNA binding has a modest effect in limiting the cellular transport of Antibody X.

在hTfR1 KI小鼠中測試單價抗體X-siRNA結合物在靜脈內投與後敲低CNS中之HPRT mRNA之藥效學活性。靜脈內投與4 mg/kg siRNA或40 mg/kg hIgG莫耳當量之每週四劑量siRNA或抗體-siRNA結合物，其中靜脈內抗CD4 20 mg/kg在第一及第三劑量之前，以抑制抗藥物抗體。在末次劑量後三天，處死小鼠，收穫腦，分離總RNA，且藉由液滴數位PCR (ddPCR)量測相對於ACTB mRNA之HPRT mRNA水準。單獨siRNA及對照IgG-siRNA結合物處理皆未導致腦中HPRT mRNA水準之顯著變化。然而，單價抗體X-siRNA處理導致HPRT mRNA之60%敲低( 圖16C)。此表明抗體X可將寡核苷酸(包括ASO及siRNA)以足夠濃度自全身投與轉運至CNS中，且轉運至腦細胞內之右區室以誘導顯著靶敲低。 實例9：抗體X親和力調節 The pharmacodynamic activity of monovalent antibody X-siRNA conjugates was tested in hTfR1 KI mice to knock down HPRT mRNA in the CNS after intravenous administration. Four weekly doses of siRNA or antibody-siRNA conjugates were administered intravenously at 4 mg/kg siRNA or 40 mg/kg hIgG molar equivalents, with intravenous anti-CD4 20 mg/kg before the first and third doses to inhibit anti-drug antibodies. Three days after the last dose, mice were sacrificed, brains were harvested, total RNA was isolated, and HPRT mRNA levels relative to ACTB mRNA were measured by droplet digital PCR (ddPCR). Neither siRNA alone nor control IgG-siRNA conjugate treatment resulted in significant changes in HPRT mRNA levels in the brain. However, monovalent Antibody X-siRNA treatment resulted in 60% knockdown of HPRT mRNA ( FIG. 16C ). This suggests that Antibody X can deliver oligonucleotides (including ASOs and siRNAs) into the CNS at sufficient concentrations from systemic administration and to the right compartments within brain cells to induce significant target knockdown. Example 9: Antibody X Affinity Modulation

設計抗體X之親和力變異體。抗體X Fab/TfR1/Tf三元復合物之結構分析鑑別出抗體X互補位處之特定殘基，該等殘基可經修飾以最佳化其對TfR1之親和力( 圖10)。對於降低之親和力設計，選擇藉由用抗體X互補位上之較小側鏈取代來修剪或移除與TfR1之有利接觸之突變位點。此等點突變包括重鏈之S40A、S60A、Y67S、Y110S及R111S (根據SEQ ID NO:100之AHo編號)，以及來自輕鏈之Y40N、Y57S及N69S (根據SEQ ID NO:200之AHo編號)。此等突變消除在cryoEM結構中觀察到的抗體X與TfR1之間之H鍵及凡得瓦(vander waal)相互作用。另一方法用於設計增加抗原結合界面處抗體X之側鏈之體積以降低與TfR1之總體互補性的突變。此等突變由HC: A112Y (根據SEQ ID NO:100之AHo編號)及LC: S39N (根據SEQ ID NO:200之AHo編號)組成。上述策略利用來自NCBI蛋白質資料庫中所有抗體V區之胺基酸頻率，以確保點突變並不罕見。 Design of affinity variants of Antibody X. Structural analysis of the Antibody X Fab/TfR1/Tf ternary complex identified specific residues at the complementary position of Antibody X that could be modified to optimize its affinity for TfR1 ( FIG. 10 ). For reduced affinity design, mutation sites were selected that trimmed or removed favorable contacts with TfR1 by substitution with smaller side chains at the complementary position of Antibody X. These point mutations included S40A, S60A, Y67S, Y110S, and R111S of the heavy chain (according to the AHo numbering of SEQ ID NO: 100), and Y40N, Y57S, and N69S from the light chain (according to the AHo numbering of SEQ ID NO: 200). These mutations abolished the H-bond and van der Waal interactions between Antibody X and TfR1 observed in the cryoEM structure. Another approach was used to design mutations that increased the volume of the side chains of Antibody X at the antigen binding interface to reduce the overall complementarity with TfR1. These mutations consisted of HC: A112Y (according to the AHo numbering of SEQ ID NO: 100) and LC: S39N (according to the AHo numbering of SEQ ID NO: 200). The above strategy utilized the amino acid frequencies of all antibody V regions from the NCBI protein database to ensure that point mutations were not rare.

將抗體X之單點突變體選殖為二價非糖基化hIgG1，在CHO中表現，且藉由如上文所述之標準方法純化。藉由流式細胞術評估對細胞表面人類及石蟹猴TfR1之二價親和力(在4℃下) ( 圖11)。儘管一些突變對相對於抗體X之結合(例如VH-S40A、VH-A112Y)幾乎沒有作用，但對於該組抗體X變異體觀察到寬範圍之表觀親和力(EC 50為約0.5至＞300 nM； 表10)。為定量所選抗體X突變對單價親和力之影響，藉由以下方式將抗體X變異體消化成Fab片段：首先將5 mg/ml抗體與木瓜蛋白酶(Roche 108014；10 ug/mg抗體)在含有20 mM半胱胺酸之緩衝液中在37℃下孵育4小時，用E-64蛋白酶抑制劑淬滅，接著藉由使消化物通過MabSelect SuRe樹脂來移除Fc片段及未消化抗體，且將緩衝液交換為PBS。藉由表面電漿子共振，在Biacore 8K+ (Cytiva)中之抗His捕獲晶片上以約50 RU捕獲帶組胺酸標籤之TfR1 ECD蛋白且以7.8-8000 nM (4倍稀釋系列)注射Fab片段，使用1:1動力學模型(Biacore Insight評估軟體，Cytiva)分析結合反應，來確定對人類及石蟹猴TfR ECD之單價親和力( 圖12)。在抗體X突變體中，自親和力(K D= 26 nM)稍強於親代抗體X (K D= 40nM)之VL-A112Y變異體，至K D為約5600 nM之VH-R101S突變體及觀察到結合但親和力太弱而無法定量之VH-Y110S突變體，觀察到寬範圍之單價親和力(平衡解離常數，K D)及相應解離速率常數(k d) ( 表10)。親和力變異體通常維持親代抗體X之良好人類-石蟹獼猴親和力匹配(＜3倍)，但VH-S60A突變似乎甚至進一步改良此物種交叉反應性。 表10. 二價mAb TfR-CHO 結合 Fab 親和力及動力學 表面電漿子共振 二價mAb胞吞轉送作用 二價mAb TfR降解 抗體/變異體 hu TfR EC 50(nM) cy TfR EC 50(nM) hu TfR k a(/M.s) hu TfR k d(/s) hu TfR K D(nM) cy TfR k a(/M.s) cy TfR k d(/s) cy TfR K D(nM) huTfR MDCKII 倍數/對照 Caco2 TfR1 對照% 抗體X 0.5 1.1 5.2×10 5 2.1×10 -2 40 3.7×10 5 3.7×10 -2 99 2.9 31 VH-S40A 0.8 1.7 4.9×10 5 1.6×10 -2 33 3.3×10 5 3.1×10 -2 93 4.3 29 VH-A112Y 1.0 1.6 2.5×10 5 6.4×10 -3 26 1.4×10 5 8.0×10 -3 59 n.d. 25 VL-N69S 1.4 3.5 4.6×10 5 3.4×10 -2 73 3.4×10 5 6.7×10 -2 200 5.9 31 VH-S60A 1.7 1.1 5.2×10 5 3.4×10 -2 64 3.6×10 5 2.8×10 -2 78 6.3 31 VL-S39N 6.0 23 3.4×10 5 7.9×10 -2 230 2.5×10 5 1.5×10 -1 610 7.8 43 VL-Y57S 13 67 1.7×10 5 1.3×10 -1 790 1.3×10 5 2.5×10 -1 1900 6.5 61 VH-Y67S 16 61 1.8×10 5 2.4×10 -1 1400 1.9×10 5 5.6×10 -1 3000 4.4 59 VL-Y40N 約50 約300 n.d. n.d. n.d. n.d. n.d. n.d. 2.7 64 VH-R111S 約100 約300 1.2×10 5 6.8×10 -1 5600 -- -- ＞8000 1.6 88 VH-Y110S ＞1000 ＞1000 -- -- ＞8000 -- -- ＞8000 1.3 96 Single-site mutants of Antibody X were cloned as bivalent aglycosylated hIgG1, expressed in CHO, and purified by standard methods as described above. Bivalent affinity to cell surface human and cynomolgus monkey TfR1 was assessed by flow cytometry (at 4°C) ( Figure 11 ). Although some mutations had little effect on binding relative to Antibody X (e.g., VH-S40A, VH-A112Y), a wide range of apparent affinities was observed for this panel of Antibody X variants ( EC50 from approximately 0.5 to >300 nM; Table 10 ). To quantify the effect of the selected Antibody X mutations on monovalent affinity, Antibody X variants were digested into Fab fragments by first incubating 5 mg/ml antibody with papain (Roche 108014; 10 ug/mg antibody) in buffer containing 20 mM cysteine at 37°C for 4 hours, quenching with E-64 protease inhibitor, and then removing Fc fragments and undigested antibody by passing the digest through MabSelect SuRe resin and exchanging the buffer to PBS. The monovalent affinity for human and cynomolgus monkey TfR ECD was determined by surface plasmon resonance capture of histidine-tagged TfR1 ECD protein at approximately 50 RU on an anti-His capture chip in a Biacore 8K+ (Cytiva) and injection of Fab fragments at 7.8-8000 nM (4-fold dilution series) and analysis of the binding reaction using a 1:1 kinetic model (Biacore Insight evaluation software, Cytiva) ( FIG. 12 ). Among the antibody X mutants, a wide range of monovalent affinities (equilibrium dissociation constants, KD ) and corresponding dissociation rate constants (kd) were observed (Table 10), from the VL-A112Y variant, which had a slightly stronger affinity (KD = 26 nM) than the parental antibody X ( KD = 40 nM ), to the VH-R101S mutant, for which KD was approximately 5600 nM, and the VH -Y110S mutant, for which binding was observed but the affinity was too weak to be quantified. The affinity variants generally maintained a good human-Cerithidea cynomolgus affinity match (<3-fold) to the parental antibody X, but the VH-S60A mutation appeared to improve this species cross-reactivity even further. Table 10. Bivalent mAb TfR-CHO binding Fab affinity and dynamics surface plasmon resonance Transcytosis of bivalent mAbs Bivalent mAb TfR degradation Antibodies/Variants hu TfR EC 50 (nM) cy TfR EC 50 (nM) hu TfR k a (/Ms) hu TfR k d (/s) hu TfR K D (nM) cy TfR k a (/Ms) cy TfR k d (/s) cy TfR K D (nM) huTfR MDCKII multiple/control Caco2 TfR1 control % Antibody X 0.5 1.1 5.2×10 5 2.1×10 -2 40 3.7×10 5 3.7×10 -2 99 2.9 31 VH-S40A 0.8 1.7 4.9×10 5 1.6×10 -2 33 3.3×10 5 3.1×10 -2 93 4.3 29 VH-A112Y 1.0 1.6 2.5×10 5 6.4×10 -3 26 1.4×10 5 8.0×10 -3 59 nd 25 VL-N69S 1.4 3.5 4.6×10 5 3.4×10 -2 73 3.4×10 5 6.7×10 -2 200 5.9 31 VH-S60A 1.7 1.1 5.2×10 5 3.4×10 -2 64 3.6×10 5 2.8×10 -2 78 6.3 31 VL-S39N 6.0 twenty three 3.4×10 5 7.9×10 -2 230 2.5×10 5 1.5× 10-1 610 7.8 43 VL-Y57S 13 67 1.7×10 5 1.3× 10-1 790 1.3×10 5 2.5× 10-1 1900 6.5 61 VH-Y67S 16 61 1.8×10 5 2.4×10 -1 1400 1.9×10 5 5.6× 10-1 3000 4.4 59 VL-Y40N About 50 About 300 nd nd nd nd nd nd 2.7 64 VH-R111S About 100 About 300 1.2×10 5 6.8× 10-1 5600 -- -- ＞8000 1.6 88 VH-Y110S ＞1000 ＞1000 -- -- ＞8000 -- -- ＞8000 1.3 96

如上文所述，產生抗體X之額外單點突變體(VL-S83A)且測試與人類及石蟹獼猴TfR1之結合。用VL-S83A Fab觀察到之親和力及相應結合動力學(締合速率常數(ka)及解離速率常數(kd))對於人類TfR1而言為K D= 52 nM，ka = 6.6 ×10 5/M /s，且k d= 3.5 ×10 -2/s，且對於石蟹猴TfR1而言為K D= 122 nM，ka = 6.0 ×10 5/M /s，且k d= 7.2 ×10 -2/s。 As described above, an additional single-site mutant of Antibody X (VL-S83A) was generated and tested for binding to human and cynomolgus monkey TfR1. The affinity and corresponding binding kinetics (association rate constant (ka) and dissociation rate constant (kd)) observed with VL-S83A Fab were KD = 52 nM, ka = 6.6 × 105 /M/s, and kd = 3.5 × 10-2 /s for human TfR1, and KD = 122 nM, ka = 6.0 × 105 /M/s, and kd = 7.2 × 10-2 /s for cynomolgus monkey TfR1.

為評估抗體X之親和力調節對細胞抗體轉運之影響，在人類TfR1介導之胞吞轉運作用之馬丁達比犬腎II (MDCK II)細胞模型中測試抗體。在具有IRES-嘌呤黴素抗性盒之人類EF1a啟動子下，用編碼人類TfR1基因之VSV-G假型慢病毒粒子穩定地轉導MDCK II細胞(ECACC 00062107)。選擇嘌呤黴素抗性細胞以產生MDCKII/hTfR細胞株。在流式細胞術中使用抗TfR抗體驗證人類TfR1之表現。To evaluate the effect of affinity modulation of Antibody X on cellular antibody trafficking, the antibodies were tested in the Martin Darby Canine Kidney II (MDCK II) cell model of human TfR1-mediated endocytic trafficking. MDCK II cells were stably transduced with VSV-G pseudotyped lentiviral particles encoding the human TfR1 gene under the human EF1a promoter with an IRES-puromycin resistance cassette (ECACC 00062107). Puromycin-resistant cells were selected to generate the MDCKII/hTfR cell line. The expression of human TfR1 was validated using anti-TfR antibodies in flow cytometry.

將MDCKII/hTfR細胞平鋪至Corning 0.4微米孔transwell插入物上，在完全培養基中每個插入物25,000個細胞，插入物中0.25 ml且底部室中1 ml，且保持在處於37℃及5% CO 2的孵育箱中。在第4天，將底部培養基完全更換，且將一半插入培養基更換為新鮮培養基。在第5天，將測試物品以100 nM添加至頂部孔中。在第7天，自頂部室及底部室收集樣品且分析測試物品之濃度。在中尺度發現檢定系統中使用通常可用之抗人類IgG試劑對測試物品進行定量，自同一測試物品之滴定標準曲線外推出結果。 MDCKII/hTfR cells were plated onto Corning 0.4 micron pore transwell inserts, 25,000 cells per insert in complete medium, 0.25 ml in the insert and 1 ml in the bottom chamber, and kept in an incubator at 37°C and 5% CO2 . On day 4, the bottom medium was completely replaced, and half of the insert medium was replaced with fresh medium. On day 5, the test article was added to the top well at 100 nM. On day 7, samples were collected from the top and bottom chambers and analyzed for the concentration of the test article. The test article was quantified using a commonly available anti-human IgG reagent in a mesoscale discovery assay system, and the results were extrapolated from a titration standard curve of the same test article.

抗體X減弱之親和力變異體相對於親代抗體X，通常顯示改良之細胞轉運( 表10；圖13A-圖13B)，為對照hIgG之最高達約8倍，但對親和力具有鐘形依賴性。在此檢定中，最大細胞胞吞轉送作用之最佳親和力範圍對於細胞表面TfR1之二價結合而言為EC 50約1-10 nM (在4℃下； 圖13A)，或者對於重組hTfR1 ECD而言，單價親和力為K D約100-1000nM (在25℃下； 圖13B)。較弱親和力之變異體(例如，VH-Y67S及VH-R111S)顯示減少之胞吞轉送作用，此可能歸因於hTfR1在此檢定中所測試之濃度(100 nM)下之不完全接合，但當血液中之抗體濃度較高時，仍可在活體內BBB處轉運。 Antibody X reduced affinity variants generally showed improved cellular transport relative to the parental antibody X ( Table 10; Figures 13A-13B ), up to about 8-fold over control hIgG, but with a bell-shaped dependence on affinity. In this assay, the optimal affinity range for maximal cellular endocytosis was an EC50 of about 1-10 nM for bivalent binding to cell surface TfR1 (at 4°C; Figure 13A ), or a monovalent affinity of KD of about 100-1000 nM for recombinant hTfR1 ECD (at 25°C; Figure 13B ). Variants with weaker affinity (e.g., VH-Y67S and VH-R111S) showed reduced endocytic trafficking, likely due to incomplete binding of hTfR1 at the concentrations tested in this assay (100 nM), but were still transported across the BBB in vivo when antibody concentrations in the blood were higher.

為研究親和力去成熟(affinity dematuration)對活體內生物分佈之影響，向huTfR1 KI小鼠靜脈內投與20 mg/kg (或莫耳IgG當量)劑量之抗體X變異體或對照hIgG。如上文所述，在1天或7天後收穫全血、血清及經灌注之腦半球。藉由MSD免疫檢定來定量血清及腦溶解產物中之總hIgG，且記錄每隻小鼠之全血球計數。To investigate the effect of affinity dematuration on in vivo biodistribution, huTfR1 KI mice were intravenously administered 20 mg/kg (or molar IgG equivalent) of antibody X variant or control hIgG. Whole blood, serum, and perfused brain hemispheres were harvested 1 or 7 days later as described above. Total hIgG in serum and brain lysates was quantified by MSD immunoassay, and complete blood counts were recorded for each mouse.

在靜脈內投與後一天，將抗體X之二價及單價形式之生物分佈(K D= 40 nM)與二價抗體X親和力變異體VL-S39N (K D= 230 nM)、VL-Y57S (K D= 790 nM)及VH-Y67S (K D= 1400 nM)進行比較。單價抗體X在血清中之水準最低，但在腦中之水準最高，且網狀紅血球損失最小( 圖14)。二價抗體X具有比單價抗體X更高之血清水準，且對於對hTfR1具有較弱親和力之變異體，血清水準進一步改良。然而，所有二價抗體之腦水準皆為單價抗體X之大約一半，與親和力無關。存在對hTfR1之親和力越弱而網狀紅血球耗竭越不嚴重之趨勢。 One day after intravenous administration, the biodistribution of the bivalent and monovalent forms of Antibody X ( K = 40 nM) was compared to the bivalent Antibody X affinity variants VL-S39N ( K = 230 nM), VL-Y57S ( K = 790 nM), and VH-Y67S ( K = 1400 nM). Monovalent Antibody X had the lowest levels in serum, but the highest levels in the brain, with minimal reticulocyte loss ( Figure 14 ). Bivalent Antibody X had higher serum levels than monovalent Antibody X, and serum levels were further improved for variants with weaker affinity for hTfR1. However, brain levels for all bivalent antibodies were approximately half of those for monovalent Antibody X, regardless of affinity. There was a trend that the weaker the affinity for hTfR1, the less severe the reticulocyte depletion.

在靜脈內投與後7天，對TfR之親和力減弱對抗體在血清及腦中之持續性有明顯作用，同時亦將對網狀紅血球之影響降至最低( 圖14)。按親和力等級次序，二價抗體X在血清中之水準比對照hIgG低約1000倍，但VL-S39N、VL-Y57S、VH-Y67S及VH-R111S之血清水準皆高得多。在血清中，對於TfR1具有約5600 nM之單價K D之二價VH-R111S變異體僅比對照hIgG低約2倍。在靜脈內投與後7天，三種親和力最弱之變異體在腦溶解產物中之水準亦升高，比對照hIgG高4-6倍，證實減弱之對TfR之親和力在持續腦暴露中之價值。此外，親和力最弱之變異體VH-R111S在此7天時間點亦對網狀紅血球計數無顯著影響，儘管其仍存在於血清中且在TfR1介導之腦攝取中起作用。 Seven days after intravenous administration, the reduced affinity for TfR had a significant effect on the persistence of the antibody in serum and brain, while also minimizing the effect on reticulocytes ( Figure 14 ). In order of affinity, the level of bivalent antibody X in serum was about 1000-fold lower than that of control hIgG, but the serum levels of VL-S39N, VL-Y57S, VH-Y67S, and VH-R111S were all much higher. In serum, the bivalent VH-R111S variant, which had a monovalent KD of about 5600 nM for TfR1, was only about 2-fold lower than that of control hIgG. At 7 days after intravenous administration, the levels of the three weakest affinity variants in brain lysates were also elevated, 4-6 times higher than control hIgG, confirming the value of reduced affinity for TfR in sustained brain exposure. In addition, the weakest affinity variant, VH-R111S, also had no significant effect on reticulocyte counts at this 7-day time point, although it was still present in serum and played a role in TfR1-mediated brain uptake.

理解抗TfR抗體對TfR1之細胞運輸及表現穩定性之作用對於估量TfR1介導之對治療劑之腦遞送的安全性及持久性至关重要。為了定量活體外細胞系統中之此等效應，將Caco2細胞(ECACC 86010202)以30,000個細胞/孔接種於12孔板中，培養4天，接著與1000 nM之抗體X或對照hIgG孵育24小時。抗體處理後，將細胞在含有磷酸酶及蛋白酶抑制劑(PhosSTOP及cOmplete；Sigma)之RIPA緩衝液(Cell Signaling Technology 9806S)中在冰上溶解30分鐘，以13,000 rpm離心15分鐘，且收集上清液並藉由BCA (Pierce 23227)分析總蛋白。接著將溶解產物以100 ug/mL蛋白質作正規化，且藉由毛細管西方墨點(Jess, ProteinSimple)分析，利用化學發光二級抗體探測TfR1 (H68.4; Thermo 13 -6800)及β肌動蛋白(MAB8929; R&D Systems)。Understanding the effects of anti-TfR antibodies on the cellular trafficking and expression stability of TfR1 is critical to assessing the safety and durability of TfR1-mediated brain delivery of therapeutics. To quantify these effects in an in vitro cell system, Caco2 cells (ECACC 86010202) were seeded at 30,000 cells/well in 12-well plates, cultured for 4 days, and then incubated with 1000 nM Antibody X or control hIgG for 24 hours. After antibody treatment, cells were lysed on ice for 30 min in RIPA buffer (Cell Signaling Technology 9806S) containing phosphatase and protease inhibitors (PhosSTOP and cOmplete; Sigma), centrifuged at 13,000 rpm for 15 min, and supernatants were collected and analyzed for total protein by BCA (Pierce 23227). Lysates were then normalized to 100 ug/mL protein and analyzed by capillary Western blot (Jess, ProteinSimple) using chemiluminescent secondary antibodies to probe TfR1 (H68.4; Thermo 13-6800) and β-actin (MAB8929; R&D Systems).

與用對照hIgG處理相比，抗體X (二價Fab-Fc)處理導致細胞溶解產物中顯著更少之TfR1 ( 圖15A)。在處理期間藉由納入ATP酶抑制劑巴弗洛黴素(100 nM)完全逆轉TfR1之此損失，表明抗體X導致TfR1之溶酶體運輸及降解。為理解抗體型式如何影響TfR1降解，亦測試用單價Fab-Fc、二價Fc-Fab及單價Fc-Fab抗體X型式之細胞處理之TfR1水準，且與二價Fab-Fc型式之抗體X之作用進行比較。與二價抗體型式相比，單價抗體處理導致細胞溶解產物中保留的TfR1水準高得多。藉由二價抗體引起之溶酶體運輸增加及TfR1損失可歸因於受體交聯或歸因於親合力之親和力增加。為檢查經由誘變降低親和力是否可將抗體誘導之TfR1損失降至最低，針對細胞TfR1降解測試抗體X親和力變異體(呈二價型式)。實際上，由抗體X親和力變異體引起之TfR1降解程度與細胞表面TfR1結合之EC 50密切相關( 表10；圖15B)。此外，抗體X之型式變異體及親和力變異體似乎皆遵循TfR1降解與細胞TfR1結合親和力之相同相關性(R2 = 0.89)，表明細胞表面TfR結合為溶酶體運輸之充分預測因子，與化合價無關。 Treatment with Antibody X (bivalent Fab-Fc) resulted in significantly less TfR1 in the cell lysate compared to treatment with control hIgG ( FIG. 15A ). This loss of TfR1 was completely reversed by inclusion of the ATPase inhibitor bafilomycin (100 nM) during treatment, indicating that Antibody X results in lysosomal trafficking and degradation of TfR1. To understand how antibody format affects TfR1 degradation, TfR1 levels in cells treated with monovalent Fab-Fc, bivalent Fc-Fab, and monovalent Fc-Fab Antibody X formats were also tested and compared to the effect of the bivalent Fab-Fc format of Antibody X. Monovalent antibody treatment resulted in much higher levels of TfR1 remaining in the cell lysate compared to the bivalent antibody format. Increased lysosomal trafficking and loss of TfR1 by the bivalent antibody can be attributed to receptor cross-linking or to increased affinity due to avidity. To examine whether lowering affinity by induction could minimize antibody-induced TfR1 loss, the antibody X affinity variant (in bivalent form) was tested for cellular TfR1 degradation. Indeed, the extent of TfR1 degradation caused by the antibody X affinity variant correlated closely with the EC50 of cell surface TfR1 binding ( Table 10; Figure 15B ). Furthermore, both format variants and affinity variants of Antibody X appear to follow the same correlation of TfR1 degradation and cellular TfR1 binding affinity (R2 = 0.89), indicating that cell surface TfR binding is a sufficient predictor of lysosomal trafficking, independent of valency.

在活體內，誘導TfR1降解可對抗體跨BBB之持續轉運產生影響，但亦對細胞功能及存活產生影響。網狀紅血球耗竭為全身投與抗TfR1抗體之已知結果，似乎即使在具有最小Fc效應子功能之情況下亦會發生，但取決於TfR1親和力( 圖14)。將所選抗體X變異體之活體內網狀紅血球損失與活體外誘導之TfR1降解進行比較( 圖15C)表明，可能存在支持TfR1降解作為網狀紅血球耗竭機制之相關性，且可使用單價變異體或親和力降低之變異體，而無顯著有害作用。 實例10：藉由組合誘變之抗體X親和力細化 In vivo , inducing TfR1 degradation can have effects on sustained antibody transport across the BBB, but also on cell function and survival. Reticulocyte depletion is a known consequence of systemic administration of anti-TfR1 antibodies and appears to occur even with minimal Fc effector function, but depends on TfR1 affinity ( Figure 14 ). Comparison of in vivo reticulocyte loss of selected Antibody X variants with in vitro induced TfR1 degradation ( Figure 15C ) suggests that there may be a correlation supporting TfR1 degradation as a mechanism of reticulocyte depletion, and that monovalent variants or variants with reduced affinity can be used without significant deleterious effects. Example 10: Refinement of Antibody X Affinity by Combinatorial Mutation

鑑於親和力調節對抗TfR抗體X之功能性質之影響，設計第二組變異體用於細化結合性質。在第一輪抗體X誘變之指導下，第二組併入了VH-S60A突變(其導致對人類及石蟹猴TfR1之密切匹配之親和力(分別為K D= 64 nM及78 nM))與VL-S39N、VL-Y57S、VH-Y67S及VH-R111S突變(其在K D為約200-6000 nM範圍內減弱對人類TfR1之親和力)之組合。此等雙突變體作為二價及單價(Fab片段)抗體產生。藉由流式細胞術測試二價抗體與表現人類或石蟹猴TfR1之CHO細胞之結合，而藉由表面電漿子共振測試Fab片段結合人類或石蟹猴TfR1 ECD之親和力及動力學，如先前實例中所述。結果示於 表11中。 Given the impact of affinity modulation on the functional properties of anti-TfR antibody X, a second set of variants was designed to refine the binding properties. Guided by the first round of mutagenesis of antibody X, the second set incorporated a combination of the VH-S60A mutation, which resulted in closely matched affinities for human and cynomolgus monkey TfR1 ( K = 64 nM and 78 nM, respectively), and the VL-S39N, VL-Y57S, VH-Y67S, and VH-R111S mutations, which reduced affinity for human TfR1 within the K range of approximately 200-6000 nM. These double mutant variants were generated as bivalent and monovalent (Fab fragment) antibodies. The binding of the bivalent antibodies to CHO cells expressing human or cynomolgus monkey TfR1 was tested by flow cytometry, while the affinity and kinetics of the Fab fragments binding to human or cynomolgus monkey TfR1 ECD were tested by surface plasmon resonance as described in the previous examples. The results are shown in Table 11 .

所有四種雙突變體二價抗體之細胞表面結合EC 50在人類與石蟹猴TfR1之間的差異皆小於2倍，範圍為約50 nM至約250 nM (在4℃下)。對於VH-S60A / VL-S39N及VH-S60A / VL-Y57S雙突變體，Fab親和力(藉由SPR在25℃下之K D)對於人類及石蟹猴TfR1胞外域而言亦極密切匹配。然而，不能準確量測VH-S60A/Y67S及VH-S60A/R111S雙突變體Fab對兩種TfR1 ECD之親和力，此乃因在檢定中測試之最高濃度(6000 nM，由於非特異性結合之限制)下，結合似乎遠低於飽和。如本實例中所述評估額外雙突變體二價抗體(VH-S60A/S61A)。此雙突變體導致人類親和力弱於石蟹猴親和力：對於VH-S60A/S61A Fab而言，K D= 235 nM (hu)及127 nM (cy)，而對於VH-S60A Fab而言，K D= 64 nM (hu)及78 nM (cy)。 The cell surface binding EC50s of all four bivalent mutant antibodies differed less than 2-fold between human and cynomolgus monkey TfR1, ranging from about 50 nM to about 250 nM (at 4°C). For the VH-S60A/VL-S39N and VH-S60A/VL-Y57S bivalent mutants, the Fab affinities ( KD by SPR at 25°C) were also very closely matched for the human and cynomolgus monkey TfR1 ectodomains. However, the affinity of the VH-S60A/Y67S and VH-S60A/R111S double mutant Fabs to both TfR1 ECDs could not be accurately measured, as binding appeared to be well below saturation at the highest concentration tested in the assay (6000 nM, due to limitations of nonspecific binding). An additional double mutant bivalent antibody (VH-S60A/S61A) was evaluated as described in this Example. This double mutant resulted in human affinities that were weaker than the cynomolgus monkey affinity: K = 235 nM (hu) and 127 nM (cy) for VH-S60A/S61A Fab, and K = 64 nM (hu) and 78 nM (cy) for VH-S60A Fab.

總之，應用用於匹配物種之間親和力之VH-S60A突變與改變親和力而不調節物種交叉反應性之額外突變的組合，以產生相對於單點突變體抗體X變異體具有改良性質之此組細化的減弱親和力抗體。 表11. 抗體X雙點突變變異體與人類及石蟹猴TfR1之結合. 二價mAb TfR-CHO 結合 單價Fab TfR1 ECD 親和力 抗體變異體 hu TfR EC 50(nM) cy TfR EC 50(nM) hu TfR K D(nM) cy TfR K D(nM) VH-S60A (SEQ ID NO: 102) / VL-S39N (SEQ ID NO: 201) 48 82 550 730 VH-S60A (SEQ ID NO: 102) / VL-Y57S (SEQ ID NO: 203) 86 130 約1300 約1500 VH-S60A/Y67S (SEQ ID NO: 107) 120 150 約3000 n.d. VH-S60A/R111S (SEQ ID NO: 108) 170 約250 n.d. n.d. 實例11：抗體X之親和力變異體在雙特異性型式下具有改良之藥物動力學及網狀紅血球耗竭效應 In summary, the VH-S60A mutation for matching interspecies affinity was applied in combination with additional mutations that altered affinity without modulating species cross-reactivity to generate this organized reduced affinity antibody with improved properties relative to the single point mutant Antibody X variant. Table 11. Binding of Antibody X double point mutation variants to human and cynomolgus monkey TfR1. Bivalent mAb TfR-CHO binding Monovalent Fab TfR1 ECD affinity Antibody variants hu TfR EC 50 (nM) cy TfR EC 50 (nM) hu TfR K D (nM) cy TfR K D (nM) VH-S60A (SEQ ID NO: 102) / VL-S39N (SEQ ID NO: 201) 48 82 550 730 VH-S60A (SEQ ID NO: 102) / VL-Y57S (SEQ ID NO: 203) 86 130 About 1300 About 1500 VH-S60A/Y67S (SEQ ID NO: 107) 120 150 About 3000 nd VH-S60A/R111S (SEQ ID NO: 108) 170 About 250 nd nd Example 11: Affinity variants of Antibody X in bispecific form with improved pharmacokinetic and reticulocyte depletion effects

在短期生物分佈研究中，與使用二價抗體X之相應應用(甚至在寬的二價親和力範圍內)相比，當呈單價型式(作為單特異性抗體( 圖14)、併入雙特異性抗體中( 圖8)或作為寡核苷酸結合物( 圖9))時，抗體X顯示出優異的腦暴露。為檢查單價TfR1靶向是否可進一步最佳化抗TfR抗體介導之向CNS之遞送及所得腦暴露，將單價型式之抗體X之親和力變異體併入雙特異性抗體中。將治療性抗體Z與抗體X變異體之Fab片段組合作為2:1雙特異性抗體(二價抗體Z:單價抗體X；參見圖18A)。設計此雙特異性抗體之四種形式(全部併入二價抗體Z)，其中每一抗體X變異體之Fab片段皆遺傳融合(經由2xG4S多肽鏈)至非靶向二價抗體Z之一條重鏈之C末端(使用額外恆定域突變以確保適當輕鏈配對及Fc異二聚化)。將此等抗體以3 mg/kg莫耳IgG當量在hTfR KI小鼠中靜脈內投與，以研究以次飽和劑量單價靶向hTfR1時親和力與生物分佈之間的關係。另外，由於此等雙特異性抗體經設計具有完全效應子功能(糖基化hIgG1 Fc)，因此密切監測對網狀紅血球動力學之影響。針對每種抗體分析兩個小鼠终末群組(cohort)，一個群組在靜脈內投與後一天處死且另一個群組在靜脈內投與後七天處死，且在心臟灌注後，收集腦樣品，溶解並分析hIgG攝取。 In short-term biodistribution studies, Antibody X showed superior brain exposure when in monovalent format (as a monospecific antibody ( FIG. 14 ), incorporated into a bispecific antibody ( FIG. 8 ), or as an oligonucleotide conjugate ( FIG. 9 ) compared to corresponding applications using the bivalent Antibody X, even across a wide range of bivalent affinities. To examine whether monovalent TfR1 targeting could further optimize anti-TfR antibody-mediated delivery to the CNS and resulting brain exposure, affinity variants of Antibody X were incorporated into the bispecific antibody in monovalent format. Therapeutic Antibody Z was combined with Fab fragments of Antibody X variants as a 2:1 bispecific antibody (bivalent Antibody Z:monovalent Antibody X; see FIG. 18A ). Four versions of this bispecific antibody were designed (all incorporated into bivalent antibody Z) in which the Fab fragment of each antibody X variant was genetically fused (via 2xG4S polypeptide chains) to the C-terminus of one heavy chain of the non-targeting bivalent antibody Z (using additional homeodomain mutations to ensure proper light chain pairing and Fc heterodimerization). These antibodies were administered intravenously in hTfR KI mice at 3 mg/kg molar IgG equivalents to investigate the relationship between affinity and biodistribution when targeting hTfR1 monovalently at subsaturating doses. In addition, because these bispecific antibodies were designed with full effector function (glycosylated hIgG1 Fc), the effects on reticulocyte kinematics were closely monitored. Two terminal cohorts of mice were analyzed for each antibody, one cohort was sacrificed one day after iv administration and the other cohort was sacrificed seven days after iv administration, and after cardiac perfusion, brain samples were collected, lysed and analyzed for hIgG uptake.

併入抗體X變異體Fab片段之雙特異性抗體之血清藥物動力學高度依賴於對hTfR1之親和力。清除率與TfR1親和力成比例增加，且對於最弱雙特異性變異體(VL-Y57S)而言，幾乎等效於單特異性hIgG1對照之清除率( 圖18B；表12)。此等雙特異性抗體之腦暴露遵循對TfR1親和力之更複雜依賴性。在靜脈內投與後一天評估之短期腦暴露隨著對TfR1之親和力降低而降低，而對於對TfR1具有中等親和力之VH-S60A/VL-S39N變異體而言，在靜脈內投與後一周評估之長期腦暴露最高( 圖18C)。為估計總體腦暴露，計算血清暴露之曲線下面積(AUC)且乘以第1天及第7天之平均腦:血清比，得到各雙特異性抗體變異體之近似腦AUC值( 表12)。最後，不同雙特異性抗體變異體對循環網狀紅血球具有差異影響，其中相對於較弱親和力變異體或不結合TfR1之hIgG對照抗體，較高親和力變異體在第1天顯示更多網狀紅血球耗竭且在第7天顯示更高回升(網狀紅血球升高) ( 圖18D)。此分析支持以下觀點：抗體X變異體具有可取決於藥理學應用來利用之不同腦暴露概況，且變異體之選擇亦可幫助減輕此等抗體對血液學之影響。 表12. 併入單價抗體X變異體之2:1雙特異性抗體之生物分佈及網狀紅血球耗竭性質. 2:1 bsAb 變異體 K D(nM) huTfR K D(nM) cyTfR 血清CL (mL/天/kg) 腦AUC (nM*天) 網狀紅血球耗竭(24h) hIgG對照 - - 15.1 9 0 VH-S60A 69 80 70.5 32 -60% VL-S39N 232 611 46.5 30 -43% VH-S60A/VL-S39N 571 592 21.4 46 -20% VL-Y57S 787 1910 16.2 29 -3% 其他實施例 The serum pharmacokinetics of bispecific antibodies incorporating the antibody X variant Fab fragment were highly dependent on affinity for hTfR1. Clearance increased proportionally with TfR1 affinity and was nearly equivalent to that of the monospecific hIgG1 control for the weakest bispecific variant (VL-Y57S) ( Figure 18B; Table 12 ). Brain exposure of these bispecific antibodies followed a more complex dependence on TfR1 affinity. Short-term brain exposure assessed one day after intravenous administration decreased with decreasing affinity for TfR1, while long-term brain exposure assessed one week after intravenous administration was highest for the VH-S60A/VL-S39N variant with intermediate affinity for TfR1 ( Figure 18C ). To estimate overall brain exposure, the area under the curve (AUC) of serum exposure was calculated and multiplied by the average brain: serum ratio on days 1 and 7 to obtain the approximate brain AUC value for each bispecific antibody variant ( Table 12 ). Finally, different bispecific antibody variants had differential effects on circulating reticulocytes, with higher affinity variants showing more reticulocyte depletion at day 1 and greater recovery (elevated reticulocytes) at day 7 relative to weaker affinity variants or a hIgG control antibody that does not bind TfR1 ( Figure 18D ). This analysis supports the notion that Antibody X variants have different brain exposure profiles that can be exploited depending on the pharmacological application, and that the choice of variant can also help mitigate the hematological effects of these antibodies. Table 12. Biodistribution and reticulocyte depletion properties of 2:1 bispecific antibodies incorporating monovalent Antibody X variants. 2:1 bsAb variant K D (nM) huTfR K D (nM) cyTfR Serum CL (mL/day/kg) Brain AUC (nM*day) Reticulocyte depletion (24h) hIgG control - - 15.1 9 0 VH-S60A 69 80 70.5 32 -60% VL-S39N 232 611 46.5 30 -43% VH-S60A/VL-S39N 571 592 21.4 46 -20% VL-Y57S 787 1910 16.2 29 -3% Other embodiments

儘管已結合本發明之詳細描述描述了本發明，但前述描述意欲說明而非限制本發明之範圍，本發明之範圍由所附發明申請專利範圍之範疇界定。其他態樣、優點及修改在所附發明申請專利範圍之範疇內。Although the present invention has been described in conjunction with the detailed description of the present invention, the foregoing description is intended to illustrate rather than limit the scope of the present invention, which is defined by the scope of the appended invention patents. Other aspects, advantages and modifications are within the scope of the appended invention patents.

圖1A為描繪藉由表面電漿子共振量測之抗體X及人類運鐵蛋白受體1胞外域(ECD)之Fab結合反應(共振單位，RU)的圖。 圖1B為描繪藉由表面電漿子共振量測之抗體X及石蟹猴運鐵蛋白受體1胞外域之Fab結合反應(共振單位，RU)的圖。 圖1C為描繪表現人類TfR1之CHO細胞在冰上孵育1 h後藉由流式細胞術量測的所指示之抗體X型式的結合分數(正規化之平均螢光強度，MFI)的圖。 圖2A為描繪單獨或與全人類運鐵蛋白(holoTf)組合之抗體X與CHO細胞上表現之hTfR1之結合抗體(MFI)的圖。 圖2B為描繪抗體X、運鐵蛋白阻斷抗體(抗體1)或holo-Tf對來自hTfR1 CHO細胞之經AlexaFluor647 (AF647)標記之人類Tf之替換(displacement)的圖。 圖3A為在3.6 Å解析度下內置於低溫EM密度(cryo-EM density)中的在內源配位體運鐵蛋白存在下結合至人類運鐵蛋白受體之抗體X Fab的原子模型。 圖3B為石蟹猴TfR及人類TfR之TfR1頂端及蛋白酶樣域序列之部分比對，其中如在 圖3A之低溫EM結構中所觀察到的在抗體X之4 Å內之殘基以灰色突出顯示且抗體X抗原決定基內之保守R364K突變(hu＞石蟹猴TfR1)以粗體顯示。嚴格保守殘基由星號表示，強保守殘基由冒號表示，且中等保守殘基由句點表示。序列來源：人類(智人( Homo sapiens)，Uniprot P02786.2)；石蟹猴(石蟹獼猴( Macaca fascicularis)，NCBI參考序列：XP_045243212.1)。 圖4A為描繪在pH 7.4及pH 5.5下量測之單價Fab-Fc型式之抗體X及hTfR1 ECD之Fab結合反應(共振單位，RU；y軸)的圖。 圖4B為描繪所指示抗體型式之抗體X之Caco2胞吞轉送作用的圖。 圖5A為描繪在hTfR KI小鼠中靜脈內投與20 mg/kg抗體X (正方形)或對照hIgG (圓形)後在所指示時間之血清hIgG濃度(nM)的圖。 圖5B為描繪在hTfR KI小鼠中靜脈內投與20 mg/kg抗體X (正方形)或對照hIgG (圓形)後在所指示時間之腦hIgG濃度(nM)的圖。 圖5C為來自投與抗體X或對照hIgG後三天之hTfR KI小鼠之腦樣品的hIgG免疫組織化學影像。 圖5D為來自投與抗體X之hTfR KI小鼠之大腦皮質中hIgG之免疫組織化學染色的影像。 圖6A為描繪在用靜脈內20 mg/kg hIgG或抗體X處理後48 h之石蟹猴腦生檢切片(biopsy)中之hIgG水準的圖。 圖6B為石蟹猴腦生檢切片之影像，顯示在靜脈內投與20 mg/kg陰性對照或抗體X後48 h，藉由免疫組織化學對海馬迴、小腦及大腦皮質之hIgG染色。 圖6C為在投與抗體X後石蟹猴腦生檢切片之影像，顯示大腦皮質之錐體神經元內之囊泡。 圖7為描繪對照hIgG (左)、抗BACE1 (左起第二個)、抗BACE1/二價抗體X (左起第三個)及抗BACE1/單價抗體X (右)的一系列草圖。 圖8A為描繪靜脈內投與50 mg/kg莫耳IgG當量之hIgG (正方形)、抗BACE (圓形)、抗BACE1/二價抗體X (三角形，指向下方)或抗BACE1/單價抗體X (三角形，指向上方)之hTfR KI小鼠中在所指示時間點之血漿hIgG濃度(nM)的圖。 圖8B為描繪靜脈內投與50 mg/kg莫耳IgG當量之hIgG (正方形)、抗BACE (圓形)、抗BACE1/二價抗體X (三角形，指向下方)或抗BACE1/單價抗體X (三角形，指向上方)之hTfR KI小鼠中在所指示時間點之腦hIgG濃度(nM)的圖。 圖8C為描繪靜脈內投與50 mg/kg莫耳IgG當量之hIgG (正方形)、抗BACE (圓形)、抗BACE1/二價抗體X (三角形，指向下方)或抗BACE1/單價抗體X (三角形，指向上方)之hTfR KI小鼠中在所指示時間點之腦澱粉樣蛋白β1-40 (Aβ40；對照%)的圖。 圖9A為描繪投與所指示構築體(參見 圖9E中之圖例)後24 h之hTfR KI小鼠之血清中總hIgG濃度(nM)的圖。 圖9B為描繪投與所指示構築體(參見 圖9E中之圖例)後24 h之hTfR KI小鼠之脑中之總hIgG濃度(nM)的圖。 圖9C為描繪投與所指示構築體(參見 圖9E中之圖例)後24 h之hTfR KI小鼠之血清中游离ASO濃度(nM)的圖。 圖9D為描繪投與每週四劑量1.5 mg/kg ASO當量之所指示構築體(參見 圖9E中之圖例)後三天的hTfR KI小鼠之腦中游離ASO濃度(nM)的圖。 圖9E為描繪投與每週四劑量1.5 mg/kg ASO當量之所指示構築體後三天的hTfR KI小鼠之腦、脊髓、脛骨前肌及肝臟中相對於Actb作正規化之Malat1 RNA (相對於DPBS媒劑小鼠之百分比)的圖。 圖10為使用抗體X Fab/TfR1/Tf三元復合物( 圖3A中所示)之低溫電子顯微術密度構建之抗體X可變域的原子模型。 圖11為描繪所指示抗體X突變體與表現人類(hu)或石蟹猴(cy) TfR1之CHO細胞之抗體結合(MFI)的圖。 圖12為描繪藉由表面電漿子共振在pH 7.4及pH 5.5下量測之抗體X變異體之Fab片段與hTfR1 ECD之Fab結合反應(共振單位，RU；y軸)的圖。 圖13A為描繪二價抗體X及抗體X突變體或對照hIgG之胞吞轉送作用的圖。 圖13B為描繪針對hTfR1之單價親和力作圖的抗體X突變體之二價胞吞轉送作用的圖。 圖14為描繪在hTfR KI小鼠中以20 mg/kg IgG當量投與所指示構築體后1天(左行)或7天(右行)時血清(頂列)或腦(中間列)中之hIgG濃度(nM)及全血(底列)中之網狀紅血球百分比的一系列圖。 圖15A為在用1 uM所指示抗體處理後之Caco2細胞溶解產物中hTfR1之毛細管西方墨點(Western blot)的影像。Baf：巴弗洛黴素(bafilomycin)。 圖15B為描繪針對各抗體之hTfR1-CHO細胞結合之EC 50作圖的用1 uM所指示構築體處理24 h之Caco2細胞中之TfR1水準(對照%)的圖。 圖15C為描繪針對用1 uM所指示構築體(參見圖15B中之圖例)處理24 h後之Caco2中之TfR1水準作圖的靜脈內投與20 mg/kg此等抗體後24 h之hTfR KI小鼠中之網狀紅血球水準(對照%)的圖。 圖16A為描繪用各種濃度之單獨siRNA或所指示結合物處理72 h後之HEK293T細胞中總HPRT mRNA (%)的圖。 圖16B為描繪在有及無siRNA之情況下單價抗體X或對照hIgG之胞吞轉送作用的圖。 圖16C為描繪在以4 mg/kg siRNA當量投與每週四劑量siRNA或siRNA結合物後三天的hTfR KI小鼠之腦中相對於Actb作正規化的HPRT RNA (相對於DPBS媒劑小鼠之百分比)的圖。 圖17A為描繪在投與hIgG對照(白色圓形)或單價抗體X (黑色圓形)後1天之hTfR KI小鼠中在所指示劑量(3、10、30及90 mg/kg靜脈內莫耳IgG當量)下之血清中hIgG濃度(nM)的圖。 圖17B為描繪在投與hIgG對照(白色圓形)或單價抗體X (黑色圓形)後1天之hTfR KI小鼠中在所指示劑量(3、10、30及90 mg/kg靜脈內莫耳IgG當量)下之腦hIgG濃度(nM)的圖。 圖17C為描繪在投與hIgG對照或單價抗體X後1天之hTFR KI小鼠中在所指示劑量(3、10、30及90 mg/kg靜脈內莫耳IgG當量)下表示為相較於對照之倍數之腦hIgG濃度(nM)的圖。 圖18A為描繪對照hIgG (左)及包含抗體Z (二價)/抗體X (單價)之雙特異性抗體(右)的一系列草圖。 圖18B為描繪靜脈內投與3 mg/kg莫耳IgG當量之hIgG對照(正方形)或四種併入抗體X變異體Fab片段之雙特異性抗體(二價抗體Z/單價抗體X (VH-S60A) (三角形，指向上方)、二價抗體Z/單價抗體X (VL-S39N) (三角形，指向下方)、二價抗體Z/單價抗體X (VH-S60A/VL-S39N) (菱形)或二價抗體Z/單價抗體X (VL-Y57S) (圓形))之hTfR KI小鼠中在所指示時間點之血清hIgG濃度(nM)的圖。 圖18C為描繪靜脈內投與3 mg/kg莫耳IgG當量之hIgG對照(正方形)或四種併入抗體X變異體Fab片段之雙特異性抗體(二價抗體Z/單價抗體X (VH-S60A) (三角形，指向上方)、二價抗體Z/單價抗體X (VL-S39N) (三角形，指向下方)、二價抗體Z/單價抗體X (VH-S60A/VL-S39N) (菱形)或二價抗體Z/單價抗體X (VL-Y57S) (圓形))之hTfR KI小鼠中在所指示時間點之腦hIgG濃度(nM)的圖。 圖18D為描繪靜脈內投與3 mg/kg莫耳IgG當量之hIgG對照(正方形)或四種併入抗體X變異體Fab片段之雙特異性抗體(二價抗體Z/單價抗體X (VH-S60A) (三角形，指向上方)、二價抗體Z/單價抗體X (VL-S39N) (三角形，指向下方)、二價抗體Z/單價抗體X (VH-S60A/VL-S39N) (菱形)或二價抗體Z/單價抗體X (VL-Y57S) (圓形))之hTfR KI小鼠中在所指示時間點之網狀紅血球水準(對照%)的圖。 Figure 1A is a graph depicting the Fab binding reaction of Antibody X and human transferrin receptor 1 extracellular domain (ECD) measured by surface plasmon resonance (resonance units, RU). Figure 1B is a graph depicting the Fab binding reaction of Antibody X and cynomolgus monkey transferrin receptor 1 extracellular domain (resonance units, RU) measured by surface plasmon resonance. Figure 1C is a graph depicting the binding fraction (normalized mean fluorescence intensity, MFI) of the indicated Antibody X versions measured by flow cytometry after incubation of CHO cells expressing human TfR1 for 1 h on ice. Figure 2A is a graph depicting the binding antibody (MFI) of Antibody X alone or in combination with holohuman transferrin (holoTf) to hTfR1 expressed on CHO cells. Figure 2B is a diagram depicting the displacement of AlexaFluor647 (AF647) labeled human Tf from hTfR1 CHO cells by Antibody X, ferritin blocking antibody (Antibody 1), or holo-Tf. Figure 3A is an atomic model of Antibody X Fab bound to the human ferritin receptor in the presence of the endogenous ligand ferritin embedded in cryo-EM density at 3.6 Å resolution. FIG3B is a partial alignment of the TfR1 top and protease-like domain sequences of cynomolgus monkey TfR and human TfR, wherein residues within 4 Å of Antibody X as observed in the cryoEM structure of FIG3A are highlighted in gray and the conserved R364K mutation within the antigenic determinant of Antibody X (hu>cynomolgus monkey TfR1) is shown in bold. Strictly conserved residues are indicated by asterisks, strongly conserved residues by colons, and moderately conserved residues by periods. Sequence sources: human ( Homo sapiens , Uniprot P02786.2); cynomolgus monkey ( Macaca fascicularis , NCBI reference sequence: XP_045243212.1). Figure 4A is a graph depicting the Fab binding reaction (resonance units, RU; y-axis) of monovalent Fab-Fc formats of Antibody X and hTfR1 ECD measured at pH 7.4 and pH 5.5. Figure 4B is a graph depicting Caco2 endocytosis of Antibody X of the indicated antibody formats. Figure 5A is a graph depicting serum hIgG concentrations (nM) at the indicated times following intravenous administration of 20 mg/kg Antibody X (squares) or control hIgG (circles) in hTfR KI mice. Figure 5B is a graph depicting brain hIgG concentrations (nM) at the indicated times following intravenous administration of 20 mg/kg Antibody X (squares) or control hIgG (circles) in hTfR KI mice. FIG. 5C is an image of hIgG immunohistochemistry of brain samples from hTfR KI mice three days after administration of Antibody X or control hIgG. FIG. 5D is an image of immunohistochemistry staining of hIgG in the cerebral cortex from hTfR KI mice administered Antibody X. FIG. 6A is a graph depicting hIgG levels in cynomolgus monkey brain biopsy sections 48 h after treatment with 20 mg/kg hIgG or Antibody X intravenously. FIG. 6B is an image of cynomolgus monkey brain biopsy sections showing hIgG staining of the hippocampus, cerebellum, and cerebral cortex by immunohistochemistry 48 h after intravenous administration of 20 mg/kg negative control or Antibody X. FIG6C is an image of a cynomolgus monkey brain biopsy section after administration of Antibody X, showing vesicles within pyramidal neurons of the cerebral cortex. FIG7 is a series of sketches depicting control hIgG (left), anti-BACE1 (second from left), anti-BACE1/bivalent Antibody X (third from left), and anti-BACE1/monovalent Antibody X (right). FIG8A is a graph depicting plasma hIgG concentrations (nM) at the indicated time points in hTfR KI mice intravenously administered 50 mg/kg molar IgG equivalents of hIgG (squares), anti-BACE (circles), anti-BACE1/bivalent Antibody X (triangles, pointing down), or anti-BACE1/monovalent Antibody X (triangles, pointing up). Figure 8B is a graph depicting brain hIgG concentrations (nM) at the indicated time points in hTfR KI mice intravenously administered 50 mg/kg molar IgG equivalents of hIgG (squares), anti-BACE (circles), anti-BACE1/bivalent Antibody X (triangles, pointing down), or anti-BACE1/monovalent Antibody X (triangles, pointing up). Figure 8C is a graph depicting brain amyloid β1-40 (Aβ40; % control) at the indicated time points in hTfR KI mice intravenously administered 50 mg/kg molar IgG equivalents of hIgG (squares), anti-BACE (circles), anti-BACE1/bivalent Antibody X (triangles, pointing down), or anti-BACE1/monovalent Antibody X (triangles, pointing up). Figure 9A is a graph depicting total hIgG concentration (nM) in serum of hTfR KI mice 24 h after administration of the indicated constructs (see the legend in Figure 9E ). Figure 9B is a graph depicting total hIgG concentration (nM) in brain of hTfR KI mice 24 h after administration of the indicated constructs (see the legend in Figure 9E ). Figure 9C is a graph depicting free ASO concentration (nM) in serum of hTfR KI mice 24 h after administration of the indicated constructs (see the legend in Figure 9E ). Figure 9D is a graph depicting free ASO concentration (nM) in brain of hTfR KI mice three days after administration of 1.5 mg/kg ASO equivalent of the indicated constructs (see the legend in Figure 9E ) every Thursday. Figure 9E is a graph depicting Malat1 RNA normalized to Actb (percentage relative to DPBS vehicle mice) in the brain, spinal cord, tibialis anterior muscle, and liver of hTfR KI mice three days after administration of 1.5 mg/kg ASO equivalents of the indicated constructs every four weeks. Figure 10 is an atomic model of the variable domain of Antibody X constructed using cryo-electron microscopy density of the Antibody X Fab/TfR1/Tf ternary complex (shown in Figure 3A ). Figure 11 is a graph depicting antibody binding (MFI) of the indicated Antibody X mutants to CHO cells expressing human (hu) or cynomolgus monkey (cy) TfR1. Figure 12 is a graph depicting the Fab binding reaction of Fab fragments of Antibody X variants to hTfR1 ECD measured by surface plasmon resonance at pH 7.4 and pH 5.5 (resonance units, RU; y-axis). Figure 13A is a graph depicting the endocytosis of bivalent Antibody X and Antibody X mutants or control hIgG. Figure 13B is a graph depicting the bivalent endocytosis of Antibody X mutants plotted for monovalent affinity to hTfR1. Figure 14 is a series of graphs depicting hIgG concentrations (nM) in serum (top row) or brain (middle row) and the percentage of reticulocytes in whole blood (bottom row) at 1 day (left row) or 7 days (right row) after administration of the indicated constructs at 20 mg/kg IgG equivalents in hTfR KI mice. FIG. 15A is an image of a capillary Western blot of hTfR1 in Caco2 cell lysates after treatment with 1 uM of the indicated antibodies. Baf: bafilomycin. FIG. 15B is a graph depicting the levels of TfR1 in Caco2 cells treated with 1 uM of the indicated constructs for 24 h (% of control) plotted against the EC 50 for hTfR1-CHO cell binding of each antibody. FIG. 15C is a graph depicting the levels of reticulocytes in hTfR KI mice 24 h after intravenous administration of 20 mg/kg of these antibodies (% of control) plotted against the levels of TfR1 in Caco2 after 24 h treatment with 1 uM of the indicated constructs (see legend in FIG. 15B ). Figure 16A is a graph depicting total HPRT mRNA (%) in HEK293T cells 72 h after treatment with various concentrations of siRNA alone or the indicated conjugates. Figure 16B is a graph depicting endocytosis of monovalent Antibody X or control hIgG with and without siRNA. Figure 16C is a graph depicting HPRT RNA normalized to Actb (percentage relative to DPBS vehicle mice) in the brains of hTfR KI mice three days after administration of siRNA or siRNA conjugates at 4 mg/kg siRNA equivalents at four doses per week. Figure 17A is a graph depicting hIgG concentrations (nM) in serum at the indicated doses (3, 10, 30, and 90 mg/kg intravenous nemolar IgG equivalents) in hTfR KI mice 1 day after administration of hIgG control (white circles) or monovalent antibody X (black circles). Figure 17B is a graph depicting hIgG concentrations (nM) in brain at the indicated doses (3, 10, 30, and 90 mg/kg intravenous nemolar IgG equivalents) in hTfR KI mice 1 day after administration of hIgG control (white circles) or monovalent antibody X (black circles). Figure 17C is a graph depicting brain hIgG concentration (nM) expressed as a multiple of control at the indicated doses (3, 10, 30, and 90 mg/kg intravenous nemolar IgG equivalents) in hTFR KI mice 1 day after administration of hIgG control or monovalent Antibody X. Figure 18A is a series of sketches depicting control hIgG (left) and bispecific antibodies comprising Antibody Z (bivalent)/Antibody X (monovalent) (right). 18B is a graph depicting serum hIgG concentrations (nM) at the indicated time points in hTfR KI mice intravenously administered 3 mg/kg molar IgG equivalents of hIgG control (squares) or four bispecific antibodies incorporating Antibody X variant Fab fragments (Bivalent Antibody Z/Monovalent Antibody X (VH-S60A) (triangles, pointing up), Bivalent Antibody Z/Monovalent Antibody X (VL-S39N) (triangles, pointing down), Bivalent Antibody Z/Monovalent Antibody X (VH-S60A/VL-S39N) (diamonds), or Bivalent Antibody Z/Monovalent Antibody X (VL-Y57S) (circles)). 18C is a graph depicting brain hIgG concentrations (nM) at the indicated time points in hTfR KI mice intravenously administered 3 mg/kg molar IgG equivalents of hIgG control (squares) or four bispecific antibodies incorporating Antibody X variant Fab fragments (Bivalent Antibody Z/Monovalent Antibody X (VH-S60A) (triangles, pointing up), Bivalent Antibody Z/Monovalent Antibody X (VL-S39N) (triangles, pointing down), Bivalent Antibody Z/Monovalent Antibody X (VH-S60A/VL-S39N) (diamonds), or Bivalent Antibody Z/Monovalent Antibody X (VL-Y57S) (circles)). 18D is a graph depicting reticulocyte levels (% of control) at the indicated time points in hTfR KI mice intravenously administered 3 mg/kg molar IgG equivalents of hIgG control (squares) or four bispecific antibodies incorporating Antibody X variant Fab fragments (Bivalent Antibody Z/Monovalent Antibody X (VH-S60A) (triangles, pointing up), Bivalent Antibody Z/Monovalent Antibody X (VL-S39N) (triangles, pointing down), Bivalent Antibody Z/Monovalent Antibody X (VH-S60A/VL-S39N) (diamonds), or Bivalent Antibody Z/Monovalent Antibody X (VL-Y57S) (circles)).

TW202517685A_113140918_SEQL.xmlTW202517685A_113140918_SEQL.xml

Claims (42)

一種結合至人類運鐵蛋白受體之抗體，其包含有包含VH互補決定區(CDR)1、VH CDR2及VH CDR3之重鏈可變區(VH)及包含VL CDR1、VL CDR2及VL CDR3之輕鏈可變區(VL)，其中： 該VH CDR1包含胺基酸序列GFTFSSYX 1MN (SEQ ID NO:18)或胺基酸序列SYX 1MN (SEQ ID NO:26)，其中X 1為S或A； 該VH CDR2包含胺基酸序列SISX 2SSSX 3IYYADSVKG (SEQ ID NO:19)，其中X 2為S或A，且其中X 3為Y或S；且 該VH CDR3包含胺基酸序列KX 4X 5X 6GDFDY (SEQ ID NO:20)，其中X 4為Y或S，其中X 5為R或S，且其中X 6為A或Y； 該VL CDR1包含胺基酸序列RASQSVSSX 7X 8LA (SEQ ID NO:21)，其中X 7為S或N，且其中X 8為Y或N； 該VL CDR2包含胺基酸序列GASX 9RAT (SEQ ID NO:22)，其中X 9為N或S；且 該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)。 An antibody that binds to a human transferrin receptor comprises a heavy chain variable region (VH) comprising a VH complement determining region (CDR) 1, a VH CDR2 and a VH CDR3, and a light chain variable region (VL) comprising a VL CDR1, a VL CDR2 and a VL CDR3, wherein: the VH CDR1 comprises the amino acid sequence GFTFSSYX 1 MN (SEQ ID NO: 18) or the amino acid sequence SYX 1 MN (SEQ ID NO: 26), wherein X 1 is S or A; the VH CDR2 comprises the amino acid sequence SISX 2 SSSX 3 IYYADSVKG (SEQ ID NO: 19), wherein X 2 is S or A, and wherein X 3 is Y or S; and the VH CDR3 comprises the amino acid sequence KX 4 X 5 X 6 GDFDY (SEQ ID NO: 20), wherein X wherein X4 is Y or S, wherein X5 is R or S, and wherein X6 is A or Y; the VL CDR1 comprises the amino acid sequence RASQSVSSX 7 X 8 LA (SEQ ID NO:21), wherein X7 is S or N, and wherein X8 is Y or N; the VL CDR2 comprises the amino acid sequence GASX 9 RAT (SEQ ID NO:22), wherein X9 is N or S; and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8). 如請求項1之抗體，其中VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及/或VL CDR3中之至少一者選自表1中所描繪之突變體CDR，且未選自表1中所描繪之該等突變體CDR之VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2或VL CDR3中之任一者選自表1中所描繪之親代CDR。The antibody of claim 1, wherein at least one of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3 is selected from the mutant CDRs described in Table 1, and any of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 or VL CDR3 not selected from the mutant CDRs described in Table 1 is selected from the parent CDRs described in Table 1. 如請求項1之抗體，其中VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2或VL CDR3中之一者選自表1中所描繪之該等突變體CDR且VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及/或VL CDR3中之五者選自表1中所描繪之該等親代CDR。The antibody of claim 1, wherein one of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 or VL CDR3 is selected from the mutant CDRs described in Table 1 and five of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3 are selected from the parent CDRs described in Table 1. 如請求項1之抗體，其中VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及/或VL CDR3中之兩者選自表1中所描繪之該等突變體CDR且VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及/或VL CDR3中之四者選自表1中所描繪之該等親代CDR。The antibody of claim 1, wherein two of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3 are selected from the mutant CDRs described in Table 1 and four of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3 are selected from the parent CDRs described in Table 1. 如請求項1之抗體，其中VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及/或VL CDR3中之三者選自表1中所描繪之該等突變體CDR且VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及/或VL CDR3中之三者選自表1中所描繪之該等親代CDR。The antibody of claim 1, wherein three of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3 are selected from the mutant CDRs described in Table 1 and three of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3 are selected from the parent CDRs described in Table 1. 如請求項1之抗體，其中VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及/或VL CDR3中之四者選自表1中所描繪之該等突變體CDR且VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及/或VL CDR3中之兩者選自表1中所描繪之該等親代CDR。The antibody of claim 1, wherein four of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3 are selected from the mutant CDRs described in Table 1 and two of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3 are selected from the parent CDRs described in Table 1. 如請求項1之抗體，其中VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2及/或VL CDR3中之五者選自表1中所描繪之該等突變體CDR且VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2或VL CDR3中之一者選自表1中所描繪之該等親代CDR。The antibody of claim 1, wherein five of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3 are selected from the mutant CDRs described in Table 1 and one of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 or VL CDR3 is selected from the parent CDRs described in Table 1. 如請求項1之抗體，其中： 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)；或 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)。 The antibody of claim 1, wherein: the VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); or the VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); the VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8). 如請求項1之抗體，其中： 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)，其中該VL包含Y57S保守取代； 該VH CDR1包含胺基酸序列GFTFSSYAMN (SEQ ID NO:9)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)，其中該VL包含Y57S保守取代； 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSNYLA (SEQ ID NO:15)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISSSSSSIYYADSVKG (SEQ ID NO:11)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KSRAGDFDY (SEQ ID NO:12)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYSAGDFDY (SEQ ID NO:13)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRYGDFDY (SEQ ID NO:14)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISASSSSIYYADSVKG (SEQ ID NO:25)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；該VH CDR3包含胺基酸序列KYSAGDFDY (SEQ ID NO:13)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSNYLA (SEQ ID NO:15)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSNLA (SEQ ID NO:16)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列GFTFSSYSMN (SEQ ID NO:3)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASSRAT (SEQ ID NO:17)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)，其中該VL包含Y57S保守取代； 該VH CDR1包含胺基酸序列SYAMN (SEQ ID NO:24)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)，其中該VL包含Y57S保守取代； 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSNYLA (SEQ ID NO:15)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISSSSSSIYYADSVKG (SEQ ID NO:11)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KSRAGDFDY (SEQ ID NO:12)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYSAGDFDY (SEQ ID NO:13)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRYGDFDY (SEQ ID NO:14)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISASSSSIYYADSVKG (SEQ ID NO:25)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISASSSYIYYADSVKG (SEQ ID NO:10)；該VH CDR3包含胺基酸序列KYSAGDFDY (SEQ ID NO:13)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSNYLA (SEQ ID NO:15)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)； 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSNLA (SEQ ID NO:16)；該VL CDR2包含胺基酸序列GASNRAT (SEQ ID NO:7)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)；或 該VH CDR1包含胺基酸序列SYSMN (SEQ ID NO:23)；該VH CDR2包含胺基酸序列SISSSSSYIYYADSVKG (SEQ ID NO:4)；該VH CDR3包含胺基酸序列KYRAGDFDY (SEQ ID NO:5)；該VL CDR1包含胺基酸序列RASQSVSSSYLA (SEQ ID NO:6)；該VL CDR2包含胺基酸序列GASSRAT (SEQ ID NO:17)；且該VL CDR3包含胺基酸序列QQQSSSPPT (SEQ ID NO:8)。 The antibody of claim 1, wherein: the VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8), wherein the VL comprises a Y57S conservative substitution; the VH CDR1 comprises the amino acid sequence GFTFSSYAMN (SEQ ID NO:9); the VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); the VH The CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); NO:8); the VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8), wherein the VL comprises a Y57S conservative substitution; the VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSNYLA (SEQ ID NO:15); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence SISSSSSSIYYADSVKG (SEQ ID NO:11); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH The CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KSRAGDFDY (SEQ ID NO:12); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); The VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KYSAGDFDY (SEQ ID NO:13); the VL The CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence SISSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KYRYGDFDY (SEQ ID NO:14); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence SISASSSSIYYADSVKG (SEQ ID NO:25); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); the VH CDR3 comprises the amino acid sequence KYSAGDFDY (SEQ ID NO:13); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSNYLA (SEQ ID NO:15); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH The VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSNLA (SEQ ID NO:16); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); The VH CDR1 comprises the amino acid sequence GFTFSSYSMN (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL The CDR2 comprises the amino acid sequence GASSRAT (SEQ ID NO:17); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); the VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8), wherein the VL comprises a Y57S conservative substitution; the VH CDR1 comprises the amino acid sequence SYAMN (SEQ ID NO:24); the VH The CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); the VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); the VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8), wherein the VL comprises a Y57S conservative substitution; the VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); the VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSNYLA (SEQ ID NO:15); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); the VH CDR2 comprises the amino acid sequence SISSSSSSIYYADSVKG (SEQ ID NO:11); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); the VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KSRAGDFDY (SEQ ID NO:12); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); the VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KYSAGDFDY (SEQ ID NO:13); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); the VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KYRYGDFDY (SEQ ID NO:14); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH The CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); the VH CDR2 comprises the amino acid sequence SISASSSSIYYADSVKG (SEQ ID NO:25); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); The VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); the VH CDR2 comprises the amino acid sequence SISASSSYIYYADSVKG (SEQ ID NO:10); the VH CDR3 comprises the amino acid sequence KYSAGDFDY (SEQ ID NO:13); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); the VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSNYLA (SEQ ID NO:15); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); the VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); the VH The VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSNLA (SEQ ID NO:16); the VL CDR2 comprises the amino acid sequence GASNRAT (SEQ ID NO:7); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8); or the VH CDR1 comprises the amino acid sequence SYSMN (SEQ ID NO:23); the VH CDR2 comprises the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO:4); the VH CDR3 comprises the amino acid sequence KYRAGDFDY (SEQ ID NO:5); the VL CDR1 comprises the amino acid sequence RASQSVSSSYLA (SEQ ID NO:6); the VL CDR2 comprises the amino acid sequence GASSRAT (SEQ ID NO:17); and the VL CDR3 comprises the amino acid sequence QQQSSSPPT (SEQ ID NO:8). 如前述請求項中任一項之抗體，其中： (i) 該VH與SEQ ID NO:100-108中之任一者至少80%、85%、90%、95%、99%或100%一致；且 (ii) 該VL與SEQ ID NO:200-204中之任一者至少80%、85%、90%、95%、99%或100%一致。 An antibody as claimed in any of the preceding claims, wherein: (i) the VH is at least 80%, 85%, 90%, 95%, 99% or 100% identical to any of SEQ ID NOs: 100-108; and (ii) the VL is at least 80%, 85%, 90%, 95%, 99% or 100% identical to any of SEQ ID NOs: 200-204. 如請求項1之抗體，其中該VH包含SEQ ID NO:100-108中任一者之胺基酸序列且該VL包含SEQ ID NO:200-204中任一者之胺基酸序列。The antibody of claim 1, wherein the VH comprises the amino acid sequence of any one of SEQ ID NOs: 100-108 and the VL comprises the amino acid sequence of any one of SEQ ID NOs: 200-204. 如前述請求項中任一項之抗體，其中該抗體為(a)單價的且對hTfR1具有＞ 10 nM之單價親和力(K D)，或為二價的且對hTfR1具有＞100 nM之單價親和力(K D)，及/或(b)具有＞= 0.01/s之解離速率(k d)。 The antibody of any of the preceding claims, wherein the antibody is (a) monovalent and has a monovalent affinity ( KD ) of >10 nM for hTfR1, or is bivalent and has a monovalent affinity ( KD ) of >100 nM for hTfR1, and/or (b) has a dissociation rate ( kd ) of >= 0.01/s. 如請求項1之抗體，其中： 該VH包含該SEQ ID NO:100之胺基酸序列且該VL包含該SEQ ID NO:200之胺基酸序列。 The antibody of claim 1, wherein: the VH comprises the amino acid sequence of SEQ ID NO:100 and the VL comprises the amino acid sequence of SEQ ID NO:200. 如請求項1之抗體，其中： 該VH包含該SEQ ID NO:101之胺基酸序列且該VL包含該SEQ ID NO:200之胺基酸序列； 該VH包含該SEQ ID NO:102之胺基酸序列且該VL包含該SEQ ID NO:200之胺基酸序列； 該VH包含該SEQ ID NO:102之胺基酸序列且該VL包含該SEQ ID NO:201之胺基酸序列； 該VH包含該SEQ ID NO:102之胺基酸序列且該VL包含該SEQ ID NO:203之胺基酸序列； 該VH包含該SEQ ID NO:103之胺基酸序列且該VL包含該SEQ ID NO:200之胺基酸序列； 該VH包含該SEQ ID NO:104之胺基酸序列且該VL包含該SEQ ID NO:200之胺基酸序列； 該VH包含該SEQ ID NO:105之胺基酸序列且該VL包含該SEQ ID NO:200之胺基酸序列； 該VH包含該SEQ ID NO:106之胺基酸序列且該VL包含該SEQ ID NO:200之胺基酸序列； 該VH包含該SEQ ID NO:107之胺基酸序列且該VL包含該SEQ ID NO:200之胺基酸序列； 該VH包含該SEQ ID NO:108之胺基酸序列且該VL包含該SEQ ID NO:200之胺基酸序列； 該VH包含該SEQ ID NO:100之胺基酸序列且該VL包含該SEQ ID NO:201之胺基酸序列； 該VH包含該SEQ ID NO:100之胺基酸序列且該VL包含該SEQ ID NO:202之胺基酸序列； 該VH包含該SEQ ID NO:100之胺基酸序列且該VL包含該SEQ ID NO:203之胺基酸序列；或 該VH包含該SEQ ID NO:100之胺基酸序列且該VL包含該SEQ ID NO:204之胺基酸序列。 The antibody of claim 1, wherein: the VH comprises the amino acid sequence of SEQ ID NO:101 and the VL comprises the amino acid sequence of SEQ ID NO:200; the VH comprises the amino acid sequence of SEQ ID NO:102 and the VL comprises the amino acid sequence of SEQ ID NO:200; the VH comprises the amino acid sequence of SEQ ID NO:102 and the VL comprises the amino acid sequence of SEQ ID NO:201; the VH comprises the amino acid sequence of SEQ ID NO:102 and the VL comprises the amino acid sequence of SEQ ID NO:203; the VH comprises the amino acid sequence of SEQ ID NO:103 and the VL comprises the amino acid sequence of SEQ ID NO:200; the VH comprises the amino acid sequence of SEQ ID NO:104 and the VL comprises the amino acid sequence of SEQ ID NO:200; The VH comprises the amino acid sequence of SEQ ID NO:105 and the VL comprises the amino acid sequence of SEQ ID NO:200; The VH comprises the amino acid sequence of SEQ ID NO:106 and the VL comprises the amino acid sequence of SEQ ID NO:200; The VH comprises the amino acid sequence of SEQ ID NO:107 and the VL comprises the amino acid sequence of SEQ ID NO:200; The VH comprises the amino acid sequence of SEQ ID NO:108 and the VL comprises the amino acid sequence of SEQ ID NO:200; The VH comprises the amino acid sequence of SEQ ID NO:100 and the VL comprises the amino acid sequence of SEQ ID NO:201; The VH comprises the amino acid sequence of SEQ ID NO:100 and the VL comprises the amino acid sequence of SEQ ID NO:202; The VH comprises the amino acid sequence of SEQ ID NO:107 and the VL comprises the amino acid sequence of SEQ ID NO:200; The VH comprises the amino acid sequence of SEQ ID NO:108 and the VL comprises the amino acid sequence of SEQ ID NO:200; The VH comprises the amino acid sequence of SEQ ID NO:100 and the VL comprises the amino acid sequence of SEQ ID NO:201; The VH comprises the amino acid sequence of SEQ ID NO:100 and the VL comprises the amino acid sequence of SEQ ID NO:202 NO:100 and the VL comprises the amino acid sequence of SEQ ID NO:203; or the VH comprises the amino acid sequence of SEQ ID NO:100 and the VL comprises the amino acid sequence of SEQ ID NO:204. 如請求項1至14中任一項之抗體，其為多特異性抗體、雙特異性抗體、單鏈抗體、Fab片段、F(ab') 2片段、Fab'片段、Fsc片段、Fv片段、scFv、sc(Fv) 2、或雙價抗體。 The antibody of any one of claims 1 to 14, which is a multispecific antibody, a bispecific antibody, a single chain antibody, a Fab fragment, a F(ab') 2 fragment, a Fab' fragment, a Fsc fragment, a Fv fragment, scFv, sc(Fv) 2 , or a bivalent antibody. 如請求項1至14中任一項之抗體，其包含恆定重鏈(CH)域及恆定輕鏈(CL)域。The antibody of any one of claims 1 to 14, comprising a constant heavy chain (CH) domain and a constant light chain (CL) domain. 如請求項1之抗體，其中該抗體包含： (a) 包含400中所列出之胺基酸序列之重鏈，及包含401中所列出之胺基酸序列之輕鏈； (b) SEQ ID NO: 401及402中所列出之胺基酸序列； (c) SEQ ID NO: 401、404及406中所列出之胺基酸序列； (d) SEQ ID NO:400及401中所列出之胺基酸序列； (e) SEQ ID NO: 401、405及406中所列出之胺基酸序列；或 (f) SEQ ID NO: 401及403中所列出之胺基酸序列。 An antibody as claimed in claim 1, wherein the antibody comprises: (a) a heavy chain comprising the amino acid sequence listed in 400, and a light chain comprising the amino acid sequence listed in 401; (b) the amino acid sequences listed in SEQ ID NOs: 401 and 402; (c) the amino acid sequences listed in SEQ ID NOs: 401, 404 and 406; (d) the amino acid sequences listed in SEQ ID NOs: 400 and 401; (e) the amino acid sequences listed in SEQ ID NOs: 401, 405 and 406; or (f) the amino acid sequences listed in SEQ ID NOs: 401 and 403. 一種核酸或多種核酸，其編碼如請求項1至17中任一項之抗體。A nucleic acid or a plurality of nucleic acids encoding the antibody of any one of claims 1 to 17. 一種表現載體或多種表現載體，其包含可操作地連接至啟動子之如請求項18之該核酸或該等核酸。An expression vector or a plurality of expression vectors comprising the nucleic acid or nucleic acids of claim 18 operably linked to a promoter. 一種經分離細胞，其包含如請求項18之該核酸或該等核酸或如請求項19之該表現載體或該等表現載體。An isolated cell comprising the nucleic acid or nucleic acids of claim 18 or the expression vector or expression vectors of claim 19. 一種經分離細胞，其包含：包含可操作地連接至啟動子之第一核酸之第一表現載體，該第一核酸編碼包含如請求項1至17中任一項之抗體之VH之第一多肽；及包含可操作地連接至啟動子之第二核酸之第二表現載體，該第二核酸編碼包含如請求項1至17中任一項之抗體之VL之第二多肽。A kind of isolated cell, comprising: a first expression vector comprising a first nucleic acid operably linked to a promoter, the first nucleic acid encoding a first polypeptide comprising the VH of any one of claims 1 to 17; and a second expression vector comprising a second nucleic acid operably linked to a promoter, the second nucleic acid encoding a second polypeptide comprising the VL of any one of claims 1 to 17. 一種製備如請求項1至17中任一項之抗體的方法，其包含培養如請求項20或21之細胞且分離該抗體。A method for preparing the antibody of any one of claims 1 to 17, comprising culturing the cell of claim 20 or 21 and isolating the antibody. 一種醫藥組合物，其包含如請求項1至17中任一項之抗體及醫藥學上可接受之載劑。A pharmaceutical composition comprising the antibody of any one of claims 1 to 17 and a pharmaceutically acceptable carrier. 一種結合物，其包含如請求項1至17中任一項之抗體及劑。A conjugate comprising the antibody of any one of claims 1 to 17 and an agent. 如請求項24之結合物，其中該劑為抗體、蛋白質或肽。The conjugate of claim 24, wherein the agent is an antibody, a protein or a peptide. 如請求項24之結合物，其中該劑為抗β澱粉樣蛋白抗體。The conjugate of claim 24, wherein the agent is an anti-β-amyloid antibody. 如請求項26之結合物，其中抗β澱粉樣蛋白抗體為阿杜卡單抗(aducanumab)、巴匹珠單抗(bapineuzumab)、甘特魯單抗(gantenerumab)、索拉珠單抗(solanezumab)、多奈單抗(donanemab)或雷卡奈單抗(lecanemab)。The conjugate of claim 26, wherein the anti-β-amyloid antibody is aducanumab, bapineuzumab, gantenerumab, solanezumab, donanemab or lecanemab. 如請求項24之結合物，其中該劑為抗τ抗體、抗α突觸核蛋白抗體、抗TDP-43抗體、抗LINGO-1抗體、抗LINGO-2抗體、抗LINGO-3抗體、抗LINGO-4抗體、抗TREM2抗體或抗C9orf72二肽重複聚GA抗體。The conjugate of claim 24, wherein the agent is an anti-tau antibody, an anti-α-synuclein antibody, an anti-TDP-43 antibody, an anti-LINGO-1 antibody, an anti-LINGO-2 antibody, an anti-LINGO-3 antibody, an anti-LINGO-4 antibody, an anti-TREM2 antibody, or an anti-C9orf72 dipeptide repeat poly-GA antibody. 如請求項24之結合物，其中該劑為蛋白質。The conjugate of claim 24, wherein the agent is a protein. 如請求項29之結合物，其中該蛋白質為顆粒蛋白前體。The conjugate of claim 29, wherein the protein is a granular protein precursor. 如請求項24之結合物，其中該劑為酶。The conjugate of claim 24, wherein the agent is an enzyme. 如請求項31之結合物，其中該酶為葡糖腦苷脂酶。The conjugate of claim 31, wherein the enzyme is glucocerebrosidase. 如請求項24至32中任一項之結合物，其中該結合物為包含該抗體及該劑之重組融合蛋白。The conjugate of any one of claims 24 to 32, wherein the conjugate is a recombinant fusion protein comprising the antibody and the agent. 如請求項24之結合物，其中該劑為核酸。The conjugate of claim 24, wherein the agent is a nucleic acid. 如請求項34之結合物，其中該核酸為mRNA、siRNA、反義寡核苷酸、微小RNA (miRNA)、嚮導RNA (gRNA)或胺基磷酸酯嗎啉基寡聚物(PMO)。The conjugate of claim 34, wherein the nucleic acid is mRNA, siRNA, antisense oligonucleotide, micro RNA (miRNA), guide RNA (gRNA) or phosphoamino morpholino oligomer (PMO). 如請求項34或35之結合物，其中該核酸經由連接子連接至該抗體。The conjugate of claim 34 or 35, wherein the nucleic acid is linked to the antibody via a linker. 如請求項24之結合物，其中該劑為奈米粒子、脂質體或病毒載體。The conjugate of claim 24, wherein the agent is a nanoparticle, a liposome or a viral vector. 一種經由胞吞轉送作用跨血腦障壁轉運劑之方法，該方法包含向人類個體投與如請求項24至37中任一項之結合物。A method for transporting an agent across the blood-brain barrier via endocytosis, the method comprising administering the conjugate of any one of claims 24 to 37 to a human subject. 一種活體內遞送劑之方法，該方法包含向人類個體投與如請求項24至37中任一項之結合物。A method for in vivo delivery, comprising administering the conjugate of any one of claims 24 to 37 to a human subject. 如請求項39之方法，其中該人類個體患有神經病症且該方法將該劑遞送至腦組織。The method of claim 39, wherein the human subject suffers from a neurological disorder and the method delivers the agent to brain tissue. 如請求項39之方法，其中該神經病症為阿茲海默氏病(Alzheimer’s disease)、帕金森氏病(Parkinson’s disease)、額顳葉失智症、ALS、杭丁頓氏病(Huntington’s disease)、多發性硬化症、脊髓性肌萎縮、肌肉營養不良、脊髓損傷、中風、眼科疾患、急性或慢性視神經炎、精神異常(psychiatric disorder)、圖雷特氏病(Tourette's disease)腦損傷、腦腫瘤或癲癇。The method of claim 39, wherein the neurological disorder is Alzheimer's disease, Parkinson's disease, frontotemporal dementia, ALS, Huntington's disease, multiple sclerosis, spinal muscular atrophy, muscular dystrophy, spinal cord injury, stroke, ophthalmological disease, acute or chronic optic neuritis, psychiatric disorder, Tourette's disease, brain injury, brain tumor, or epilepsy. 一種治療有需要之人類個體之阿茲海默氏病之方法，其包含向該人類個體投與治療有效量之如請求項26或27之結合物。A method for treating Alzheimer's disease in a human subject in need thereof, comprising administering to the human subject a therapeutically effective amount of the conjugate of claim 26 or 27.
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Family Cites Families (15)

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WO1991004753A1 (en) 1989-10-02 1991-04-18 Cetus Corporation Conjugates of antisense oligonucleotides and therapeutic uses thereof
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US9598496B2 (en) * 2011-05-09 2017-03-21 Perseus Proteomics Inc. Antibody capable of specifically recognizing transferrin receptor
US20150353639A1 (en) * 2012-05-21 2015-12-10 Genentech, Inc. Methods for improving safety of blood-brain barrier transport
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MA45328A (en) 2016-04-01 2019-02-06 Avidity Biosciences Llc NUCLEIC ACID-POLYPEPTIDE COMPOSITIONS AND USES THEREOF
WO2017221883A1 (en) 2016-06-20 2017-12-28 武田薬品工業株式会社 Antibody-drug conjugate
KR20250148679A (en) 2017-12-06 2025-10-14 어비디티 바이오사이언시스 인크. Compositions and methods of treating muscle atrophy and myotonic dystrophy
IL319265A (en) * 2018-12-21 2025-04-01 Avidity Biosciences Inc Anti-transferrin receptor antibodies and uses thereof
KR20210108961A (en) * 2018-12-28 2021-09-03 쿄와 기린 가부시키가이샤 Bispecific Antibodies that Bind to TfR
EP4185315A4 (en) * 2020-07-23 2024-07-31 Dyne Therapeutics, Inc. Anti-transferrin receptor (tfr) antibody and uses thereof
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